At the annual American Chemical Society spring meeting in 2010, researchers described how active pharmaceutical ingredients (APIs) contained within medications that are applied topically to the skin may enter wastewater streams—and potentially wind up in drinking water—as a result of bathing and showering.1 These APIs include steroids such as cortisone and testosterone, acne medications, antimicrobials, and narcotics.
This study follows other high-profile reports on the presence of “emerging contaminants” in drinking water. In 2008, a study by The Associated Press2 reported that drugs such as antibiotics, anticonvulsants, mood stabilizers, and cholesterol-lowering medications were found in the drinking water of more than 40 million Americans. A similar study by the U.S. Geological Survey3 found an average of 20 different drugs in the wastewater streams—everything from caffeine to over-the-counter medications such as ibuprofen to rare, but potent, cancer chemotherapy drugs.
In addition to pharmaceuticals, contaminants in drinking water such as perchlorates, pesticides, herbicides, endocrine disrupting chemicals, brominated flame retardants, and personal care products make for a steady stream of news about what is in our water supply.4 While such contaminants can be found in drinking water, should they be a concern for researchers? Are these contaminants making their way from the tap into the high-purity water used in the laboratory?
Emerging contaminants are considered to be substances characterized by a real or perceived threat to human health or the environment, for which there is no published health standard or a standard is currently being developed. These substances can include nanoparticles, pharmaceuticals, personal care products, endocrine disrupting compounds, and chemicals used in products and packaging.
Despite being referred to as “emerging contaminants,” many of these compounds have been in use for decades. Their presence in water is not new. What is new, however, is our ability to measure these contaminants at the very low concentrations at which they exist in our water supply. Analytical laboratories monitoring the presence of such emerging contaminants must ensure their laboratory water is purified to the highest degree possible, so that even minute amounts of contaminant in the purified water do not interfere with trace-level analyses.
Other laboratories that require a similar standard of purity for water are those that develop sensitive methods for the detection of emerging contaminants and their metabolites in various matrices and those that focus on toxicity testing.
Emerging contaminants may also have an impact on cell-based or other biological assays. Effects of these contaminants on experimental outcomes may be so subtle, however, that the cause of unexpected results might not be immediately traced back to the water, leading to wasted time and effort in tracking down the culprit.
The need for vigilance
Figure 1 - A combination of purification technologies removes a wide variety of emerging contaminants that may be present in tap water.
Millipore (Billerica, MA) water purification systems (Figure 1) remove a wide variety of contaminants by combining various purification technologies such as reverse osmosis, electrodeionization, activated carbon, ion-exchange resins, and ultraviolet radiation. Point-of-use (POU) disposable cartridges have been designed to answer the needs of scientists requiring water free of specific contaminants. The company routinely monitors reports of emerging contaminants and evaluates whether they are effectively removed by water purification systems or perhaps require additional purification steps. Two recent examples include perchlorate and endocrine disrupter chemicals (EDCs).
Perchlorate is an emerging contaminant used in explosives, solid rocket fuel, matches, and air bags that is receiving a great deal of attention. Perchlorate is chemically inert under most conditions, and until recently was not considered to be a hazardous substance; as such, the compound was commonly disposed of in wastewater systems. In the late 1990s, however, development of a sensitive method for perchlorate detection in ground and surface water showed widespread contamination. The growing number of environmental laboratories monitoring perchlorate in water and food require perchlorate-free analytical-grade water.
Millipore developed an ion chromatography method to analyze perchlorate at the nanogram-per-liter level in high-purity water and assessed the removal efficiency of various combinations of water purification techniques.5 Because perchlorate was not present in the tap water used to feed the water purification systems, it was added to assess removal efficiencies. A company study showed that reverse osmosis alone removed 97% of the added perchlorate, while ion exchange resins and electrodeionization removed all remaining traces. A water purification system that combines these technologies ensures that high-purity water used in the laboratory is free of perchlorate.
EDCs6 are natural and synthetic substances that alter the function of the endocrine system and consequently cause adverse effects in an organism or its progeny. Substances suspected of being EDCs include organohalogens (chloroform, dioxins), chemicals used in pesticides (DDT), and plastics (bisphenol A, phthalates). Research groups are actively designing sensitive analytical methods to identify and quantify EDCs for which high-purity water is a requirement. In addition to EDCs entering the laboratory water from the tap, the materials used in the water purification system itself may contaminate water. Plastic materials that can leach EDCs include filtration membranes, resin housings, and plastic piping.
Millipore developed a point-of-use cartridge optimized for EDC removal.7 The cartridge, which contains a specific type of activated carbon, is made of materials selected to prevent recontamination of the purified water. GC-MS analysis showed that water purified using a combination of technologies commonly used in Millipore water purification systems with the addition of the specific carbonaceous POU cartridge contained no measurable amounts of EDCs such as phthalates and dioxins.
The possible impact
Awareness of these emerging contaminants—and others that will emerge in the future—must remain high for both developers of laboratory water purification systems and researchers. New contaminants will certainly be identified and may require novel purification techniques, while concentrations of known contaminants that currently do not pose a concern for most laboratories may rise to levels that have a widespread impact on analyses and experimentation.
Posing a further challenge is the increased sensitivity of analytical methods. As these methods become more sensitive, the likelihood also increases that existing contaminants—not just the ones making news—may become detectable and interfere with or confound results. These methods, i.e., HPLC, LC-MS, polymerase chain reaction (PCR), and microarrays, to name a few, all share the requirement for one critical reagent—water. Used as blanks, for dissolution and dilution of samples, dilution of standards, preparation of mobile phases, and media and buffer preparation, water is central to a laboratory's productivity and success.
Few factors affect HPLC analyses more than contaminants in the water used for the mobile phase. While poor water quality is one of the easiest problems to fix, it is one of the least-understood factors in an analytical laboratory. Reports indicate that 70-80% of HPLC performance issues are ultimately attributed to water quality in eluents, samples, and standards.8
Emerging contaminants can be organic molecules or ions. Contamination of water in the mobile phase used in reversed-phase HPLC by organic molecules or ions is known to cause baseline shifts and the appearance of extraneous peaks that can interfere with the spectral identification and quantitation of low-level analytes.
In one study,9 commercially available HPLC-grade bottled water without total oxidizable carbon (TOC) specifications was compared with freshly delivered ultrapure water with a TOC level below 5 ppb. (TOC is a simple measure of organic contamination in water.) When bottled water was used as a mobile phase in HPLC separations, baseline variability and poor chromatographic performance were observed, as compared to the results observed when ultrapure water was used.
Ion chromatography (IC) also requires water free of any contaminants. The presence of ions will result in higher background, which translates to poor sensitivity. Trace analysis of ions is carried out in many laboratories, and this requires water that is free of ions that could compromise the quality of data obtained. In addition, the presence of organic contaminants in water used for eluent preparation could damage the resins in the analytical column, reducing its separation efficiency.
As the sensitivity of analytical techniques keeps improving and new contaminants emerge, there is a constant need for laboratory water to reach new levels of purity. The emergence of new contaminants and the ability to detect existing ones in water at extremely low levels present an ongoing challenge to both scientists and developers of water purification systems. The scientific community must remain mindful of how these contaminants might impact laboratory analyses and the concentration levels that may be cause for concern. Scientists and water system manufacturers must work together to ensure existing purification technologies are effectively removing contaminants that can affect laboratory results and design new strategies as needed.
- Donn, J.; Mendoza, M.; Pritchard, J. AP probe finds drugs in drinking water. The Associated Press, Mar 9, 2008.
- Dove, A. Drugs down the drain. Nature Med.2006, 12, 376-7.
- Fono, L.J.; MacDonald, H.S. Emerging compounds: a concern for water and wastewater utilities. J. Am. Waterworks Assoc. Nov 2008, 100(11), 50-7.
- Castillo, E.; Riche, E.; Kano, I.; Mabic, S. Trace analysis of perchlorate: analytical method and removal efficiency of purification technologies. LC·GC: The Peak2008, 21-9.
- Colborn, T.; Dumanoski, D.; Myers, J.P. Our Stolen Future: Are We Threatening Our Fertility, Intelligence, and Survival? A Scientific Detective Story. Dutton: New York, NY, 1996.
- Riche, E.; Ishii, N.; Mabic, S. Generating high-purity water for endocrine disrupter analysis. The Column July 2006, 14-21.
- Mabic, S.; Regnault, C.; Krol, J. The misunderstood laboratory solvent: reagent water for HPLC. LC·GC North America2005, 23(1), 74-82.
- Tarun, M.; Monferran, C.; Devaux, C.; Mabic, S. Improving chromatographic performance by using freshly delivered ultrapure water in the mobile phase. LC·GC: The Peak June 2009, 7-14.
Dr. Riche is an Applications Support Scientist, and Dr. Tarun is an Applications Scientist, Millipore Corp., 290 Concord Rd., Billerica, MA 01821, U.S.A.; tel.: 978-715-4321; e-mail: Estelle_riche@millipore.com.
Sidebar: Keeping a watch on water
Purified water is one of the most commonly utilized reagents in the
laboratory. It is used throughout experimental protocols in virtually
every type of application. Researchers must remain aware of possible
contaminants present in laboratory water, whether classified as emerging
or not, and how these contaminants may impact their studies.
Genomics and gene expression
The presence of bacteria or bacterial by-products such as nucleases can
also have a significant impact on experiments. When conducting
genomics studies, scientists require nuclease-free water in order to
prevent sample degradation and maintain the integrity of RNA and
DNA. Nucleases in a laboratory's water can interfere with experiments
employing PCR and other methods that measure or manipulate
these nucleic acids.
DNA microarrays are now a ubiquitous tool for measuring the variation or
expression levels of thousands of genes in a single experiment.
The presence of organic compounds and ions in the water can adversely
affect hybridization and interfere with the detection and measurement
of genes using fluorescence.
High-purity water is essential for proteomics studies. It is required
during sample preparation to prevent degradation or contamination of
the sample and to successfully separate and identify proteins using
electrophoresis or chromatography. High-purity water is also needed for
protein or peptide analyses that use mass spectrometry to avoid
contamination of the ionization chambers and minimize interferences in
enzymes that mimic the activity of reagents such as alkaline
phosphatases can interfere with immunoassays. Endotoxins in a laboratory
water supply can disrupt cell growth and function in tissue cultures
and cell-based assays.
A number of compounds found in tap water can impact cell cultures.
Bacterial contamination can cause sudden changes in media pH.
Most Gram-negative bacteria release endotoxin, a complex
lipopolysaccharide that can cause macrophages and mononuclear phagocytes
to release a variety of proinflammatory cytokines. Endotoxins also
affect other types of cells and can lead to changes in growth and
cloning efficiency, and production of recombinant proteins.
Heavy metals such as mercury, lead, zinc, nickel, chromium, and cadmium
are toxic to various cells, including glial and neural cells.
Magnesium inhibits glucose-6-phosphate dehydrogenase and DNA nuclease
II. Manganese causes DNA polymerase to incorporate ribose
instead of deoxyribose into nascent DNA chains.
Small organic molecules are commonly present in raw water supplies
(humic acids, tannins, pesticides, endocrine disrupters, etc.) and may
remain in tap water. These substances are known to affect the
development of cells and should therefore be removed by suitable
during the production of laboratory-grade water.