, Volume 15, Issue 7, pp 536-553
Date: 21 Oct 2008

Changes in toxicity and Ah receptor agonist activity of suspended particulate matter during flood events at the rivers Neckar and Rhine — a mass balance approach using in vitro methods and chemical analysis

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Background, aim, and scope

As a consequence of flood events, runoff and remobilized sediments may cause an increase of ecotoxicologically relevant effects from contaminant reservoirs. Aquatic and terrestrial organisms as well as cattle and areas of settlement are exposed to dislocated contaminants during and after flood events. In this study, the impacts of two flood events triggered by intense rain at the rivers Neckar and Rhine (Southern Germany) were studied. Effects in correlation to flood flow were assessed at the river Neckar using samples collected at frequent intervals. River Rhine suspended particulate matter (SPM) was sampled over a longer period at normal flow and during a flood event. Three cell lines (H4L1.1c4, GPC.2D.Luc, RTL-W1) were used to compare Ah receptor agonist activity in different biotest systems. Multilayer fractionation was performed to identify causative compounds, focusing on persistent organic contaminants.

Materials and methods

Native water and SPM of flood events were collected at the river Neckar and at the monitoring station (Rheinguetestation, Worms, Germany) of the river Rhine. Water samples were XAD-extracted. SPM were freeze-dried and Soxhlet-extracted using acetone and finally dissolved in dimethyl sulfoxide. Resulting crude extracts were analyzed for cytotoxicity with the neutral red assay. Aryl hydrocarbon receptor (AhR) agonist activity was measured in a set of biological test systems (DR-CALUX, GPC.2D, and ethoxyresorufin-O-deethylase (EROD) assay) and different cell lines. In addition, crude extracts were fractionated using a combined method of multilayer (sequence of acidified silica layers) and carbon fractionation. Fractions from the multilayer fractionation contained persistent organic compounds (polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), polychlorinated biphenyls (PCBs), and some polycyclic aromatic hydrocarbon (PAHs)); fractions from the carbon fractionation were separated into a PCDD/F and a PCB fraction. Dioxin-like activity of multilayer and carbon fractions was determined in the EROD assay and expressed as biological toxicity equivalency concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (bio-TEQs). The calculation of chemical equivalency concentrations (chem-TEQs) and comparison to bio-TEQ values allowed the determination of the contribution of the analyzed persistent compounds to the total biological effects measured.


Soluble compounds in native and extracted water samples resulted in no or minor activity in the toxicity tests, respectively. Filter residues of native water caused increased AhR-mediated activity at the peak of the flood. Activities of SPM of the river Neckar correlated well with the flow rate indicating a flood-dependent increase of toxicity culminating at the peak of flow. River Rhine SPM showed a decrease of activity regarding an SPM sample of the flood event compared to a long-term sample. Excellent correlations with AhR agonistic activity were determined for DR-CALUX and EROD assay, while the GPC.2D assay did not correlate with both other biotests. The activity of persistent dioxin-like acting compounds in multilayer and carbon fractionated PCDD/F and PCB fractions was low if compared to corresponding crude extracts. The congener pattern of PCDD/F revealed that the contaminations mainly originated from products and productions of the chlorine and organochlorine industries.


Native and extracted water samples could be shown to contain little or no cytotoxic or AhR agonistic compounds. In contrast, particle-bound compounds were shown to be the relevant effect-causing fraction, as indicated by the activities of filter residues of native water and SPM. Compounds other than fractionated persistent PCBs and PCDD/Fs were more relevant to explain AhR-mediated activities of crude flood SPM at both rivers assessed. Biologically detected activities could at least in part be traced back to chemically analyzed and quantified compounds.


The calculation of the portion of persistent PCBs and PCDD/Fs in multilayer fractions causing the high inductions in the EROD assay in combination with chemical analysis provides a suitable tool to assess dioxin-like activity of persistent compounds in SPM sampled over the course of flood events. Depending on the catchment area and annual course of flood events, end points may either indicate an increase or a decrease of activity. In order to determine the ecological hazard potential of mobilized contaminants during flood events, the focus should be set on particle-bound pollutants. Furthermore, PCDD/Fs and PCBs, commonly expected to be the most relevant pollutants in river systems, could be shown to contribute only to a minor portion of the overall AhR-mediated activity. However, they might be most relevant for human exposure when considering persistence and bioaccumulation–biomagnification in the food chain.

Recommendations and perspectives

As a consequence of climate change, flood events will increase in frequency and intensity at least in some regions such as Central Europe. Thus, it is crucial to identify the potential hazard of (re-)mobilized contaminants from reservoirs dislocated via floods and threatening especially aquatic organisms and cattle grazing in flood plains. Since other less persistent compounds seem to be more relevant to explain AhR-mediated activities in flood SPM, nonconventional PAHs and more polar compounds also need to be considered for risk assessment. Effect-directed analysis using broad-range fractionation methods taking into account compounds from polar to nonpolar should be applied for identification of pollutants causing biological effects, thus integrating biological and chemical parameters.

Responsible editors: Roland Weber, Mats Tysklind, Caroline Gaus