Ronald S. Tjeerdema

Evaluation of metabolite extraction strategies from tissue samples using NMR metabolomics

Metabolomic analysis of tissue samples can be applied across multiple fields including medicine, toxicology, and environmental sciences. A thorough evaluation of several metabolite extraction procedures from tissues is therefore warranted. This has been achieved at two research laboratories using muscle and liver tissues from fish. Multiple replicates of homogenous tissues were extracted using the following solvent systems of varying polarities: perchloric acid, acetonitrile/water, methanol/water, and methanol/chloroform/water. Extraction of metabolites from ground wet tissue, ground dry tissue, and homogenized wet tissue was also compared. The hydrophilic metabolites were analyzed using 1-dimensional (1D) 1H nuclear magnetic resonance (NMR) spectroscopy and projections of 2-dimensional J-resolved (p-JRES) NMR, and the spectra evaluated using principal components analysis. Yield, reproducibility, ease, and speed were the criteria for assessing the quality of an extraction protocol for metabolomics. Both laboratories observed that the yields of low molecular weight metabolites were similar among the solvent extractions; however, acetonitrile-based extractions provided poorer fractionation and extracted lipids and macromolecules into the polar solvent. Extraction using perchloric acid produced the greatest variation between replicates due to peak shifts in the spectra, while acetonitrile-based extraction produced highest reproducibility. Spectra from extraction of ground wet tissues generated more macromolecules and lower reproducibility compared with other tissue disruption methods. The p-JRES NMR approach reduced peak congestion and yielded flatter baselines, and subsequently separated the metabolic fingerprints of different samples more clearly than by 1D NMR. Overall, single organic solvent extractions are quick and easy and produce reasonable results. However, considering both yield and reproducibility of the hydrophilic metabolites as well as recovery of the hydrophobic metabolites, we conclude that the methanol/chloroform/water extraction is the preferred method.

NMR-derived developmental metabolic trajectories: an approach for visualizing the toxic actions of trichloroethylene during embryogenesis

Fish embryo toxicity tests for chemical risk assessment have traditionally been based upon non-specific endpoints including morphological abnormalities, hatching success, and mortality. Here we extend the application of 1H NMR-based metabolomics in environmental toxicology by adding a suite of metabolic endpoints to the Japanese medaka (Oryzias latipes) embryo assay, with the goal to provide more sensitive, specific and unbiased biomarkers of toxicity. Medaka were exposed throughout embryogenesis to five concentrations of trichloroethylene (TCE; 0, 8.76, 21.9, 43.8, 87.6, 175xa0mg/L) and the relative sensitivities of the traditional and metabolomic endpoints compared. While the no-observable-adverse-effect-level for hatching success, the most sensitive traditional indicator, was 164xa0mg/L TCE, metabolic perturbations were detected at all exposure concentrations. Principal components analysis (PCA) highlighted a dose-response relationship between the NMR spectra of medaka extracts. In addition, 12 metabolites that exhibited highly significant dose-response relationships were identified, which indicated an energetic cost to TCE exposure. Next, embryos were exposed to 0, 0.88, 8.76xa0mg/L TCE and sampled on each of the 8xa0days of development. Projections of 66 two-dimensional J-resolved NMR spectra were obtained, and PCA revealed developmental metabolic trajectories that characterized the basal and TCE-perturbed changes in the entire NMR-visible metabolome throughout embryogenesis. Although no significant increases in mortality, gross deformity or developmental retardation were observed relative to the control group, TCE-induced metabolic perturbations were observed on day 8. In conclusion, these results support the continued development of NMR-based metabolomics as a rapid and reproducible tool for biomarker discovery and environmental risk assessment.

Solid‐phase sediment toxicity identification evaluation in an agricultural stream

The lower Santa Maria River watershed provides important aquatic habitat on the central California coast and is influenced heavily by agricultural runoff. As part of a recently completed water quality assessment, we conducted a series of water column and sediment toxicity tests throughout this watershed. Sediment from Orcutt Creek, a tributary that drains agricultural land, consistently was toxic to the amphipod Hyalella azteca, which is a resident genus in this river. Toxicity identification evaluations (TIEs) were conducted to determine cause(s) of toxicity. We observed no toxicity in sediment interstitial water even though concentrations of chlorpyrifos exceeded published aqueous toxicity thresholds for H. azteca. In contrast to interstitial water, bulk sediment was toxic to H. azteca. In bulk-phase sediment TIEs, the addition of 20% (by volume) coconut charcoal increased survival by 41%, implicating organic chemical(s). Addition of 5% (by volume) of the carbonaceous resin Ambersorb 563 increased survival by 88%, again suggesting toxicity due to organic chemicals. Toxicity was confirmed by isolating Ambersorb from the sediment, eluting the resin with methanol, and observing significant toxicity in control water spiked with the methanol eluate. A carboxylesterase enzyme that hydrolyzes synthetic pyrethroids was added to overlying water, and this significantly reduced toxicity to amphipods. Although the pesticides chlorpyrifos, DDT, permethrin, esfenvalerate, and fenvalerate were detected in this sediment, and their concentrations were below published toxicity thresholds for H. azteca, additivity or synergism may have occurred. The weight-of-evidence suggests toxicity of this sediment was caused by an organic contaminant, most likely a synthetic pyrethroid.

Evidence of pesticide impacts in the santa maria river watershed, California, USA

The Santa Maria River provides significant freshwater and coastal habitat in a semiarid region of central California, USA. We conducted a water and sediment quality assessment consisting of chemical analyses, toxicity tests, toxicity identification evaluations, and macroinvertebrate bioassessments of samples from six stations collected during four surveys conducted between July 2002 and May 2003. Santa Maria River water samples collected downstream of Orcutt Creek (Santa Maria, Santa Barbara County, CA, USA), which conveys agriculture drain water, were acutely toxic to cladocera (Ceriodaphnia dubia), as were samples from Orcutt Creek. Toxicity identification evaluations (TIEs) suggested that toxicity to C. dubia in Orcutt Creek and the Santa Maria River was due to chlorpyrifos. Sediments from these two stations also were acutely toxic to the amphipod Hyalella azteca, a resident invertebrate. The TIEs conducted on sediment suggested that toxicity to amphipods, in part, was due to organophosphate pesticides. Concentrations of chlorpyrifos in pore water sometimes exceeded the 10-d median lethal concentration for H. azteca. Additional TIE and chemical evidence suggested sediment toxicity also partly could be due to pyrethroid pesticides. Relative to an upstream reference station, macroinvertebrate community structure was impacted in Orcutt Creek and in the Santa Maria River downstream of the Creek input. This study suggests that pesticide pollution likely is the cause of ecological damage in the Santa Maria River.

Chemistry and Fate of Simazine

Simazine, first introduced in 1956, is a popular agricultural herbicide used to inhibit photosynthesis in broadleaf weeds and grasses. It is a member of the triazine family, and according to its physicochemical properties, it is slightly soluble in water, relatively nonvolatile, capable of partitioning into organic phases, and susceptible to photolysis. Sorption and desorption studies on its behavior in soils indicate that simazine does not appreciably sorb to minerals and has the potential to leach in clay and sandy soils. The presence of organic matter in soils contributes to simazine retention but delays its degradation. The primary sorptive mechanism of simazine to OM has been proposed to be via partitioning and/or by the interaction with functional groups of the sorbent. Farming practices directly influence the movement of simazine in soils as well. Tilled fields lower the runoff of simazine when compared to untilled fields, but tilling can also contribute to its movement into groundwater. Planting cover crops on untilled land can significantly reduce simazine runoff. Such practices are important because simazine and its byproducts have been detected in groundwater in The Netherlands, Denmark, and parts of the U.S. (California, North Carolina, Illinois, and Wisconsin) at significant concentrations. Concentrations have also been detected in surface waters around the U.S. and United Kingdom. Although the physicochemical properties of simazine do not support volatilization, residues have been found in the atmosphere and correlate with its application. Although at low concentrations, simazine has also been detected in precipitation in Pennsylvania (U.S.), Greece, and Paris (France). Abiotically, simazine can be oxidized to several degradation products. Although hydrolysis does not contribute to the dissipation of simazine, photolysis does. Microbial degradation is the primary means of simazine dissipation, but the process is relatively slow and kinetically controlled. Some bacteria and fungal species capable of utilizing simazine as a sole carbon and nitrogen source at a fast rate under laboratory conditions have been identified. Metabolism of simazine in higher organisms is via cytochrome P-450-mediated oxidation and glutathione conjugation.

Navigating the tmdl process: sediment toxicity

A key step in the development of Total Maximum Daily Load (TMDL) allocations for water bodies impaired due to sediment toxicity is the identification of chemicals responsible for toxicity. Sediment toxicity identification evaluation procedures (TIEs) are one of the primary tools used in this process. This project evaluated standardized and recently developed sediment TIE methods developed by the US EPA and others, to determine their utility for identifying chemicals responsible for toxicity. In this study, formulated sediments were spiked with five chemicals: copper, fluoranthene, tetrachlorobenzene, nonylphenol and ammonia. Experiments were conducted using sediments spiked with single chemicals and chemical mixtures. Toxicity tests used the estuarine amphipod Eohaustorius estuarius and the freshwater amphipod Hyalella azteca. TIEs were conducted using solid-phase (whole sediment) and sediment interstitial waters.nnOur results indicate that the TIE methods are sufficient to characterize and identify toxicity due to single and multiple chemicals using spiked sediments. One key finding from the spiked sediment experiments is that methods to elute chemicals from extraction media used in solid-phase and interstitial water TIEs require further refinement to ensure complete and consistent elution of sorbed chemicals. An additional finding is that both solid-phase and interstitial water TIE procedures provide useful lines of evidence and that both approaches should be used in a weight-of-evidence approach in the sediment TIE process. In the final phase of this project, we evaluated the TIE procedures using ambient samples collected from three marine sites and three freshwater sites. Marine sediments were collected from Switzer Creek in San Diego Bay, Upper Newport Bay in Newport Beach, and Consolidated Slip, in Long Beach Harbor, all of which are sites in southern California. Freshwater sediments were collected from two additional sites in California: San Diego Creek, in Newport Beach, and Alisal Slough, in Salinas. The final freshwater sediment was collected from a site in Indiana Harbor. Multiple solid-phase and interstitial water TIEs were conducted on each sediment and results of these were combined with chemical analyses and other lines-of-evidence to evaluate the methods.nnThe results indicate that sediment TIE methods are sufficiently developed to characterize toxicity due to general classes of chemicals such as cationic metals, organic chemicals, and ammonia. Methods to improve extraction and elution of metal and organic chemicals in highly toxic sediments require further refinement in order to allow identification of specific chemicals responsible for toxicity as part of the Phase II TIE process. These refinements include determining the appropriate masses of solid-phase extraction media to allow complete removal of toxic chemicals from sediments and interstitial waters. In addition, we recommend that additional studies be designed to determine optimal equilibration periods to maximize removal of toxic chemicals using resin amendments in solid-phase sediment TIEs. As in the spiked-sediment TIEs, we observed inconsistent elution of toxic chemicals from extraction media used in solid-phase and interstitial water Phase II TIEs with the ambient samples. These procedures require additional work to ensure that chemicals responsible for toxicity are completely eluted from extraction media. Improvement of the Phase II TIE elution and solvent handling steps should facilitate better identification of chemicals responsible for toxicity, particularly in situations where toxicity is due to highly insoluble chemicals. When these considerations are satisfactorily addressed, the methods evaluated are capable of providing useful lines of evidence that can be combined to successfully identify sediment toxicants.nnThis title belongs to WERF Research Report Series nnISBN: 9781843397663 (Print)nnISBN: 9781780403786 (eBook)