736 (HAR-1117-819173) Pesticide Occurance in Selected South Florida Canals and Biscayne Bay During High Agicultural Activity.
Start time: 1:50 PM
Harman-Fetcho, J¹, McConnell, L¹, Potter, T², Rice, C¹, Sadeghi, A¹, Sefton, K¹, Schaffer, B³, Curry, R⁴, Smith, R¹, Hapeman, C¹, ¹ USDA-ARS-Environmental Quality Lab, Beltsville, Maryland, USA² USDA-ARS-Southeastern Watershed Research Lab, Tifton, GA, USA³ University of Florida-Tropical Research and Education Center, Homestead, Florida, USA⁴ National Park Service - Biscayne National Park, Homestead, Florida, USA
Climate and soil conditions in South Florida along with an extensive canal system facilitate movement of agricultural pesticides into surface waters. In a two-year study (2002-2004) of the currently-used pesticides in South Florida, 91 surface water samples were analyzed for 39 pesticides. Atrazine, endosulfan, metolachlor, chlorpyrifos, and chlorothalonil were the most frequently detected in the canals and in Biscayne Bay, with average concentrations of 16, 11, 9.0, 2.6 and 6.0 ng/L, respectively. Concentrations of atrazine and chlorpyrifos were highest near corn production. Chlorothalonil and endosulfan concentrations were highest near vegetable production, with no clear trend for metolachlor which is used on multiple crops. Concentration data were used to calculate an aquatic life hazard potential for the planting period (November) versus the harvest period (March). This analysis indicated that a higher hazard potential occurs during harvest, primarily from the use of endosulfan. These data will also serve to document canal conditions prior to implementation of the Comprehensive Everglades Restoration Plan (CERP).

737 (WAC-1117-728134) Temporal variation in mixture toxicity of chlorpyrifos and atrazine on Pimephales promelas and Xenopus laevis.
Start time: 2:10 PM
Wacksman, M¹, Lydy, M¹, ¹ Southern Illinois University Carbondale, Carbondale, IL, USA
Chlorpyrifos is often used in agricultural and residential settings to control worms, grubs, cockroaches, beetles and other insects. Atrazine is widely used in agriculture being the most heavily applied herbicide in the United States. These chemicals are often found in run-off water at elevated levels, causing exposure to non-target species. Past research has shown greater than additive toxicity when invertebrates were exposed to mixtures of atrazine and chlorpyrifos. This study expands this research to include vertebrate species, specifically fathead minnows and African clawed frogs. Groups of known age fathead minnows, Pimephales promelas, and African clawed frogs, Xenopus laevis were exposed to chlorpyrifos in binary mixtures with atrazine. These tests were performed using static renewal methods with 48 h pre-exposure to atrazine followed by 48 h of chlorpyrifos and atrazine exposure. Temporal variations were seen in the organisms' response to chlorpyrifos in a laboratory setting. The affect of atrazine on chlorpyrifos toxicity was shown in this study. These findings have been supported by acetylcholinesterase activity of whole fish and tadpole homogenates. A time-dependent acetylcholinesterase curve has also been examined for both species. These curves show low activity in young control organisms; with activity increasing during the organisms' maturation. These results may have ramifications concerning early life cycle testing with fathead minnows, since atrazine affects the cholinesterase system, therefore mixture toxicity may be underestimated until the organism's enzymatic system is fully developed.

738 (LYD-1117-808488) Hazard of Pesticide Mixtures in Corn-Belt Surface Water.
Start time: 2:30 PM
Lydy, M¹, Belden, J¹, ¹ Southern Illinois University, Carbondale, IL, USA
Although numerous studies have shown that mixtures of toxicants generally result in greater toxicity than would be expected from any of the single components, little work has been conducted quantifying the increased hazard posed in actual environmental samples. In the current study, a database containing pesticide-monitoring data was evaluated using a toxic unit approach. Four model species including duckweed, green algae, water fleas, and bluegill were used as indicators of toxicity. Toxic unit values (TU) calculated based on all pesticides and pesticide classes were used to evaluate the risk posed by each sample. In each case, the toxic unit value of the mixture was compared to the individual pesticide that occurred at the highest concentration. Few samples had a mixture TU value of greater than 0.1 for water fleas and bluegill, while many samples had TU values above 0.1 for duckweed and green algae. For duckweed and green algae, only a few pesticides, and only two classes of herbicides, the triazines and chloroacetanilides generally dominated the magnitudes of the toxic unit values. Although consideration of mixtures increased the toxic unit value, and thus the potential hazard, the increase was always less than 2-fold. The small increase in TU value indicates that in this pesticide-usage landscape consideration of mixtures may have minimal impact on the overall risk assessment of pesticides.

739 (BEL-1117-809450) Occurrence of pesticide mixtures in corn-belt surface water.
Start time: 2:50 PM
Belden, J¹, Lydy, M¹, ¹ Southern Illinois University, Carbondale, IL, USA
Monitoring of surface water has indicated that pesticides, usually occurring as mixtures, are ubiquitous contaminants throughout agricultural regions of the United States. Thus, it can be assumed that the majority of aquatic organisms are exposed to pesticide mixtures during their lifetime. Understanding the hazards posed by mixtures is a complicated task because a large number of different pesticides may occur as co-contaminants. Testing all of the possible mixtures is not feasible for even simple systems. Although toxicity-modeling techniques have improved recently, there is still a great deal of uncertainty in regard to modeling, especially for chronic endpoints. Therefore, techniques are needed for determining priority mixtures that are most likely to have an environmental impact. In our approach, occurrence of pesticide mixtures were determined by defining a pesticide-usage landscape (corn/soybean row-crops) and toxicologically censoring the monitoring data collected in the landscape. To sensor the data, toxicological endpoints for duckweed, green algae, water fleas, and bluegill were used to generate levels of interest for each species. Pesticide concentrations below the level of interests were not included in further analyses. Thus, species-dependent sub-sets of the monitoring database were generated that only included pesticide concentrations that are likely to be important toxicologically for a given reference species. Through this approach, the prevalence and types of mixtures that are important were determined. The composition of pesticide mixtures as determined by this approach was much simpler than would have been predicted statistically, with only a few specific mixtures accounting for most contamination patterns. Nearly all of the problematic mixtures identified in this study were composed of herbicides from one of two classes, triazine and chloroacetanilide, and were identified using duckweed or green algae biological endpoints in the censoring process.

(59366) Break.
Start time: 3:10 PM

740 (BIA-1117-747693) Characterization of tissue specific protein and gene expression changes in response to binary mixtures of pesticides.
Start time: 3:50 PM
Biales, A¹, Bencic, D¹, Lazorchak, J¹, Flick, R¹, Lattier, D¹, ¹ USEPA/ORD/NERL/EERD/MIRB, Cincinnati, OH, USA
Aquatic organisms and other receptors in the Nation's watersheds are exposed to complex environmental mixtures of natural and synthetic origins. Components of these mixtures vary based on their ecological context. Efforts have focused on establishing relatively quick, sensitive biological methods that are capable of distinguishing mixture constituents; however, little progress has been made. Here we use 2-Dimensional (2-D) protein gel electrophoresis to identify changes in protein abundance in adult and juvenile fathead minnows (Pimephales promelas) exposed to a binary mixture of ecologically related chemical pesticides at environmentally relevant concentrations. Because expressed proteins are directly responsible for mediating cellular responses, changes in their abundance are considered to have more biological relevance and have been shown to be less variable among individuals than transcriptional profiles. Chemicals were selected because they are commonly used in agricultural settings throughout the United States and are therefore likely to co-occur in aquatic ecosystems. We first characterized the differential protein responses to these chemicals individually in five tissues known to be commonly involved in toxicological pathways: liver, gill, kidney, brain and gonads and responsive proteins were identified by mass spectrometry. Following characterization of the individual chemicals, protein expression responses of binary mixtures were analyzed in the same five tissues using 2-D protein gel electrophoresis. In the next phase of this study we will synthesize QPCR oligonucleotide primers against amino acid sequences for responsive proteins. These QPCR primers will then be used to correlate changes in transcription of individual genes with abundance changes in associated proteins, in order to create useful transcription-based indicators of both individual and mixture specific exposures. This will be among the first projects to measure global biological changes as a response to the complex interactions of mixture components.′

741 (TRI-1117-727128) Effects of triazine herbicides on organophosphate insecticide toxicity in Chironomus tentans and Hyalella azteca.
Start time: 4:10 PM
Trimble, A¹, Schuler, L¹, Belden, J¹, Lydy, M¹, ¹ Southern Illinois University, Carbondale, Illinois, USA
The purpose of this research was to examine the effects of select triazine herbicides on organophosphate insecticide toxicity. The acute toxicities of two organophosphate insecticides, chlorpyrifos and diazinon, were measured separately and in binary mixtures with 10 different triazine herbicides using 4^th instar larvae of the aquatic midge, Chironomus tentans, and the juvenile aquatic amphipod, Hyalella azteca. Preliminary studies with the triazine herbicides indicated no significant toxicity effects to either organism when dosed alone. However, with nearly every triazine, binary mixture studies showed greater than additive effects relative to the toxicity of the insecticides alone. These effects were greater overall to C. tentans compared to H. azteca, with some of the triazines causing a doubling in the toxicities of the organophosphates. In addition to the acute analyses, chronic toxicity effects to chlorpyrifos toxicity were examined using three of the most commonly detected active-use triazine herbicides. Results of these chronic tests will be presented and compared to the acute effects obtained previously. These results should be of interest to water managers responsible for policy decisions because it provides important information on acceptable concentrations of commonly occurring pesticides that do not follow the concentration addition model.

742 (GRU-1117-923296) Response of pre-spawn coho salmon to pesticides in urban streams in western Washington.
Start time: 4:30 PM
grue, christian¹, Hearsey, James¹, Grassley, James¹, ¹ University of Washington, seattle, WA, USA
Recent monitoring suggests that pre-spawn mortality of coho salmon (Onchorynchus kisutch) has increased in natural waters, particularly urban streams in western Washington. Reasons are not known, but it has been suggested that pre-spawn mortality and pesticides in surface waters are linked. In fall 2004, we exposed green male and female coho to a chemical cocktail representative of that reported in urban streams and monitored their time to death, the ability of females to ripen, and effects of the chemical cocktail on brain cholinesterase activity. Treatments consisted of two pairs of fish (2 males + 2 females) given a continuous exposure to the chemical cocktail or clean water. Two 700 gal tanks per treatment were used and new green fish of the appropriate sex were added to the tanks when fish in the tanks died. Nominal concentrations (ppb active ingredient) of 8 herbicides, 2 insecticides and a fungicide were the maximum reported after stormwater events in either spring or fall. With the exception of the fungicide, formulated products (single ai) were used and if possible were selected from those available at retail outlets. Ten or more fish of each sex were exposed to the two treatments over a 6-week period (> 40 total fish). Time to death for each sex was similar between the treatments (mean = ca. 8.3 days); all but 1 treated female and 1 control female ripened, and none of the overt symptoms that have been suggested to be associated with pre-spawn mortality were observed in treated fish. Brain cholinesterase activity was not inhibited in either treated males or females. Results suggest that other factors (general water quality, habitat, or other contaminants) may be responsible for the mortality that has been observed in the field.

743 (GRU-1117-925040) Response of juvenile rainbow trout to pesticides in urban streams in western Washington.
Start time: 4:50 PM
Curran, Catherine¹, Bogard, Sara¹, Bricker, Jacki¹, Lowery, Erin¹, Menard, Cara¹, Shavey, Christie¹, Grassley, James¹, grue, christian¹, ¹ University of Washington, seattle, WA, USA
Recent efforts to document pesticide concentrations within surface waters have generated a significant amount of information. Unfortunately, these reports uniformly suffer from a lack of adequate biological interpretation. Although levels reported are low (most <1.0 ppb), the presence of these chemicals has generated concerns, particularly their potential effects on salmonids. We exposed juvenile (ca. 10 g) rainbow trout (Oncorhynchus mykiss) to a chemical cocktail representative of urban streams in western Washington. Nominal concentrations (ppb active ingredient) of 9 herbicides, 3 insecticides, an insecticide breakdown product, and a fungicide were the maximum reported during peak storm flow events (hereafter 1X). With the exception of the fungicide and the breakdown product, formulated products (single ai) were used and if possible were selected from those available at retail outlets. Two experiments were conducted. In the first, 20 trout were individually exposed to the 1 X cocktail or clean dechlorinated water for 96-h under static conditions at 12 C. In the second, 10 trout were individually exposed to a 1X, 3.3X or 10X cocktail or clean dechlorinated water under the same conditions. Behavior and water quality were assessed at 6, 24, 48, 72 and 96 h. Survivors were euthanized and frozen (-40C) for subsequent brain acetylcholinesterase assays. All fish survived, except for 1 fish exposed to the 10X cocktail. Fish exposed to the chemical cocktails were more lethargic than controls, particularly those exposed to the 10X cocktail. Brain AChE activity was not inhibited in fish exposed to the 1X cocktail in either experiment, but was in the 3.3X (23%) and 10X (84%) cocktails. Concentrations tested are >2 orders of magnitude greater than mean (geometric) concentrations of the pesticides reported during storm events.

744 (AAA-1117-093838) Including mixtures in the determination of acute and chronic water-quality criteria for pesticides.
Start time: 5:10 PM
Chèvre, N¹, Loepfe, C¹, Fenner, K¹, Singer, H¹, Stamm, C¹, Escher, B, ¹ EAWAG, Duebendorf, Switzerland
Monitoring programs throughout America and Europe have demonstrated the widespread presence of pesticides in surface water and public concern is focused on the possible environmental impact of such pollution. The comparison of acute and chronic Water Quality Criteria (AQC, CQC) with environmental concentrations gives an indication of the pressure that each pesticide puts on the aquatic system and allows identification of those that present a risk for aquatic life. However, in the environment, aquatic organisms are not only exposed to single pesticides but typically to mixtures of these substances. It is therefore of particular importance to take mixtures into account when defining AQC/CQC. We recently developed a method to define consistent and comparable AQC/CQC for pesticides having similar mode of action. The AQC/CQC of the single compounds can be combined to define acute and chronic risk quotientsfor mixture of pesticides with similar mode of action. Following the concentration addition model, which typically applies for compounds with similar mode of action, the risk quotient of the mixture can be expressed as the sum of the ratios between the measured environmental concentration and the AQC/CQC for each pesticide. This risk should stay below 1 to ensure low risk for the aquatic life. The proposed approach has the advantage of being easy to calculate and communicate, and will be proposed to replace the present limit value of 0.1 g/l for pesticides in Switzerland. The AQC/CQC have been defined for more than 20 pesticides (herbicides, insecticides and fungicides) grouped to various mode of action classes and will be presented here.