The interactive effects of the antifouling herbicides Irgarol 1051 and Diuron on the seagrass Zostera marina (L.).

The herbicides Irgarol 1051 (2-(tert-butylamino)-4-cyclopropylamino)-6-(methylthio)-1,3,5-triazine) and Diuron (3-(3',4'-dichlorophenyl)-1,1-dimethylurea) are commonly incorporated into antifouling paints to boost the efficacy of the compound towards algae. Previous investigations have identified environmental concentrations of these herbicides as being a threat to non-target organisms, such as seagrasses. Their individual toxicity has been assessed, but they can co-occur and interact, potentially increasing their toxicity and the threat posed to seagrass meadows. Chlorophyll fluorescence (Fv:Fm) and leaf specific biomass ratio (representing plant growth) were examined in Zostera marina L. after a 10-day exposure to the individual herbicides. The EC20 for each herbicide was determined and these then used in herbicide mixtures to assess their interactive effects. Irgarol 1051 was found to be more toxic than Diuron with lowest observable effect concentrations for Fv:Fm reduction of 0.5 and 1.0 +/- microg/l and 10-day EC50 values of 1.1 and 3.2 microg/l, respectively. Plants exposed to Irgarol 1051 and Diuron showed a significant reduction in growth at concentrations of 1.0 and 5.0 microg/l, respectively. When Z. marina was exposed to mixtures, the herbicides commonly interacted additively or antagonistically, and no significant further reduction in photosynthetic efficiency was found at any concentration when compared to plants exposed to the individual herbicides. However, on addition of the Diuron EC20 to varying Irgarol 1051 concentrations and the Irgarol 1051 EC20 to varying Diuron concentrations, significant reductions in Fv:Fm were noted at an earlier stage. The growth of plants exposed to Diuron plus the Irgarol 1051 EC20 were significantly reduced when compared to plants exposed to Diuron alone, but only at the lower concentrations. Growth of plants exposed to Irgarol 1051 and the Diuron EC20 showed no significant reduction when compared to the growth of plants exposed to Irgarol 1051 alone. Despite the addition of the EC20 not eliciting a further significant reduction when compared to the herbicides acting alone for most of the mixtures, the lowest observable significant effect concentration for growth and photosynthetic efficiency decreased to 0.5 microg/l for both herbicides. Irgarol 1051 and Diuron have been shown to occur together in concentrations above 0.5 microg/l, suggesting that seagrasses may be experiencing reduced photosynthetic efficiency and growth as a result.

The influence of UV-B radiation on the reproductive cells of the intertidal macroalga, Enteromorpha intestinalis.

The relative sensitivity of the reproductive cells of the seaweed Enteromorpha intestinalis to UV-B was assessed by measuring in vivo chlorophyll fluorescence (F(v) variable fluorescence), germination success and growth rates. Zoospores (the asexual reproductive cells) exhibit up to a 6 fold higher sensitivity to UV-B exposure than the mature thalli (measured as chlorophyll fluorescence, F(v)), and differences in growth rates were also found. Consistent patterns emerged throughout these experiments in variable fluorescence, germination success and growth rates, indicating a greater sensitivity in the sexual reproductive phase of the life cycle compared with the asexual phase. Inhibition of germination success (up to 50%) and growth rates (up to 16.4%) of settled gametes and zoospores after 1-h exposures to elevated levels of UV-B (equivalent to 27 and 31% ozone depletion) showed that damage to the reproductive cells was irreversible. In conclusion, the ecological significance of elevated UV-B exposure in the marine environment may be seriously under-estimated if effects on the early lifestages of algae are not considered.

Hsp70 expression in Enteromorpha intestinalis (Chlorophyta) exposed to environmental stressors.

Numerous studies have indicated that stress proteins have potential as biomarkers of exposure to environmental contaminants. Analysis of stress proteins in animals, especially Hsp70, has dominated this research area despite increasing interest in the use of plants as pollution monitors. In the present study Hsp70 expression in Enteromorpha intestinalis has been investigated under exposure to a variety of stressors, to evaluate the potential use of Hsp70 as a biomarker of contaminant exposure in this seaweed. E. intestinalis was exposed to thermal stress, copper under differing conditions of nutrient availability and the triazine herbicide Irgarol 1051. Measurements were also taken to compare Hsp70 with conventional sublethal endpoints of toxicity such as growth and fluorescence induction parameters such as Fv/Fm. E. intestinalis exhibited a typical heat shock response. Hsp70 expression was increased with copper exposure, but proved to be a relatively insensitive biomarker of copper exposure compared to growth measurements. Nutrient limitation enhanced copper toxicity and significantly impaired growth, Fv/Fm and Hsp70 production. Fv/Fm and growth were strongly affected by Irgarol 1051 exposure, but Hsp70 levels were unaltered following exposure to the herbicide. The implications of using Hsp70 expression in E. intestinalis as a biomarker are discussed.

A comparison of the kinetic properties of phosphoenolpyruvate carboxylase from guard-cell and mesophyll-cell protoplasts of Commelina communis.

Some kinetic properties of partially purified phosphoenolpyruvate carboxylase (PEPCase) from guard-cell and mesophyll-cell protoplasts of Commelina communis are described. The PEPCase activity inherent to each cell type was determined and the apparent K m (phosphoenolpyruvate) and K i (malate) were compared. Malate sensitivity was much higher (K i malate 0.4 mol m(-3)) in the extract of guard-cell protoplasts than in that of mesophyllcell protoplasts (K i malate 4.2 mol m(-3)). The stimulation of activity by glucose-6-phosphate in the presence of malate ('deinhibition') was also investigated in extracts from both cell types and was found to be similar to previously reported results with epidermal tissue. The effect of contamination of an extract of guard-cell protoplasts with mesophyll-cell protoplasts was measured in the presence and absence of malate. It was found that a small amount to mesophyll-cell contaminant appears to desensitize the malate inhibition of PEPCase from guard-cell protoplasts. It is concluded that experiments which use epidermal tissue to study guardcell PEPCase may give misleading information as a consequence of mesophyll contamination.

A study of the in-vitro regulation of phosphoenolpyruvate carboxylase from the epidermis of Commelina communis by malate and glucose-6-phosphate.

Phosphoenolpyruvate (PEP) carboxylase activity in epidermal extracts of Commelina communis has been compared in the presence of malate and glucose-6-phosphate. The activity of PEP carboxylase was inhibited by increasing malate concentrations at several substrate (PEP) concentrations and changes in both the apparent K m (PEP) and V max values in the presence of malate suggested the occurence of mixed-type inhibiton. In the presence of glucose-6-phosphate no increase in enzyme activity was observed, although there was a slight decrease in the K m (PEP). However, glucose-6-phosphate appeared to alleviate the inhibition caused by malate. The possible implications of these properties in the control of malate production in guard cells is discussed.