Measuring variability in phytotoxicity testing using crop and wild plant species.

A series of experiments was conducted to assess the level of variability in phytotoxicity testing and to investigate factors that may explain some of the observed uncertainties and inconsistencies. The work was conducted in greenhouse or growth chamber environments with plants growing individually in pots and harvested 28 d after spraying with two herbicides, glyphosate and atrazine, as formulated products. Between six and 10 doses were used on five or six replicates, necessitating over 4,500 individually growing plants. In the first set of experiments, several ecotypes (originating from different areas of the world) of eight wild plant species were tested. Significant differences in sensitivity to atrazine and glyphosate were found among ecotypes of most species tested. In the second suite of experiments, the reproducibility of results during different seasons (when growing conditions vary) was investigated using three crops and four wild plant species. Results showed that seasonal variability elicited a pronounced discrepancy in response between plants tested at different times of the year. It was found that no consistent effects could be attributed to the biotic or abiotic factors investigated. Several ecotypes of the same species differed in their seed size, percentage germination, or germination requirements, as well as in growth patterns, but these differences could not explain differences in herbicide sensitivity. Likewise, differences in phytotoxicity could not be attributed to factors such as temperature, light intensity, and sunlight duration. The present study supports the inclusion of an uncertainty factor in risk assessments to account for the intrinsic variability in plant sensitivity to herbicides.

Germination requirements for 29 terrestrial and wetland wild plant species appropriate for phytotoxicity testing.

BACKGROUND: Species selected for phytotoxicity testing have been limited to a few standard crop species owing to restrictive recommendations at the regulatory level. However, guidelines by the Organisation for Economic Development and Cooperation (OECD) were recently amended in 2006 to include a list of herbaceous non-crop plant species suitable for testing. The objective of this study was to outline the optimum germination requirements for a selection of wild species for which seeds were readily available from commercial suppliers. RESULTS: Of the 29 herbaceous terrestrial and wetland species included in this study, all achieved 50% germination and 23 reached > 70% germination to meet the criterion outlined in the OECD guidelines. Most species attained their maximum germination within 14 days or less. Cold stratification of imbibed seeds improved germination for 14 species. Increasing sowing soil depth did not improve seed germination. The variance attained in this experiment between replicates was low, especially for species with > 70% germination (standard error approximately 5%). CONCLUSION: The present study showed that 23 of the 29 species tested required minimal pretreatments and produced consistent, reliable and uniform germination reaching at least 70%. The inclusion of wild plant species in regulatory testing should be given real consideration.

Herbicidal effects on nontarget vegetation: investigating the limitations of current pesticide registration guidelines.

The impact of herbicide exposure on nontarget vegetation within agroecosystems has sparked extensive research that revealed that current pesticide registration guidelines may be inadequate at predicting the effects of herbicides on wild plants and habitats. This study extends the current interest by presenting three experiments highlighting some of the limitations to current phytotoxicity testing guidelines. Several crops and wild plant species were grown under greenhouse conditions following standard protocol for phytotoxicity testing. Plants were sprayed with five different herbicides at the four- to six-leaf stage, and biomass was recorded at 28 d after spray. Results showed that current regulatory protocol will likely underestimate herbicide phytotoxicity if testing does not include data for the complete tank-mix formulation. The present study also showed that the range in herbicide sensitivity among cultivars of the same crop can be quite extensive and that, depending on the cultivar included in a risk assessment, conclusions regarding the phytotoxicity of any given herbicide may differ. Although no significant differences in sensitivity were found between crops and related wild species, results revealed that current guidelines are too rigid in terms of species selection. Considering the variability among crop cultivars, coupled with the ecological importance and the ease of germination of many noncrop plant species, pesticide regulatory guidelines would be improved if wild species were included in testing. Findings of the present study indicate that current pesticide regulatory guidelines require modifications to ensure a more accurate assessment of herbicide effects on nontarget plant species.

Removing UV-A and UV-C radiation from UV-B fluorescent lamp emissions. Differences in the inhibition of photosynthesis in the marine alga Dunaliella tertiolecta using chromate versus cellulose acetate-polyester filters.

Ultraviolet-B (UV-B; 280-320 nm)-emitting lamps unavoidably emit ultraviolet-A (UV-A; 320-400 nm) and ultraviolet-C (UV-C; <280 nm) radiation. Short-wavelength-blocking filters are generally used to limit the wave bands of UV under investigation. The widespread use of such filters means that all exposures to UV-B radiation will have a significant UV-A component. Therefore, the physiological effects unique to UV-B exposure are difficult to clearly isolate. This study presents a method to remove the UV-A and UV-C "contamination" using a liquid potassium chromate (K(2)CrO(4)) filter, thus allowing more direct assessment of the effects of UV-B exposure. Cultures of the green marine alga Dunaliella tertiolecta were grown in the absence of UV radiation. Sunlamps supplied the UV radiation for a 24 h exposure (solar radiation was not used in this study). The UV radiation was filtered either by the standard method (i.e. cellulose acetate (CA) with polyester = Mylar controls) or by a liquid filter of potassium chromate. Photosynthetic responses were compared. Major decreases in the ratio of variable to maximal fluorescence in dark-adapted cells and photosynthetic capacity were observed in CA-filtered cultures, whereas no change was observed in cells exposed to the same UV-B flux with the UV-A removed by K(2)CrO(4). The use of a CA filter with a Mylar control does not link results unequivocally to UV-B radiation. Such results should be interpreted with caution.

Long-term hyposaline and hypersaline stresses produce distinct antioxidant responses in the marine alga Dunaliella tertiolecta.

Tolerance to salinity stress in higher plants correlates to levels of antioxidant enzymes and/or substrates. Do hyperosmotic and hypoosmotic stress induce antioxidant responses in salt tolerant algae, and if so, are these responses the same for both excess and minimal salinity? To answer these questions, cultures of the marine alga Dunaliella tertiolecta (Chlorophyta) were grown in seven salinities covering a 60-fold range from 0.05 to 3.0 mol/L NaCl. Long-term effects of salinity on growth and antioxidant parameters were determined. Growth rates were reduced at the salinity extremes (0.05 mol/L NaCl and 3 mol/L NaCl) indicating the cultures were stressed. The levels of six antioxidant enzymes and three antioxidant substrates were quantified at these growth salinities. Compared to growth at optimum salinities (i.e. 0.2-0.5 mol/L NaCl), high salinities produced a 260% increase in monodehydroascorbate reductase, a doubling of ascorbate peroxidase activity and a three-fold increase in the rate of dark respiration. Cells acclimated to low growth salinities (hyposaline stress, i.e. < 0.2 mol/L NaCl) showed major increases in glutathione and alpha-tocopherol coupled with decreases in Fv/Fm ratios and in total and reduced ascorbate compared to moderate and high external salinities. Cell volumes remained unchanged, except at the lowest salinity where they doubled. Catalase, superoxide dismutase, dehydroascorbate reductase and glutathione reductase activities were not altered by extreme salinities. The involvement of oxidative stress at both salinity extremes is implied by the alterations in antioxidant enzymes and substrates, but the specific changes are very different between hypo and hypersaline stresses.

Contrasting effects of UV-A and UV-B on photosynthesis and photoprotection of beta-carotene in two Dunaliella spp.

Photosynthetic and antioxidant responses following exposure to either ultraviolet-A or ultraviolet-B were contrasted in two species of the unicellular green alga, DUNALIELLA: Species selection was based on the ability of Dunaliella bardawil (UTEX 2538) to accumulate inter-thylakoid beta-carotene when subjected to environmental stress while Dunaliella salina (UTEX 200) lacks this ability. Cells were cultured in high and low levels of visible light (150 and 35 micro mol photons m(-2 )s(-1), respectively) and then either ultraviolet-A (320-400 nm) or ultraviolet-B (290-320 nm) was added to visible light for 24-h exposure. A potassium chromate solution was found to be an ideal screen for removal of ultraviolet-A and ultraviolet-C from ultraviolet-B radiation. There were no significant changes in photosynthetic or antioxidant parameters following exposure to ultraviolet-B. Ultraviolet-A exposure significantly decreased photosynthetic parameters (>70% decrease in Fv/Fm and the ratio of light-limited to light-saturated photosynthesis in low beta-carotene cells) and resulted in 50% increases in ascorbate peroxidase activity and ascorbate concentrations. The results suggest exposure to ultraviolet-A (but not ultraviolet-B) directly affects photosynthesis, observed as a loss of photosystem II electron transport efficiency and increased radical formation. This research indicates that the accumulated beta-carotene in D. bardawil prevents UV-related photosynthetic damage through blue-light/ultraviolet-A absorption (supported by trends observed for antioxidant enzyme responses).