Thinking Big by Thinking Small: Tracy Farmer Institute Scientists Focusing on the Health Risks of Nanoparticles - 2011

The nano-explosion is on. Nanoparticles, far lighter but stronger than steel and a million times smaller than the head of a pin, are now being used in 1,500 consumer products, according to Paul Bertsch, UK professor of environmental chemistry and toxicology, and director of the UK Tracy Farmer Institute for Sustainability and the Environment. Plastic imbued with clay nanoparticles helps make Miller Brewing Co. beer bottles less likely to break and improves how long the brew lasts in storage. Simply H’s Toddler Health nutritional drink mix includes 300-nanometer (300 billionths of a meter) iron particles. Other products that contain nanoparticles? Odorless socks, hockey sticks, drug-delivery systems..., and the list keeps growing. A wide range of cooking and cleaning items, for example, now employ nanosize silver particles to kill microbes. Nanoparticles’ usefulness has been established. But what will these materials manipulated by humans do to the environment?And do they pose a health risk to animals and humans? At the Tracy Farmer Institute, researchers in the institute’s Nanoscience Working Group are involved in various projects to try to answer these questions. Bertsch points out that normal use of nano-enhanced products—such as washing your clothes—can release nanoparticles, “downstream,” into wastewater treatment plants, where they end up in sewage sludge. About 3 million tons of dried-out sludge is subsequently mixed into agricultural soil each year, as fertilizer. Beyond any environmental damage these particles can cause, there is the direct risk to animals and, possibly, humans. A 2005 study in Environmental Science & Technology showed that zinc oxide nanoparticles were toxic to human lung cells in lab tests even at low concentrations. Other studies have shown that tiny silver particles (15 nanometers) killed liver and brain cells from rats. Three particles in particular are currently raising the most concern with regards to public health: nanosilver, titanium dioxide, and carbon nanotubes. “The nanotechnology revolution will change all aspects of our life and transform the global economy,” says Bertsch. “It is critical that risks to the environment and human health be understood and properly managed so that this promise is realized.”

Nanoparticles Worming Their Way Up the Food Chain

In the past year, members of the Nanoscience Working Group have completed two projects to examine how nanoparticles move through soil and into an animal model. Environmental toxicologist Jason Unrine in the UK plant and soil sciences department headed up a team of scientists, including Bertsch and Olga Tsyusko, who wanted to know if nanoparticles in fertilizer spread onto fields eventually made their way into the food chain. "Because we expected the nanomaterials to aggregate onto soil particles, we were initially very skeptical that organisms could take them up from the soil," Unrine states. To find out, his team mixed earthworms—organisms near the bottom of the food chain—into artificial soil tainted with gold nanoparticles. "We used gold nanoparticles because they're stable, insoluble and easily detected," Unrine explains. "They make a good tracer."

After 28 days, the researchers examined the worms’ tissues to look for uptake of nanoparticles. The scientists first performed laser ablation (removal of material from the surface of an object by vaporization) coupled with inductively  coupled plasma mass spectrometry to map the total gold distribution in earthworm tissues. Then Unrine’s team worked with a technician at the National Synchrotron Light Source at Brookhaven National Laboratory on Long Island, New York, who performed X-ray microanalysis. “Using these various techniques, we found gold nanoparticles distributed throughout the worm’s body”, Unrine says. Although the nanoparticles didn’t significantly affect earthworm mortality, exposed worms produced up to 90 percent fewer offspring. “Our research provided the first unequivocal evidence that engineered nanomaterials can be taken up from the soil and enter food webs,” Unrine says. This project, which has received considerable attention in the scientific media, was supported by funding from the Environmental Protection Agency and the National Science Foundation.

More Evidence, with a Little Help from Tobacco and Hornworms

To further explore nanoparticle absorption in the food chain, Bertsch headed up a team that cultivated tobacco plants in a nutrient solution and then were grown in a nano-suspension for a week in a College of Ag hydroponic greenhouse. While the plants grew, the team added gold nanoparticles to the water to mimic consumer nanoparticles in wastewater sludge. The plants were placed in enclosures containing hornworms, caterpillars which love to eat tobacco leaves. After the worms had had time to digest their meal, Bertsch's group examined various tissues of the hornworm to measure the presence of the particles, which were purposely made to be easily traceable. Though Bertsch was the PI on this grant, he credits Ph.D. graduate student Jonathan Judy for doing the lion's share of the work, in particular for developing the exposure methods and fine-tuning the experiments. The findings?  The worms failed to excrete the particles that had been suspended in the water and drawn through the roots of the plant. And the particles were found to have made their way into the tissues of the caterpillars at an even greater concentration than in the plant the caterpillars had eaten: the hornworms accumulated concentrations of the nanomaterials about 6 to 12 times higher than in the plant. “We expected nanoparticles to bioaccumulate——but not to biomagnify like that,” says Bertsch, whose team published this study last December in Environmental Science & Technology. The findings from this study were themselves amplified by a separate but related study led by Patricia Holden, an environmental microbiologist at University of California, Santa Barbara. In this work, predatory microbes also built up concentrated levels of cadmium selenide nanoparticles that ingested them. “It’s really interesting for all of us doing this work to see two different models using two different nanoparticles arrive at conclusions reinforcing each other,” says Bertsch, who adds that these findings add further concern to how nanoparticles might affect and endanger agricultural crops. “While heavy metals and other toxins in sludge are federally regulated, manmade nanoparticles are not. That may be a cause for concern as farms increasingly mix sludge into their soils, where, we now know, nanoparticles build up over time.” “This experiment was conducted under highly specific artificial conditions, so it is important not to try to apply these findings too broadly,” Judy says.  “Many more experiments like ours need to be conducted before we will have a clear picture of the risks to terrestrial food webs posed by nanotechnology.  Specifically, investigations into the bioavailability of nanoparticles to higher trophic levels and to plants from soil would be highly informative.” There’s some evidence that nanoparticles are toxic under lab-controlled conditions, Bertsch says, but realistically assessing risks to health and the environment demands more advanced models. He and other scientists—three groups in England—have begun collaboration on an experiment at Cranfield University in England that will use the institution’s wastewater stream as a real-life laboratory. “This experimental wastewater treatment facility actually uses the sewage generated by the university and surrounding town,” Bertsch explains. “We add nanomaterials to actual sewage to producing biosolids, then look at the resulting transformations.”  Organisms are then exposed to these biosolids that contain nanomaterials. “It’s a real-world approach to measuring particle uptake.” “We’re still at an early stage of accumulating information that will hopefully lead quickly to paradigms for predicting the environmental fate, bioavailability, and toxicity of manufactured nanomaterials,” Bertsch adds.

Can Nanoparticles Find Their Way into Our Brains?

In separate but related work, another UK nanoparticles team has been investigating how the sizes and shapes of nanoparticles affect their ability to enter the brain. The project is being led by Robert Yokel, a professor in the College of Pharmacy, whose team includes scientists from UK’s Center for Applied Energy Research, chemistry, engineering and the department of anatomical sciences at the University of Louisville’s School of Medicine.

This group has focused on the potential health impacts of nano-sized cerium oxide, a material used as a diesel fuel additive in Europe. Cerium oxide is reputed to improve fuel efficiency, suppress soot from exhaust and reduce the concentration of other ultra-fine particles in air that have known health effects. The project is backed by a $4 million, four-year grant, with $2 million coming from the EPA, the largest EPA Science to Achieve Results (STAR) grant ever awarded to the University of Kentucky. “We’re working with this diesel fuel additive in part because it’s going into the environment,” says Yokel. “It’s also electron-dense, which helps, because we can see it.” Yokel admits that there are major challenges working with nanotechnology. “First, we need to know what we’re working with, to try to characterize individual nanoparticles.”  One trait of the material Yokels’ group has worked with is that it agglomerates clumps together, which affect how the body deals with it. Another challenge in determining the material’s properties, he explains, is the fact that when nanomaterials enter an animal’s system—Yokel and his team are using rats—the substance gets coated with protein, which changes the material’s surface properties. In addition to understanding the basic makeup of these particles, the researchers wanted to find out where the material goes once it's introduced into an animal system, and what the material does when it gets there. In tracking these nanoparticles, Yokel's group made two surprising and important discoveries: nanoparticles do not get into the brain (they can't penetrate the blood-brain barrier) and that the material tends to accumulate in the liver, spleen, bone marrow, and kidneys. "It goes to these organs and stays there," Yokel says. "It doesn't seem to be cleared." This "persistence," as Yokel puts it, of the material was seen in both a 30-day study and a 90-day study, and this finding may have some health implications. "One of our team members found some tissue damage, some granuloma in liver," Yokel says. "The concern is that cerium is highly reactive, so while it's sitting there, it's chemically active and may be forming free radicals." The research team has recently begun the fourth and final year of the project, and will publish their findings in 2012.

What's Next for UK's Nanoscience Group?

Bertsch says that the one sure thing about future nanomaterials research is that it will continue to be challenging. "The diversity of nanomaterials from the standpoint of chemical composition, size, shape, and surface functionalization is overwhelming," says Bertsch. "Our studies to date have just begun to scratch the surface. We need to understand what properties of nanoparticles influence bioavailability and toxicity so that we can begin to develop guiding principles for predicting the hazard or risk." Bertsch adds that this work will lead into a new area of research—designing nanomaterials to be biologically benign while still useful. "Also, our work with the consortium in England will move into more environmentally relevant systems with the biosolids generated from the large-scale experimental wastewater treatment plant." These studies will also examine the potential for nanomaterials to enter the human food chain. “It’s easy to imagine designing a nanoparticle so environmentally benign that it doesn’t offer any commercial value over what is otherwise being done today—and that’s no good for innovation,” Bertsch says. “You can also envision synthesizing nanoparticles that are fantastic in products but incredibly toxic—that’s obviously no good for the environment. The question is, can you design something with the best of both worlds in mind?” — Jeff Worley

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