Credit: Courtesy of Jason Biggs

Christopher Reddy vividly remembers the first time he heard about a perplexing organohalogen compound found by researchers in seabird eggs along the Pacific and Atlantic coasts, as well as in the Great Lakes region. He was sitting in the audience at an American Chemical Society meeting in the late 1990s listening to Sheryl Tittlemier, then a graduate student at Carleton University, present her findings.

Structurally, the halogenated bipyrrole discovered by the Carleton team resembled polychlorinated biphenyls (PCBs), a class of synthetic molecules used as coolants, stabilizers, insulators, and flame retardants whose production was banned in the U.S. in the late 1970s because of concerns over their toxicity and persistence in the environment. But there was no record of this particular compound having ever been synthetically produced. Tittlemier, now a scientist at the Canadian Grain Commission, said she suspected the compound might be a natural product (Environ. Sci. Technol. 1999, DOI: 10.1021/es980646f).

Tittlemier’s supposition wasn’t far-fetched: Decades earlier, scientists had discovered a surprising variety of organohalogen compounds in the environment, produced by bacteria, fungi, plants, marine organisms such as the acorn worm, and more. Some resembled notorious organic pollutants made by humans, including the pesticide DDT, halogenated dioxins, and brominated flame retardants.

When scientists find molecules like these in the environment, though, it’s not always easy to tell whether they are natural or synthetic in origin. Reddy, then a postdoc at Woods Hole Oceanographic Institution (WHOI), immediately thought he could help Tittlemier figure out the compound’s origin using radiocarbon dating techniques in his lab. If the compound was synthetic, it would have been made from petrochemical building blocks formed on Earth hundreds of millions of years ago. Any of the radioactive carbon-14 that was once in the confounding molecule would have long ago decayed. In contrast, if the compound was produced by an organism in the environment recently, it would still contain a certain amount of the isotope. Reddy approached Tittlemier after her presentation, and the two agreed to collaborate. They eventually determined that the compound—1,1´-dimethyl-3,3´,4,4´-tetrabromo-5,5´-dichloro-2,2´-bipyrrole—was indeed naturally produced (Environ. Sci. Technol. 2004, DOI: 10.1021/es030568i).

The molecule joined the list of thousands of other natural organohalogens that scientists had identified, a list that today is approaching 6,000 compounds, according to Dartmouth College’s Gordon W. Gribble, author of “Naturally Occurring Organohalogen Compounds: A Comprehensive Update.”

“You could call them naturally produced persistent organic pollutants,” says Reddy, now a marine chemist at WHOI. There’s a public perception that humans have produced more halogenated compounds than nature has, he says. “That’s not necessarily true.”

Like their synthetic counterparts, natural organohalogens bioaccumulate in food webs, especially marine ones, where they are sometimes much more abundant than synthetic pollutants. For example, Gribble says, 3.5 million tons of chloromethane are produced naturally per year versus the 10,000 tons produced industrially per year.

And evidence suggests that people eating a marine diet may be exposed to some natural organohalogens. These include molecules structured similarly to brominated flame retardants and a PCB-like natural product called Q1 that was found in the breast milk of several women in the Faroe Islands who regularly ate whale blubber. Although the toxicity of these compounds is still unknown, some researchers worry that it may be similar to that of the human-made pollutants they resemble. Unlike those pollutants, these natural compounds can’t be banned. So scientists are now using genetic and other analytical techniques to figure out what organisms are making the natural products, how and why they do it, and what risk the molecules might pose.

Marine sponges are one of the most abundant sources of natural organohalogens in the ocean, as the late D. John Faulkner, natural products chemist at Scripps Institution of Oceanography, discovered decades ago. In benthic filter-feeding sponges of the family Dysideidae, a staggering 10% or more of the dry weight of the organism is made of polybrominated compounds. Recently, scientists made a major breakthrough in understanding how these compounds are made—and it turns out, the sponges aren’t the ones doing the synthesizing.

“These compounds are like a flame retardant with very subtle differences,” including additional hydroxyl or methoxyl groups, says Vinayak Agarwal of Georgia Institute of Technology, who began studying the compounds while working as a postdoc with Bradley Moore at Scripps.

The flame retardants these compounds resemble, polybrominated diphenyl ethers (PBDEs), were first made commercially in the 1970s, replacing PCBs for fire prevention. But they also turned out to be toxic to humans and some wildlife. “There is a substantial amount of research showing that human-made PBDE flame retardants may contribute to cancer or disrupt normal hormone function, lowering birth weight or disrupting neurological development,” says Frederick Tyson, a scientific program director at the National Institute of Environmental Health Sciences (NIEHS). The manufacturer of two major classes of these compounds voluntarily phased out U.S. production in 2004; the European Union has also banned these PBDE classes.

Agarwal says that when he first found out about the natural analogs of PBDEs in sponges, “it was baffling to me that nature would synthesize such a molecule.” He wondered, was the sponge itself or something associated with it making the compounds?

Faulkner and other researchers had suspected that a filamentous cyanobacteria living in the sponges made the compounds, because the molecules abounded in the areas of the sponge occupied by the microbes. In fact, a few years ago, Agarwal and colleagues showed that other, free-living bacteria in the ocean produce similar brominated organochemicals. But the symbiotic bacteria in the sponge, Hormoscilla spongeliae, could not be cultured in the lab, so it was not clear if the microbes were making the molecules.

To solve this puzzle, Agarwal and colleagues used a technique called marine metagenomics, which involves screening the DNA of one or many organisms and their associated microbiomes—the fungi, bacteria, and other microbes living with them—for certain genetic signatures and then using analytical tricks to trace the genes back to their owners.

The team harvested Dysideidae sponges in Guam and ground them up to extract DNA from the marine creatures and their microbiomes. Then the researchers sequenced the entire mix of DNA and searched for genes they thought could be involved in the biosynthesis of these compounds. They had identified several of these biosynthetic genes in the free-living bacteria, so they started by looking for homologs of the genes.

After a great deal of screening, they found a handful of enzymes in the mix that are similar to those in the free-living bacteria. Finally, they confirmed in several ways that the enzymes came from Hormoscilla spongeliae bacteria. One way was by sequencing the 16S rRNA genes in the sample; these genes are like an ID badge for different types of bacteria (Nat. Chem. Biol. 2017, DOI: 10.1038/nchembio.2330).

Agarwal, and many other scientists studying naturally produced organohalogen compounds, think bacteria make them for self-defense. In the ocean, Agarwal explains, “there’s a chemical war going on.” Bacteria use chemicals to protect themselves from threats and to taste bad to predators. Intriguingly, the compounds may also play a structural role for the sponges because they make up the long, needlelike structures that form sponges’ internal skeleton.

“This work is absolutely critical,” WHOI’s Reddy says, because it reveals some of the skill sets these microbes used to evolve. Nature was making a chemical deterrent to predators way before Monsanto, Dow, and other companies began making organohalogen pesticides, he says. “It’s interesting we came to the same conclusions about halogen compounds and potency.”

Agarwal’s findings may also offer insights into how other microbes evolved the ability to break down synthetic pollutants in the environment, Reddy says, pointing to PCBs found in the Hudson River downstream of two General Electric plants that released them from the 1940s through the late 1970s. The area is now a Superfund site where some naturally occurring microbes effectively remediate the pollutants, Reddy says. “Where did they learn to break them down? I don’t think that the genes evolved over 30 to 40 years.”

Now that Agarwal knows the signature of some genes involved in the biosynthesis of these naturally produced PBDE-like compounds, he and his team can more easily search for other sources of them in the ocean. So far, they have found other cyanobacteria carrying similar genes, including one associated with seagrass and another with a marine snail.

His team plans to amp up the quest with an upcoming metagenomics study that will survey many different parts of the ocean to identify hot spots where the compounds are produced and reveal how the molecules bioaccumulate in the ocean food web.

Scientists have found some of the hydroxylated PBDEs Agarwal studies in humans. For example, researchers found 6-OH-BDE-47 in the blood serum of pregnant women in South Korea and in the umbilical cord blood serum of their fetuses at average levels of about 18 and 30 pg/g of serum, respectively (Environ. Sci. Technol. 2010, DOI: 10.1021/es1002764). The U.S. Environmental Protection Agency has set a safe daily exposure level for the structurally similar synthetic PBDE compound BDE‑47 at 0.1 µg/kg body weight. Blood serum levels probably underestimate whole body levels because the compound may also be in other tissues, says toxicologist Margaret James of the University of Florida. But to give a sense of the relative magnitude of these values, the levels in the study are equivalent to a 65-kg woman having about 0.1 µg of the compound in all the blood in her body, compared with an EPA safe daily exposure level for this woman of 6.5 µg in her whole body.

Other naturally produced organohalogen compounds have also appeared in pet food and pets (Environ. Sci. Technol. 2017, DOI: 10.1021/acs.est.7b01009). In both cases, the researchers think the concentrations found in pet food and seafood indicate that the exposure may be linked with a seafood-based diet.

To complicate matters, synthetic PBDEs sometimes become hydroxylated when they are metabolized by humans and animals. So it’s often not clear if the compounds represent natural products or metabolized versions of synthetic ones. And researchers are only beginning to learn how toxic the naturally occurring compounds are, at what levels people might encounter them, and their potential impact on human health, NIEHS’s Tyson says.

The toxicity of natural, hydroxylated PBDEs would likely be comparable to that of the synthetic ones, says James, who is collaborating with Agarwal to examine these issues. Structurally similar synthetic compounds like the antibacterial agent triclosan, a hydroxylated chlorinated diphenyl ether, can interfere with sulfotransferase enzymes that are important in ensuring balanced levels of thyroid hormone, estrogens, and dopamine in the human body, James says. “Initially we want to look at the natural compounds’ effects on activity of the enzymes and determine how potent the molecules are,” she says.

Although the sponges harbor molecules that resemble toxic synthetic chemicals, they may also offer a bright side. Agarwal is screening their diverse microbiomes for molecules that may benefit human health, including potential anticancer agents.