HUMIC SUBSTANCES • SERIES • RESEARCH ARTICLE Different natural organic matter isolates cause similar stress response patterns in the freshwater amphipod, Gammarus pulex Darya S. Bedulina & Maxim A. Timofeyev & Martin Zimmer & Elke Zwirnmann & Ralph Menzel & Christian E. W. Steinberg Received: 9 January 2009 / Accepted: 22 June 2009 / Published online: 21 July 2009 # Springer-Verlag 2009 Abstract Background, aim, and scope Dissolved humic substances (HSs) are exogenous stressors to aquatic plants and animals which activate a variety of transcriptional and biochemical reactions or block photosynthesis. While there are consis- tent indications which structures may lead to reduced photosynthetic activity, there is much less clear information available on which HS structures or building blocks act as stressors in animals. Consequently, this work was designed to comparatively study the impact of natural organic matter (NOMs) from different sources on major anti-stress mechanisms in one single animal. We utilized major antioxidant responses and relative expression levels of stress proteins (small HSPs and HSP70) and expected that different HSs provoke different response patterns. Materials and methods We tested the freshwater amphipod Gammarus pulex which was collected from several shallow creeks in Northern Germany. All specimens were maintained in aerated 5-L tanks with filtered water from their natural environment at 10°C with prior acclimation. Animals were fed ad libitum with a commercial preparation once every second day. The exposure water was exchanged with the same frequency. NOMs were isolated from three different sources: two from small brown-water lakes in Northern Germany by reverse osmosis and the third one as an aqueous extract from a black layer of a Brazilian sandbar soil (State of Rio de Janeiro). The rationale was to apply NOMs of contrasting quality. Chemical fingerprint features of the NOMs were taken by high-performance size exclusion chromatography. As stress parameters in the animals, the activities of peroxidase and catalase were recorded quantita- tively, and stress proteins, HSP70, as well as small α- crystalline HSPs were analyzed semiquantitatively. Results The three NOMs clearly differed in molecular masses, humic substance contents, the moieties of poly- saccharides, and low-molecular-weight substances. With the exception of one short-term response, the peroxidase activity increased after 3 to 12 h exposure, whereas the catalase activity did not show any significant modulation. With one exception, the stress protein expression increased after 30 min exposure in a biphasic pattern, and the sHSPs responded less strongly than HSP70. Discussion Although the quality of the exposed NOMs differs significantly, a rather uniform response pattern appears in the animals. Obviously, the contrasting contents Responsible editors: Christian E. W. Steinberg and Markus Hecker D. S. Bedulina : M. A. Timofeyev (*) Irkutsk State University, Ul. Karl-Marx 1, 664003 Irkutsk, Russia e-mail: m.a.timofeyev@gmail.com M. A. Timofeyev Baikalian Research Centre, Ul. Karl-Marx 5-10, 664003 Irkutsk, Russia D. S. Bedulina : M. Zimmer Zoologisches Institut, Christian-Albrechts-Universität, Am Botanischen Garten 9, 24118 Kiel, Germany E. Zwirnmann Leibniz Institute of Freshwater Ecology and Inland Fisheries, Müggelseedamm 310, 12587 Berlin, Germany R. Menzel : C. E. W. Steinberg Department of Biology, Humboldt-Universität zu Berlin, Arboretum, Späthstraße 80/81, 12437 Berlin, Germany Environ Sci Pollut Res (2010) 17:261–269 DOI 10.1007/s11356-009-0222-5