295 (ROS-1117-841648) Why is Aquatic Macrophyte Toxicity Testing Important?
Start time: 8:00 AM
Roshon, R.¹, ¹ Stantec Consulting Ltd., Guelph, ON, Canada
Rooted aquatic macrophytes are an important ecosystem component, but are not required for regulatory testing. Several toxicity tests with rooted aquatic macrophytes have been published with ASTM (E-1841 and E-1913). These test methods can be used to determine the toxicity of pesticides, metals, and organic chemicals to non-target aquatic plants, including Oryza sativa, and Myriophyllum sibiricum. There are advantages and disadvantages of these toxicity tests, as compared to the currently accepted Lemna, algae, fish and invertebrate tests. The most obvious drawback is the additional infrastructure and cost associated with the rooted aquatic macrophyte testing, as compared with the simpler fish and invertebrate toxicity studies. Toxicity tests with aquatic macrophytes are replicable and repeatable systems. Features of these toxicity tests that enhance standardization include a chemically defined nutrient solution and an artificial rooting substrate. Due to their ecological relevance and sensitivity, these partial life cycle laboratory toxicity tests would be a beneficial addition to the regulatory data set.

296 (POO-1117-836626) Development of a standard growth protocol for the submersed macrophyte Myriophyllum spicatum to test non-target effects of chemicals in aquatic systems.
Start time: 8:20 AM
Poovey, A.¹, Getsinger, K.¹, ¹ US Army Engineer Research and Development Center, Vicksburg, MS, USA
Myriophyllum spicatum L. is a rooted submersed macrophyte native to Eurasia, however, it is invasive in North America. Because M. spicatum reproduces through vegetative fragmentation, grows rapidly and is easily cultivated, it has potential as a test species for non-target effects of chemicals in aquatic systems. Growth of this plant was investigated in a series of studies under controlled conditions using combinations of both natural and artificial sediments, fertilization rates, and a variety of aqueous media. The objective was to develop a test system that would support healthy shoot and root growth over a short period, while minimizing algal contamination. Apical shoots were planted in beakers filled with sediment, placed in aquaria containing an aqueous medium, and allowed to grow for 21 d. Initial results demonstrated that plants generated longer shoots and produced more highly developed roots in light intensities of 7000 versus 14,000 lux. As growth in fertilized artificial sediments (e.g. OECD chironomid sediment) was similar to that in a fertilized natural lake sediment, a modified OECD sediment was chosen for subsequent studies. OECD sediment in combination with an aqueous medium modified from the OECD daphnia reproduction test provided acceptable growth when amended with 300 mg L^-1 of both NH₄Cl and Na₂HPO₄ providing a total shoot length increase of 41.6 cm and weight increase of 1.4 g. This sediment and medium combination generally had little algal contamination (chlorophyll a ≤4 mg m^-3). Rooted macrophtyes, such as M. spicatum, effectively mine N and P from the sediments. Aqueous media high in N and P may stimulate phytoplankton blooms rather than contribute to macrophyte nutrition. Phytoplankton blooms developed in aquaria with unfertilized OECD sediment and 1x AAP growth media where chlorophyll a was 12 to 17 mg m^-3, and shoot growth was unacceptable (length increase = 2.8 cm, weight increase = -0.03 g). Based on this work M. spicatum is a candidate for non-target testing of chemicals in aquatic systems; using the sediment and media described, doubling of shoot length has been obtained in 14 days. Further work is needed to refine the method and identify additional causes of algal contamination.

297 (GAU-1117-828260) Are Duckweed and Algae Equivalent Indicators of Phytotoxicity?
Start time: 8:40 AM
Gausman, M¹, Belanger, S¹, Guckert, J¹, ¹ The Procter & Gamble Company, Cincinnati, OH, USA
An extensive amount of literature exists on the use of the floating macrophyte, duckweed (Lemna species) that, as with algae, can potentially give insight into the phytotoxicity of chemicals. However, information on toxicity of consumer product and other high volume chemicals to duckweed is lacking. Traditionally, algae, especially the chlorophytes Scenedesmus (=Desmodemsus) subspicatus and Selenastrum (=Pseudokirchneriella) capricornutum have been used in this application. The objectives of this research are to (1) compare sensitivity of the two test organisms, (2) explore advantages and disadvantages of using duckweed and algae in toxicity testing of chemicals, and (3) experimentally evaluate responses of both algae and duckweed to various surfactants. Literature was compiled for a broad comparison between duckweed species and green algae to understand similarities and differences with respect to toxicity responses, test conditions and experimental designs. An experimental program was also developed to assess the comparative sensitivity of duckweed and green algae. Duckweed and algal data were both available for over 40 different compounds in nearly 200 tests. The majority of chemicals were herbicides (10), metals (7), and pharmaceuticals (10). Only one surfactant comparison was available. For herbicides, duckweed and algae were similarly sensitive; for metals, algae were more sensitive than duckweed; and for pharmaceuticals, duckweed was less sensitive and algae sensitivity varied greatly (possibly reflecting a wider array of modes of toxic action). When algal and duckweed responses were regressed against each other for all compounds combined, algae were somewhat more sensitive (slope = 0.63, r-square = 0.5). For new experimental comparisons of algal and duckweed responses to surfactants it appears that algae are more sensitive to cationic surfactants, but duckweed is more sensitive to anionic surfactants. Results of the literature assessment will be reviewed in detail along with a comparison of the strengths and limitations of both assays.

298 (REI-1117-727426) Toxicity assessment for fluorinated organics using Lemna minor oxygen production.
Start time: 9:00 AM
Reinhold, D¹, Saunders, F¹, ¹ Georgia Institute of Technology, Atlanta, GA, USA
To determine the relationship between uptake and activity by L. minor at non-inhibitory to toxic concentrations of fluorinated organics, a novel oxygen-production assessment was developed and optimized. Assessment of O₂-production by L. minor was optimized to increase sensitivity and reliability of response while decreasing assessment time. Normalized O₂-production (M O₂/h-g L. minor) was measured in sealed reactors using manometers and partial pressure of gases with GC-TCD. Dark reactors were used to account for microbial and plant respiration. O₂-production by L. minor was negatively correlated with media pH and greater than 13 mM phosphate buffer was required to maintain neutral pH. Increasing carbonate concentration did not increase O₂-production by L. minor, indicating that media contained sufficient carbon for photosynthesis. Optimized O₂-production assessment allowed for easy quantification of L. minor activity under high-plant-density growth conditions similar to natural ecosystems. Toxicity studies using 24-h exposure and 6-h O₂-production assessment yielded toxicities similar to single-frond, 7-d chlorosis measurements. As such, O₂-production by L. minor was a viable, rapid alternative for assessing toxicity of contaminants in conjunction with uptake experiments. Uptake experiments with 10 M fluorinated organic yielded pseudo-first-order uptake rate coefficients of 0.2-0.7 d^-1, indicating 19-50% uptake from aqueous phase in 24-h. Uptake rate coefficients drastically decreased with increasing concentration of fluorinated organics at concentrations of 10-100 M. A gradual decrease in uptake rate coefficients was observed at concentrations greater than 100 M. Dependence of uptake rates on concentration did not correspond with toxicity. This presentation will illustrate the ability of aquatic plants to sequester contaminants, demonstrate independence of uptake on toxicity, and discuss methodology for sensitive toxicity assessment using L. minor O₂-production with reference to standardized duckweed toxicity assessments.

(58050) COFFEE BREAK.
Start time: 9:20 AM

299 (GRE-1117-737934) Molecular and physiological responses of the aquatic plant Lemna gibba to metals and PAHs.
Start time: 10:00 AM
Akhtar, Tariq ¹, Lees, Hazel¹, Lampi, Mark¹, Greenberg, Bruce ¹, ¹ Department of Biology, University of Waterloo, Waterloo, ON, Canada
Lemna gibba can assimilate contaminants from the aquatic phases of the environment in which it resides. We have examined the mechanisms of how metals and polycyclic aromatic hydrocarbons exert their toxicity on this higher aquatic plant. It was found that copper, cadmium and 1,2-dihydroxyanthraquinone (1,2-dhATQ) negatively impact Lemna gibba, at relatively low levels. Further, they are synergistically toxic via a reactive oxygen species mechanism. We have now examined how Lemna gibba responds to these contaminants at a molecular and physiological level. Specifically, both copper and 1,2-dhATQ appeared to disrupt photosynthetic electron transport causing an overall reduced plastoquionone pool. We further demonstrate that the redox state of the plastoquinone pool can effectively trigger a variety of stress responses. In particular, copper and 1,2-dhATQ causes numerous changes in gene expression as well as accumulation of flavonoids, which could both be traced back to a reduced plastoquinone pool. Interestingly, by oxidizing the plastoquinone pool many of these responses were reversed. Other environmental stresses such as temperature fluctuations and UV-B radiation effectively caused similar stress responses in concert with a reduced plastoquinone pool. We therefore conclude that the redox balance of the cell (possibly via the plastoquinone pool) acts as a general sensor for environmental stress in Lemna gibba.

300 (BRA-1117-850085) Characterizing the herbicidal effects of statin pharmaceuticals in Lemna gibba.
Start time: 10:20 AM
Brain, R¹, Reitsma, T¹, Bestari, K¹, Solomon, K¹, ¹ University of Guelph, Guelph, Ontario, Canada
Statins are synthetic lipid-lowering agents which competitively inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) in the treatment of high cholesterol. HMG-CoA is one of the most highly regulated enzymes in mammals as well as plants. In plant systems HMG-CoA is the rate determining step in the cytosolic mevalonic acid pathway where isopentenyl diphosphate (IPP) is synthesized. IPP provides the basic building blocks of the terpenoid biosynthetic pathway. An autonomous pathway in the chloroplast has also been characterized which synthesizes IPP, however, although there is some crosstalk between pathways, the anabolic products are considered largely distinct. The mevalonic acid pathway is responsible for the synthesis of sterols and the side-chain of ubiquinone, which are critical for membrane fluidity and electron transport, respectively. The plastidic pathway is responsible for the synthesis of carotenoids, chlorophylls, and plastoquinone, which are critical components of the photosynthetic pathway. Statin pharmaceuticals have previously been considered not to have significant impacts on anabolic plant processes through the inhibition of HMG-CoA, however our laboratory studies with Lemna gibba indicate that statins do cause concentration-dependent toxicity via reduction of mevalonate (HMG-CoA mediated) derived products. This presentation will discuss the concentration-dependent effects of atorvastatin and lovastatin on the levels of plastoquinone, ubiquinone, -sitosterol, and stigmasterol in L. gibba, quantified using HPLC-UV/Vis. Extraction and quantification protocols developed for quinones and sterols as well as atorvastatin and lovastatin will be detailed.

301 (TRO-1117-827266) Uptake of Ionizable Organic Contaminants by the Aquatic Plant L. minor.
Start time: 10:40 AM
Tront, J¹, Reinhold, D¹, Saunders, F, ¹ School of Civil and Environmental Engineering, Georgia Tech, Atlanta, GA, USA
Previous research demonstrated that plants take up, metabolize and sequester organic pollutants. However, limited studies have investigated uptake of ionizable contaminants or factors which affect uptake by aquatic plants. This study examined key factors affecting uptake of a class of ionizable organic contaminants (halogenated phenols) by Lemna minor. Factors such as initial plant activity and contaminant speciation were examined in batch systems using 2,4,5-trichlorophenol (pK_a = 7.0) as the model contaminant. Initial plant activity, measured as the oxygen production rate, was found to positively correlate with contaminant uptake rate. Experimentation at a range of pH values (6 − 9) and uptake rates were linearly correlated to percent of contaminant in protonated form. The impact of halogen number, type, and positioning on uptake was investigated using a suite of halogenated phenols in batch reactors with media buffered so that aqueous-phase speciation was not a variable. Halogenated phenol uptake was quantified with pseudo-first-order rate coefficients. Data gathered for mono-, di-, and trihalogenated phenols revealed that uptake rates: (i) were not affected by halogen substituent type, (ii) were significantly affected by substituent position, and (iii) did not vary with increasing number of halogen substituents. Relationships between uptake rates and physiochemical properties (e.g., log K_ow: 1.77 − 4.13; pK_a: 5.96 − 9.54) were investigated. Data analysis comparing hydrophobicity with uptake rate indicated that hydrophobicity was not a critical variable in uptake rate of ionizable contaminants. Furthermore, uptake data did not follow Gaussian relationships previously defined for uptake by terrestrial plants (Briggs et al., 1982; Burken and Schnoor, 1998). The relationship between uptake by L. minor and fraction protonated at cytosolic pH was also examined. Therefore, results demonstrated that factors such as plant metabolic activity, percent protonated (in aqueous phase and in the cytosol) play a critical role in determining rate of ionizable organic contaminant uptake by plants.

302 (TOR-1117-597531) Transgenic Microalgae as Heavy Metal Biosensors.
Start time: 11:00 AM
Torres, M.A.¹, Falcao, V.R¹, Colepicolo, P.¹, Rajamani, S.², Ewalt, J.², Sayre, R.T.², ¹ Chemistry Institute, Departament of Biochemistry, University of Sao Paulo, Sao Paulo, Sao Paulo, Brazil² Department of Plant Cellular and Molecular Biology, Ohio State University, Columbus, OH, USA
Here we describe the design and properties of a heavy metal biosensor for the measurement of bio-available heavy metals in aqueous environments. The biosensor protein includes an N-terminal, cyan variant of the green fluorescent protein (mCFP), an intervening metallothionein (MT), and a C-terminal yellow variant of the green fluorescent protein (mYFP) from jellyfish. The genes encoding these proteins were codon-optimized for expression in Chlamydomonas and the modified green-fluorescent variants were engineered to reduce pH effects on fluorescence emission quantum yield and to reduce end-to-end dimerization. In the absence of heavy metals MT assumes an unfolded, random-coil structure. Following metal binding MT folds into a compact dumbbell shaped protein. The metal metal-induced folding of MT brings the two fluorescent proteins mCFP (excitation = 440 nm: emission = 485 nm) and mYFP (excitation = 515 nm: emission = 527nm) within 70 of each other facilitating fluorescence resonance energy transfer (FRET) between the two fluorophores, resulting in a shift of the relative ratio of yellow and blue fluorescence. We have expressed this heavy metal biosensor in a cell wall-less and arg- strain of Chlamydomonas reinhardtii. Fluorescence analyses confirm that the protein is expressed in the algae. When grown in TAP media the ratio of yellow to blue fluorescence is 1.0. Experiments show the effects of Cd²⁺, Cu²⁺ and Pb²⁺ ions on the fluorescence emission spectra of the biosensor. These results point microalgal biosensors like important organisms and could be used in biomonitoring of heavy metals in various water bodies.

303 (HAN-1117-816785) Aquatic Plants: Now and in the future.
Start time: 11:20 AM
Hanson, M¹, ¹ Department of Environment and Geography, University of Manitoba, Winnipeg, Manitoba, Canada
Aquatic plants can act as integrators and indicators of water quality, permitting characterization of both direct and indirect effects of a chemical contaminant or a physico-chemical change. Despite this utility, their environmental relevance, specifically as primary producers, and the efforts of various government agencies, there has been no adoption of any aquatic plants in the pesticide registration process in North America aside from Lemna spp. Based on a review of the current literature, it is clear that aquatic plants can be a sensitive and cost effective route to understanding the effects of contaminants and assessing ecosystem health. Aquatic plants demonstrate a range of sensitivities, have a variety of exposure routes, exhibit low statistical variability when compared with other organisms, and are easily manipulated in the field and laboratory. This presentation will expand on each of these points with relevant illustrations from the literature and propose where we need to go in the future, including studies that integrate plant populations and community interactions.