news
Measuring estrogen conjugates in sewage extrapolated to determine the amount of estrogen conjugates present. According to Brownawell, the measurements can be questionable. The investigators used HPLC/ESIMS/MS to directly measure steroid conjugates. A major problem was ion suppression by the matrix. The researchers overcame the problem by extensively purifying the samples by solid-
OH PALM BEACH COUNTY REEF RESCUE
Endocrine-disrupting compounds wreak havoc in the environment. Detecting these compounds is a challenge because they are usually present at trace levels in complex mixtures. In this issue of Analytical Chemistry (pp 7032–7038), Sharanya Reddy, Charles Iden, and Bruce Brownawell at Stony Brook University have developed a method that measures minute quantities of estrogen conjugates in sewage. Naturally occurring estrogens and ethynylestradiol, the synthetic estrogen found in oral contraceptives, are some of the more potent endocrine-disrupting compounds discharged into the environment. They enter the aquatic environment in the form of the more hydrophilic glucuronide and sulfate conjugates via the urine of humans and animals. Although the conjugates aren’t biologically active, research suggests that they are converted back to their estrogenic forms in sewage. The biologically active estrogens can interfere with the reproductive development of aquatic wildlife and cause the feminization of male and sexually immature fish. Because of their potency, there is a push to understand how these conjugates of estrogen and other steroids are distributed throughout the environment. But analyzing these steroid conjugates is easier said than done. The difficulty in quantifying the estrogen conjugates, as Brownawell explains, is that “you’re looking at very trace levels, on the order of parts per trillion and below, that are environmentally significant. The challenge is [that] these [conjugates] are relatively polar compounds in a sea of naturally occurring organic matter that’s also similarly polar.” A few groups have recently reported methods to directly measure estrogen conjugates in wastewater samples. But the majority of the quantitations have been done indirectly: The amount of free estrogen in a sample before and after hydrolysis is determined and then
HO Estrogen conjugates in sewage can release estrogens into aquatic environments, where they can disrupt the reproductive development of fish.
phase extraction and anion-exchange chromatography. The investigators also added known amounts of stable isotope standards to the samples during the purification procedure. To account for matrix suppression effects during ESI and the amount of sample lost during the experiment, they tracked isotope-labeled standards that were spiked into the samples. Further, the researchers used acid to preserve their stored samples. “One thing that set us back in the study is that we were losing our isotope standards when we spiked them in the field,” says Brownawell. Bacteria present in feces secrete glucuronidases. In sewage, where urine and feces are mixed, the bacterial enzymes degrade the steroid glucuronides in urine, confounding the measurement of steroid compounds. Addition of bacterial poisons like sodium azide, mercury, or formaldehyde didn’t prevent the breakdown of the es-
trogen glucuronides, because “presumably the bacteria had already secreted glucuronidase,” explains Brownawell. “The standing stock of [glucuronidase] was at high enough levels that it was hydrolyzing our samples. We were [still] seeing generation of free estrogen with [the addition of] those poisons but not when we acidified [the samples].” The investigators found that the concentrations of the estrogen sulfates were ~100 greater than those of estrogen glucuronides in sewage influents. The quantitation demonstrated that the sulfate conjugates were less labile than the glucuronide conjugates. A small amount of the estrogen sulfates was detectable in sewage effluents, even after biological treatment in the plant. The finding suggests that the sulfates can persist after cleanup procedures and be released into the environment, where the sulfates can act as a source of free estrogens. Although the investigators’ analytical method is similar to those reported recently by other groups (Chromatographia 2002, 56, 25–32; Sci. Total. Environ. 2003, 302, 199–209), they improved the detection limits for the estrogen glucuronides with the acid preservation of the samples. Their method also allowed a larger volume of sample to be processed, which in turn permitted better sensitivity by ESI-MS/MS. The results from the work could have implications for sewage treatment. “An interesting speculation is that with the type of sewage systems we mostly have in the developed world, where we combine feces and urine, these glucuronides don’t last very long. But the Europeans have been working on approaches of separating urine streams from feces. I think it’s interesting to speculate that you might get greater persistence and release [of steroid conjugates] into the environment if urine isn’t commingled with intestinal bacteria,” says Brownawell. a —Rajendrani Mukhopadhyay
N O V E M B E R 1 , 2 0 0 5 / A N A LY T I C A L C H E M I S T R Y
411 A
