Gene expression and pathologic alterations in juvenile rainbow trout due to chronic dietary TCDD exposure.

The goal of this project was to use functional genomic methods to identify molecular biomarkers as indicators of the impact of TCDD exposure in rainbow trout. Specifically, we investigated the effects of chronic dietary TCDD exposure on whole juvenile rainbow trout global gene expression associated with histopathological analysis. Juvenile rainbow trout were fed Biodiet starter with TCDD added at 0, 0.1, 1, 10 and 100 ppb (ngTCDD/g food), and fish were sampled from each group at 7, 14, 28 and 42 days after initiation of feeding. 100 ppb TCDD caused 100% mortality at 39 days. Fish fed with 100 ppb TCDD food had TCDD accumulation of 47.37 ppb (ngTCDD/g fish) in whole fish at 28 days. Histological analysis from TCDD-treated trout sampled from 28 and 42 days revealed that obvious lesions were found in skin, oropharynx, liver, gas bladder, intestine, pancreas, nose and kidney. In addition, TCDD caused anemia in peripheral blood, decreases in abdominal fat, increases of remodeling of fin rays, edema in pericardium and retrobulbar hemorrhage in the 100 ppb TCDD-treated rainbow trout compared to the control group at 28 days. Dose- and time-dependent global gene expression analyses were performed using the cGRASP 16,000 (16K) cDNA microarray. TCDD-responsive whole body transcripts identified in the microarray experiments have putative functions involved in various biological processes including growth, cell proliferation, metabolic process, and immune system processes. Nine microarray-identified genes were selected for QPCR validation. CYP1A3 and CYP1A1 were common up-regulated genes and HBB1 was a common down-regulated gene among each group based on microarray data, and their QPCR validations are consistent with microarray data for the 10 and 100 ppb TCDD treatment groups after 28 days exposure (p<0.05). In addition, in the 100 ppb group at 28 days, expression of complement component C3-1 and trypsin-1 precursor have a more than 10-fold induction from the microarray experiments, and their QPCR validations are consistent and showed significant induction in the 100 ppb group at 28 days (p<0.05). Overall, lesion in nasal epithelium is a novel and significant result in this study, and TCDD-responsive rainbow trout transcripts identified in the present study may lead to the development of new molecular biomarkers for assessing the potential impacts of environmental TCDD on rainbow trout populations.

A fresh look at road salt: aquatic toxicity and water-quality impacts on local, regional, and national scales.

A new perspective on the severity of aquatic toxicity impact of road salt was gained by a focused research effort directed at winter runoff periods. Dramatic impacts were observed on local, regional, and national scales. Locally, samples from 7 of 13 Milwaukee, Wisconsin area streams exhibited toxicity in Ceriodaphnia dubia and Pimephales promelas bioassays during road-salt runoff. Another Milwaukee stream was sampled from 1996 to 2008 with 72% of 37 samples exhibiting toxicity in chronic bioassays and 43% in acute bioassays. The maximum chloride concentration was 7730 mg/L. Regionally, in southeast Wisconsin, continuous specific conductance was monitored as a chloride surrogate in 11 watersheds with urban land use from 6.0 to 100%. Elevated specific conductance was observed between November and April at all sites, with continuing effects between May and October at sites with the highest specific conductance. Specific conductance was measured as high as 30,800 µS/cm (Cl = 11,200 mg/L). Chloride concentrations exceeded U.S. Environmental Protection Agency (USEPA) acute (860 mg/L) and chronic (230 mg/L) water-quality criteria at 55 and 100% of monitored sites, respectively. Nationally, U.S. Geological Survey historical data were examined for 13 northern and 4 southern metropolitan areas. Chloride concentrations exceeded USEPA water-quality criteria at 55% (chronic) and 25% (acute) of the 168 monitoring locations in northern metropolitan areas from November to April. Only 16% (chronic) and 1% (acute) of sites exceeded criteria from May to October. At southern sites, very few samples exceeded chronic water-quality criteria, and no samples exceeded acute criteria.

Aquatic toxicity of airfield-pavement deicer materials and implications for airport runoff.

Concentrations of airfield-pavement deicer materials (PDM) in a study of airport runoff often exceeded levels of concern regarding aquatic toxicity. Toxicity tests on Vibrio fischeri, Pimephales promelas, Ceriodaphnia dubia, and Pseudokirchneriella subcapitata (commonly known as Selenastrum capricornutum) were performed with potassium acetate (K-Ac) PDM, sodium formate (Na-For) PDM, and with freezing-point depressants (K-Ac and Na-For). Results indicate that toxicity in PDM is driven by the freezing-point depressants in all tests except the Vibrio fisheri test for Na-For PDM which is influenced by an additive. Acute toxicity end points for different organisms ranged from 298 to 6560 mg/L (as acetate) for K-Ac PDM and from 1780 to 4130 mg/L (as formate) for Na-For PDM. Chronic toxicity end points ranged from 19.9 to 336 mg/L (as acetate) for K-Ac PDM and from 584 to 1670 mg/L (as formate) for Na-For PDM. Sample results from outfalls at General Mitchell International Airport in Milwaukee, WI (GMIA) indicated that 40% of samples had concentrations greater thanthe aquatic-life benchmarkfor K-Ac PDM. K-Ac has replaced urea during the 1990s as the most widely used PDM at GMIA and in the United States. Results of ammonia samples from airport outfalls during periods when urea-based PDM was used at GMIA indicated that 41% of samples had concentrations exceeding the U.S. Environmental Protection Agency (USEPA) 1-h water-quality criterion. The USEPA 1-h water-quality criterion for chloride was exceeded in 68% of samples collected in the receiving stream, a result of road-salt runoff from urban influence near the airport. Results demonstrate that PDM must be considered to comprehensively evaluate the impact of chemical deicers on aquatic toxicity in water containing airport runoff.

Aquatic toxicity of nine aircraft deicer and anti-icer formulations and relative toxicity of additive package ingredients alkylphenol ethoxylates and 4,5-methyl-1H-henzotriazoles.

Characterization of the effects of aircraft deicer and anti-icer fluid (ADAF) runoff on aquatic organisms in receiving streams is a complex issue because the identities of numerous toxic additives are proprietary and not publicly available. Most potentially toxic and endocrine disrupting effects caused by ADAF are due to the numerous additive package ingredients which vary among manufacturers and types of ADAF formulation. Toxicity investigations of nine ADAF formulations indicate that endpoint concentrations for formulations of different manufacturers are widely variable. Type IV ADAF (anti-icers) are more toxic than Type I (deicers) for the four organisms tested (Vibrio fischeri, Pimephales promelas, Ceriodaphnia dubia, and Selenastrum capricornutum). Acute toxicity endpoint concentrations ranged from 347 to 7700 mg/L as ADAF for Type IV and from 1550 to 45,100 mg/L for Type I formulations. Chronic endpoint concentrations ranged from 70 to 1300 mg/L for Type IV and from 37 to 18,400 mg/L for Type I formulations. Alkylphenol ethoxylates and tolyltriazoles are two known classes of additives. Nonylphenol, nonylphenol ethoxylates, octylphenol, octylphenol ethoxylates, and 4,5-methyl-1H-benzotriazoles were quantified in the nine ADAF formulations, and toxicity tests were conducted with nonylphenol ethoxylates and 4,5-methyl-1H-benzotriazoles. Toxicity units computed for glycol and these additives, with respect to toxicity of the ADAF formulations, indicate that a portion of ADAF toxicity can be explained by the known additives and glycols, but much of the toxicity is due to unidentified additives.

Impacts of aircraft deicer and anti-icer runoff on receiving waters from Dallas/Fort Worth International Airport, Texas, U.S.A.

From October 2002 to April 2004, data were collected from Dallas/Fort Worth (DFW) International Airport (TX, U.S.A.) outfalls and receiving waters (Trigg Lake and Big Bear Creek) to document the magnitude and potential effects of aircraft deicer and anti-icer fluid (ADAF) runoff on water quality. Glycol concentrations at outfalls ranged from less than 18 to 23,800 mg/L, whereas concentrations in Big Bear Creek were less because of dilution, dispersion, and degradation, ranging from less than 18 to 230 mg/L. Annual loading results indicate that 10 and 35% of what was applied to aircraft was discharged to Big Bear Creek in 2003 and 2004, respectively. Glycol that entered Trigg Lake was diluted and degraded before reaching the lake outlet. Dissolved oxygen (DO) concentrations at airport outfalls sometimes were low (<2.0 mg/L) but typical of what was measured in an urban reference stream. In comparison, the DO concentration at Trigg Lake monitoring sites was consistently greater than 5.5 mg/L during the monitoring period, probably because of the installation of aerators in the lake by DFW personnel. The DO concentration in Big Bear Creek was very similar at sites upstream and downstream of airport influence (>5.0 mg/L). Results of toxicity tests indicate that effects on Ceriodaphnia dubia, Pimephales promelas, and Selanastrum capricornutum are influenced by type IV ADAF (anti-icer), not just type I ADAF (deicer) as is more commonly assumed.

Characterization of aircraft deicer and anti-icer components and toxicity in airport snowbanks and snowmelt runoff.

Snowbank samples were collected from snowbanks within a medium-sized airport for four years to characterize aircraft deicer and anti-icer (ADAF) components and toxicity. Concentrations of ADAF components varied with median glycol concentrations from individual sampling periods ranging from 65 to 5940 mg/L. Glycol content in snowbanks ranged from 0.17 to 11.4% of that applied to aircraft. Glycol, a freezing point depressant, was selectively removed during melt periods before snow and ice resulting in lower glycol concentrations after melt periods. Concentrations of ADAF components in airport runoff were similar during periods of snowmelt as compared to active ADAF application periods; however, due to the long duration of snowmelt events, greater masses of glycol were transported during snowmelt events. Alkylphenol ethoxylates (APEO), selected APEO degradation products, and 4- and 5-methyl-1H-benzotriazole were detected in snowbank samples and airport snowmelt. Concentrations of APEO parent products were greater in snowbank samples than in runoff samples. Relative abundance of APEO degradation products increased in the downstream direction from the snowbank to the outfalls and the receiving stream with respect to APEO parent compounds and glycol. Toxicity in Microtox assays remained in snowbanks after most glycol had been removed during melt periods. Increased toxicity in airport snowbanks as compared to other urban snowbanks was not explained by additional combustion or fuel contribution in airport snow. Organic markers suggest ADAF additives as a possible explanation for this increased toxicity. Results indicate that glycol cannot be used as a surrogate for fate and transport of other ADAF components.

Toxicity of ambient atmospheric particulate matter from the Lake Michigan (USA) airshed to aquatic organisms.

Short-term chronic and acute aquatic bioassays are valuable tools in screening a variety of environmental samples. However, only a limited number of studies have used these methods for testing the toxicity of atmospheric particulate matter samples. Previous studies have shown that compounds known to have adverse biological effects, such as polycyclic aromatic hydrocarbons, are deposited in significant quantities into Lake Michigan (USA); however, these compounds comprise a small portion of the total particulate matter deposition. In the present study, a method is described for using Ceriodaphnia dubia, Selenastrum capricornutum (green algae), and MitoScan bioassays to compare the toxicities of reconstituted hard freshwater and methylene chloride extracts of atmospheric particulate matter collected at three locations around the southern shore of Lake Michigan in August 2000. The locations include an urban/industrial site in Milwaukee (WI, USA), an urban-impacted/industrial site in Porter (IN, USA), and a rural site in Bridgman (MI, USA). The bulk chemistry, including organic and elemental carbon, sulfate, nitrate, ammonium, and chloride, shows regional similarities over the sampling event, but the toxicities vary spatially by site, by extraction solvent, and by bioassay. Thus, the bioassays are sufficiently sensitive to show differences in toxicity among the atmospheric particulate matter extracts and have significantly different responses to the samples to enable an initial comparison of toxicity from the different sites.

Modifications to the fathead minnow (Pimephales promelas) chronic test method to remove mortality due to pathogenic organisms.

Whole effluent toxicity testing is used to evaluate the discharge of materials that may be harmful to indigenous aquatic life. Unlike most environmental analyses, receiving water (the water body into which the effluent is discharged) often is used as dilution water in whole effluent toxicity tests to simulate the aquatic environment into which the effluent is introduced. In approximately 26% of whole effluent toxicity tests conducted by Wisconsin (USA) certified labs between 1988 and 1998, a pathogenic effect caused failure of the receiving water controls during the fathead minnow chronic test (i.e., > 20% mortality). We performed microbiological work to isolate pathogenic organisms from receiving waters, the fish, and their food. We found pathogenic organisms such as Flexibacter columnaris, Aeromonas hydrophila, and Flavobacter spp. to be ubiquitous and were not able to remove them from the test (e.g., through decontamination of the fish food and carefully following sterility procedures). To eliminate the pathogenic effect, we evaluated manipulations of the sample and the test method including filtering receiving water, irradiating receiving water, using older fish (48 h), using clean test beakers each day of the test, and using smaller test beakers (30 ml) with two fish per beaker. In samples demonstrating the pathogenic effect, most of these manipulations significantly reduced mortality. The use of smaller tests cups was significantly better at reducing the effect than all of the other sample and method manipulations. These results indicate that a simple method modification to the fathead minnow chronic test will improve test reliability when diluting effluents with receiving waters.

Studies of the environmental fate and effect of aircraft deicing fluids: detection of 5-methyl-1H-benzotriazole in the fathead minnow (Pimephales promelas).

This paper presents the results of a number of field and laboratory studies to evaluate the environmental impact of aircraft deicing and anti-icing fluids (ADAFs) on aquatic systems. Both 5-methyl-1H-benzotriazole (5-MeBt) and 4-methyl-1H-benzotriazole (4-MeBt), known additives to ADAFs, were found in whole-tissue extracts from minnows placed downstream of an effluent outfall that receives ADAF contaminated runoff from General Mitchell International Airport (Milwaukee, WI, USA). Neither of these compounds was detected in tissues from minnows placed upstream from the airport. A toxicity assessment of water collected during the minnow exposure studies utilizing Hyalella azteca, Pimephales promelas, and Ceriodaphnia dubia showed greater toxicity in a secondary airport outfall containing ADAFs when compared to upstream non-ADAF-contaminated samples. In two 28-d static renewal tests using 5-MeBt laboratory-fortified waters, 5-MeBt was detected in whole-tissue extracts of minnows at all concentrations tested. In studies using laboratory water fortified with 5-MeBt, the median lethal concentration (LC50) of 5-MeBt for P. promelas was found to be 22.0 mg/L. The LC50 for C. dubia to 5-MeBt laboratory-fortified water was found to be 81.3 mg/L. The 25% inhibition concentration (IC25) of 5-MeBt for the green alga Selenastrum capricornutum was 23.2 mg/L, and the average median effective concentration (EC50) for Microtox was 4.25 mg/L. The results of these field and lab studies indicate that additives, other than glycols, used in aircraft deicing fluids can be found in aquatic systems and may be of greater risk than previously believed.