Acute and reproductive toxicity of nano-sized metal oxides (ZnO and TiO2) to earthworms (Eisenia fetida).

An increase in nanomaterial applications will likely lead to an increased probability of environmental exposures, raising concerns regarding the safety of these materials. Recent studies have indicated that manufactured nanomaterials, such as metal oxides, have the potential to be harmful to aquatic and terrestrial organisms. The majority of nano-metal oxide research addressing potential toxicological issues has been focused in aquatic environments with very little terrestrial data. This study characterized the acute and reproductive toxicity of zinc oxide (ZnO) and titanium dioxide (TiO(2)) to earthworms (Eisenia fetida) in a terrestrial system. Following a 14 d exposure, nano-sized ZnO on filter paper was acutely toxic to E. fetida, while nano-sized TiO(2) did not exhibit acute toxicity. In contrast, neither nano-sized ZnO nor TiO(2) exhibited acute toxicity to earthworms in sand. Both nano-sized ZnO and TiO(2), following a 4 week exposure, caused reproductive effects in earthworms in artificial soil. Overall, nano-sized ZnO exhibited greater toxicity than nano-sized TiO(2) in Eisenia fetida.

Uptake of 17α-ethynylestradiol and triclosan in pinto bean, Phaseolus vulgaris.

Pharmaceuticals and personal care products (PPCPs) have emerged as a group of potential environmental contaminants of concern. PPCPs in soil may enter terrestrial food webs via plant uptake. We evaluated uptake of 17α-ethynylestradiol (EE2) and triclosan in bean plants (Phaseolus vulgaris) grown in sand and soil. The extent of uptake and accumulation of EE2 and triclosan in plants grown in sand was higher than in plants grown in soil. In sand (conditions of maximum contaminant bioavailability), bioconcentration factors (BCFs) of EE2 and triclosan in roots (based on dry weights) were 1424 and 16,364, respectively, whereas BCFs in leaves were 55 for EE2 and 85 for triclosan. In soil, the BCF of EE2 decreased from 154 in the first week to 32 in the fourth week while it fluctuated in leaves from 18 to 20. The BCF for triclosan in plants grown in soil increased over time to 12 in roots and 8 in leaves. These results indicate that the potential for uptake and accumulation of PPCPs in plants exists. This trophic transfer pathway should be considered when assessing exposure to certain PPCPs, particularly with the use of recycled wastewater for irrigation.

Acute effects of Fe2O3, TiO2, ZnO and CuO nanomaterials on Xenopus laevis.

Metal oxide nanomaterials have exhibited toxicity to a variety of aquatic organisms, especially microbes and invertebrates. To date, few studies have evaluated the toxicity of metal oxide nanomaterials on aquatic vertebrates. Therefore, this study examined effects of ZnO, TiO(2), Fe(2)O(3), and CuO nanomaterials (20-100 nm) on amphibians utilizing the Frog Embryo Teratogenesis Assay Xenopus (FETAX) protocol, a 96 h exposure with daily solution exchanges. Nanomaterials were dispersed in reconstituted moderately hard test medium. These exposures did not increase mortality in static renewal exposures containing up to 1,000 mg L(-1) for TiO(2), Fe(2)O(3), CuO, and ZnO, but did induce developmental abnormalities. Gastrointestinal, spinal, and other abnormalities were observed in CuO and ZnO nanomaterial exposures at concentrations as low as 3.16 mg L(-1) (ZnO). An EC(50) of 10.3 mg L(-1) ZnO was observed for total malformations. The minimum concentration to inhibit growth of tadpoles exposed to CuO or ZnO nanomaterials was 10 mg L(-1). The results indicate that select nanomaterials can negatively affect amphibians during development. Evaluation of nanomaterial exposure on vertebrate organisms are imperative to responsible production and introduction of nanomaterials in everyday products to ensure human and environmental safety.

Effects of ZnO nanomaterials on Xenopus laevis growth and development.

The objectives of this study were to quantify uptake and developmental effects of zinc oxide nanomaterials (nano-ZnO) on Xenopus laevis throughout the metomormosis process. To accomplish this, X. laevis were exposed to aqueous suspensions of 40-100 nm nano-ZnO beginning in-ovo and proceeding through metamorphosis. Nanomaterials were dispersed via sonication methods into reconstituted moderately hard water test solutions. A flow-through system was utilized to decrease the likelihood of depletion in ZnO concentration. Exposure to 2 mg/L nano-ZnO significantly increased mortality incidence to 40% and negatively affected metamorphosis of X. laevis. Tadpoles exposed to 2 mg/L nano-ZnO developed slower as indicated by tadpoles with an average stage of 56 at the conclusion of the study which was significantly lower than the control tadpole stages. No tadpoles exposed to 2 mg/L of nano-ZnO completed metamorphosis by the conclusion of the study. Tadpoles exposed to 0.125 mg/L nano-ZnO experienced faster development along with larger body measurements indicating that low dose exposure to nano-ZnO can stimulate growth and metamorphosis of X. laevis.

Bacterial community dynamics in high and low bioavailability soils following laboratory exposure to a range of hexahydro-1,3,5-trinitro-1,3,5-triazine concentrations.

Few studies have examined the potential long-term effects of high concentrations of hexahydro-1,3,5-trinitro-1,3,5-triaxine (RDX) on bacterial communities in soil. In the present study, a sandy loam soil and a silt loam soil (high and low bioavailability, respectively) were artificially contaminated with RDX (0, 50, 500, 1,500, 5,000, 10,000, and 15,000 mg/kg soil). Bacterial communities from each treatment were monitored over 63 d to characterize the effects of RDX exposure on bacterial activity, biomass, functional diversity (Biolog microtiter plates), and structural diversity (denaturing gradient gel electrophoresis of 16S rDNA). Bacterial communities native to the high bioavailability soil were inherently different than bacterial communities native to the silt loam soil, not only in terms of bacterial activity and biomass, but also in terms of bacterial community functional and structural diversity. Soil RDX contamination was correlated with decreased bacterial biomass in the silt loam soil treatments and with decreased bacterial activity in the sandy loam soil treatments on day 7. Soil RDX contamination did not cause a significant shift in the functional diversity of the bacterial communities native to the silt loam soil, but was correlated with a shift in identities of substrates used by bacterial communities native to the sandy loam soil on day 7. Bacterial community structure was insensitive to the gradient of RDX concentrations at the beginning of the incubation. However, the identities of carbon substrates used by bacterial communities in both soil types were affected by long-term incubation with RDX.

Effects of functionalized and nonfunctionalized single-walled carbon nanotubes on root elongation of select crop species.

Single-walled carbon nanotubes have many potential beneficial uses, with additional applications constantly being investigated. Their unique properties, however, create a potential concern regarding toxicity, not only in humans and animals but also in plants. To help develop protocols to determine the effects of nanotubes on plants, we conducted a pilot study on the effects of functionalized and nonfunctionalized single-walled carbon nanotubes on root elongation of six crop species (cabbage, carrot, cucumber, lettuce, onion, and tomato) routinely used in phytotoxicity testing. Nanotubes were functionalized with poly-3-aminobenzenesulfonic acid. Root growth was measured at 0, 24, and 48 h following exposure. Scanning-electron microscopy was used to evaluate potential uptake of carbon nanotubes and to observe the interaction of nanotubes with the root surface. In general, nonfunctionalized carbon nanotubes affected root length more than functionalized nanotubes. Nonfunctionalized nanotubes inhibited root elongation in tomato and enhanced root elongation in onion and cucumber. Functionalized nanotubes inhibited root elongation in lettuce. Cabbage and carrots were not affected by either form of nanotubes. Effects observed following exposure to carbon nanotubes tended to be more pronounced at 24 h than at 48 h. Microscopy images showed the presence of nanotube sheets on the root surfaces, but no visible uptake of nanotubes was observed.

Metals and organochlorine pesticides in caudal scutes of crocodiles from Belize and Costa Rica.

Despite high animal diversity in the Neotropics and the largely unregulated use and disposal of pesticides and industrial chemicals in Central America, few data exist regarding accumulation of environmental contaminants in Central American wildlife. In this study we examined accumulation of metals and organochlorine (OC) pesticides in caudal scutes of crocodiles from Belize and Costa Rica. Scutes from Morelet's crocodiles (Crocodylus moreletii) from two sites in northern Belize were analyzed for metals, and scutes from American crocodiles (C. acutus) from one site in Costa Rica were analyzed for metals and OC pesticides. All scutes (n=25; one scute from each of 25 individuals) contained multiple contaminants. Mercury was the predominant metal detected, occurring in all scutes examined from both species. Other metals detected include cadmium, copper, lead, and zinc. American crocodile scutes from Costa Rica contained multiple OC pesticides, including endrin, methoxychlor, p,p'-DDE, and p,p'-DDT, all of which occurred in 100% of scutes analyzed (n=6). Mean metal and OC concentrations varied in relation to those previously reported in crocodilian scutes from other localities in North, Central, and South America. OC concentrations in American crocodile scutes were generally higher than those previously reported for other Costa Rican wildlife. Currently, caudal scutes may serve as general, non-lethal indicators of contaminant accumulation in crocodilians and their areas of occurrence. However, a better understanding of the relationships between pollutant concentrations in scutes, internal tissues, and environmental matrices at sample collection sites are needed to improve the utility of scutes in future ecotoxicological investigations.

Organochlorine contaminants in complete clutches of Morelet's crocodile (Crocodylus moreletii) eggs from Belize.

Seven complete clutches of Morelet's crocodile (Crocodylus moreletii) eggs were collected in northern Belize and examined for organochlorine (OC) pesticide residues. The primary OC detected, p,p-DDE, was found in every egg analyzed (n=175). Other OCs detected included p,p-DDT, p,p-DDD, methoxychlor, aldrin, and endosulfan I. Concentrations of individual OCs ranged from 4 ppb (ng chemical/g egg wet weight) to greater than 500 ppb. A statistical evaluation of p,p-DDE levels in three complete clutches was used to derive the minimum number of eggs needed from a clutch to precisely determine the mean p,p-DDE concentration representative of that clutch. Sample sizes of 8 (80% confidence level) and 11 (90% confidence level) were determined to yield an accurate estimate of contaminant levels in a full clutch of eggs. The statistically recommended sample size of 11 eggs (at 90% confidence level) was successfully tested on the four additional clutches.

Effects of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) metabolites on cricket (Acheta domesticus) survival and reproductive success.

The effect of two major hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) metabolites, hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX), on cricket (Acheta domesticus) survival and reproduction was studied. RDX metabolites did not have adverse effects on cricket survival, growth, and egg production. However, MNX and TNX did affect egg hatching. MNX and TNX were more toxic in spiked-sand than in topical tests. TNX was more toxic to egg than MNX. Developmental stage and exposure time affected hatching. After 30 days exposure to MNX or TNX, the EC20, EC50, and EC95 were 47, 128, and 247 microg/g for TNX, and 65, 140, and 253 microg/g for MNX in topical tests. The ECs for 20, 50, and 95 were 21, 52, and 99 microg/g for MNX, and 12, 48, and 97 microg/g for TNX in sand. No gross abnormalities in cricket nypmhs were observed in all experiments indicating that neither TNX or MNX is teratogenic in this assay.

Development of an extraction method for perchlorate in soils.

Perchlorate originates as a contaminant in the environment from its use in solid rocket fuels and munitions. The current US EPA methods for perchlorate determination via ion chromatography using conductivity detection do not include recommendations for the extraction of perchlorate from soil. This study evaluated and identified appropriate conditions for the extraction of perchlorate from clay loam, loamy sand, and sandy soils. Based on the results of this evaluation, soils should be extracted in a dry, ground (mortar and pestle) state with Milli-Q water in a 1 ratio 1 soil ratio water ratio and diluted no more than 5-fold before analysis. When sandy soils were extracted in this manner, the calculated method detection limit was 3.5 microg kg(-1). The findings of this study have aided in the establishment of a standardized extraction method for perchlorate in soil.

Effects of perchlorate on earthworm (Eisenia fetida) survival and reproductive success.

The effects of perchlorate on earthworm, Eisenia fetida, survival and reproductive success were evaluated using three types of tests: dermal contact (filter paper), sand, and artificial soil. All studies utilized a range of perchlorate concentrations in order to simulate levels that are likely to occur in the environment under different scenarios (typical soil levels vs. spill levels). The OECD filter paper contact test involved exposing earthworms to the test compound on moist filter paper for 14 days to evaluate earthworm survival under a worst-case dermal exposure scenario. A similar test involved exposing the earthworms to perchlorate-contaminated sand, where earthworm survival decreased as concentration of perchlorate increased, with no worms surviving 14 days at the highest treatment concentrations (>2000 microg/g). However, the perchlorate concentrations that affected the survival of E. fetida are likely to occur only under extreme conditions (e.g. spills). The effect of perchlorate on the reproductive success (cocoon production) of E. fetida over a 4-week test period in artificial soil and a 3-week test period in sand was also examined. Production of cocoons was observed in soil containing up to 100 microg/g perchlorate, with no production in the uppermost treatment groups (1000 microg/g). Cocoon production was highest in the control group, although overall cocoon production appeared to be low. In contrast to the acute toxicity tests, perchlorate did affect earthworm reproduction at environmentally relevant soil concentrations. In addition, preliminary data suggest that cocoons produced under perchlorate contamination did not hatch as well as cocoons produced in control soil despite incubation of both sets of cocoons in clean soil or sand.

Optimization of operating conditions for the determination of perchlorate in biological samples using preconcentration/preelution ion chromatography.

Perchlorate originates as a contaminant in the environment from the use of salts in the manufacture of solid rocket fuels and munitions. Monitoring potential perchlorate contamination in the environment is of interest, however, very few analytical methods have been developed for perchlorate determination in biological samples. Analysis of complex samples by ion chromatography is complicated by matrix components that can interfere with perchlorate determination. However, a recently developed preconcentration/preelution (PC/PE) ion chromatography method has demonstrated the capability to analyze certain complex samples such as high salinity water, milk, and hydroponic fertilizers. The ability of this method to reduce sample background and lower detection limits in ion chromatography for various biological samples was evaluated in this study. The PC/PE method was applicable to the analysis of kidneys, livers, zebrafish, quail eggs, lettuce, and urine. Optimal operating conditions were determined for each matrix. Ranges of optimal wash volumes were shorter when 15 mM NaOH prewash solutions were used compared with 10mM and good recovery was achieved for most matrices with an injection period > or =60s. Prewash solution concentration did not appear to significantly affect matrix background. The PC/PE method was capable of reducing sample background when compared to EPA Method 314.0, which resulted in detection limits, with the exception of zebrafish and urine, that were two-fold lower than those achieved with EPA Method 314.0.

Challenges in determining perchlorate in biological tissues and fluids: implications for characterizing perchlorate exposure.

The ability to measure environmental contaminants in biological tissues and fluids is important in the characterization of exposure. However, the analysis of certain contaminants in these matrices presents significant challenges. Perchlorate (ClO4-) has emerged as a potential contaminant of concern primarily in drinking water and also in contaminated food. Significant advances have been made in the analysis of perchlorate in environmental matrices (water, soil) by ion chromatography (IC). In contrast, the analysis of perchlorate in extracts of biological tissues and fluids (vegetation, organs, milk, blood, urine, etc.) presents several challenges including small sample sizes, extracts with high matrix conductivity, and co-elution of other ions during IC analysis. To be able to detect low concentrations of perchlorate in biological samples, interferences must be removed or minimized, such as through the use of preparative chromatography cleanup techniques and/or alternative analytical methods less susceptible to common interferences (preconcentration or mass spectrometric detection). We present discussion and examples of the challenges encountered in the analysis of tissue extracts and fluids for perchlorate by IC and how some of those analytical challenges have been overcome.

A cleanup method for perchlorate determination in urine.

There is increasing concern about perchlorate exposure because of perchlorate's potential effects on organisms as a thyroid hormone disruptor, as well as its contamination of the environment being much more widespread than previously thought. Perchlorate is excreted primarily into urine, therefore, evaluating perchlorate residues in urine should be a reasonable approach for determining exposure and if successful could be used as an effective biomarker of perchlorate exposure. Since the presence of ions and other biomolecules in matrices like urine usually confounds accurate determination of perchlorate by ion chromatography, it is necessary to develop efficient methods for perchlorate determination in these matrices. We developed a method that reduces the background signal of urine, which is typically the problem with the analysis of biological fluids and tissues by ion chromatography. Relatively high recovery of perchlorate was shown. In cow urine samples spiked with perchlorate at 2.5, 10, and 100mug/L, perchlorate recoveries were 67%+/-2.5, 77%+/-3.6, and 81%+/-1.7 (mean+/-S.D.), respectively. In addition, the detection limit was as low as 12.6, 12.3, and 18.7mug/L in cow, vole, and human urine samples, respectively.

Patterns of genotoxicity and contaminant exposure: evidence of genomic instability in the marsh frogs (Rana ridibunda) of Sumgayit, Azerbaijan.

The wetlands of Sumgayit in the Azerbaijan Republic contain complex mixtures of contaminants, including polycyclic aromatic hydrocarbons (PAHs), mercury, organochlorine pesticides, and polychlorinated biphenyls. Marsh frogs (Rana ridibunda) were collected from several contaminated wetlands within the city as well as from two reference sites outside the city. Sediment samples revealed heterogeneous patterns of PAH and mercury concentrations throughout Sumgayit, with the highest levels occurring east of the Sumgayit River, within the industrial zone. Flow cytometry and micronucleus assay revealed elevated estimates of genetic damage in frogs from the wetlands east of the Sumgayit River compared to frogs from the reference sites. Flow cytometric data showed a significant correlation with sediment mercury concentrations, whereas population micronucleus frequencies were significantly correlated with high-molecular-weight PAHs.

Organochlorine pesticides and mercury in cottonmouths (Agkistrodon piscivorus) from northeastern Texas, USA.

Despite their ecological importance and global decline, snakes remain poorly studied in ecotoxicology. In this study, we examined organochlorine (OC) pesticide and mercury accumulation in cottonmouths (Agkistrodon piscivorus) living on a contaminated site in northeastern Texas (USA). Mercury and p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) were detected in all snakes examined. Other OCs, including p,p'-dichlorodiphenyltrichloroethane (p,p'-DDT), methoxychlor, aldrin, and heptachlor, also were detected, but less frequently. Concentrations of p,p'-DDE were higher in fat than in liver, while mercury concentrations were highest in liver, followed by kidney and tail clips. One animal contained the highest mercury concentration yet reported for a snake (8,610 ng/g). Mercury concentrations in liver and kidney were higher in males than females, while no intersex differences in p,p'-DDE concentrations were observed. Concentrations of p,p'-DDE in fat were correlated positively with body size in male cottonmouths but not females, suggesting a slower rate of accumulation in females. Body size strongly predicted mercury concentrations in liver, kidney, and tail clips of both sexes. Tail clips were strong predictors of mercury in liver and kidney in males but not females, suggesting possible sex-dependent differences in mercury toxicokinetics. Both long-term field studies and controlled laboratory investigations are needed to adequately assess the response of cottonmouths to chronic contaminant exposure.

Preconcentration/preelution ion chromatography for the determination of perchlorate in complex samples.

The determination of perchlorate in complex matrices by ion chromatography (IC) with an online preconcentration and preelution technique is discussed. The method was applied to different sample types containing large concentrations of matrix anions that would otherwise interfere with analysis via conventional IC. The present approach was highly effective in removing most of the matrix anions and was thus resistant to the interferences commonly encountered in a high ionic strength background. Method performance was evaluated by analyzing for low-level perchlorate in synthetic high ionic strength solutions, tissue extracts, and hydroponic nitrate fertilizer samples. Not only is it easier to practice the present method compared to USEPA Method 314.0, but for most of these samples the present approach provided equal to or better recovery of perchlorate than Method 314.0. With a sample of specific conductance 12,650muScm(-1), for example, the present method provided a perchlorate recovery of 101% at the 25mugL(-1) level versus 89% by EPA Method 314.0. Method detection limits of perchlorate in hydroponic fertilizer samples with this method (130-190mugkg(-1)) are the lowest thus far reported.

Uptake of perchlorate in terrestrial plants.

Cucumber (Cucumis sativus L.), lettuce (Lactuca sativa L.), and soybean (Glycine max) were used to determine uptake of the perchlorate anion (100 ppb) from sand. Plants were watered with different ratios of Hydrosol (a diluted solution of Peters All-Purpose Plant Food) to Milli-Q water (18 MOmega) to determine if the presence of other nutrients (such as nitrate) influenced perchlorate uptake. Perchlorate concentrations in sand and plant tissues were determined weekly. Perchlorate uptake was observed in all three plant species. In most experiments, perchlorate was completely depleted from sand in which plants were growing. Perchlorate concentrations in lettuce were also significantly higher than those in cucumber and soybean (P < 0.0001). Perchlorate concentrations in sand decreased at a higher rate at lower ratios of Hydrosol to Milli-Q, indicating that plant (cucumber) uptake of perchlorate is influenced by the presence of external nutrients. The results of an 8-week uptake study in cucumber and a 6-week uptake study in lettuce suggest that a threshold perchlorate concentration is reached: for cucumber, 150 ppm and for lettuce, 750 ppm. Although the presence of external nutrients decreases the rate of perchlorate uptake by plants, significant concentrations of perchlorate occur in aboveground plant tissues even after relatively short periods of growth. The potential for trophic transfer of perchlorate from soil to higher organisms through plants exists.

Organochlorine contaminants in eggs: the influence of contaminated nest material.

Snake eggs were placed in a synthetic nest contaminated with known concentrations of six organochlorines (OCs) to evaluate whether OCs from contaminated nest material accumulate in eggs. It was hypothesized that contaminated nest material may have contributed to OC burdens in eggs observed previously. The six OCs tested included lindane, heptachlor, aldrin, dieldrin, endrin, and DDT. Eggs were removed at 0, 4, and 6 weeks and analyzed using gas chromatography with electron capture detection. Lindane was found at the highest concentration (153 ng/g at 4 weeks and 162 ng/g at 6 weeks). The next highest uptake was for endrin (25 ng/g at 4 weeks and 106 ng/g at 6 weeks). Heptachlor, aldrin, and dieldrin were also taken up into the eggs, but DDT was not detected in any of the eggs at any sampling period. The concentration of OCs increased from week 4 to week 6 for all the OCs except DDT. Structure-activity relationships were examined to determine which physicochemical properties of the OCs tested could be used as predictors of uptake into the eggs. A variety of physicochemical properties were evaluated including vapor pressure and molecular connectivity (a numerical description of topology). Octanol-water partition coefficient (Kow) was a good predictor (r2 = 0.63, p = 0.06) of OC uptake into the eggs using this limited data set.