Ecotoxicological effects of copper oxide nanoparticles (nCuO) on the soil microbial community in a biosolids-amended soil.

This study represents a holistic approach in assessing the effects of copper oxide nanoparticles (nCuO) on microbial health and community structure in soil amended with municipal biosolids. The biosolids were amended with nCuO (<50 nm) and mixed into a sandy loam soil at measured Cu concentrations of 27, 54, 123, 265 and 627 mg Cu kg-1 soil. A suite of tests were used to assess the potential impact of nCuO on microbial growth, activity, and diversity. Microbial growth was determined by the heterotrophic plate count (HPC) method, while microbial diversity was assessed using both community level physiological profiling (CLPP) and 16S ribosomal DNA (rDNA) sequencing. Microbial activity was assessed by examining soil nitrification, organic matter decomposition, soil respiration (basal and substrate induced) and soil enzyme assays for dehydrogenase, phosphatase and ß-glucosidase activities. As a readily soluble positive control, copper sulfate (CuSO4) was used at measured Cu concentrations of 65, 140, 335 and 885 mg Cu kg-1 soil for select tests, and at the highest concentration for the remaining tests. Analysis on Cu bioavailability revealed that extractable Cu2+ was higher in CuSO4-spiked soils than nCuO-spiked soils. At a nCuO exposure concentration of ≤265 mg Cu kg-1 soil, stimulatory effects were observed in nitrification, ß-glucosidase and community level physiological profiling (CLPP) tests. nCuO showed no significant inhibitory effects on the soil microbial growth, activity or diversity at the highest concentration (i.e. 627 mg Cu kg-1 soil), with the exception of the dehydrogenase (i.e. ≥27 mg Cu kg-1 soil) and phosphatase (i.e. 627 mg Cu kg-1 soil) enzyme activities. In contrast, inhibition from CuSO4 at 885 mg Cu kg-1 soil was observed in all tests with the exception of ß-glucosidase enzyme activity. The growth of a Cu tolerant bacterium, Rhodanobacter sp., was observed at 885 mg Cu kg-1 soil (CuSO4).

Extracting Metallic Nanoparticles from Soils for Quantitative Analysis: Method Development Using Engineered Silver Nanoparticles and SP-ICP-MS.

The lack of an efficient and standardized method to disperse soil particles and quantitatively subsample the nanoparticulate fraction for characterization analyses is hindering progress in assessing the fate and toxicity of metallic engineered nanomaterials in the soil environment. This study investigates various soil extraction and extract preparation techniques for their ability to remove nanoparticulate Ag from a field soil amended with biosolids contaminated with engineered silver nanoparticles (AgNPs), while presenting a suitable suspension for quantitative single-particle inductively coupled plasma mass spectroscopy (SP-ICP-MS) analysis. Extraction parameters investigated included reagent type (water, NaNO3, KNO3, tetrasodium pyrophosphate (TSPP), tetramethylammonium hydroxide (TMAH)), soil-to-reagent ratio, homogenization techniques as well as procedures commonly used to separate nanoparticles from larger colloids prior to analysis (filtration, centrifugation, and sedimentation). We assessed the efficacy of the extraction procedure by testing for the occurrence of potential procedural artifacts (dissolution, agglomeration) using a dissolved/particulate Ag mass ratio and by monitoring the amount of Ag mass in discrete particles. The optimal method employed 2.5 mM TSPP used in a 1:100 (m/v) soil-to-reagent ratio, with ultrasonication to enhance particle dispersion and sedimentation to settle out the micrometer-sized particles. A spiked-sample recovery analysis shows that 96% ± 2% of the total Ag mass added as engineered AgNP is recovered, which includes the recovery of 84.1% of the particles added, while particle recovery in a spiked method blank is ∼100%, indicating that both the extraction and settling procedure have a minimal effect on driving transformation processes. A soil dilution experiment showed that the method extracted a consistent proportion of nanoparticulate Ag (9.2% ± 1.4% of the total Ag) in samples containing 100%, 50%, 25%, and 10% portions of the AgNP-contaminated test soil. The nanoparticulate Ag extracted by this method represents the upper limit of the potentially dispersible nanoparticulate fraction, thus providing a benchmark with which to make quantitative comparisons, while presenting a suspension suitable for a myriad of other characterization analyses.

Single Particle-Inductively Coupled Plasma Mass Spectroscopy Analysis of Metallic Nanoparticles in Environmental Samples with Large Dissolved Analyte Fractions.

There is an increasing interest to use single particle-inductively coupled plasma mass spectroscopy (SP-ICPMS) to help quantify exposure to engineered nanoparticles, and their transformation products, released into the environment. Hindering the use of this analytical technique for environmental samples is the presence of high levels of dissolved analyte which impedes resolution of the particle signal from the dissolved. While sample dilution is often necessary to achieve the low analyte concentrations necessary for SP-ICPMS analysis, and to reduce the occurrence of matrix effects on the analyte signal, it is used here to also reduce the dissolved signal relative to the particulate, while maintaining a matrix chemistry that promotes particle stability. We propose a simple, systematic dilution series approach where by the first dilution is used to quantify the dissolved analyte, the second is used to optimize the particle signal, and the third is used as an analytical quality control. Using simple suspensions of well characterized Au and Ag nanoparticles spiked with the dissolved analyte form, as well as suspensions of complex environmental media (i.e., extracts from soils previously contaminated with engineered silver nanoparticles), we show how this dilution series technique improves resolution of the particle signal which in turn improves the accuracy of particle counts, quantification of particulate mass and determination of particle size. The technique proposed here is meant to offer a systematic and reproducible approach to the SP-ICPMS analysis of environmental samples and improve the quality and consistency of data generated from this relatively new analytical tool.

Toxicity and metal bioaccumulation in Hordeum vulgare exposed to leached and nonleached copper amended soils.

Soil leaching has been proposed as a way to reduce soil-spiking artifacts (i.e., increased acidity, metal solubility) that occur in soils amended with trace metal salts. Leaching metal-spiked samples prior to ecotoxicity testing is therefore expected to reduce toxicity; however, leaching not only removes excess amounts of the trace metal being tested, but also reduces the concentrations of cations that could decrease the toxic effects of the metal of interest. To clarify these conflicting processes, the effects of leaching on toxicity and bioaccumulation of Cu, Ca, and Al were investigated using 14-d plant assays conducted on leached and nonleached, Cu-spiked soils. The median effective concentration (EC50) to root elongation ranged from 78 µg/g to 589 µg/g. Leaching was found to reduce toxicity by 1.2-fold to 2.1-fold. The Cu(2+) activity predicted toxicity better than root or shoot Cu concentrations, which were generally not affected by leaching. Plant uptake of Ca increased with increasing Cu dose in nonleached samples, which likely contributed to the hormesis-like response observed in these samples, whereas Ca uptake in the leached samples was more consistent with that of the control except at the largest Cu doses for which Ca uptake decreased. Surprisingly, Al uptake in the most acidic soil was greater in leached than nonleached samples, which may have contributed to the greater toxicity exhibited in this soil than was predicted by Cu(2+) activity. These findings have implications for predicting trace metal toxicity in nutrient-stressed, acidic soils. Environ Toxicol Chem 2013;32:1800-1809. © 2013 SETAC.

Comparing soil chemistries of leached and nonleached copper-amended soils.

Leaching metal-spiked samples has been proposed as a means to reduce the artifacts of the spiking procedure (e.g., salt effect, increased metal solubility) that can artificially increase metal bioaccessibility and toxicity in laboratory ecotoxicity tests. The effects on soil chemistry from leaching Cu-spiked samples were investigated by comparing chemistries of freshly spiked samples to samples that underwent the spike/leach procedure. Chemical parameters investigated included electrical conductivity (EC), pH, ethylenediaminetetraacetic acid- and CaCl(2) -extractable Cu, soil-solution Cu, Cu(2+) activity (estimated using Visual MINTEQ), and other solution parameters (dissolved organic carbon [DOC], Ca, Mg, Al). In leached samples, the electrical conductivity values of the spiked samples did not vary significantly from those of the control samples (p > 0.05), confirming that the leaching procedure had sufficiently minimized the salt effect. In the range of soil Cu concentrations where Cu ecotoxicity is expected, the pH in freshly spiked samples was as much as 0.52 units lower than the pH from leached samples at the same total-soil Cu concentration. The CaCl(2) -extractable fraction was up to 2.3-fold smaller in leached samples and inversely related to the pH of the spiked soil. Despite little to no difference in soil-solution Cu, up to 100-fold less Cu(2+) activity was observed in leached samples. Reduced Cu(2+) activity was related to less Al(3+) competition for DOC. Leaching resulted in solution chemistries that were more consistent with those of the control samples and reduced the artifacts of traditional soil-spiking procedures.