They are captured from the wild, piled inside plastic bins, and hauled into labs where they are strapped to a stand. A hypodermic needle is inserted into the membrane surrounding their heart, causing their blood to leak into a glass bottle below. Once the blood stops dripping (when about 30 percent of it has been drained) they are thrown back into the ocean. An estimated 10–30 percent do not survive this ordeal. The bleeding itself does not usually kill them; rather, death is caused by the additive effects of capture, transport, handling, and time spent out of the water, all of which cause stress and sometimes injury. Of those who survive, welfare is nonetheless compromised: For several weeks after the bleeding, they are slower, lethargic, and have weaker immune systems, making them more susceptible to disease; they are also less likely to spawn that season.
This is the fate of hundreds of thousands of horseshoe crabs each year in the United States at the hands of the biomedical industry. The animals’ blood—bright blue because it contains copper rather than iron like ours—is collected for one key reason: It clots in the presence of “endotoxins.” Endotoxins are shed when gram-negative bacteria, such as E. coli or salmonella, die. If high levels of endotoxins were to enter the bloodstream or spinal fluid, they could cause fever, respiratory problems, or death.
For this reason, every medical substance and device that is injected or placed inside a human or animal body—for example, vaccines, insulin, injectable antibiotics, pacemakers, joint replacements, and any water used in their manufacturing process—is tested to ensure that it isn’t contaminated with endotoxins. Since the 1970s, the standard method to check for the presence of endotoxins has been the horseshoe crab blood–derived limulus amebocyte lysate (LAL) test.
A synthetic, animal-free alternative known as recombinant factor C (rFC) has been available commercially since 2004. Yet, since that time, the number of horseshoe crabs captured and bled each year has continued to rise. Between 2004 and 2012, the number of crabs used increased by 85 percent. In 2019, over 637,000 crabs were bled—a 30 percent increase from the year before. With millions of coronavirus vaccine batches being developed, trialed, and produced in the last year and a half in the United States alone, the number of crabs used by the biomedical industry is likely to have increased again. In March 2020, 241 vaccines and other therapies were in product development; that number had jumped to 838 by August 2021.
Why is the synthetic rFC test seldom used in place of the animal-derived LAL test? Regulations are partly to blame. In the United States, companies conducting endotoxin testing are directed by the Food and Drug Administration to follow the standards laid out by the US Pharmacopeia (USP). Pharmacopeias are independent scientific organizations that set the standards for medicines, dietary supplements, and food ingredients worldwide. The USP currently allows the use of rFC, but only if scientists do their own validation study for each new compound they want to use the test for; the time and cost associated with this requirement mean that rFC is rarely used.
The USP was expected to approve the rFC test without restrictions, but reversed this decision in May 2020 on the grounds that more evidence was needed to prove that rFC was effective. Fouad Atouf, USP vice president of global biologics, told the Washington Post in August 2020 that among industry stakeholders consulted, no broad consensus emerged that enough data existed to confirm its effectiveness.
One industry stakeholder and vocal opponent of rFC is Charles River Laboratories, the largest manufacturer of the lucrative crab blood–derived LAL test. The company claims that rFC is not as safe as LAL. Jay Bolden, an expert in endotoxin detection at the pharmaceutical giant Eli Lilly, disagrees. Bolden asserts that rFC testing is not only as effective as LAL, but is also more efficient, more cost-effective, and yields fewer false positives. In 2016, his company decided to use rFC for all new injectable products despite the extra work this entails under current USP guidance.
Recently, Bolden and other members of the Parenteral Drug Association Leadership Forum published a review of existing studies comparing the efficacy of LAL and rFC to detect endotoxins. Their conclusion: The two tests are comparable in protecting patient safety. Bolden told the Washington Post, “We looked at all the available scientific literature on rFC, and we found a dozen studies that say exactly what we think the [USP] would need to make those kind of judgments.” He said only one study, performed by Charles River, found rFC to be inferior, and that this was because they used unfiltered water, which can skew the results. In an article published in the American Pharmaceutical Review, endotoxin expert Kevin Williams argued that unfiltered water samples should not be included in validation studies, because unfiltered water is not part of routine endotoxin testing and therefore falls outside the USP’s validation requirements. Meanwhile, for the past year, rFC has been officially recognized by the European Pharmacopeia as a standard method. The change was proposed in late 2018, and came into effect on January 1, 2021.
Scientists have an obligation to use effective animal-free methods when these exist. The continued use of horseshoe crabs to produce LAL not only harms individual crabs but also puts a strain on the species and other species that rely on them. American horseshoe crabs are listed as vulnerable on the IUCN Red List, and their populations are in decline. Moreover, red knot shorebirds, a species listed as threatened, depend on horseshoe crabs for their own survival: On their 9,000-mile migration route from the southern tip of Argentina (where they winter) to the Canadian Arctic (where they breed), red knots make a crucial stop on the eastern US seaboard, where they double their weight by feeding on nutrient-rich horseshoe crab eggs.
The continued use of crabs to produce a test for which there is an equivalent, if not superior, alternative, is unacceptable. People’s health is also at risk. “As it is now, the entire supply chain for endotoxin testing of drugs rests upon the harvest of a vulnerable or near extinct sea creature,” Williams told the Washington Post, “As prudent as the pharmaceutical industry is, this seems to be a current blind spot.”