TO EARLIER GENERATIONS OF FISHERMEN, the notion of horseshoe crabs as a scarce resource would have been lunacy. Through the mid-20th century, the crabs were harvested by the millions from Delaware Bay and stacked onshore to dry, stinking up beaches in the process. The dried carcasses were ground into a fertilizer called cancerine (“derived from crabs”). Science gave the crabs a break when chemical manufacturers commercialized synthetic fertilizer after World War II.

At the same time, however, researchers at the Marine Biological Laboratory in Woods Hole, Massachusetts, were analyzing intriguing properties in horseshoe crab blood.

Scientists had determined in the early 1900s that the crabs’ blood tended to clot quickly in the presence of foreign bacteria. In 1964 hematologist Jack Levin and pathobiologist Frederik Bang described the clotting mechanism and showed it was triggered by endotoxins, a component of the cell membrane of Gram-negative bacteria. When the bacteria are killed or damaged, they release the toxins.

As little as one milligram of intravenous endotoxins can be fatal, so federal regulations require all drugs and medical devices delivered through the skin or implanted in the body, as well as the water and raw materials used to make them, to be tested for bacterial contamination.

In the 1940s drug manufacturers began testing their products for endotoxin contamination by injecting them into live rabbits and waiting four to six hours to see whether the animals developed fevers. Levin and Bang developed an alternative test, the limulus amebocyte lysate (LAL) assay, that used the cells responsible for the clotting mechanism in horseshoe crab blood. (The cells are called amebocytes for their ability to move and change shape, like amoebas.) In their original gel clot method, which is still widely used today, a technician mixes LAL and a test sample, such as an injectable drug, in a test tube and incubates the solution at 37°C for 60 minutes. Then they invert the tube. If the sample contains clinically significant levels of endotoxin, a dense clot sticks to the bottom of the tube. No detectable endotoxin, no clot. More sophisticated methods can indicate endotoxin concentrations.

As studies showed LAL was cheaper and more sensitive than using rabbits, companies formed to manufacture it according to FDA specifications. In 1980 the U.S. Pharmacopeia, an independent scientific organization that sets standards for medicines and other products, published guidelines for bacterial endotoxin testing using LAL. Horseshoe crabs—once piled up to rot—became the centerpiece of a widespread laboratory process. Today as many as 100 million endotoxin tests are conducted worldwide every year.

The impact this shift had on horseshoe crabs wasn’t obvious right away. It’s not easy to estimate population size or trends for a species that spends most of its life crawling across the seafloor like an aquatic Roomba.

Coincidentally, as the crabs’ new biomedical role expanded, fishermen started to target less-widely-sought species, such as conch and eels, and collected more crabs as bait. Yearly horseshoe crab landings along the Atlantic coast, which generally hovered between one and two million pounds through the 1980s, surged to more than six million pounds in the mid-1990s. Egg density on beaches began to decline, reducing food for birds. Counts of red knots stopping at Delaware Bay during migration plummeted, and studies showed the birds were having trouble gaining weight there for the rest of their journey north.

In response to lobbying from conservation groups, federal regulators established yearly state quotas for the bait fishery starting in 2000. LAL producers avoided similar ceilings, but beginning in 2004 they were required to report how many crabs they were using and estimate how many had died from being bled or during transport.

Initially, the number of crabs caught for bait dwarfed the total taken for biomedical use. But as bait fishing quotas were tightened, crab harvest for LAL increased. By 2010 it approached 500,000 crabs per year, compared to some 700,000 crabs fished for bait. Starting in 2020, worldwide demand for COVID-19 vaccines and then Ozempic and other GLP-1 weight loss drugs boosted demand for testing, and thus LAL. As a result, biomedical collection of horseshoe crabs has overtaken bait landings. In 2023 fishermen reported landing 738,789 crabs for bait, compared with 1,113,644 crabs collected solely for bleeding. (Some crabs fished for bait are bled first, then killed.)

Obviously, bait crabs suffer 100% mortality; for biomedical bleeding, the Atlantic States Marine Fisheries Commission estimates about 15% of crabs harvested for blood die, although some studies have documented rates as high as 30%. Other studies of bled crabs have shown so-called sublethal effects—for example, mating less frequently than animals not bled—that can last for several weeks after they are returned to the ocean.

“Bleeding is a stressor. So are exposure to higher temperatures and being kept out of the water,” says Plymouth State University neurobiologist Christopher Chabot, who has coauthored multiple studies examining how biomedical bleeding affects horseshoe crabs. In his view, industry standards probably were less protective a decade ago than they are today. Many crabs likely weren’t kept cool or wet during transport and could well be held out of the water for more than a day.

Chabot estimates that today the lethality of bleeding probably aligns with the commission’s estimates of 15% or less if labs follow current standards. However, he notes that the companies are very secretive about their processes. Assuming a 15% mortality rate, biomedical bleeding would have killed 155,801 crabs in 2023.

Even if LAL production kills only one-fifth as many crabs as bait fishing, there are other reasons to question it—notably, animal welfare. Many consumers might be startled to learn that drug safety testing calls for strapping live horseshoe crabs to “bleeding stations,” inserting 14-gauge needles into their hearts, and draining 30% of their blood supply, although LAL companies stress that the procedure is quick and the crabs tolerate it well.

The Three Rs principles—guidelines developed more than 50 years ago to promote ethical use of animals in science and product testing—advise scientists to (1) replace animals where possible; (2) reduce the number of animals used; and (3) refine methods to minimize pain, suffering, and distress for animals that are used. LAL advanced animal welfare by moving the drug industry away from rabbit testing, in which rabbits were used in multiple injection tests and then killed.

But this shift raised its own ethical and ecological questions, including the effects on both the crabs and species that rely on them for food. Since it became the standard, discussion has centered around improving methods for handling the crabs and reducing the amount of crude LAL required for each assay. That still leaves out the many consumers who choose to avoid health products and cosmetics tested on animals but who have no alternative for injectable drugs other than opting out entirely. Now, though, there’s a real prospect of removing animals from endotoxin testing altogether.

IN 2003 LONZA, a Swiss-based producer, commercialized the first synthetic substitute for LAL. Based on research by biologists Bow Ho and Jeak Ling Ding, a husband-and-wife team at the National University of Singapore, the technology is known as recombinant factor C (rFC) because it uses a cloned version of limulus factor C, the enzyme in amoebocyte cells that initiates the endotoxin clotting reaction. Other scientists later developed another technology, called recombinant cascade reagent (rCR), which simulates the clotting reaction using engineered versions of three horseshoe crab blood proteins.

These advances came amid rising concern for migratory birds, particularly red knots, that relied on horseshoe crab eggs to refuel. In 2005, nine conservation groups petitioned the U.S. Fish and Wildlife Service to list a subspecies of red knots, called the rufa red knot, as endangered. The groups called for immediate action, citing studies that projected the birds could go extinct in less than a decade if trends continued. The authors noted that “the leading known cause of the red knot’s decline—loss of their main food supply at Delaware Bay due to reductions in horseshoe crabs—continues unabated” and demanded action to reduce horseshoe crab harvests.

After years of review, the agency listed red knots as threatened (a step below endangered) in 2014. It subsequently designated nearly 700,000 acres along U.S. coastlines from Massachusetts to Texas as critical habitat for the birds. This step helps protect places that are necessary to conserve an at-risk species, whether the species is currently found in those areas or not. Federal agencies are barred from permitting, funding, or carrying out actions that are likely to destroy or harm these areas.

The red knot’s plight caught the public’s attention. Books and documentaries described the small bird’s epic journey and its tight ecological link with the horseshoe crab. Birders latched onto the issue, among them Jay Bolden, a senior biologist at pharmaceutical giant Eli Lilly. In 2013, when Bolden learned horseshoe crab populations in Asia were also declining, he realized there was a business case as well as a conservation argument for reducing reliance on LAL.

Other drug manufacturers in the United States and Europe were also starting to look at synthetic alternatives, but the process wasn’t simple. The FDA had announced in 2012 that companies could use rFC for endotoxin testing but refused to give a blanket approval; companies had to prove rFC worked as well or better than the LAL assay for each drug and device before switching over. Bolden persuaded executives at Eli Lilly that the company should try to make that case and coauthored a string of peer-reviewed analyses that validated rFC’s effectiveness in multiple products.

“We would not have made this change without data showing that it will protect human health and produce safe medications,” Bolden says. His research found that rFC was both cost-effective and performed more consistently from batch to batch than LAL.

Other key factors in the company’s decision-making included supply chain security and being able to use the same equipment all over the world. “We’re in a better position to deliver high-quality products to our customers without depending on a wild resource,” Bolden says.

IN A LONG-AWAITED STEP, the U.S. Pharmacopeia approved a set of guidelines called Chapter 86 in the summer of 2024 that officially declared rFC and rCR tests effective and safe alternatives to LAL assays. The guidelines went into effect on May 1, 2025, removing the last major barrier to adoption.

To assess industry progress in moving to animal-free test methods, several environmental groups and drug companies developed a scorecard and surveyed 50 major corporations in early 2025. Eleven companies responded, reporting that they had moved anywhere from 15% to 85% of the way toward transitioning off LAL. The scorecard assigned points in five categories, such as ending use of TAL (a reagent derived from endangered Asian horseshoe crabs), reducing use of LAL, and transitioning various categories of products to the new tests.

Reflecting Bolden’s internal advocacy, Eli Lilly led the way at 85%, followed by GSK (formerly GlaxoSmithKline) at 65%. The rest of the group, in descending order of progress, included Amgen, Sanofi, Bristol Myers Squibb, AstraZeneca, Merck, Roche, Novartis, Moderna, and Novo Nordisk.

“We don’t want this to be a shaming exercise—we want to help drug companies remove horseshoe crabs from the supply chain,” including by putting them in touch with each other, says Elizabeth Bennett, communications director at Revive & Restore, a participating conservation group that supports “genetic rescue” of extinct and endangered species using cloning and gene-editing technologies.

Ending use of LAL is an ambitious goal for the heavily regulated pharmaceutical industry, which isn’t typically known for turning on a dime.

“Industry adoption in pharma is a massive undertaking,” Bennett acknowledges. “Reputation management is a very big concern.” In a large corporation, changing a central process in this way can involve many divisions, including R&D teams, process teams, and companies’ environmental, social, and governance programs.

Another factor slowing uptake is more straightforward. Drug safety testing is a lucrative industry. Companies that produce LAL have made major investments in the technology for more than 40 years. Today, they assert that they are careful stewards of horseshoe crabs. One producer, Charles River Laboratories, contends that LAL production actually protects the crabs. “Without the need for limulus amebocyte lysate (LAL), the legal protection of the horseshoe crab is not guaranteed, and they would again fall prey to overfishing,” a company website states.

“Biotech companies are all about innovation until they have established market share. Then they cling to it relentlessly and don’t want to change,” says Kristoffer Whitney, an associate professor of science, technology, and society at Rochester Institute of Technology who is writing a book about biomedical use of horseshoe crabs. He observes that the U.S. Pharmacopeia finally approved synthetic LAL alternatives, after years of study, when all LAL producers had developed recombinant products. “I’m not suggesting a conspiracy, but it didn’t hurt that all producers now had an LAL alternative,” he points out.

Even if every major drug company ends use of LAL, horseshoe crabs will face other stresses. The bait fishery still takes hundreds of thousands of crabs per year, and coastal development has consumed many prime Atlantic spawning beaches over the past half-century.

“People may be overly optimistic about how much difference rFC and rCR will make for horseshoe crabs’ survival,” says Fordham University biologist Mark Botton, who co-chairs the Horseshoe Crab Specialist Group at the International Union for the Conservation of Nature. “No change in regulations will ever lead to an immediate change in population size, because the crabs take 9 to 10 years to reach sexual maturity.”

Federal fishery regulators describe crab stocks as stable in New England, poor in the New York region, and good in Delaware Bay and the Southeast. For its part, Botton’s conservation group contends that all four existing species of horseshoe crabs are imperiled by overharvesting for use as food, bait, and biomedical bleeding, and by habitat lost to shoreline development. In 2024, more than two dozen conservation and animal welfare groups petitioned the federal government to declare American horseshoe crabs as threatened or endangered; their request remains pending before the Trump administration.  

Still, arguments for shifting away from LAL are coming into alignment. The Atlantic coast is frequently hit by hurricanes, which are becoming larger and more destructive with climate change. A direct hit on an important spawning zone like Delaware Bay could threaten crab harvests for multiple seasons.

Other countries are already making the change. The European Union adopted guidelines for rFC in 2020, as did China. Britain followed suit in 2024. “The pharmaceutical industry is conservative by nature, but a lot of barriers have fallen just in the past two or three years. There’s definitely a lot of momentum now,” says Bolden.

And again, there are those three Rs, starting with “replace.”

Maybe, after more than 50 years of bloodletting, horseshoe crabs have done enough for human health. And maybe, thousands of years from now, their role in medical testing will be just a blip in the long saga of existential threats that this enduring species has weathered.