Impact of heterophylly on the sensitivity of Myriophyllum aquaticum biotests

Background Parrot feather ( Myriophyllum aquaticum ) is an amphibious macrophyte with an inducible heterophylly. M. aquaticum plants adapted to the emersed state have leaves with a distinct cuticle and water repellent properties. In contrast M. aquaticum plants adapted to the submerged state have leaves typical for submerged hydrophytes with a strongly reduced cuticle. The aim of the study was to evaluate if this heterophylly of M. aquaticum affects the results of macrophyte biotests. Therefore, the two model substances atrazine and 2,4-dichlorophenoxyacetic acid (2,4-D) were tested each with three M. aquaticum biotests, the only modified parameter being the adaptation time to submergence (0, 7, 28 days). Results Root length was the most sensitive growth endpoint regarding the test substances atrazine and 2,4-D. Biotests with plants adapted to the submerged state show three times more sensitive results (EC50 7d 142.2 μg/L, EC50 28d 154.5 μg/L) than biotests with plants without an adaptation phase (EC50 0d 458.8 μg/L) in case of atrazine and five times more sensitive results (EC50 28d 46.9 μg/L, EC50 0d 246.3 μg/L) in case of 2,4-D. Apart from the differences in sensitivity, the differently adapted M. aquaticum plants show a completely different growth behavior. The growth rates based on shoot length were nearly ten times higher in the biotests with not adapted M. aquaticum plants than in the biotests with plants 28 days adapted to submergence. Additional measurements of the quantum yield of PSII could demonstrate that rapid growth in length is not based on photosynthetic carbon assimilation. Conclusions The heterophylly of M. aquaticum affects significantly the sensitivity of aquatic macrophyte biotests and should be taken into account in the development of a standardized test design. The 2,4-D results show the importance of an additional macrophyte biotest to the Lemna test, where the no observed effect concentration (NOEC) value is more than 30 times lower (7 μg/L) than the NOEC value of Lemna in the literature (270 μg/L). Furthermore, the growth rate endpoint in macrophyte biotests should not be misinterpreted. Rapid shoot elongation of amphibious macrophytes, which become submerged, is mainly caused by ethylene-triggered endogenous processes that are not connected to photosynthetic carbon assimilation and appear to be part of a stress reaction to avoid adverse environmental conditions.