Green at the Bench

What are the alternatives? There are several products on the market. Shepard suggests Invitrogen's SYBR Safe—a proprietary DNA stain thought to be less toxic. At 59 cents per minigel, SYBR Safe 10,000x concentrate it is more expensive than ethidium bromide, which runs about 1 cent per minigel.

According to Invitrogen, SYBR Safe is less mutagenic than ethidium bromide, as determined by the Ames test, the industry standard for mutagenicity. It's not classified as a hazardous waste, and it meets the requirements of the Clean Water Act and the National Pollutant Discharge Elimination System (NPDES). It's also excited by visible blue light, which is safer than UV and eliminates the possibility of sample damage by UV radiation.

But because Invitrogen will not release the active ingredients in SYBR Safe, a white paper documenting third-party tests is the primary verification of such claims. "We still treat it as a dangerous compound, and I'm not sure if its safety has been determined, but it's likely less toxic and we have stuck with it," says Bruce Paster, a professor at Forsyth who began using the product after Shepard taught a series of workshops about ethidium bromide alternatives as a graduate student there.

Other alternatives to ethidium bromide include GelRed and GelGreen (Biotium, Hayward, Calif.) and MegaFluor (Euroclone, Siziano, Italy). GelRed and GelGreen can also be excited with a common UV transilluminator, and they're nonmutagenic and noncytotoxic. They can be used as either a precast gel stain or a post gel stain; they are just as sensitive as SYBR Safe and may be even more durable, as some users say they degrade less quickly. Products like CarolinaBLU DNA Stain and the generic Nile Blue stain allow DNA to be visible while it runs through the gel, and BioRad's Fast Blast is a good alternative for schools and small labs. All of these products are less mutagenic than ethidium bromide, and depending on your building's waste disposal system, they can often be dumped down the drain, so check with your environmental health and safety team.

Lab job: Used when working with histology samples to wash away residual stains and dyes.

Just how bad for your health is it? Xylene ($20 a gallon) is a volatile organic compound, easily introduced into the air lab workers breath. Exposure can cause headaches, muscle coordination problems, confusion, and memory difficulties, as well as eye, nose, and throat irritation. Both the Environmental Protection Agency (EPA) and the International Agency for Research on Cancer (IARC) believe there is too little information to say whether the chemical causes cancer. Some studies, however, have reported harm to the liver and kidneys.

What are the alternatives? It's not uncommon for a lab to burn through 800 gallons of xylene in a single year. According to Catherine Zimmer of the University of Minnesota Technical Assistance Program, AmeriClear by Stephens Scientific ($70 per gallon) is one of the most popular alternatives. "It's non-flammable, less toxic, and can be easily swapped into the typical process," she says.

Some critics, however, say xylene alternatives make samples more brittle, and also raise their own safety concerns. AmeriClear and its sister product Histoclear ($52.80 per gallon) contain D-limonene, a chemical that causes skin irritation and a wide range of allergic reactions. "These products may be safer, but they are not completely safe," says René Buesa, emeritus lab manager at Mount Sinai Medical Center in Miami, Fla.

The active ingredients in other xylene alternatives are considered proprietary. For example, some are based on alkane, a class of saturated hydrocarbons including kerosene and other flammable petroleum products, but many manufacturers don't release the specific alkane they use. "This makes it hard to verify safety claims," says Buesa. "I won't use something unless I know for sure what it is. This is like playing Russian Roulette."

Perhaps the safest and most environmentally friendly alternative to xylene is simple mineral oil—the primary ingredient in baby lotions (purified Penreco's Drakeol—$9.00 a gallon in 55-gallon drums). Buesa recently compared the safety and quality of more than 57 different xylene alternatives (Ann Diagn Pathol, EPUB, Feb 5, 2009), and found that nontoxic mineral oil offers a brighter image and higher-quality sample preparation. A mixture of isopropanol and mineral oil must be prepared first, but the overall staining process is otherwise uninterrupted.

Xylene can also be recycled in a distiller, which reduces the amount shipped off as toxic waste. "It won't minimize the amount of exposure for employees, but recycling is better for the environment," says Zimmer.

Lab job: A heavy metal found in trace amounts in a surprising span of lab reagents, from acetic acid and ethanol to antibodies and enzyme preparations. One source is Thimerosal, an organomercury compound used as a preservative in antibody preparations, vaccines, and other reagents. It's usually added during the manufacturing process—most often as an antiseptic or antifungal agent—so researchers often don't know it's there. "It may not be required that thimerosal is listed, and when it is [named on the ingredients list], many researchers don't recognize it as a mercury- containing chemical," says Jamie Harvie, mercury coordinator for the Arlington, Va.– based nonprofit Health Care Without Harm.

Mercury also creeps into lab reagents during the sodium hydroxide manufacturing process, when electrodes that contain mercury are used to run current through salt water. The sodium is then used to make other chemical ingredients, but it often contains trace mercury that might not be listed on the label. "[Companies] are transitioning to mercury-free processes," says Harvie, "but it's hard to know which [approach] a manufacturer uses."

Just how bad for your health is it? Exposure to mercury can cause nervous disorders, severe kidney damage, and acute poisoning, but the low levels found in lab chemicals are more likely to impact the environment. "Mercury is very bad for fish and wildlife," says Shepard. "This is why products that contain mercury should be treated as hazardous waste, and not sent down the drain."

What are the alternatives? Since reading the list of ingredients won't necessarily help researchers avoid mercury, Harvie recommends taking a more active approach. "Ask vendors for a certificate of analysis that indicates the concentration of mercury in a product, and what tests are used to determine this," he says. Universities can also add contaminant transparency clauses to their purchasing contracts. "This will help prevent research facilities from getting docked for having mercury in their water, when they are simply product end-users," he says. Lists of select mercury-free products can be found on the University of Minnesota Technical Assistance website (http://www.mntap.umn.edu), and at http://www.sustainablehospitals.org.

Lab job: A mixture of water, formaldehyde, and sometimes methanol used to crosslink proteins, allowing sections of tissue to be sliced and placed on a microscope slide.

Just how bad for your health is it? Formaldehyde is highly toxic and can cause acute renal failure and respiratory problems. It's classified as a probable human carcinogen.

What are the alternatives? While no replacement chemicals are currently on the market, many labs reuse formalin. Finely porous filters get rid of residue after use, and formalin can be run through a distiller that removes impurities through boiling and condensation. Companies such as CBG Biotech (Columbus, Ohio) sell devices that purify multiple reagents, allowing formalin, xylene, and alcohols to be recycled ($20,000–$25,000 per recycler). "It was hard to recycle formalin 10 years ago, but the technology has now improved significantly," says Zimmer.

Yet formalin recycling is not without its critics. Recycling and distillation may reduce the environmental footprint, but it increases exposure for the average worker, says Buesa. Even though Buesa worked tirelessly to find an alternative for xylene, he opted against recycling formalin. "The more you handle formalin, the more likely you are to be exposed," he says. "Even if recycling reduces waste and saves money, you can't put a price on the health of the worker."

Instead, Buesa encourages labs to make sure they dispose of formalin according to toxic waste guidelines. Not all labs consider formalin to be a toxic waste, and as little as 20 years ago formalin was considered safe enough to dump down the drain. "Luckily, more facilities are being prohibited from sending toxic things like formalin to the sewer," says Zimmer. "This is a trend I have seen."

Search MIT's Green Alternatives Wizard for reagent alternatives (MIT's Green Alternatives Wizard).

• Bio-Rad offers nonhazardous Bio-safe Coomassie stain ($49.00), which does not require the use of methanol and acetic acid for destaining.

• Use cadmium- and heavy metal–free biohazard bags (available from Bio-Elite, Stericycle, and Tyco).

• Buy centrifuge and other tubes in Styrofoam- free packing bags; reuse pipette tip racks by autoclaving and refilling, or buy tips packaged in paper boxes (Sorenson Bio, $596 for case of five 10mL, 200mL, and 1000mL tubes).

• Step outside your purchasing contracts and get your buyers on board for green office supplies at outlets such as http://www. thegreenoffice.com/, http://greenearthofficesupply.stores.yahoo.net/index.html, and http://www.sustainablegroup.net/.