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Bottled Water Quality Investigation : Methodology

October 15, 2008


The University of Iowa’s Hygienic Laboratory (UHL) analyzed samples of ten popular brands of bottled water for a broad array of contaminants ranging from chemicals regulated in tap water to chemicals that may have leached from the plastic bottles themselves. The University of Missouri also conducted bioassays on samples of the same brands in order to identify any estrogenic activity. The information below describes the components of this study, detailing the water sampling procedures, the analytical methods, and the quality assurance and quality control provisions included in the study design.

Bottled Water sample acquisition

Environmental Working Group (EWG) acquired samples of ten brands of bottled water from grocery stores and other retailers in 8 states and the District of Columbia. For each of the ten brands, EWG shipped 24 liters (or 6 gallons) to the University Hygienic Laboratory (Iowa City, IA) and 0.5 liters to the University of Missouri (Columbia, MO).

Analysis of Estrogenic Activity

The University of Missouri performed an estrogen-dependent cell proliferation assay in a 96-well robotic format to determine if there was any estrogenic activity associated with any of the bottled water samples. Using the Tomtec robotics unit, the laboratory seeded MCF-7 cells in 96-well plates and allowed them to adapt for 3 days in culture. Then, the cells were incubated for 4 days with extracts from water samples as well as a serial dilution of estrogen standards (i.e., bisphenol A). At the end of the 4-day exposure, the medium was removed and assayed for DNA, which was used to calculate the number of cells present.

Analysis of Pharmaceuticals, Antibiotics, Ingredients of Personal Care Products (LC/MS/MS)

UHL developed a method for analyzing pharmaceuticals, antibiotics and common personal care product ingredients used for this study. Samples were first prepared in accordance with UHL's PHARMA LC-1 methodology. Following this, as detailed by the testing method, ammonium carbonate was added to 100 mL samples of each brand of bottled water to increase their pH. The samples were then passed through a preconditioned Water Oasis HLB SPE column to extract the test analytes. Ethyl acetate was used to wash the analytes from the SPE column; this solvent was later removed from the samples. 0.1 mL of acetonitrile was then added to the samples, which were analyzed using LC/MS/MS.

Analysis of Fluoride (SM 4500-F C)

For each brand, a 4 mL sample was prepared and tested for fluoride in accordance with the standard methodology for testing water and wastewater (Standard Method SM 4500-F C). A buffer was added to the samples and concentration measurements were conducted using a fluoride electrode and a standard reference electrode.

Analysis of Ammonia (SM 4500-NH3 B, LAC10-107-06-1J)

For the analysis of ammonia, UHL scientists prepared 100 mL samples from each brand of bottled water using standard methodology SM 4500-NH3 B. They then determined the ammonia concentration in the samples using method LAC10-107-06-1J. In accordance with this methodology, alkaline phenol and sodium hypochlorite were added to the samples, and sodium nitroprusside (nitroferricyanide) was added to the mixture to increase detection sensitivity. The absorbance of the reaction product was measured at 630 nm. This absorbance is directly proportional to the original ammonia concentration in the sample.

Analysis of Volatile Contaminants (GC/MS, EPA 524.2)

UHL prepared and tested bottled water samples for volatile contaminants in accordance with the Environmental Protection Agency’s methodology (EPA 524.2). The target contaminants included six chemicals detected in this study - four different trihalomethanes, acetaldehyde and toluene. Following sample preparation, inert gas was bubbled through 25 mL samples, trapping volatile organic compounds (VOCs) with low water solubility in a tube containing suitable sorbent materials. Once the purging process was complete, the tube was heated and backflushed with helium to desorb the trapped compounds into a capillary gas chromatography (GC) column. The contaminant concentrations were measured from the trap using GC/MS. The laboratory also utilized the GC/MS to identify other non-target compounds that were present in the samples.

Analysis of Nitrate (EPA 300.0)

Using EPA methodology (EPA 300.0), the laboratory prepared and tested water samples for nitrate. In accordance with the methodology, a 100 uL aliquot of the samples were introduced into an Ion Chromatograph device. The ions of interest were separated and their concentrations measured using a system comprised of a guard column, a separator column, a suppressor device and a conductivity detector.

Analysis of Haloacetic Acids (EPA 552.2)

Using EPA methodology (EPA 552.2), the laboratory prepared and tested 40 mL samples of each of the bottled water brands for haloacetic acids (HAAs). The samples were acidified to a pH of less than 0.5 with concentrated sulfuric acid. The acidified samples were extracted with 4 mL of methyl-tert-butyl ether (MTBE). Acidic methanol was then added and the samples were slightly heated. The samples were neutralized by back extraction with a saturated solution of sodium bicarbonate, and a capillary gas chromatography system equipped with an electron capture detector (ECD) was used to identify and measure the target analytes.

Analysis of Extractable Contaminants (GC/MS, EPA 525.2)

An EPA-approved methodology (EPA 525.2) was used to analyze the bottled water for extractable contaminants. Organic analytes were extracted from 1 L samples of water with a liquid-solid extraction (LSE) method, in which the samples were passed through a cartridge coated with a solid matrix containing a chemically bonded C18 organic phase. The organic compounds were extracted from the LSE cartridge by washing it with small quantities of ethyl acetate, followed by methylene chloride. The extract was dried and concentrated to a volume of 1 mL. Analysts then separated, measured and identified chemical contaminants using GC/MS.

Analysis of Heterotrophic Plate Count (HPC) (SM 9215B)

UHL prepared and tested the bottled water samples for heterotrophic bacteria using standard methodology SM 9215B. In accordance with this methodology, 1 mL of each sample was diluted with phosphate buffer and pipetted onto heterotrophic plate count media which was then incubated at 35 degrees Celsius for 48 hours. At the end of the incubation period, the number of bacterial colonies formed was noted.

Analysis of Total Coliform (MMO-MUG, SM 9223)

UHL used standard methodology SM 9223 to analyze the bottled water samples for total coliform. Colilert reagent was added to 100 mL of each sample and the mixture was incubated at 35 degrees Celsius for 24 hours. After incubation, the samples were examined for a color change and total coliform was recorded as most probably number (MPN).

Procedures for quality assurance and quality control (QA/QC)

UHL conducted all analyses in accordance with their stringent QA/QC program. For all methods, they ran an initial calibration with pre-set laboratory sample matrices, and also performed continuing calibrations during sample runs. Each analysis included a method blank and a control sample with defined concentration, as appropriate for each specific methodology.