Toxicity of pesticides and their mixture to primary producers

Abstract

The earth’s aquatic ecosystems are threatened by the contamination with a multitude of anthropogenic chemical pollutants. Pesticides are one important group of environmental contaminants. They are frequently detected in our surface waters and occur as single substances and in mixtures of various compositions. Copper (Cu) is often used as fungicide and herbicide in orcharding and viniculture. Cu toxicity is generally linked to the bioavailable fraction, i.e. to the concentration of the free Cu2+. In phototrophic organisms, one toxic mode of action of Cu is due to the inhibition of photosynthesis. Phytotoxicity of Cu was also found to be related to the generation of reactive oxygen species (ROS). However, until now, it is not clear, whether ROS are a mere consequence of Cu toxicity or the primary cause. One objective of this dissertation thesis was thus to investigate the role of ROS in the toxicity of Cu to phototrophic organisms to gain a better understanding of its toxicity mechanism. The two freshwater green algal species Pseudokirchneriella subcapitata and Chlorella vulgaris were chosen as test organisms. Cu-induced ROS formation was investigated in relation to short-term effects on photosynthetic activity and long-term effects on growth of P. subcapitata and C. vulgaris. Photosynthetic activity was determined as in vivo chlorophyll fluorescence. Exposure to 30 nM and 300 nM Cu resulted in a light and time dependent increase in ROS concentrations in P. subcapitata and C. vulgaris. The potential of Cu to induce ROS was comparable in both algae but the effect on photosynthesis differed with 300 nM Cu leading to a 12 % reduction of photosynthetic activity in P. subcapitata but not C. vulgaris after 24 h. This indicates that species-specific sensitivities were not caused by differences in ROS content but more likely resulted from differences in each ROS defence systems. The ROS scavenger N-tert-butyl-α-phenylnitrone (BPN) diminished Cu induced ROS production to control levels and completely restored Cu inhibiting effects on photosynthetic activity of P. subcapitata. This implies that ROS may play a primary role in the mechanism of copper toxicity to photosynthesis in algal cells. Further experiments revealed a time-dependent ROS release process across the plasma membrane. More than 90 % of total ROS were determined to be extracellular in P. subcapitata, indicating an efficient way of cellular protection against oxidative stress. Besides Cu, the triazine atrazine and the phenyl urea herbicides isoproturon and diuron are frequently detected in our surface waters. In contrast to Cu, phytotoxicity of these three pesticides is due to one specific mode of action which has been thoroughly investigated. Atrazine, isoproturon, and diuron inhibit photosynthesis by interrupting electron transport through photosystem II (PSII). Laboratory studies demonstrated that mixture toxicity of triazine and phenylurea herbicides to single species and communities is predictable by the concept of concentration addition. However, there is a lack of studies that verify the applicability of these concepts for natural communities exposed under complex environmental conditions. A further objective of this thesis was thus to verify if the concept of concentration addition can be applied also for natural communities exposed under realistic environmental conditions. Therefore, a phytoplankton community and the three submersed macrophytes Myriophyllum spicatum, Elodea canadensis, and Potamogeton lucens were studied in freshwater outdoor mesocosms. The three PSII inhibitors atrazine, isoproturon, and diuron were chosen as test substances and applied as single substances and in a mixture. In the single treatments the 30 % hazardous concentrations (HC30) of the three substances derived from species sensitivity distribution (SSD) curves were used. The SSD curves were established on the basis of EC50 growth inhibition data obtained from laboratory tests with different algal and plant species. The herbicide mixture comprised one third of the HC30 of each individual herbicide. If the concept of concentration holds true the herbicide mixture was expected to elicit the same toxic effects as the HC30 of three herbicides alone. Herbicide concentrations and effects on phytoplankton and macrophytes were investigated during a five-week period of constant concentrations and a subsequent fivemonth post-treatment period when the herbicides dissipated from the water phase. Photosynthetic efficiency of phytoplankton and the three macrophytes was selected as an endpoint directly linked to the mode of action of the three test substances. Moreover, effects on abundance, diversity, and species composition of phytoplankton as well as on growth of the two macrophytes E. canadensis and M. spicatum were examined. In the period of constant concentrations averaged herbicide water concentrations were determined to be in the range of target concentrations ± 20 %. In the post-treatment period the dissipation of the herbicides was described by first order kinetics. Half-lives corresponded to 107 d for atrazine, 35 d for isoproturon, and 43 d for diuron. At the end of the experiment atrazine concentrations in the water phase still reached approximately 40 % of the target concentration whereas isoproturon and diuron had nearly completely disappeared. In the constant exposure period the single herbicides were shown to be equitoxic due to comparable effects on photosynthetic efficiency, abundance, and diversity of phytoplankton. Furthermore, the herbicides were found to act concentration additive since the effects of the mixture on photosynthetic efficiency, abundance, and diversity were similar to those of the single substances. Because of different sensitivities of a few algal species towards the herbicides, species composition of the communities in the various treatments developed differently during the post-treatment period. Diuron and isoproturon treated algal communities did not differ considerably from the untreated communities concerning photosynthetic efficiency, diversity, and species composition already a short time after the end of the period of constant exposure. This might be linked to the rapid decrease in herbicide concentrations in the water phase. However, tolerance of single algal species towards the herbicides might have also contributed to the recovery of diuron and isoproturon treated phytoplankton. Photosynthetic efficiency and species composition of atrazine and mixture treated phytoplankton was found to be adversely affected and considerably different compared to the untreated communities until the end of the post-treatment period. Continuous exposure to persisting atrazine concentrations resulted in a different succession of phytoplankton in these two treatments. In the three submersed macrophytes inhibition of photosynthesis was determined in a short time window from day 2 to 5 after first application only indicating a rapid adaptation of the macrophytes towards herbicide stress. The observed short term effects of the herbicides and their mixture on photosynthesis might also explain that growth of M. spicatum and E. canadensis was not affected. In addition, the single herbicide concentrations turned out to be equitoxic in M. spicatum since they elicited similar effects on photosynthetic efficiency of this macrophyte. Concentration addition of atrazine, isoproturon, and diuron could also be verified for this macrophyte since the mixture inhibited photosynthetic efficiency comparable to the single substances. In E. canadensis and P. lucens the HC30 of atrazine, isoproturon, and diuron did not stimulate a similar inhibition of photosynthesis and were thus found to be not equitoxic. For this reason, any conclusions on concentration addition of the herbicides in these two macrophytes could not be drawn. This case study confirmed the applicability of the concept of concentration addition for three PSII inhibitors when considering their effects on a natural algal community and on the macrophyte M. spicatum under environmental conditions. The results can thus contribute to the current discussion concerning the incorporation of mixture toxicity in the regulation of surface water quality to adequately protect aquatic communities from pesticide impact and to guarantee a sustained management of the aquatic ecosystems.