The Toxicogenome of Hyalella azteca: A Model for Sediment Ecotoxicology and Evolutionary Toxicology.

Hyalella azteca is a cryptic species complex of epibenthic amphipods of interest to ecotoxicology and evolutionary biology. It is the primary crustacean used in North America for sediment toxicity testing and an emerging model for molecular ecotoxicology. To provide molecular resources for sediment quality assessments and evolutionary studies, we sequenced, assembled, and annotated the genome of the H. azteca U.S. Lab Strain. The genome quality and completeness is comparable with other ecotoxicological model species. Through targeted investigation and use of gene expression data sets of H. azteca exposed to pesticides, metals, and other emerging contaminants, we annotated and characterized the major gene families involved in sequestration, detoxification, oxidative stress, and toxicant response. Our results revealed gene loss related to light sensing, but a large expansion in chemoreceptors, likely underlying sensory shifts necessary in their low light habitats. Gene family expansions were also noted for cytochrome P450 genes, cuticle proteins, ion transporters, and include recent gene duplications in the metal sequestration protein, metallothionein. Mapping of differentially expressed transcripts to the genome significantly increased the ability to functionally annotate toxicant responsive genes. The H. azteca genome will greatly facilitate development of genomic tools for environmental assessments and promote an understanding of how evolution shapes toxicological pathways with implications for environmental and human health.

How TK-TD and population models for aquatic macrophytes could support the risk assessment for plant protection products.

This case study of the Society of Environmental Toxicology and Chemistry (SETAC) workshop MODELINK demonstrates the potential use of mechanistic effects models for macrophytes to extrapolate from effects of a plant protection product observed in laboratory tests to effects resulting from dynamic exposure on macrophyte populations in edge-of-field water bodies. A standard European Union (EU) risk assessment for an example herbicide based on macrophyte laboratory tests indicated risks for several exposure scenarios. Three of these scenarios are further analyzed using effect models for 2 aquatic macrophytes, the free-floating standard test species Lemna sp., and the sediment-rooted submerged additional standard test species Myriophyllum spicatum. Both models include a toxicokinetic (TK) part, describing uptake and elimination of the toxicant, a toxicodynamic (TD) part, describing the internal concentration-response function for growth inhibition, and a description of biomass growth as a function of environmental factors to allow simulating seasonal dynamics. The TK-TD models are calibrated and tested using laboratory tests, whereas the growth models were assumed to be fit for purpose based on comparisons of predictions with typical growth patterns observed in the field. For the risk assessment, biomass dynamics are predicted for the control situation and for several exposure levels. Based on specific protection goals for macrophytes, preliminary example decision criteria are suggested for evaluating the model outputs. The models refined the risk indicated by lower tier testing for 2 exposure scenarios, while confirming the risk associated for the third. Uncertainties related to the experimental and the modeling approaches and their application in the risk assessment are discussed. Based on this case study and the assumption that the models prove suitable for risk assessment once fully evaluated, we recommend that 1) ecological scenarios be developed that are also linked to the exposure scenarios, and 2) quantitative protection goals be set to facilitate the interpretation of model results for risk assessment.

An ecosystem services approach to pesticide risk assessment and risk management of non-target terrestrial plants: recommendations from a SETAC Europe workshop.

The registration of plant protection products (PPPs) in the EU is under Regulation 1107/2009, which recommends a tiered approach to assessing the risk to non-target terrestrial plants (NTTPs). However, little information is provided on how to perform and implement higher tier studies or how to use them to refine the risk assessments. Therefore, a stakeholder workshop was organized to consolidate current knowledge and expertise to aid the further development of testing and assessment procedures for NTTPs. This brief communication highlights the agreed recommendations of the workshop, which relate to the three main themes, i.e. specific protection goals, risk assessment and mitigation. The participants of the workshop adopted the European Food Safety Authority (EFSA) approach of using an ecosystem services framework for identifying specific protection goals. First, delivery and protection of ecosystem services were discussed for in-crop, in-field and off-crop, and off-field areas. Second, lower and higher tier risk assessment methods, including modelling approaches, were evaluated. Third, options for risk mitigation of spray drift and run-off were discussed and evaluated. Several important knowledge gaps were identified, and specific data collation and literature-based tasks were actioned to begin to address them. A full workshop report is planned for the fall of 2014.

Silver nanoparticle-specific mitotoxicity in Daphnia magna.

Silver nanoparticles (Ag NPs) are gaining popularity as bactericidal agents in commercial products; however, the mechanisms of toxicity (MOT) of Ag NPs to other organisms are not fully understood. It is the goal of this research to determine differences in MOT induced by ionic Ag(+) and Ag NPs in Daphnia magna, by incorporating a battery of traditional and novel methods. Daphnia embryos were exposed to sublethal concentrations of AgNO3 and Ag NPs (130-650 ng/L), with uptake of the latter confirmed by confocal reflectance microscopy. Mitochondrial function was non-invasively monitored by measuring proton flux using self-referencing microsensors. Proton flux measurements revealed that while both forms of silver significantly affected proton efflux, the change induced by Ag NPs was greater than that of Ag(+). This could be correlated with the effects of Ag NPs on mitochondrial dysfunction, as determined by confocal fluorescence microscopy and JC-1, an indicator of mitochondrial permeability. However, Ag(+) was more efficient than Ag NPs at displacing Na(+) within embryonic Daphnia, based on inductively coupled plasma-mass spectroscopy (ICP-MS) analysis. The abnormalities in mitochondrial activity for Ag NP-exposed organisms suggest a nanoparticle-specific MOT, distinct from that induced by Ag ions. We propose that the MOT of each form of silver are complementary, and can act in synergy to produce a greater toxic response overall.

A spatially and temporally explicit risk assessment for salmon from a prey base exposed to agricultural insecticides.

This risk assessment applied a framework for determining probable co-occurrence of juvenile spring Chinook salmon (Oncorhynchus tshawytscha) with agricultural pesticides in the Willamette Basin, Oregon (Teply et al. this issue) to characterize risk to the threatened population. The assessment accounted for spatial and temporal distribution of 6 acetylcholinesterase-inhibiting insecticides in salmonid habitat within the basin and their relative contributions to mixture toxicity estimated from chemical monitoring data. The 6 insecticides were chlorpyrifos, diazinon, malathion, carbaryl, carbofuran, and methomyl. Seasonal distributions of the juvenile salmon prey base across the basin were determined and compared to co-occurrence with the insecticide mixture to determine the probability of prey reduction and reduced production of juvenile fish. Probability of effect on freshwater aquatic invertebrates was based on acute toxicity species sensitivity distributions (normalized to the most potent compound, chlorpyrifos) using a novel approach to apply the toxicological concept of concentration addition to species sensitivity distributions with differing slopes. The chlorpyrifos distribution was then used to determine relative sensitivity among various species tested within the important taxa making up the prey base. A prey base index was devised, incorporating diet composition and prey availability, to evaluate the indirect effects of the insecticide mixture on juvenile salmon production occurring as a result of a reduction in the prey base. Our analysis targeted fish use of backwater and off-channel habitat units, because they generally coincide with agricultural lands in lowlands and represent shallow habitat with limited water exchange. The percentage of agricultural land use within 300 m of critical habitat stream reaches was used to scale chemical measurement data from a site with high agricultural land use across the full extent of the basin to provide estimates of chemical exposure in each reach. Seasonal impacts were evaluated from mean monthly concentrations. Stressor impact on 5 key taxa was evaluated at each time step and for each reach, and the outcome was compared to a conservation threshold assigned to the prey base index. Only 13% of juveniles reared in backwater, off-channel habitat within 300 m of agricultural land. Percent reduction of carrying capacity as a consequence of reduced prey was estimated to be 5% over the entire brood year. This can be considered lost capacity that is probably compensated elsewhere via increased occupancy (emigration to other habitat units within the reach), which is not accounted for in the model.

Pilot evaluation of a vacuum-assisted biopsy instrument for percutaneous renal biopsy in dogs.

Kidney biopsies in dogs are commonly obtained using automated spring-loaded biopsy instruments. Interpretation of biopsies from dogs with glomerular disease requires examination of at least 5-10 glomeruli, with at least two biopsies usually required for full evaluation. The purpose of this study was to compare quality and interpretability of renal biopsies obtained from healthy dogs with a large-gauge, vacuum-assisted biopsy instrument versus two biopsies obtained with a spring-loaded biopsy needle. Twenty dogs were randomized into two groups, and percutaneous, ultrasound-guided renal biopsies were evaluated using standard criteria. There were no significant differences in the number of biopsies that contained renal tissue, cortex, or medulla. Biopsies obtained with either instrument contained an adequate number of glomeruli and an equivalent number of arterioles and severity of tissue compression. Differences included easier penetration of the renal capsule and collection of sufficient tissue for interpretation with only one instrument pass when using the vacuum-assisted device (vs two passes required with the spring-loaded instrument). Before use in client-owned dogs, future studies should evaluate whether these differences are clinically relevant advantages in the diagnostic evaluation of dogs with kidney disease, and determine the prevalence and severity of complications when using this larger gauge device.

Use of GC × GC/TOF-MS and LC/TOF-MS for metabolomic analysis of Hyalella azteca chronically exposed to atrazine and its primary metabolite, desethylatrazine.

Atrazine is one of the most commonly detected contaminants in the U.S. Little information is available on one of atrazine's metabolites, desethylatrazine (DEA). Two-dimensional gas chromatography and liquid chromatography coupled with time of flight- mass spectrometry were used to examine metabolite profiles of Hyalella azteca chronically exposed to 30 µg/L atrazine and DEA. The majority of identified metabolites were by-products of ß-oxidation of fatty acids suggesting possible disruption in energy metabolism. Eicosanoids increased in exposed females suggesting possible perturbations in neuropeptide hormonal systems. Overall, this research demonstrates the feasibility of utilizing metabolomic profiling of invertebrate species exposed to environmental contaminants as a way to determine mechanisms of toxicity.

Bioaccumulation and biomagnification of polybrominated diphenyl ethers in a food web of Lake Michigan.

Polybrominated diphenyl ethers are hydrophobic chemicals and can biomagnify in food chains. Little is known about the biomagnification of PBDEs in the Lake Michigan food web. Plankton, Diporeia, lake whitefish, lake trout, and Chinook salmon were collected from Lake Michigan in 2006 between April and August. Fish liver and muscle and whole invertebrates were analyzed for six PBDEs (BDE-47, 99, 100, 153, 154, and 209). Carbon and nitrogen stable isotope ratios (delta(13)C and delta(15)N) were also quantified in order to establish the trophic structure of the food web. Geometric means of Sigma PBDE concentrations in fish ranged from 0.562 to 1.61 microg/g-lipid. BDE-209 concentrations ranged from 0.184 to 1.23 microg/g-lipid in all three fish species. Sigma BDE-47, 99, and 209 comprised 80-94% of Sigma PBDE molar concentration. Within each fish species, there were no significant differences in PBDE concentrations between liver and muscle. The highest concentration of BDE-209 (144 microg/g-lipid) was detected in Diporeia. Based on analysis of delta(15)N and PBDE concentrations, BDE-47 and 100 were found to biomagnify, whereas BDE-209 did not. A significant negative correlation between BDE-209 and trophic level was found in this food web. Biomagnification factors were also calculated and again BDE-47 and 100 biomagnified between food web members whereas BDE-209 did not. Diporeia could be one of the main dietary sources of BDE-209 for fish in Lake Michigan; BDE-47 and 100 biomagnified within this food chain; the concentration of BDE-209 decreased at higher trophic levels, suggesting partial uptake and/or biotransformation of BDE-209 in the Lake Michigan food web.

Bioaccumulation and biotransformation of decabromodiphenyl ether and effects on daily growth in juvenile lake whitefish (Coregonus clupeaformis).

Decabromodiphenyl ether (BDE 209) is the main congener in the commonly used commercial flame retardant mixture, "deca-BDE". There is evidence showing that fish can debrominate BDE 209 into potentially more toxic congeners. The objective of this study was to evaluate BDE 209 uptake and its potential effects on juvenile lake whitefish (Coregonus clupeaformis). Lake whitefish were fed BDE 209 at four nominal concentrations (control, 0.1, 1, and 2 microg/g-diet) for 30 days. Livers and carcasses were analyzed for 11 polybrominated diphenyl ether (PBDE) congeners (BDE 47, 99, 100, 153, 154, 196, 197, 206, 207, 208, and 209) and daily otolith increment width was measured as an estimate of growth before and after exposure. Four congeners (BDE 206, 207, 208, and 209) were detected in livers and carcasses. Hepatic BDE 209 concentrations in the 1 and 2 microg/g treatments were significantly higher than in the control group (1.25 and 5.80 nmol/g-lipid compared to 0.183 nmol/g-lipid). The concentration of BDE 209 detected in the tissues of the control group resulted from BDE 209 in the base diets. Concentrations of all congeners from the 1 and 2 microg/g groups were higher in livers than carcasses, indicating the liver was the primary organ of BDE 209 accumulation. Compared to the fraction in diets, the molar fraction of BDE 209 was lower in livers and carcasses, whereas the fractions of BDE 206, 207, and 208 were higher. These different distributions of PBDE congeners resulted from differential adsorption and metabolism. One congener, BDE 206, could be a major metabolite from BDE 209 debromination. Otolith increment widths were narrower in fish from the highest diet concentration administered, suggesting BDE 209 may have affected growth rates. In conclusion, this in vivo study with lake whitefish showed that BDE 209 was debrominated into lower PBDE congeners and that exposure to 2 microg/g may have affected fish growth.

Drinking-water herbicide exposure in Indiana and prevalence of small-for-gestational-age and preterm delivery.

BACKGROUND: Atrazine and other corn herbicides are routinely detected in drinking water. Two studies on potential association of atrazine with small-for-gestational-age (SGA) and preterm birth prevalence found inconsistent results. Moreover, these studies did not control for individual-level potential confounders. OBJECTIVES: Our retrospective cohort study evaluated whether atrazine in drinking water is associated with increased prevalence of SGA and preterm birth. METHODS: We developed atrazine concentration time series for 19 water systems in Indiana from 1993 to 2007 and selected all births (n = 24,154) based on geocoded mother's residences. Log-binomial models were used to estimate prevalence ratios (PRs) for SGA and preterm delivery in relation to atrazine concentrations during various periods of the pregnancy. Models controlled for maternal demographic characteristics, prenatal care and reproductive history, and behavioral risk factors (smoking, drinking, drug use). RESULTS: Atrazine in drinking water during the third trimester and the entire pregnancy was associated with a significant increase in the prevalence of SGA. Atrazine in drinking water > 0.1 microg/L during the third trimester resulted in a 17-19% increase in the prevalence of SGA compared with the control group (< 0.1 microg/L). Mean atrazine concentrations over the entire pregnancy > 0.644 microg/L were associated with higher SGA prevalence than in the control group (adjusted PR = 1.14; 95% confidence interval, 1.03-1.24). No significant association was found for preterm delivery. CONCLUSIONS: We found that atrazine, and perhaps other co-occurring herbicides in drinking water, is associated with an increased prevalence of SGA, but not preterm delivery.

Effect of furosemide and furosemide-carbazochrome combination on exercise-induced pulmonary hemorrhage in Standardbred racehorses.

The objective was to quantify the effect of furosemide and carbazochrome on exercise-induced pulmonary hemorrhage (EIPH) in Standardbred horses using red blood cell count and hemoglobin concentration in bronchoalveolar lavage (BAL) fluid. Six healthy Standardbred horses with prior evidence of EIPH performed a standardized treadmill test 4 h after administration of placebo, furosemide, or furosemide-carbazochrome combination. Red blood cell (RBC) counts and hemoglobin concentrations were determined on the BAL fluid. The RBC count in BAL ranges were (2903-26,025 cells/microL), (45-24,060 cells/microL), and (905-3045 cells/microL) for placebo, furosemide, and furosemide-carbazochrome, respectively. Hemoglobin concentration ranges were (0.03-0.59 mg/mL), (0.01-0.55 mg/mL), and (0.007-0.16 mg/mL) for placebo, furosemide, and furosemide-carbazochrome groups, respectively. No significant differences were detected among treatments. However, there was great variability among horses, suggesting that a larger sample size or better selection of horses was needed.

Acute and chronic toxicity of atrazine and its metabolites deethylatrazine and deisopropylatrazine on aquatic organisms.

Little is known about the toxicity of the atrazine (ATRZ) metabolites desethylatrazine (DEA) and deisopropylatrazine (DIA). We evaluated the acute and chronic toxicity of ATRZ, DEA, and DIA on the amphipods Hyalella azteca and Diporeia spp., and the unicellular algae Pseudokirchneriella subcapitata. In general, acute and chronic toxicity was ranked ATRZ > DEA > DIA. All 96-h median inhibition concentrations (IC(50)) were above concentrations found in the environment (>1,500 microg/L), and sensitivity was highest for the algae. When amphipods were exposed chronically (21 days), Diporeia was several orders of magnitude more sensitive compared to H. azteca. Neither ATRZ nor DEA altered H. azteca sex ratios. In conclusion, our results suggest that short-term exposures of these chemicals to algae and amphipods to concentrations routinely detected in surface waters are unlikely to be a cause of concern. However, the unexpected high sensitivity of Diporeia spp. to these herbicides deserves further attention considering the declining status of this amphipod in the Great Lakes basin.

Risk of limb birth defects and mother's home proximity to cornfields.

BACKGROUND: Although previous studies have linked proximity to crops and birth defects, they lacked individual-level exposure data and none was based on using planted area instead of linear proximity to crops as the exposure metric. We studied birth defects in relation to the area of corn or soybeans within 500 m of the mother's residence. METHODS: We selected all singleton births from rural areas conceived during the 2000-2004 spring-summer months (n=48,216). We determined whether the area with corn or soybeans around the home was associated with birth defects using multiple unconditional logistic regression. RESULTS: We found that limb birth defects (ICD-9-CM 754.5, 755) increased in relation to cornfields (Adjusted OR=1.22; 95 % CI=1.01, 1.47 per additional 10 ha planted with corn within 500 m). None of the birth defect types studied was associated with soybeans. CONCLUSIONS: In the Midwest, a significant and expanding proportion of the population is now living in close proximity to cornfields. Our results suggest that additional studies should be conducted to identify which factor(s) associated with cornfields are behind the observed increase in limb birth defects.

Toxicity of soybean rust fungicides to freshwater algae and Daphnia magna.

Soybeans are intensively grown over large swaths of land in the Midwestern US. Introduction of the pathogenic fungus responsible for Soybean Rust (Phakopsora pachyrhizi) will likely result in a significant increase in the environmental load of strobilurin and conazole fungicides. We determined the toxicity of six such fungicides to the unicellular algae Pseudokirchneriella subcapitata and the aquatic invertebrate, Daphnia magna. We found that levels of concern of some fungicides were lower than annual average runoff concentrations predicted for Indiana. Our results suggest that pyraclostrobin and propiconazole, and to a lesser extent tebuconazole, may cause impacts to algae and daphnids in areas where soybeans are intensively grown. More studies are needed to describe the ecological effects of sublethal exposures to these fungicides, as well as monitoring environmental concentrations in watersheds where these fungicides are applied to soybeans.

Seasonal energetics of northern phocid seals.

The metabolic rate of harp (Pagophilus groenlandicus), harbor (Phoca vitulina), and ringed seals (Pusa hispida) was measured at various temperatures in air and water to estimate basal metabolic rates (BMRs) in these species. The basal rate and body composition of three harp seals were also measured throughout the year to examine the extent to which they vary seasonally. Marine mammalian carnivores generally have BMRs that are over three times the rates expected from body mass in mammals generally, both as a response to a cold-water distribution and to carnivorous food habits with the basal rates of terrestrial carnivores averaging about 1.8 times the mean of mammals. Phocid seals, however, have basal rates of metabolism that are 30% lower than other marine carnivores. Captive seals undergo profound changes in body mass and food consumption throughout the year, and after accounting for changes in body mass, the lowest rate of food intake occurs in summer. Contrary to earlier observations, harp seals also have lower basal rates during summer than during winter, but the variation in BMR, relative to mass expectations, was not associated with changes in the size of fat deposits. The summer reduction in energy expenditure and food consumption correlated with a reduction in BMR. That is, changes in BMR account for a significant portion of the seasonal variation in energy expenditure in the harp seal. Changes in body mass of harp seals throughout the year were due not only to changes in the size of body fat deposits, but also to changes in lean body mass. These results suggest that bioenergetics models used to predict prey consumption by seals should include time-variant energy requirements.

Oxygen flux as an indicator of physiological stress in fathead minnow (Pimephales promelas) embryos: a real-time biomonitoring system of water quality.

The detection of harmful chemicals and biological agents in real time is a critical need for protecting freshwater ecosystems. We studied the real-time effects of five environmental contaminants with differing modes of action (atrazine, cadmium chloride, pentachlorophenol, malathion, and potassium cyanide) on respiratory oxygen consumption in 2-day postfertilization fathead minnow (Pimephales promelas) eggs. Our objective was to assess the sensitivity of fathead minnow eggs using the self-referencing micro-optrode technique to detect instantaneous changes in oxygen consumption after brief exposures to low concentrations of contaminants. Oxygen consumption data indicated that the technique is indeed sensitive enough to reliably detect physiological alterations induced by four of the five contaminants. After 2 h of exposure, we identified significant increases in oxygen consumption upon exposure to pentachlorophenol (100 and 1000 microg/L), cadmium chloride (0.0002 and 0.002 microg/L), and atrazine (150 microg/L). In contrast, we observed a significant decrease in oxygen flux after exposuresto potassium cyanide (44 and 66 microg/L) and atrazine (1500 microg/L). No effects were detected after exposures to malathion (200 and 340 microg/L). Our work is the first step in development of a new technique for physiologically coupled biomonitoring as a sensitive and reliable tool for the detection of environmental toxicants.