Fatal interactions

Carbon nanotubes combined with lead or pesticides increase toxic effects on fish

Tilapia in experiments performed at the Fishing Institute in Cananeia

EDUARDO CESARTilapia in experiments performed at the Fishing Institute in CananeiaEDUARDO CESAR

Studies conducted by a partnership between researchers at the University of Campinas (Unicamp), the National Nanotechnology Laboratory (LNNano) and the Fishing Institute of the State of São Paulo, in Cananeia, on the southern coast of the state, show that when carbon nanotubes come into contact with toxic substances like lead and pesticides in aquatic environments, there is a significant increase in toxicity for fish such as Nile tilapia (Oreochromis niloticus), fresh-water shrimp and other species. The latest results of the research that evaluated the interaction between these nanomaterials and carbofuran—a highly toxic pesticide used in Brazilian agriculture—were published online in the journal Ecotoxicology and Environmental Safety in November 2014 and will appear in the print edition in January 2015. “In combination with the nanotubes, there was a five-fold increase in carbofuran toxicity in the tilapia,” says Professor Oswaldo Alves of the Solid State Chemistry Laboratory (LQES) of the Unicamp Institute of Chemistry.  “This is a clear indication that the nanostructure is enhancing the toxic effect of the pesticide.” Similarly, it also acts as an excellent concentrator of pesticides, metals and hormones. That means that nanotubes are materials with properties that could potentially be used in water treatment system filters and sensors. “However, we need to assess how we will dispose of these materials and think about the future environmental implications,” says Alves.

Among the tests carried out by Professor Edison Barbieri at the Fishing Institute—in partnership with Diego Stéfani Teodoro Martinez, Alves’ former doctoral student and postdoctoral researcher and currently a researcher at LNNano in Campinas—are oxygen consumption, one of the measures used to assess metabolism, and the swimming capacity of the fish. The findings indicate that the carbon nanostructures can act as pesticide carriers and affect the behavior as well as the survival of fish.

The tests showed that concentrations of up to 2 milligrams (mg) of nanotubes per liter of water made no difference in oxygen consumption when compared to the control group. When just carbofuran was placed in the water, there was initially an increase in oxygen consumption and then a decrease, indicating that the fish were beginning to die. “In the experiments carried out with 0.5 mg, 1 mg and 2 mg of this substance, combined with (1 mg) carbon nanotubes, oxygen consumption declined rapidly, indicating a clear difference compared to the control group,” says Barbieri, coordinator of the research at the Fishing Institute. Swimming capacity decreased over time when the pesticide and the nanotubes were in the water.

microscopic image of test using nanotubes and lead shows that gill filaments are deformed and swollen...

UNICAMPMicroscopic image of test using nanotubes and lead shows that gill filaments are deformed and swollen…UNICAMP

Begun in 2010, the study sought to understand the interaction between nanomaterials and common pollutants such as lead, which in Cananeia, for example, poses a serious environmental problem. “There, outcrops of lead from galenas [minerals containing lead sulfide] are natural and occur when there is a lot of rain, resulting in leaching of the soil,” says Barbieri. The study began with the exposure of tilapia to carbon nanotubes and lead in different concentrations for up to 96 hours. The results were presented by Martinez in November 2012 at an international conference on nanomaterial safety called NanoSafe, held every two years in Grenoble, France. Nanotubes increase the acute toxicity of lead to tilapia by up to a factor of five. In the experiment in which the fish were exposed only to these carbon nanostructures, there was no sign of acute toxicity up to a level of 2 mg per liter.

The first phase of the tests consisted of carrying out the control experiment using just water. Then, tests were performed with the nanotubes and lead separately, in different concentrations up to 2 mg per liter, before finally placing the two materials in the tank together. “Oxygen consumption decreased in all cases in which both were present,” says Barbieri. A scientific article containing the results of the study was published in the March 2013 issue of the Journal of Physics: Conference Series. “Few studies in the international scientific literature address the interaction between environmental pollutants and carbon nanotubes, focusing instead on the impacts on the physiology and behavior of fish,” says Martinez. Moreover, there is a lack of conclusive evidence on the long-term effects of these nanomaterials when disposed of in the environment. This means that research has not kept up with the market growth of these nanostructures with special physical and chemical properties, which have increased yearly.

... compared to the control group exposed to no toxic substances

UNICAMP… compared to the control group exposed to no toxic substancesUNICAMP

The interaction between lead and carbon nanotubes and their toxic effects on the gills of tilapia was also studied by Edison Barbieri’s master’s students at the Fishing Institute. They studied the effects of this combination on the gills, the main organ responsible for gas exchange and the excretion of ammonia, and the mechanism by which aquatic animals purge toxic waste such as ammonia, urea and salts from their bodies. The gills are responsible for maintaining internal equilibrium, known as homeostasis. “In the control experiment you can see that all the filaments of the gills are preserved, but with the addition of carbon nanotubes and lead, together or separately, there is swelling and deformation of the cells in the lining of the gills, which are responsible for gas exchange” reports Barbieri. The tests with carbofuran and nanotubes also had similar results.

Before the experiments began, the materials were carefully purified and characterized in order to ensure strict quality control and convergent results. “Part of Diego’s dissertation work involved learning to purify carbon nanotubes,” says Alves. “He devoted four years to this and is now benefiting from that work because he has quality material for biological and toxicological tests. Alves explains that the biological tests need to be done using proven, well-known material. “The nanotubes made by one company are different than those produced by another.” To ensure that the final material was of the same quality, the researchers ordered nanotubes from a South Korean company, and then applied a refined purification process to them before their initial use. “Each lot of material leaving the LQES is accompanied by a technical report that identifies the sample and describes its characteristics.”

Around 20 metric tons of nanotubes are produced yearly, with 600 different types available on the market for applications that include nanocomposites, concrete, special inks, energy, electronics and even medicine and the environment. Europe and South Korea top the list of the largest producers. “Although no Brazilian companies are producing these substances on a large scale, we need to be pro-active and think to the future and to the regulation of this technology,” says Alves. “We need to plan for disposal, and for this we need to understand the material’s entire life cycle, which requires a lot of research.” Currently, all carbon nanotube-based material is imported into Brazil simply as carbon material. This category includes everything from the activated carbon used in filters to drugs, since there is no specific regulation for nanomaterials. The Organization for Economic Cooperation and Development (OECD), for example, points out that nanostructured materials have interesting properties, but care is needed when incorporating them into products, especially those for biological use. “The organization warns that they should only be used if there is laboratory data on their toxicity or some assessment on the possible effects on the body,” says Alves, whose group receives funding from the National Institute of Science, Technology and Innovation (INCT) in Functional Complex Materials (Inomat), which receives funding from FAPESP and the Ministry of Science, Technology and Innovation (MCTI).

Transmission electron microscope image shows nanotubes produced at the Unicamp Institute of Chemistry

LQES/UNICAMPTransmission electron microscope image shows nanotubes produced at the Unicamp Institute of Chemistry LQES/UNICAMP

Research now focuses on the influence of the organic material in the water of rivers and lakes on nanostructures. “We want to know if, in the presence of organic materials, the pesticides stop interacting with the nanotubes,” says Martinez. “The proposal is to develop a platform for all classic and emerging environmental pollutants, including hormones and antibiotics.” This will allow assessment of the interactions between various pollutants and nanostructured samples. “The work performed up to now indicates that these nanostructures should not be allowed to reach rivers or the ocean.” We still need to carry out studies on their disposal in soil and the effect on plants, as we already know that their environmental impacts are long-lasting.

Regulation on the agenda
Event in France addresses production and safe use of nanomaterials

The issue of regulation has mobilized researchers and companies all over the world that are interested in the many possible uses of nanostructures. On November 5–6, 2014, for example, Brazilian representatives attended a major event in the Netherlands, led by the European Union, which resulted in Brazil’s adoption of one of the most important clusters related to international regulation of nanotechnology. Another important event, in the scientific arena, was NanoSafe 2014, an international conference on the production and safe use of nanomaterials, held in Grenoble, France on November 18–20, 2014.

The fourth time the conference was held—it has taken place every two years since 2008—more than 300 researchers from 30 countries participated, presenting 160 talks and 86 posters, in addition to 12 exhibitors, including companies and organizations. “Since the first conference, the event has sought to address several issues related to nanomaterial safety in a variety of sessions,” says Professor Oswaldo Alves, of the University of Campinas (Unicamp), who attended the event. He presented the results of a project he coordinates on the increase of the toxicity of carbon nanotubes to fish when lead is also present.

Graphical representations of nanomaterials: nanotubes above; functional nanostructures to the left

VIN CRESPI / PENNSYLVANIA STATE UNIVERSITY Graphical representations of nanomaterials: nanotubes above; functional nanostructures to the leftVIN CRESPI / PENNSYLVANIA STATE UNIVERSITY

Among the topics discussed at NanoSafe 2014 were new applications of nanomaterials; nanotoxicology, based on studies involving the respiratory tract, brain and skin; interaction with the environment; the release of nanomaterials; industrial manufacturing and prevention; life-cycle analysis; regulation and standardization; and responsible development. Brazil, represented by researchers from institutions such as Unicamp, the University of São Paulo (USP), the Federal University of São Paulo (Unifesp), the Federal University of the ABC (UFABC), the Federal University of Minas Gerais (UFMG) and the Federal University of Juiz de Fora (UFJF), presented 15 studies. One of the novelties shown by some companies was a portable device that allows monitoring of the presence of nanoparticles in industrial facilities, at construction sites and in other environments.

Regulation and standardization of nanomaterials are essential to the commercial development of nanotechnology. “The issue was placed on the agenda by the European Commission, based on the European Strategy for Nanotechnology, which is supported by three pillars: safety, integration and responsibility,” says Alves. One of these pillars includes standardization. “The European Parliament has highlighted the importance of standardization as a way to track the introduction of nanomaterials on the market. It believes that this will facilitate the implementation of effective regulation,” he explains. Some countries, such as Belgium, France and Denmark, have implemented regulations on specific nanomaterials.

“In the United States, the Environmental Protection Agency (EPA) published two resolutions in 2008, within the scope of the Toxic Substances Control Act (TSCA), clearly indicating a change of attitude on the issue of nanotechnology regulation in the United States,” says Alves. The FDA, the US agency that controls food and drugs, also deals with nanotechnology applications in public health and has held public consultations since 2011. Alves stresses that, despite the fact that standardization of nanotechnology is at the center of discussions in Grenoble, it is a highly complex task. “We know that the very nature of nanomaterials, characterized by a lack of homogeneity, is a huge obstacle.” In his opinion, these difficulties will be overcome not only by developing new methods for manufacturing nanostructured materials and new equipment, but also through new analysis and tracking procedures. This will allow us to obtain the validations needed for standardization.

National Institute of Science, Technology and Innovation in Functional Complex Materials (Inomat) (nº 2008/57867-8); Grant Mechanism Research grant—Thematic Project; Principal investigator Fernando Galembeck (Unicamp/LNNano); Investment R$2,085,423.04 (FAPESP).

Scientific articles
CAMPOS-GARCIA, J. et al. Ecotoxicological effects of carbofuran and oxidised multiwalled carbon nanotubes on the freshwater fish Nile tilapia: Nanotubes enhance pesticide ecotoxicity. Ecotoxicology and Environmental Safety. v. 111, p. 131-7. Jan. 2015
Martinez, D. S. T. et al. Carbon nanotubes enhanced the lead toxicity on the freshwater fish. Journal of Physics: Conference Series. v. 429, n. 012043 Mar. 2013.