Tag Archives: zebrafish

Smaller (20nm vs 110nm) silver nanoparticles are more likely to absorbed by fish

An Oct. 8, 2015 news item on Nanowerk offers some context for why researchers at the University of California at Los Angeles (UCLA) are studying silver nanoparticles and their entry into the water system,

More than 2,000 consumer products today contain nanoparticles — particles so small that they are measured in billionths of a meter.

Manufacturers use nanoparticles to help sunscreen work better against the sun’s rays and to make athletic apparel better at wicking moisture away from the body, among many other purposes.

Of those products, 462 — ranging from toothpaste to yoga mats — contain nanoparticles made from silver, which are used for their ability to kill bacteria. But that benefit might be coming at a cost to the environment. In many cases, simply using the products as intended causes silver nanoparticles to wind up in rivers and other bodies of water, where they can be ingested by fish and interact with other marine life.

For scientists, a key question has been to what extent organisms retain those particles and what effects they might have.

I’d like to know where they got those numbers “… 2,000 consumer products …” and “… 462 — ranging from toothpaste to yoga mats — contain nanoparticles made from silver… .”

Getting back to the research, an Oct. 7, 2015 UCLA news release, which originated the news item, describes the work in more detail,

A new study by the University of California Center for Environmental Implications of Nanotechnology has found that smaller silver nanoparticles were more likely to enter fish’s bodies, and that they persisted longer than larger silver nanoparticles or fluid silver nitrate. The study, published online in the journal ACS Nano, was led by UCLA postdoctoral scholars Olivia Osborne and Sijie Lin, and Andre Nel, director of UCLA’s Center for Environmental Implications of Nanotechnology and associate director of the California NanoSystems Institute at UCLA.

Nel said that although it is not yet known whether silver nanoparticles are harmful, the research team wanted to first identify whether they were even being absorbed by fish. CEIN, which is funded by the National Science Foundation, is focused on studying the effects of nanotechnology on the environment.

In the study, researchers placed zebrafish in water that contained fluid silver nitrate and two sizes of silver nanoparticles — some measuring 20 nanometers in diameter and others 110 nanometers. Although the difference in size between these two particles is so minute that it can only be seen using high-powered transmission electron microscopes, the researchers found that the two sizes of particles affected the fish very differently.

The researchers used zebrafish in the study because they have some genetic similarities to humans, their embryos and larvae are transparent (which makes them easier to observe). In addition, they tend to absorb chemicals and other substances from water.

Osborne said the team focused its research on the fish’s gills and intestines because they are the organs most susceptible to silver exposure.

“The gills showed a significantly higher silver content for the 20-nanometer than the 110-nanometer particles, while the values were more similar in the intestines,” she said, adding that both sizes of the silver particles were retained in the intestines even after the fish spent seven days in clean water. “The most interesting revelation was that the difference in size of only 90 nanometers made such a striking difference in the particles’ demeanor in the gills and intestines.”

The experiment was one of the most comprehensive in vivo studies to date on silver nanoparticles, as well as the first to compare silver nanoparticle toxicity by extent of organ penetration and duration with different-sized particles, and the first to demonstrate a mechanism for the differences.

Osborne said the results seem to indicate that smaller particles penetrated deeper into the fishes’ organs and stayed there longer because they dissolve faster than the larger particles and are more readily absorbed by the fish.

Lin said the results indicate that companies using silver nanoparticles have to strike a balance that recognizes their benefits and their potential as a pollutant. Using slightly larger nanoparticles might help make them somewhat safer, for example, but it also might make the products in which they’re used less effective.

He added that data from the study could be translated to understand how other nanoparticles could be used in more environmentally sustainable ways.

Nel said the team’s next step is to determine whether silver particles are potentially harmful. “Our research will continue in earnest to determine what the long-term effects of this exposure can be,” he said.

Here’s an image illustrating the findings,

Courtesy ACS Nano

Courtesy ACS Nano

Here’s a link to and a citation for the paper,

Organ-Specific and Size-Dependent Ag Nanoparticle Toxicity in Gills and Intestines of Adult Zebrafish by Olivia J. Osborne, Sijie Lin, Chong Hyun Chang, Zhaoxia Ji, Xuechen Yu, Xiang Wang, Shuo Lin, Tian Xia, and André E. Nel. ACS Nano, Article ASAP DOI: 10.1021/acsnano.5b04583 Publication Date (Web): September 1, 2015

Copyright © 2015 American Chemical Society

This paper is behind a paywall.

Copper nanoparticles, toxicity research, colons, zebrafish, and septic tanks

Alicia Taylor, a graduate student at UC Riverside, surrounded by buckets of effluent from the septic tank system she used for her research. Courtesy: University of California at Riverside

Alicia Taylor, a graduate student at UC Riverside, surrounded by buckets of effluent from the septic tank system she used for her research. Courtesy: University of California at Riverside

Those buckets of efflluent are strangely compelling. I think it’s the abundance of orange. More seriously, a March 2, 2015 news item on Nanowerk poses a question about copper nanoparticles,

What do a human colon, septic tank, copper nanoparticles and zebrafish have in common?

They were the key components used by researchers at the University of California, Riverside and UCLA [University of California at Los Angeles] to study the impact copper nanoparticles, which are found in everything from paint to cosmetics, have on organisms inadvertently exposed to them.

The researchers found that the copper nanoparticles, when studied outside the septic tank, impacted zebrafish embryo hatching rates at concentrations as low as 0.5 parts per million. However, when the copper nanoparticles were released into the replica septic tank, which included liquids that simulated human digested food and household wastewater, they were not bioavailable and didn’t impact hatching rates.

A March 2, 2015 University of California at Riverside (UCR) news release (also on EurekAlert), which originated the news item, provides more detail about the research,

“The results are encouraging because they show with a properly functioning septic tank we can eliminate the toxicity of these nanoparticles,” said Alicia Taylor, a graduate student working in the lab of Sharon Walker, a professor of chemical and environmental engineering at the University of California, Riverside’s Bourns College of Engineering.

The research comes at a time when products with nanoparticles are increasingly entering the marketplace. While the safety of workers and consumers exposed to nanoparticles has been studied, much less is known about the environmental implications of nanoparticles. The Environmental Protection Agency is currently accessing the possible effects of nanomaterials, including those made of copper, have on human health and ecosystem health.

The UC Riverside and UCLA [University of California at Los Angeles] researchers dosed the septic tank with micro copper and nano copper, which are elemental forms of copper but encompass different sizes and uses in products, and CuPRO, a nano copper-based material used as an antifungal agent to spray agricultural crops and lawns.

While these copper-based materials have beneficial purposes, inadvertent exposure to organisms such as fish or fish embryos has not received sufficient attention because it is difficult to model complicated exposure environments.

The UC Riverside researchers solved that problem by creating a unique experimental system that consists of the replica human colon and a replica two-compartment septic tank, which was originally an acyclic septic tank. The model colon is made of a custom-built 20-inch-long glass tube with a 2-inch diameter with a rubber stopper at both ends and a tube-shaped membrane typically used for dialysis treatments within the glass tube.

To simulate human feeding, 100 milliliters of a 20-ingredient mixture that replicated digested food was pumped into the dialysis tube at 9 a.m., 3 p.m. and 9 p.m. for five-day-long experiments over nine months.

The septic tank was filled with waste from the colon along with synthetic greywater, which is meant to simulate wastewater from sources such as sinks and bathtubs, and the copper nanoparticles. The researchers built a septic tank because 20 to 30 percent of American households rely on them for sewage treatment. Moreover, research has shown up to 40 percent of septic tanks don’t function properly. This is a concern if the copper materials are disrupting the function of the septic system, which would lead to untreated waste entering the soil and groundwater.

Once the primary chamber of the septic system was full, liquid began to enter the second chamber. Once a week, the effluent was drained from the secondary chamber and it was placed into sealed five-gallon containers. The effluent was then used in combination with zebrafish embryos in a high content screening process using multiwall plates to access hatching rates.

The remaining effluent has been saved and sits in 30 five-gallon buckets in a closet at UC Riverside because some collaborators have requested samples of the liquid for their experiments.

Here’s a link to and a citation for the paper,

Understanding the Transformation, Speciation, and Hazard Potential of Copper Particles in a Model Septic Tank System Using Zebrafish to Monitor the Effluent* by Sijie Lin, Alicia A. Taylor, Zhaoxia Ji, Chong Hyun Chang, Nichola M. Kinsinger, William Ueng, Sharon L. Walker, and André E. Nel. ACS Nano, 2015, 9 (2), pp 2038–2048 DOI: 10.1021/nn507216f
Publication Date (Web): January 27, 2015

Copyright © 2015 American Chemical Society

This paper is behind a paywall.

* Link added March 10, 2015.

Free the rats, mice, and zebrafish from the labs—replace them with in vitro assays to test nanomaterial toxcicity

The July 9, 2012 Nanowerk Spotlight article by Carl Walkey (of the University of Toronto) focuses on research by Dr. André Nel and his coworkers at the California NanoSystems Institute (CNSI) and the University of California Los Angeles (UCLA) on replacing small animal model testing for nanomaterial toxicity with in vitro assays,

Currently, small animal models are the ‘gold standard’ for nanomaterial toxicity testing. In a typical assessment, researchers introduce a nanomaterial into a series of laboratory animals, generally rats or mice, or the ‘workhorse’ of toxicity testing – zebrafish (see: “High content screening of zebrafish greatly speeds up nanoparticle hazard assessment”). They then examine where the material accumulates, whether it is excreted or retained in the animal, and the effect it has on tissue and organ function. A detailed understanding often requires dozens of animals and can take many months to complete for a single formulation. The current infrastructure and funding for animal testing is insufficient to support the evaluation of all nanomaterials currently in existence, let alone those that will be developed in the near future. This is creating a growing deficit in our understanding of nanomaterial toxicity, which fuels public apprehension towards nanotechnology.

Dr. André Nel and his coworkers at the California NanoSystems Institute (CNSI) and the University of California Los Angeles (UCLA) are taking a fundamentally different approach to nanomaterial toxicity testing.

Nel believes that, under the right circumstances, resource-intensive animal experiments can be replaced with comparatively simple in vitro assays.  The in vitro assays are not only less costly, but they can also be performed using high throughput (HT) techniques. By using an in vitro HT screening approach, comprehensive toxicological testing of a nanomaterial can be performed in a matter of days. Rapid information gathering will allow stakeholders to make rational, informed decisions about nanomaterials during all phases of the development process, from design to deployment.

I’ve excerpted a brief description of Nel’s approach,

Rather than using in vitro systems as direct substitutes for the in vivo case, Nel is using a mechanistic approach to connect cellular responses to more complex biological responses, attempting to employ mechanisms that are engaged at both levels and reflective of specific nanomaterial properties.

“You need to align what you test at a cellular level with what you want to know at the in vivo” says Nel. “If oxidative stress at the cellular level is a key initiating element, then by screening for this outcome in cells you more are likely to yield something more predictive of the in vivo outcome. We can do a lot of our mechanistic work at an implementation level that allows development of predictive screening assays.”

By measuring many relevant mechanistic responses, and integrating the results, Nel believes that the in vivo behavior of a nanomaterial can be accurately predicted, provided that enough thinking goes into the devising the systems biology approach to safety assessment.

According to Walkey’s article, this approach could result in a ‘reverse’ nanomaterial development process,

Nel’s approach will influence not only the way in which nanomaterial toxicity is assessed, but also the way in which nanomaterials are developed. Currently, nanomaterials are designed to meet the need of a particular application. Toxicity is then evaluated retrospectively. Formulations that exhibit unacceptable toxicity at that point may be abandoned after a significant investment in development. Because Nel’s approach generates toxicity information much faster than traditional techniques, it will be possible to integrate toxicity during the design of a new nanomaterial. The proactive characterization of nanomaterial toxicity will provide feedback during the design process, producing formulations that maximize efficacy and minimize risk.

This is a very interesting article (illustrated with images and peppered with accessibly explanations of the issues) for anyone following the ‘nanomaterial toxicology’ story.

Fish and Chips: Singapore style and Australia style

A*STAR’s Institute of Bioengineering and Nanotechnology (IBN), located in Singapore, has announced a new platform for testing drug applications. From the April 4, 2012 news item on Nanowerk,

A cheaper, faster and more efficient platform for preclinical drug discovery applications has been invented by scientists at the Institute of Bioengineering and Nanotechnology (IBN), the world’s first bioengineering and nanotechnology research institute. Called ‘Fish and Chips’, the novel multi-channel microfluidic perfusion platform can grow and monitor the development of various tissues and organs inside zebrafish embryos for drug toxicity testing. This research, published recently in Lab on a Chip (“Fish and Chips: a microfluidic perfusion platform for monitoring zebrafish development”) …

From the IBN April 4, 2012 media release,

The conventional way of visualizing tissues and organs in embryos is a laborious process, which includes first mounting the embryos in a viscous medium such as gel, and then manually orienting the embryos using fine needles. The embryos also need to be anesthetized to restrict their motion and a drop of saline needs to be continuously applied to prevent the embryos from drying. These additional precautions could further complicate the drug testing results.

The IBN ‘Fish and Chips’ has been designed for dynamic long-term culturing and live imaging of the zebrafish embryos. The microfluidic platform comprises three parts: 1) a row of eight fish tanks, in which the embryos are placed and covered with an oxygen permeable membrane, 2) a fluidic concentration gradient generator to dispense the growth medium and drugs, and 3) eight output channels for the removal of the waste products (see Image 2). The novelty of the ‘Fish and Chips’ lies in its unique diagonal flow architecture, which allows the embryos to be continually submerged in a uniform and consistent flow of growth medium and drugs (…), and the attached gradient generator, which can dispense different concentrations of drugs to eight different embryos at the same time for dose-dependent drug studies.

Professor Hanry Yu, IBN Group Leader, who led the research efforts at IBN, said, “Toxicity is a major cause of drug failures in clinical trials and our novel ‘Fish and Chips’ device can be used as the first step in drug screening during the preclinical phase to complement existing animal models and improve toxicity testing. The design of our platform can also be modified to accommodate more zebrafish embryos, as well as the embryos of other animal models. Our next step will involve investigating cardiotoxicity and hepatoxicity on the chip.”

As a pragmatist I realize that, to date, we have no substitute for testing drugs on animals prior to clinical human trials so this ‘type of platform’ is necessary but it always gives me pause. Just as the relationship between human and animals did the first time I came across a ‘Fish and Chips’ project in the context of a performance at the 2001 Ars Electronica event in Linz, Austria. As I recall Fish and Chips was made up fish neurons grown on silicon chips then hooked up to hardware and software to create a performance both visual and auditory.

Here’s an image of the 2001 Fish and Chips performance at Ars Electronica,

Ars Electronica Festival 2001: Fish & Chips / SymbioticA Research Group, Oron Catts, Ionat Zurr, Guy Ben-Ary

You can find a full size version of the image here on Flickr along with the Creative Commons Licence.

The Fish and Chips performance was developed at SymbioticA (University of Western Australia). From SymbioticA’s Research page,

SymbioticA is a research facility dedicated to artistic inquiry into knowledge and technology in the life sciences.

Our research embodies:

  • identifying and developing new materials and subjects for artistic manipulation
  • researching strategies and implications of presenting living-art in different contexts
  • developing technologies and protocols as artistic tool kits.

Having access to scientific laboratories and tools, SymbioticA is in a unique position to offer these resources for artistic research. Therefore, SymbioticA encourages and favours research projects that involve hands on development of technical skills and the use of scientific tools.

The research undertaken at SymbioticA is speculative in nature. SymbioticA strives to support non-utilitarian, curiosity based and philosophically motivated research.

They list six research areas:

  • Art and biology
  • Art and ecology
  • Bioethics
  • Neuroscience
  • Tissue engineering
  • Sleep science

SymbioticA’s Fish and Chips project has since been retitled MEART, from the SymbioticA Research Group (SARG) page,

Meart – The semi-living artist

The project was originally entitled Fish and Chips and later evolved into MEART – the semi living artist. The project is by the SymbioticA Research group in collaboration with the Potter Lab.

The Potter Lab or Potter Group is located at the Georgia (US) Institute of Technology. Here’s some more information about MEART from the  Potter Group MEART page,

The Semi living artist

Its ‘brain’ of dissociated rat neurons is cultured on an MEA in our lab in Atlanta while the geographically detached ‘body’ lives in Perth. The body itself is a set of pneumatically actuated robotic arms moving pens on a piece of paper …

A camera located above the workspace captures the progress of drawings created by the neurally-controlled movement of the arms. The visual data then instructed stimulation frequencies for the 60 electrodes on the MEA.

The brain and body talk through the internet over TCP/IP in real time providing closed loop communication for a neurally controlled ‘semi-living artist’. We see this as a medium from which to address various scientific, philosophical, and artistic questions.

Getting back to SymbioticA, my most recent mention of them was in a Dec. 28, 2011 posting about Boo Chapple’s (resident at SymbioticA) Transjuicer installation at Dublin’s Science Gallery (I’ve excerpted a portion of an interview with Chapple where she describes what she’s doing),

I’m not sure that Transjuicer is so much about science as it is about belief, the economy of human-animal relations, and the politics of material transformation.

On that note I leave you with these fish and chips (from the Wikipedia essay about the menu item Fish and Chips),

Cod and chips in Horseshoe Bay, B.C., Canada, December 2010. Credit: Robin Miller

Nanomaterials and health: the good, the bad, and the ugly?

One of the things I’ve noticed about the nanomaterials safety debate is how quickly it devolves to:  nanomaterials are good (some media reporters, business and corporate lawyers) vs nanomaterials are bad (some media reporters and civil society groups). Unfortunately, we still don’t know much about nanomaterials and their possible effects on health and the environment but there is enough evidence to support a single position if you’re willing discount evidence that doesn’t support your case. There are even people (pro and con) who will use evidence that doesn’t support their case very well unless they leave out details.

Take for example, this interview with Pat Roy Mooney (executive director of the ETC Group) at the Elevate Festival, October 2009 in Austria. Much of what he has to say is quite right (more work needs to be done to ensure safety) but you might get the impression that all this nanotechnology research that’s been talked about has resulted only in consumer products such as sunscreens and cosmetics. At about 4 mins., 15 secs., the reporter challenges Mooney and points out that the research may be very helpful in cleaning water (vital in some areas of the world) and could have other benefits. Mooney concedes the point, grudgingly.

Oddly, Mooney spends quite a bit of time suggesting that gold nanoparticles are a problem. That may be  but the more concerning issue is with silver nanoparticles which are used extensively in clothing and which wash off easily. This means silver nanoparticles are ending up in the water supply and in our fish populations. Studies with zebrafish strongly suggest far more problems with silver nanoparticles than gold nanoparticles. You can check this paper (which compares the two nanoparticles), this paper (about silver only) and this paper (about silver only) or run a search.

Mooney goes on to describe problems with other nanomaterials that I’m unfamiliar with, but I don’t know how far I can trust the information he’s giving me.

Mooney isn’t the only one who likes to remove nuance and shading. In a recent interview on the Metropolitan Corporate Counsel website, one of the interview subjects, William S. Rogers, Jr., essentially dismisses concerns about carbon nanotubes with this:

Rogers: Before the EPA announcement in January, 2010 concerning the proposed SNUR, a series of studies was done beginning in the United Kingdom with a study led by Poland, et al. (2008). That study involved the injection of multi-walled nanotubes into the abdomen of mice, the mucosal lining of which is identical to the mesothelium of the pleura or chest. The injection directly into the abdomen was intended to simulate exposure of the mesothelium in the chest due to inhalation exposure. Approximately 90 days later they examined the biological changes who had taken place as a result of exposure of the abdominal mesolthelial lining to the carbon nanotubes. They reportedly found evidence of inflammation that was consistent with the type of inflammation that had traditionally been recognized in people who had inhalation exposure to asbestos fibers and who later developed mesothelioma. They did not find actual mesothelioma in the mice, but rather what were thought to be precursors to such cancers. The result of publication of these findings was an alarmist reaction that carbon nanotubes posed a danger to humans analogous to that of asbestos fibers. This became headline news.

Up to this point I could agree with him, but now Rogers goes on to point out the study’s shortcomings,

The problem with the study was that the mice were exposed to massive doses of nanotubes by injection, which is not a natural or likely cause of human exposure. The test methodologies were a poor analog for what likely human exposure would be in any setting. Many commentators criticized the study’s findings and suggested that its conclusions about a potential relationship between carbon nanotubes and asbestos fibers was flawed because it rested largely on their shape similarity (long and thin); however, for the last two years there has been talk in the popular media about whether the risks associated with all nanomaterials are akin to those associated with asbestos fibers. The only similarities between carbon nanotubes and asbestos fibers is their long aspect ratio, unlike other nanomaterials. There has been more focus on carbon nanotube toxicity than on other nanomaterial substances, which has percolated up to the EPA. EPA has now decided to treat carbon nanotubes separately from other nano-objects.

Rogers fails to mention that this was a pilot study which was intended to lay the basis for further research. Dr. Andrew Maynard, one of the authors of the study, noted in a March 26, 2009 posting on his blog (2020 Science) further work had been done,

I’m looking at an electron microscope image of a carbon nanotube – as I cannot show it here, you’ll have to imagine it. It shows a long, straight, multi-walled carbon nanotube, around 100 nanometers wide and 10 micrometers long. There is nothing particularly unusual about this. What is unusual is that the image also shows a section of the lining of a mouse’s lung. And the nanotube is sticking right through the lining, like a needle through a swatch of felt.

The image was shown at the annual Society of Toxicology meeting in Baltimore last week, and comes from a new study by researchers at the National Institute for Occupational Safety and Health (NIOSH) on the impact of inhaled multi-walled carbon nanotubes on mice. [You can find out more about the NIOSH study here]

It’s highly significant because it takes scientists a step closer to understanding whether carbon nanotubes that look like harmful asbestos fibers, could cause asbestos-like disease…

Both the carbon nanotube studies mentioned here are studies of long, multi-walled carbon nanotubes. This distinction is important as substances at the nanoscale can behave differently from each other depending on their shape and size. Both Maynard and the NIOSH researchers suggest that more study is required but clearly the evidence is mounting.

Interestingly, the Good Nano Guide (GNG)* page on carbon nanotubes mentions the Poland study but not the NIOSH Study. The page also notes that at least one study indicates issues with single-walled and multi-walled carbon nanotubes as well as C60 (fullerenes). I wonder if there’s a policy about including only studies that have been published in peer-reviewed journals.

(*a ‘best practices for nanomaterials’ wiki hosted by the International Council on Nanotechnology ETA (April 12, 2010: From Dr. Kristen Kulinowski, “As to your question about our policy for posting information at the GNG, there is no policy that states we only publish peer-reviewed papers.” Dr. KK has offered this and  more information about the GNG in the comments.)

The media also are playing a role in this discussion. I’ve noted before Andrew Schneider’s nanotechnology series for AOL News, from his article Obsession with Nanotech Growth Stymies Regulators,

Separately, the NIOSH team discovered that beyond the well-documented lung damage that comes from inhalation of carbon nanotubes, [emphasis mine] those heavily used carbon structures were causing inflammation of the brain in the test animals.

Except for the fact that “well-documented lung damage that comes from inhalation” is an over statement, Schneider’s article is a good read although as I’ve noted elsewhere I don’t know how far to trust his information. [ETA: April 21, 20010, Schneider also fails to note the the type of carbon nanotube (likely the long, multi-walled ones) on which he bases his unsubstantiated claim. ]

After writing all this, I’m torn. On the one hand,  I do think that if people like Schneider and Mooney had their way, none of us would be eating potatoes, tomatoes, or eggplants. After all, they’re members of the nightshade family and the ill effects of ingesting other members of that family, belladonna (deadly nightshade) and datura (jimson weed), are well documented. On the other hand, folks like William Rogers are all too willing dismiss some very troubling research as their clients strive to bring products to market, seemingly regardless of any consequences.

ETA: Happy Weekend!