Guritno Roesijadi

Adverse outcome pathways and ecological risk assessment: Bridging to population‐level effects

Maintaining the viability of populations of plants and animals is a key focus for environmental regulation. Population-level responses integrate the cumulative effects of chemical stressors on individuals as those individuals interact with and are affected by their conspecifics, competitors, predators, prey, habitat, and other biotic and abiotic factors. Models of population-level effects of contaminants can integrate information from lower levels of biological organization and feed that information into higher-level community and ecosystem models. As individual-level endpoints are used to predict population responses, this requires that biological responses at lower levels of organization be translated into a form that is usable by the population modeler. In the current study, we describe how mechanistic data, as captured in adverse outcome pathways (AOPs), can be translated into modeling focused on population-level risk assessments. First, we describe the regulatory context surrounding population modeling, risk assessment and the emerging role of AOPs. Then we present a succinct overview of different approaches to population modeling and discuss the types of data needed for these models. We describe how different key biological processes measured at the level of the individual serve as the linkage, or bridge, between AOPs and predictions of population status, including consideration of community-level interactions and genetic adaptation. Several case examples illustrate the potential for use of AOPs in population modeling and predictive ecotoxicology. Finally, we make recommendations for focusing toxicity studies to produce the quantitative data needed to define AOPs and to facilitate their incorporation into population modeling.

Acetone-butanol fermentation of marine macroalgae.

The objective of this study was to subject mannitol, either as a sole carbon source or in combination with glucose, and aqueous extracts of the kelp Saccharina spp., containing mannitol and laminarin, to acetone-butanol fermentation by Clostridium acetobutylicum (ATCC 824). Both mannitol and glucose were readily fermented. Mixed substrate fermentations with glucose and mannitol resulted in diauxic growth of C. acetobutylicum with glucose depletion preceding mannitol utilization. Fermentation of kelp extract exhibited triauxic growth, with an order of utilization of free glucose, mannitol, and bound glucose, presumably laminarin. The lag in laminarin utilization reflected the need for enzymatic hydrolysis of this polysaccharide into fermentable sugars. The butanol and total solvent yields were 0.12 g/g and 0.16 g/g, respectively, indicating that significant improvements are still needed to make industrial-scale acetone-butanol fermentations of seaweed economically feasible.

Adaptive responses and latent costs of multigeneration cadmium exposure in parasite resistant and susceptible strains of a freshwater snail.

Population response to anthropogenic activities will be influenced by prior adaptation to environmental conditions. We tested how parasite-resistant and -susceptible strains of the freshwater snail, Biomphalaria glabrata, responded to cadmium and elevated temperature challenges after having been exposed to low-level cadmium continuously for multiple generations. Snails exposed to cadmium for three generations were removed for the fourth generation, and challenged in the fifth generation with (1) chronic cadmium exposure over the entire life cycle; (2) lethal cadmium exposure of adults; and (3) elevated temperature challenge of adults. The parasite susceptible NMRI strain is more cadmium tolerant than the parasite resistant BS90 strain and remained more tolerant than BS90 throughout this study. Additionally, NMRI exhibited greater adaptive capacity for cadmium than BS90 and became more tolerant of both chronic and lethal cadmium challenges, while BS90 became more tolerant of lethal cadmium challenge only. Fitness costs, reflected in population growth rate, were not apparent in fifth generation snails maintained in control conditions. However, costs were latent and expressed as decreased tolerance to a secondarily imposed temperature stress. Adaptation to prior selection pressures can influence subsequent adaptation to anthropogenic stresses and may have associated costs that reduce fitness in novel environments.

FRET Imaging of Diatoms Expressing a Biosilica-Localized Ribose Sensor

Future materials are envisioned to include bio-assembled, hybrid, three-dimensional nanosystems that incorporate functional proteins. Diatoms are amenable to genetic modification for localization of recombinant proteins in the biosilica cell wall. However, the full range of protein functionalities that can be accommodated by the modified porous biosilica has yet to be described. Our objective was to functionalize diatom biosilica with a reagent-less sensor dependent on ligand-binding and conformational change to drive FRET-based signaling capabilities. A fusion protein designed to confer such properties included a bacterial periplasmic ribose binding protein (R) flanked by CyPet (C) and YPet (Y), cyan and yellow fluorescent proteins that act as a FRET pair. The structure and function of the CRY recombinant chimeric protein was confirmed by expression in E. coli prior to transformation of the diatom Thalassiosira pseudonana. Mass spectrometry of the recombinant CRY showed 97% identity with the deduced amino acid sequence. CRY with and without an N-terminal Sil3 tag for biosilica localization exhibited characteristic ribose-dependent changes in FRET, with similar dissociation constants of 123.3 µM and 142.8 µM, respectively. The addition of the Sil3 tag did not alter the affinity of CRY for the ribose substrate. Subsequent transformation of T. pseudonana with a vector encoding Sil3-CRY resulted in fluorescence localization in the biosilica and changes in FRET in both living cells and isolated frustules in response to ribose. This work demonstrated that the nano-architecture of the genetically modified biosilica cell wall was able to support the functionality of the relatively complex Sil3-CyPet-RBP-YPet fusion protein with its requirement for ligand-binding and conformational change for FRET-signal generation.

Demographic responses to multigeneration cadmium exposure in two strains of the freshwater gastropod, Biomphalaria glabrata.

A life table response experiment (LTRE) was used to quantify the population-level effects of continuous, multigeneration cadmium exposure on two strains of the freshwater gastropod, Biomphalaria glabrata: the parasite-resistant BS90 and parasite-susceptible NMRI strains. Snails were exposed to waterborne cadmium for three consecutive generations. Survival, growth, and reproduction were measured empirically and incorporated into a stage-based, deterministic population model. Cadmium significantly affected hatching success, time to maturity, and juvenile and adult survival in both strains. There were significant effects of generation on fecundity, hatching success, time to maturity and juvenile survival in NMRI, and time to maturity and adult survival in BS90. Cadmium significantly affected the population growth rate, λ, in BS90. Cadmium, generation, and the cadmium × generation interaction had significant effects on λ in NMRI. At the high cadmium exposure, λ for NMRI showed a decrease from generation 1 to generation 2, followed by an increase from generation 2 to generation 3. The λ value in high-cadmium BS90 steadily decreased over the three generations, while NMRI at this same concentration was similar to the controls. The results indicate that strain-specific differences in response to multigeneration cadmium exposure are evident in B. glabrata. Moreover, effects seen in the first generation are not necessarily indicative of effects in subsequent generations. Changes in λ over the course of the three-generation exposure suggest that acclimation and/or adaptation to cadmium may have occurred, particularly in NMRI at the high cadmium exposure level.

Photoluminescence detection of 2,4,6-trinitrotoluene (TNT) binding on diatom frustule biosilica functionalized with an anti-TNT monoclonal antibody fragment

A selective and label-free biosensor for detection of the explosive compound 2,4,6-trinitrotoluene (TNT) in aqueous solution was developed based on the principle of photoluminescence quenching of upon immunocomplex formation with antibody-functionalized diatom frustule biosilica. The diatom frustule is an intricately nanostructured, highly porous biogenic silica material derived from the shells of microscopic algae called diatoms. This material emits strong visible blue photoluminescence (PL) upon UV excitation. PL-active frustule biosilica was isolated from cultured cells of the marine diatom Pinnularia sp. and functionalized with a single chain variable fragment (scFv) derived from an anti-TNT monoclonal antibody. When TNT was bound to the anti-TNT scFv-functionalized diatom frustule biosilica, the PL emission from the biosilica was partially quenched due to the electrophilic nature of the nitro (-NO2) groups on the TNT molecule. The dose-response curve for immunocomplex formation of TNT on the scFv-functionalized diatom frustule biosilica had a half-saturation binding constant of 6.4 ± 2.4·10(-8)M and statistically-significant measured detection limit of 3.5·10(-8)M. The binding and detection were selective for TNT and TNB (trinitrobenzene) but not RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) or 2,6-DNT (2,6-dinitrotoluene).

Effects of Electromagnetic Fields on Fish and Invertebrates

In this progress report, we describe the preliminary experiments conducted with three fish and one invertebrate species to determine the effects of exposure to electromagnetic fields. During fiscal year 2010, experiments were conducted with coho salmon (Onchrohychus kisutch), California halibut (Paralicthys californicus), Atlantic halibut (Hippoglossus hippoglossus), and Dungeness crab (Cancer magister). The work described supports Task 2.1.3: Effects on Aquatic Organisms, Subtask 2.1.3.1: Electromagnetic Fields.

Antigen Binding and Site-Directed Labeling of Biosilica-Immobilized Fusion Proteins Expressed in Diatoms

The diatom Thalassiosira pseudonana was genetically modified to express biosilica-targeted fusion proteins comprising either enhanced green fluorescent protein (EGFP) or single chain antibodies engineered with a tetracysteine tagging sequence. Of interest were the site-specific binding of (1) the fluorescent biarsenical probe AsCy3 and AsCy3e to the tetracysteine tagged fusion proteins and (2) high and low molecular mass antigens, the Bacillus anthracis surface layer protein EA1 or small molecule explosive trinitrotoluene (TNT), to biosilica-immobilized single chain antibodies. Analysis of biarsenical probe binding using fluorescence and structured illumination microscopy indicated differential colocalization with EGFP in nascent and mature biosilica, supporting the use of either EGFP or bound AsCy3 and AsCy3e in studying biosilica maturation. Large increases in the lifetime of a fluorescent analogue of TNT upon binding single chain antibodies provided a robust signal capable of discriminating binding to immobilized antibodies in the transformed frustule from nonspecific binding to the biosilica matrix. In conclusion, our results demonstrate an ability to engineer diatoms to create antibody-functionalized mesoporous silica able to selectively bind chemical and biological agents for the development of sensing platforms.

Micro‐photoluminescence of single living diatom cells

Diatoms are single-celled microalgae that possess a nanostructured, porous biosilica shell called a frustule. This study characterized the micro-photoluminescence (μ-PL) emission of single living cells of the photosynthetic marine diatom Thalassiosira pseudonana in response to UV laser irradiation at 325 nm using a confocal Raman microscope. The photoluminescence (PL) spectrum had two primary peaks, one centered at 500-510 nm, which was attributed to the frustule biosilica, and a second peak at 680 nm, which was attributed to auto-fluorescence of photosynthetic pigments. The portion of the μ-PL emission spectrum associated with biosilica frustule in the single living diatom cell was similar to that from single biosilica frustules isolated from these diatom cells. The PL emission by the biosilica frustule in the living cell emerged only after cells were cultivated to silicon depletion. The discovery of the discovery of PL emission by the frustule biosilica within a single living diatom itself, not just its isolated frustule, opens up future possibilities for living biosensor applications, where the interaction of diatom cells with other molecules can be probed by μ-PL spectroscopy. Copyright

Assessing the effects of marine and hydrokinetic energy development on marine and estuarine resources

The worlds oceans and estuaries offer enormous potential to meet the nations growing demand for energy. The use of marine and hydrokinetic (MHK) devices to harness the power of wave and tidal energy could contribute significantly toward meeting federaland state-mandated renewable energy goals while supplying a substantial amount of clean energy to coastal communities. Locations along the eastern and western coasts of the United States between 40° and 70° north latitude are ideal for MHK deployment, and recent estimates of wave and current energy resource potential in the US suggest that up to 400 terawatt hours could be generated, representing about 10% of national energy demand. Because energy derived from wave and tidal devices is highly predictable, their inclusion in our energy portfolio could help balance available sources of energy production, including hydroelectric, coal, nuclear, wind, solar, geothermal, and others. As an emerging industry, MHK energy developers face many challenges associated with the siting, permitting, construction, and operation of pilot and commercial-scale facilities. As the industry progresses, it will be necessary not only to secure financial support and develop robust technologies capable of efficient, continued operation in harsh environments, but also to implement effective monitoring programs to evaluate long-term effects of device operation and assure resource agencies and members of the public that potential environmental impacts are understood and can be addressed. At this time, little is known about the environmental effects of MHK energy generation at pilotor full-scale operational scenarios. Potential effects could include changes to aquatic species behavior from exposure to electromagnetic fields or operational noise; physical interaction of marine mammals, fish, and invertebrates with operating devices or mooring cables; or changes to beach characteristics and water quality from long-term deployment of devices in coastal locations. This lack of knowledge creates a high degree of uncertainty that affects the actions of regulatory agencies, influences the opinions and concerns of stakeholder groups, affects the commitment of energy project developers and investors, and ultimately, the solvency of the industry. To address the complexity of environmental issues associated with MHK energy, PNNL has received support from the Department of Energy Office of Energy Efficiency and Renewable Energy Waterpower Program to develop research and development that draws on the knowledge of the industry, regulators, and stakeholders. Initial research has focused on 1) the development of a knowledge management database and related environmental risk evaluation system, 2) the use of hydrodynamic models to assess the effects of energy removal on coastal systems, 3) the development of laboratory and mesocosm experiments to evaluate the effects of EMF and noise on representative marine and estuarine species, and 4) collaborative interaction with regulators and other stakeholders to facilitate ocean energy devices, including participation in coastal and marine spatial planning activities. In this paper, we describe our approach for initial laboratory investigations to evaluate potential environmental effects of EMFs on aquatic resources. Testing will be conducted on species that are a) easily procured and cultured, b) ecologically, commercially, recreationally or culturally valuable, and c) reasonable surrogates for threatened or endangered species. Biological endpoints of interest are those that provide compelling evidence of magnetic field detection and have a nexus to individual, community, or population-level effects. Through laboratory, mesocosm, and limited field testing, we hope to reduce the uncertainly associated with the development of ocean energy resources, and gain regulatory and stakeholder acceptance. We believe this is the best approach for moving the science forward and provides the best opportunity for successfully applying this technology toward meeting our countrys renewable energy needs. During the project, the team will work closely with two other national laboratories (Sandia and Oak Ridge), the Northwest National Marine Renewable Energy Center at University of Washington and Oregon State University, and Pacific Energy Ventures.