Reducing bioassay variability by identifying sources of variation and controlling key parameters in assay protocol

Reducing bioassay variability by identifying sources of variation and controlling important parameters in assay protocol was demonstrated in this study. The variability of a bioassay based on a luminescent bacterium was examined as an example. This assay involved the growth of cells, storage at a low temperature, activation, and exposure to a test sample, and the assay response was bioluminescence inhibition. After determining that measurement error was small and negligible, the total assay variability was decomposed in an initial variance components study into between-batch, between-vial, and between-tube variations. Results indicated that between-vial variations accounted for the majority of the total observed variability and that reducing this type of variation would be beneficial. Five parameters in the assay protocol were determined as factors that potentially affected assay variability significantly. A split-plot design was employed to investigate the effects of these factors and some of their interactions on the assay response. One of the five factors, i.e., activation temperature, turned out to have a significant effect. The variance components study was repeated with better control of activation temperature as well as other parameters. Results indicated that the total variability of the bioassay was reduced by approximately 85%.     

Solute transport in crystalline rocks at Aspö--I: geological basis and model calibration

Water-conducting faults and fractures were studied in the granite-hosted Asp? Hard Rock Laboratory (SE Sweden). On a scale of decametres and larger, steeply dipping faults dominate and contain a variety of different fault rocks (mylonites, cataclasites, fault gouges). On a smaller scale, somewhat less regular fracture patterns were found. Conceptual models of the fault and fracture geometries and of the properties of rock types adjacent to fractures were derived and used as input for the modelling of in situ dipole tracer tests that were conducted in the framework of the Tracer Retention Understanding Experiment (TRUE-1) on a scale of metres. After the identification of all relevant transport and retardation processes, blind predictions of the breakthroughs of conservative to moderately sorbing tracers were calculated and then compared with the experimental data. This paper provides the geological basis and model calibration, while the predictive and inverse modelling work is the topic of the companion paper [J. Contam. Hydrol. 61 (2003) 175]. The TRUE-1 experimental volume is highly fractured and contains the same types of fault rocks and alterations as on the decametric scale. The experimental flow field was modelled on the basis of a 2D-streamtube formalism with an underlying homogeneous and isotropic transmissivity field. Tracer transport was modelled using the dual porosity medium approach, which is linked to the flow model by the flow porosity. Given the substantial pumping rates in the extraction borehole, the transport domain has a maximum width of a few centimetres only. It is concluded that both the uncertainty with regard to the length of individual fractures and the detailed geometry of the network along the flowpath between injection and extraction boreholes are not critical because flow is largely one-dimensional, whether through a single fracture or a network. Process identification and model calibration were based on a single uranine breakthrough (test PDT3), which clearly showed that matrix diffusion had to be included in the model even over the short experimental time scales, evidenced by a characteristic shape of the trailing edge of the breakthrough curve. Using the geological information and therefore considering limited matrix diffusion into a thin fault gouge horizon resulted in a good fit to the experiment. On the other hand, fresh granite was found not to interact noticeably with the tracers over the time scales of the experiments. While fracture-filling gouge materials are very efficient in retarding tracers over short periods of time (hours-days), their volume is very small and, with time progressing, retardation will be dominated by altered wall rock and, finally, by fresh granite. In such rocks, both porosity (and therefore the effective diffusion coefficient) and sorption K(d)s are more than one order of magnitude smaller compared to fault gouge, thus indicating that long-term retardation is expected to occur but to be less pronounced.     

Earthworm biomass as additional information for risk assessment of heavy metal biomagnification: a case study for dredged sediment-derived soils and polluted floodplain soils

The important role of earthworms in the biomagnification of heavy metals in terrestrial ecosystems is widely recognised. Differences in earthworm biomass between sites is mostly not accounted for in ecological risk assessment. These differences may be large depending on soil properties and pollution status. A survey of earthworm biomass and colonisation rate was carried out on dredged sediment-derived soils (DSDS). Results were compared with observations for the surrounding alluvial plains. Mainly grain size distribution and time since disposal determined earthworm biomass on DSDS, while soil pollution status of the DSDS was of lesser importance. Highest earthworm biomass was observed on sandy loam DSDS disposed at least 40 years ago.     

Laboratory evaluation of thermophilic-anaerobic digestion to produce Class A biosolids. 2. Inactivation of pathogens and indicator organisms in a continuous-flow reactor followed by batch treatment.

Thermophilic-anaerobic digestion in a single-stage, mixed, continuous-flow reactor is not approved in the United States as a process capable of producing Class A biosolids for land application. This study was designed to evaluate the inactivation of pathogens and indicator organisms in such a reactor followed by batch treatment in a smaller reactor. The combined process was evaluated at 53 degrees C with sludges from three different sources and at 51 and 55 degrees C with sludge from one of the sources. Feed sludge to the continuous-flow reactor was spiked with the pathogen surrogates Ascaris suum and vaccine-strain poliovirus. Feed and effluent were analyzed for these organisms and for indigenous Salmonella spp., fecal coliforms, Clostridium perfringens spores, and somatic and male-specific coliphages. No viable Ascaris eggs were observed in the effluent from the continuous reactor at 53 or 55 degrees C, with greater than 2-log removals across the digester in all cases. Approximately 2-log removal was observed at 51 degrees C, but all samples of effluent biosolids contained at least one viable Ascaris egg at 51 degrees C. No viable poliovirus was found in the digester effluent at any of the operating conditions, and viable Salmonella spp. were measured in the digester effluent in only one sample throughout the study. The ability of the continuous reactor to remove fecal coliforms to below the Class A monitoring limit depended on the concentration in the feed sludge. There was no significant removal of Clostridium perfringens across the continuous reactor under any condition, and there also was limited removal of somatic coliphages. The removal of male-specific coliphages across the continuous reactor appeared to be related to temperature. Overall, at least one of the Class A pathogen criteria or the fecal coliform limit was exceeded in at least one sample in the continuous-reactor effluent at each temperature. Over the range of temperatures evaluated, the maximum time required to meet the Class A criteria by batch treatment of the continuous-reactor effluent was 1 hour for Ascaris suum and Salmonella spp. and 2 hours for fecal coliforms.     

Controlled release, blind test of DNAPL remediation by ethanol flushing

A dense nonaqueous phase liquid (DNAPL) source zone was established within a sheet-pile isolated cell through a controlled release of perchloroethylene (PCE) to evaluate DNAPL remediation by in-situ cosolvent flushing. Ethanol was used as the cosolvent, and the main remedial mechanism was enhanced dissolution based on the phase behavior of the water-ethanol-PCE system. Based on the knowledge of the actual PCE volume introduced into the cell, it was estimated that 83 L of PCE were present at the start of the test. Over a 40-day period, 64% of the PCE was removed by flushing the cell with an alcohol solution of approximately 70% ethanol and 30% water. High removal efficiencies at the end of the test indicated that more PCE could have been removed had it been possible to continue the demonstration. The ethanol solution extracted from the cell was recycled during the test using activated carbon and air stripping treatment. Both of these treatment processes were successful in removing PCE for recycling purposes, with minimal impact on the ethanol content in the treated fluids. Results from pre- and post-flushing partitioning tracer tests overestimated the treatment performance. However, both of these tracer tests missed significant amounts of the PCE present, likely due to inaccessibility of the PCE. The tracer results suggest that some PCE was inaccessible to the ethanol solution which led to the inefficient PCE removal rates observed. The flux-averaged aqueous PCE concentrations measured in the post-flushing tracer test were reduced by a factor of 3 to 4 in the extraction wells that showed the highest PCE removal compared to those concentrations in the pre-flushing tracer test.     

Occurrence of spruce bark beetles in forest stands at different levels of air pollution stress

The spruce bark beetle, Ips typographus (L.) is the most serious pest of mature spruce stands, mainly Norway spruce, Picea abies (L.) Karst. throughout Eurasia. A complex of weather-related events and other environmental stresses are reported to predispose spruce stands to bark beetle attack and subsequent tree mortality; however the possible role of industrial pollution as a predisposing factor to attack by this species is poorly understood. The abundance and dynamics of I. typographus populations was evaluated in 60-80 year old Norway spruce stands occurring on 10 x 50 ha sites in five countries within the Carpathian range that were selected in proximity to established ozone measurement sites. Data were recorded on several parameters including the volume of infested trees, captures of adult beetles in pheromone traps, number of attacks, and the presence and relative abundance of associated bark beetle species. In several cases, stands adjacent to sites with higher ozone values were associated with higher bark beetle populations. The volume of sanitary cuttings, a reflection of tree mortality, and the mean daily capture of beetles in pheromone traps were significantly higher at sites where the O(3) level was higher. However, the mean infestation density on trees was higher in plots associated with lower O(3) levels. Captures of beetles in pheromone traps and infestation densities were higher in the zone above 800 m. However, none of the relationships was conclusive, suggesting that spruce bark beetle dynamics are driven by a complex interaction of biotic and abiotic factors and not by a single parameter such as air pollution.     

Distribution of organochlorine pesticides, polychlorinated biphenyls and alpha-HCH enantiomers in pork tissues

The distribution of HCH isomers, DDT analogues and selected PCB congeners in pork organs collected from the same individuals raised in Romanian farms was investigated. Organochlorine pesticides (HCHs and DDTs) were the principal contaminants in all samples, while PCB concentrations were low, in accordance with previously reported concentrations from Romanian animal farms. The most part of the pollutant load in the body is retained in the adipose tissue, with HCHs ranging between 16 and 27.7 ng/g lipid and with higher concentrations of DDTs ranging between 65.9 and 334.5 ng/g lipid. The highest PCB levels (up to 32 ng/g lipid) were measured in lung and liver. The lipid-normalized concentrations in the brain were lower than in all other tissues due to the presence of the blood-brain barrier or due to a lower proportion of the neutral lipids such as triglycerides. The highest concentrations of DDTs were measured in muscle and fat, with p,p'-DDE being the principal contributor and with a variable contribution of p,p'-DDD and p,p'-DDT. In liver, p,p'-DDD has a higher contribution to the sum DDTs, while in all analyzed livers, the concentration of p,p'-DDT was very low. beta-HCH was the most persistent HCH isomer in all tissues, accounting for 40-97% of sum HCHs. For all animals, the highest concentrations of beta-HCH and HCHs were found in liver, while the lowest HCH concentrations were measured in brain and spinal marrow. Additionally, the distribution of alpha-HCH enantiomers in the tissues was discussed. In all samples (except 2 brain samples), (+) alpha-HCH was depleted and (-) alpha-HCH was enantioenriched. Enantiomeric ratios in brain were the highest measured values between all organs. For all studied animals, ERs increased in the order fat < muscle < liver < brain.     

Development of a hazardous air pollutants monitoring program using the Data Quality Objectives process

To effectively reduce the environmental compliance costs associated with meeting hazardous air pollutant emission requirements, the U.S. Environmental Protection Agency's Data Quality Objective (DQO) process has been proposed as a suitable framework for establishing a defensible monitoring program. Through the use of a hazardous materials pilot study, the variability in the composite vapor pressure for regulated handwipe cleaning solvents was established. These results served as inputs to the DQO process, which identified that for facility decision-makers to claim with a 99% confidence level that the facility is in compliance with the National Emission Standards for Hazardous Air Pollutants (NESHAP), a minimum of 12 handwipe cleaning solvent compliance samples (taken at random every 6 months) must have a composite vapor pressure equal to or below the regulatory limit of 45 mmHg at 20 degrees C. Implementation of the DQO-based compliance-sampling plan eliminates the need for an affected facility to sample all regulated handwipe cleaning solvents while still maintaining a reasonably high level of confidence in the compliance status of its regulated sources. The approach described for designing a defensible compliance sampling plan can be extended to other aspects of the aerospace NESHAP rule, including compliance sampling for surface coating, chemical depainting, and hazardous waste disposal.     

Explicit and implicit coupling during solute transport through clay membrane barriers

Simulations of salt (KCl) flux through a 1-m-thick clay membrane barrier (CMB) based on coupled solute transport theory are compared to simulated fluxes based on traditional advective-dispersive transport theory. The simulations are based on measured values for the effective salt-diffusion coefficient (Ds*) and chemico-osmotic efficiency coefficient (omega) for a bentonite-based barrier material subjected to KCl solutions. The results indicate that the exit salt flux is reduced due to both explicit coupling (hyperfiltration and chemico-osmotic counter-advection) and an implicit coupling effect resulting from the decrease in Ds* due to a decrease in the apparent tortuosity factor, tau a, with an increase in omega. Implicit coupling is shown to be more significant than explicit coupling for reducing and retarding salt flux through a CMB under diffusion-dominated conditions. Failure to account for the implicit coupling effect may result in unrealistic results, such as the existence of salt flux through a perfect (ideal) clay membrane (i.e., omega=1).