Nurturing caterpillars is hard work, especially in a laboratory environment. In nature, the insects often spend their larval stage in a stand of plants or trees, so they don’t have to search hard for sustenance. But in an artificial enclosure containing a single plant, says Jonathan D. Judy, a third-year graduate student at the University of Kentucky, “you have to keep an eye on them so that they don’t get too far away from their food.”
During a recent experiment, Judy mothered about 30 tobacco hornworm caterpillars (Manduca sexta) on his benchtop, picking them up and gently nudging them back onto their plants when they fell off or wandered away. Like a caring parent, he cleaned their enclosures daily to get rid of feces and bacteria. “Caterpillars are very sensitive to disease,” the grad student explains.
All of this caretaking was necessary for an investigation of the transfer of nanoparticles within the food chain, from tobacco plant (Nicotiana tabacum) to insect. Judy and his advisers, environmental chemist Paul M. Bertsch and ecotoxicologist Jason M. Unrine, demonstrated that not only could a tobacco plant grown in a solution of gold nanoparticles accumulate the materials in its leaves, but it could also impart them to the hungry caterpillars that fed on it (C&EN, Dec. 20, 2010, page 35). Even more surprising to the researchers was that the particles biomagnified: The concentration of the materials in the guts of caterpillars was up to 12 times as high as that in the tobacco leaves.
Judy’s experiment is one of several that probe the effects of nanotechnology on the environment and its web of organisms. Concern about the safety of nanomaterials has grown over the past decade as their inclusion in consumer products has ramped up.
More than 1,000 products containing nanomaterials are on the market, according to an inventory maintained by the Project on Emerging Nanotechnologies, a joint venture of the Woodrow Wilson International Center for Scholars and Pew Charitable Trusts. Antimicrobial clothing contains silver nanoparticles; paints, coatings, and “chemical-free” sunscreens are spiked with titanium dioxide particles; and photovoltaic panels are made with quantum dots. And some of those materials are making their way into the environment, swirling down drains and into the water supply, or traveling through wastewater treatment plants into sludge that is then applied as fertilizer to farmland.
“As nanotechnology marches on, researchers need to get in front of potential environmental implications so that there’s not a big public backlash,” Bertsch says. But until now, the reverse has been true: Technology is outpacing nanomaterial-related environmental health and safety research, he says. It will be a struggle for researchers and regulators to catch up.
But they’re making valiant efforts. Federal funds allotted for nanotechnology safety research have grown from $34.8 million in fiscal 2005 to a requested $117 million in 2011. In 2008, the National Science Foundation and the Environmental Protection Agency established two research centers devoted to the environmental implications of nanotechnology, one based at Duke University—from which Bertsch obtained funding—and the other at the University of California, Los Angeles (C&EN, Oct. 20, 2008, page 53).
“Research into the potential ecological effects of nanomaterials is important and still new,” says Patricia A. Holden, an environmental scientist at UC Santa Barbara. And the investment in these studies could affect nanotechnology’s perception and regulation.
Just as Bertsch’s group did, another team, led by Holden, recently demonstrated food-chain transfer of nanomaterials. The researchers found that cadmium selenide quantum dots moved from Pseudomonas aeruginosa bacteria to predator Tetrahymena thermophila protozoa (Nat. Nanotechnol., DOI: 10.1038/nnano.2010.251).
In Holden’s aquatic food chain, as in Bertsch’s terrestrial one, the nanomaterials biomagnified: The concentration of quantum dots in the protozoa’s food vacuoles, or digestive compartments, was five times as high as that in the bacteria they ingested. In addition, using both optical and scanning transmission electron microscopy, the researchers observed an abnormally high proportion of filled food vacuoles in T. thermophila. Holden says that the quantum dots must have slowed the protozoa’s digestion and caused the buildup of the nanomaterials.
Typically, substances biomagnify in a food web because organisms excrete them inefficiently, Bertsch says. And this slow elimination often has toxic consequences. Mercury and dichlorodiphenyltrichloroethane (DDT) are just two well-known—and poisonous—examples of compounds that march up the food chain.
Bertsch’s team didn’t study nanoparticle elimination from its caterpillars, which appeared to be healthy despite having ingested the tiny materials. But in an unpublished study of earthworms, which take up the materials directly from soil, the team saw inefficient excretion of particles. Like earthworms, hornworm caterpillars take up the gold nanoparticles across their gut linings and store them in their tissues, Bertsch says. So he thinks that caterpillars might have similar problems with excretion.
Holden and her team saw clearer effects on their organisms: stunted digestion and early death of the protozoa. Before their deaths, the T. thermophila also stopped swimming. This lack of motility, combined with the protozoa’s buildup of nanomaterials, worries Holden. “They’re sitting there, ready to be eaten by the next organism in the food chain” and continue the biomagnification process, she says.
Not all food-chain-transfer studies, however, have shown biomagnification. A research team led by environmental engineer Yongsheng Chen of Georgia Institute of Technology and immunologist Yung Chang of Arizona State University, Tempe, reported last year that TiO2 nanoparticles did not increase in concentration when zebrafish ate water fleas (Daphnia magna) containing the materials (Chemosphere, DOI: 10.1016/j.chemosphere.2010.03.022). The fish also excreted all the nanomaterials within three or four days.
Chang is not surprised at the difference in results. Whether biomagnification occurs or not, she says, is “probably going to vary from particle to particle, depending on its physicochemical properties.” For instance, the TiO2 nanoparticles that she used tend to cluster into large aggregates, she says, ranging from 100 nm to several micrometers in diameter. Such aggregates can be taken up by organisms but are likely difficult to transport into cells for accumulation. These clusters are much larger than the 5- to 15-nm gold nanoparticles used by Bertsch’s group and the 5-nm quantum dots used by Holden’s team.
Another difference was that the TiO2 nanoparticles were unfunctionalized, Chen says, whereas Bertsch’s nanoparticles were coated with tannic acid, making them more hydrophobic. “Scientists functionalize nanoparticles for biomedical applications,” Chang says. “It makes them easily usable, especially for the delivery of chemicals or drugs, but it could also increase their bioaccumulation.”
Although Chen and Chang’s team did not observe biomagnification of TiO2 nanoparticles in zebrafish, the scientists did find—in a separate, non-food-chain experiment—that the nanomaterials impaired fish reproduction (Chemosphere, DOI: 10.1016/j.chemosphere.2010.12.069). After 13 weeks in a tank filled with an aqueous solution of TiO2 nanoparticles, female zebrafish produced 30% fewer eggs than normal. The aqueous solution contained 0.1 mg/L TiO2, a concentration that resembles the level of TiO2 particles in wastewater-treated sludge, according to one estimate (Environ. Sci. Technol., DOI: 10.1021/es9015553).
“That’s pretty significant,” Chen says. Current safety standards for chemicals are mainly based on acute toxicity levels, he adds. But acute toxicity might not be relevant to chronic exposure to nanomaterials, he says. Chronic exposure could affect an entire population’s dynamics—and the balance of life in the aquatic food chain. Such cascading effects could cause more damage than a one-time high-level exposure would by killing a few fish, he contends.
Despite these eyebrow-raising findings, says R. David Holbrook, a chemical engineer at the National Institute of Standards & Technology, it’s too early to draw conclusions about the safety of nanomaterials in the food chain. In 2008, Holbrook’s group was the first to report transfer of nanomaterials between two organisms (Nat. Nanotechnol., DOI: 10.1038/nnano.2008.110). “The science is just not there to make a decision about whether engineered nanomaterials can be grossly categorized as bad or good,” he says. He points out that most of the published food-chain studies have used concentrations of materials “that far exceed anything we would expect to see in the natural environment.”
Bertsch’s team, for instance, exposed its tobacco plants to gold nanoparticles at a concentration of about 100 ppm in its caterpillar study. Judy acknowledges that the concentration is “many, many times higher” than has been observed for metal nanoparticles in biosolid sludge from wastewater treatment plants.
Using such high concentrations, Holbrook explains, is by design. “Processes in nature have decades to evolve—we don’t have that luxury,” he says. “If you’re lucky, you have a four-year grant.” To speed things up, scientists often boost the nanomaterial concentrations. As analytical techniques and methodology improve, Holbrook predicts, more researchers will study environmentally relevant concentrations in the food chain.
They will also move to more complicated food webs. Through the new Transatlantic Initiative for Nanotechnology & the Environment, a consortium of U.S. and U.K. scientists, Bertsch’s team will introduce nanomaterials to a waste stream entering a pilot-scale treatment plant at Cranfield University, in England. After the plant filters and processes the stream, the researchers will use the resulting biosolids in experiments on the materials’ uptake in a chain of plants, root-colonizing bacteria, and earthworms.
Collecting meaningful data in a three-organism food chain and beyond won’t be easy, Judy says. Just keeping the organisms alive takes a lot of work, he says, recalling his tribulations with the caterpillars.
But the effort is well worth it, Bertsch says. Understanding the properties that make nanomaterials bioavailable or dangerous, he says, will enable scientists to design materials that are safer.