Toxicity of pesticides and their mixture to primary producers

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.