Botulinum neurotoxin C1 (BoNt C1) and its corresponding gene were detected in seven aquatic habitats covering a range of low (LR) to high risk (HR) avian botulism outbreak areas during a study period of 10 months. Toxin and gene in sediment and avian faecal samples were analysed before (in situ) and after cultivation (in vitro) by a newly adapted ELISA, the common mouse bioassay and by a recently described nested PCR protocol. BoNt C1 gene fragments were detected in 74% and 83% of all investigated sediment samples by in situ PCR and in vitro PCR, respectively, at comparable frequencies in HR and LR areas. Similar high values were also observed for faecal samples. No BoNt C1 could be detected in the sediment in situ, while 53% and 56% of all cultivated samples contained BoNt C1 as detected in the mouse bioassay and the ELISA, respectively. The percentage of BoNt C1 positive cultivated samples was significantly higher (2-fold) in HR areas than in LR areas. Hence, our data clearly indicate an increased ratio of potentially BoNt C1 producing clostridia to BoNt C1 genes as the frequency or likelihood of botulinum epizootics increases in the environment. In addition, the good correlation between the results from the ELISA and the mouse bioassay for all sediment and faecal samples (r=0.90, p<0.001, n=121) indicates a high potential for the ELISA to reduce/replace the mouse bioassay for the detection of BoNt C1 in environmental samples.