Rebecca Klaper

Daphnia magna mortality when exposed to titanium dioxide and fullerene (C60) nanoparticles

Nanoparticles (1-100 nm) comprise the latest technological advances designed to do everything from absorb environmental toxins to deliver drugs to a target organ. Recently, however, they have come under scrutiny for the potential to cause environmental damage. Because compounds in this miniature size range have chemical properties that differ from those of their larger counterparts, nanoparticles deserve special attention. Our main objective was to assess the potential impact that nanoparticles may have on release into aquatic environments. We prepared titanium dioxide (TiO2) and fullerene (C60) nanoparticles by filtration in tetrahydrofuran or by sonication. Daphnia magna were exposed to the four solutions using U.S. Environmental Protection Agency 48-h acute toxicity tests. Images of the particle solutions were recorded using transmission-electron microscopy, and the median lethal concentration, lowest-observable-effect concentration, and no-observable-effect concentration were determined. Exposure to filtered C60 and filtered TiO2 caused an increase in mortality with an increase in concentration, whereas fullerenes show higher levels of toxicity at lower concentrations. Exposure to the sonicated solutions caused varied mortality. Understanding the potential impacts of nanoparticles will help to identify the most appropriate nanotechnology to preserve the aquatic environment while advancing medical and environmental technology.

Pharmaceuticals and personal care products found in the Great Lakes above concentrations of environmental concern

The monitoring of pharmaceuticals and personal care products (PPCPs) has focused on the distribution in rivers and small lakes, but data regarding their occurrence and effects in large lake systems, such as the Great Lakes, are sparse. Wastewater treatment processes have not been optimized to remove influent PPCPs and are a major source of PPCPs in the environment. Furthermore, PPCPs are not currently regulated in wastewater effluent. In this experiment we evaluated the concentration, and corresponding risk, of PPCPs from a wastewater effluent source at varying distances in Lake Michigan. Fifty-four PPCPs and hormones were assessed on six different dates over a two-year period from surface water and sediment samples up to 3.2 km from a wastewater treatment plant and at two sites within a harbor. Thirty-two PPCPs were detected in Lake Michigan and 30 were detected in the sediment, with numerous PPCPs being detected up to 3.2 km away from the shoreline. The most frequently detected PPCPs in Lake Michigan were metformin, caffeine, sulfamethoxazole, and triclosan. To determine the ecological risk, the maximum measured environmental concentrations were compared to the predicted no-effect concentration and 14 PPCPs were found to be of medium or high ecological risk. The environmental risk of PPCPs in large lake systems, such as the Great Lakes, has been questioned due to high dilution; however, the concentrations found in this study, and their corresponding risk quotient, indicate a significant threat by PPCPs to the health of the Great Lakes, particularly near shore organisms.

Electron microscopy of gold nanoparticle intake in the gut of Daphnia magna

Few studies have described aquatic organisms interacting with manufactured nanoparticles. One key factor in determining these materials potential toxicity is the extent to which these particles accumulate in tissues. This may be most important for aquatic organisms as they contact large quantities of water through their feeding behavior. We examined the uptake and release of gold particles in filter-feeding Daphnia magna. Daphnia were exposed to sublethal concentrations of gold nanoparticles for 1, 6, 12, or 24 hours. Transmission Electron Microscopy was used to examine the presence and distribution of gold in gut tissue. The highest concentration of nanoparticles was found after 12 h. Particle clearance was investigated by placing organisms in fresh water and observing particles retention in the gut tissue over time. The initial trend of high gold concentration in the mouth with low levels in the tail region was reversed, suggesting clearance of particles with time.

Toxicity biomarker expression in daphnids exposed to manufactured nanoparticles: changes in toxicity with functionalization.

In previous work we have shown that the toxicity of nanomaterials to Daphnia spp. differs with the type of nanoparticle either due to the core of the particle or to the way in which a particle suspension is prepared. The purpose of this study was to investigate the toxicity and antioxidant response of Daphnia pulex in relation to a change in surface functionalization of nanomaterials with the same core material, nC60. Despite the lack of acute toxicity for various nC60 suspensions up to 100 ppm concentration, there was a significant production of the toxicity biomarkers glutathione-S-transferase and catalase, at lower concentrations indicating changes in reactive oxygen species. Nanoparticle functionalization significantly affected this response. Oxidative stress markers appear to be a good predictor of potential future toxicity of nanomaterials. Functionalization alters both toxicity and oxidative stress in whole organism assays.

Environmental concentrations of the selective serotonin reuptake inhibitor fluoxetine impact specific behaviors involved in reproduction, feeding and predator avoidance in the fish Pimephales promelas (fathead minnow).

Pharmaceuticals and personal care products (PPCPs) have been found in surface waters worldwide, but little is understood of their effects on the wildlife that inhabit these waters. Fluoxetine (Prozac; Eli Lilly), a highly prescribed selective serotonin reuptake inhibitor (SSRI), is a commonly found PPCP in surface water. The purpose of this project was to determine if environmentally relevant concentrations of fluoxetine impact behavior that is important for population survival in native fish species, including reproduction, feeding and predator avoidance. Chronic 4-week exposures were conducted with doses ranging from 100 ng/L to 100 μg/L to cover a range of environmentally relevant concentrations up to higher concentrations comparable to other published studies with the same drug that have documented various physiological impacts. Pimephales promelas (fathead minnow), a species native to North America, was used as it conducts a range of specific mating behaviors and therefore serves as an excellent model of specific impacts on brain function. Fluoxetine concentrations as low as 1 μg/L, a concentration that has been found in many freshwater environments, were found to significantly impact mating behavior, specifically nest building and defending in male fish. Males were also found to display aggression, isolation, and repetitive behaviors at higher concentrations. Female mating behavior was largely unaffected. In addition, predator avoidance behaviors in males and females were also impacted at 1 μg/L. Feeding was impacted at 10 μg/L and in the highest exposure (100 μg/L), egg production was limited by deaths of females due to significant male aggressive behaviors in the first two weeks of exposure. Specific behavioral changes occurred at each concentration (most noticeably 1 μg/L and 100 μg/L) indicating a dose dependent effect that triggered different responses at lower exposures versus higher exposures or differential impacts of dose depending on brain region. Length of exposure also had an impact on aggressive behavior. Changes in hormone levels, indicating significant neuroendocrine changes, suggested as a mechanism of response in higher dose and acute studies, were not linked to changes in behaviors at the doses used in this study. This research provides detailed data on how exposures to fluoxetine impact specific fish behaviors and reproduction and that the effects are dose dependent.

Rickettsia associated with male-killing in a buprestid beetle.

Many populations of the buprestid leaf-mining beetle, Brachys tessellatus, from central South Carolina, USA, show highly skewed sex ratios, ranging from 1.3 to 6.0 females per male. We have identified a Rickettsia bacterium that is associated with sex ratio distortion (SRD) and selective killing of male embryos in B. tessellatus. Molecular assays of infection by this bacterium are highly associated with SRD within families, and treatment with an antibiotic (tetracycline) increases the number of male eggs that hatch and develop. The 16S rDNA sequence indicates that this is a novel Rickettsia, most closely related to Rickettsia bellii (a tick-associated bacterium) and a pea-aphid Rickettsia. It is also related to a Rickettsial bacterium that causes male-killing in an unrelated ladybird beetle species. Low levels of parthenogenesis are also observed in this system (about 10% of females) and may be the result of selection due to male rarity, or a direct result of infection by the Rickettsia.

Gene Expression Changes Related to Endocrine Function and Decline in Reproduction in Fathead Minnow (Pimephales promelas) after Dietary Methylmercury Exposure

Background Methylmercury (MeHg) is a known neurotoxic agent, but the mechanisms by which MeHg may act on reproductive pathways are relatively unknown. Several studies have indicated potential changes in hormone levels as well as declines in vertebrates with increasing dietary MeHg exposure. Objectives The purpose of this study was to identify alterations in gene expression associated with MeHg exposure, specifically those associated with previously observed changes in reproduction and reproductive biomarkers. Fathead minnows, Pimephales promelas, were fed one of three diets that were similar to documented concentrations of MeHg in the diets of wild invertivorous and piscivorous fish. We used a commercial macroarray in conjunction with quantitative polymerase chain reaction to examine gene expression in fish in relation to exposure to these environmentally relevant doses of MeHg. Results Expression of genes commonly associated with endocrine disruption was altered with Hg exposure. Specifically, we observed a marked up-regulation in vitellogenin mRNA in individual Hg-exposed males and a significant decline in vitellogenin gene expression in female fish with increasing Hg concentrations. Other genes identified by the macroarray experiment included those associated with egg fertilization and development, sugar metabolism, apoptosis, and electron transport. We also observed differences in expression patterns between male and female fish not related to genes specifically associated with reproduction, indicating a potential physiological difference in the reaction of males and females to MeHg. Conclusion Gene expression data may provide insight into the mechanisms by which MeHg affects reproduction in fish and indicate how MeHg differs in its effect from other heavy metals and endocrine-disrupting compounds.

Evaluation of a model for the removal of pharmaceuticals, personal care products, and hormones from wastewater

Current wastewater treatment processes are insufficient at removing many pharmaceutical and personal care products (PPCPs) from wastewater and it is necessary to identify the chemical characteristics that determine their fate. Models that predict the fate of various chemicals lack verification using in situ data, particularly for PPCPs. BIOWIN4 is a quantitative structure-activity relationship (QSAR) model that has been proposed to estimate the removal of PPCPs from wastewater, but data verifying the accuracy of its predictions is limited. In this study, the in situ soluble and suspended solid concentrations were assessed from raw influent, primary effluent, secondary effluent, and final effluent for 54 PPCPs and hormones over six dates. When assessing the removal efficiency across the different stages of the WWTP, the majority of the removal occurred across the secondary treatment process for the majority of the compounds. The primary treatment and disinfection process had limited impacts on the removal of most PPCPs. Sorption to solids was found to influence the removal for compounds with a log octanol-water partitioning coefficient greater than 4.5 across the secondary treatment process. For other compounds, the removal of PPCPs across the secondary treatment process was significantly correlated with the biodegradation predicted by BIOWIN4. Removal efficiencies across the aerobic secondary treatment process were predicted by integrating BIOWIN4 into pseudo-first order kinetics of PPCPs and these predicted values were compared to the in situ data. This study determines that under a certain set of operating conditions, two chemical characteristics - the expected hydrophobic interaction and the modeled biological degradation from BIOWIN4 - were found to predict the removal of highly degradable and recalcitrant PPCPs from a wastewater secondary treatment process.

Daphnia as an emerging model for toxicological genomics

Daphnia are already an established model species in toxicology. This freshwater crustacean is used commonly for environmental monitoring of pollutants around the globe and plays an important role in establishing regulatory criteria by government agencies (e.g., US EPA, Environment Canada organization for Economic Cooperation and Development, Environment Agency of Japan). Consequently, daphniids represent 8% of all experimental data for aquatic animals within the toxicological databases (Denslow et al., 2007). As such, their incorporation within the new field of toxicological genomics is limited only by the advancement of genomic resources. Because the development of these technologies requires the input and feedback of a large research community that extends far beyond the boundaries of any one discipline, the Daphnia Genomics Consortium (DGC) was formed in 2001 to: (i) provide the organizational framework to coordinate efforts at developing the Daphnia genomic toolbox; (ii) facilitate collaborative research and (iii) develop bioinformatics strategies for organizing the rapidly growing database. This chapter reviews the progress in establishing Daphnia as model species for genomic studies, with emphasis on toxicological applications. As the goals of the DGC are defined largely by extending the boundaries of current biological research in light of genomic information, this chapter first reviews Daphnias unique biological attributes that make it ideal for such an expansion of research efforts. These attributes include a long tradition of ecological, evolutionary and toxicological study, culminating in the benefits provided by emerging genomic tools.

Emerging wastewater contaminant metformin causes intersex and reduced fecundity in fish

The occurrence of intersex fish, where male reproductive tissues show evidence of feminization, have been found in freshwater systems around the world, indicating the potential for significant endocrine disruption across species in the ecosystem. Estrogens from birth control medications in wastewater treatment plant effluent have been cited as the likely cause, but research has shown that endocrine disruption is not solely predictable based on hormone receptor interactions. Many other non-hormone pharmaceuticals are found in effluent at concentrations orders of magnitude higher than estrogens, yet there is little data indicating the impacts of these other medications. The widely prescribed anti-diabetic metformin is among the most abundant of pharmaceuticals found in effluent and is structurally dissimilar from hormones. However, we show here that exposing fathead minnows (Pimephales promelas) to a concentration of metformin found in wastewater effluent causes the development of intersex gonads in males, reduced size of treated male fish, and reduction in fecundity for treated pairs. Our results demonstrate that metformin acts as an endocrine disruptor at environmentally relevant concentrations.