Different natural organic matter isolates cause similar stress response patterns in the freshwater amphipod, Gammarus pulex

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 consistent 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. 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 10A degrees 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 quantitatively, and stress proteins, HSP70, as well as small alpha-crystalline HSPs were analyzed semiquantitatively. The three NOMs clearly differed in molecular masses, humic substance contents, the moieties of polysaccharides, 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. Although the quality of the exposed NOMs differs significantly, a rather uniform response pattern appears in the animals. Obviously, the contrasting contents of HSs and polysaccharides did not affect the anti-stress response of the exposed gammarids which is in contrast to previous lifespan studies with Caenorhabditis elegans. Furthermore, all NOM sources led to increased contents of both HSP70 and sHSPs. To the best of our knowledge, this is the first protein study to show that also small HSPs are expressed when the animals are exposed against humic material. Since the response patterns of the exposed gammarids, in contrast to the initial hypothesis, are rather uniform and since HSs are parts of life on Earth, we furthermore presume that they may have been a primordial exogenous trigger for the development of anti-stress systems in exposed organisms. Effect studies of chemical stresses on organisms should consider exposure to both natural triggers and xenobiotic compounds in low concentrations-in order to prospectively differentiate between these triggers and, subsequently, classify them.