We all want that medicines, vaccines, and medical devices are safe. But it is incomprehensible why 550,000 horseshoe crabs, of which up to 150,000 are estimated to die, have to suffer for this safety every year. An animal-free method has not only been available for 25 years but has also been proven to provide at least equal or even better results for safety assessments. This article sheds light on the huge profits of a concealed animal exploitation industry sustained through clever lobbying, and takes a stand for an animal that, despite its alien appearance, deserves protection from suffering and exploitation.

The horseshoe crab: strange and insignificant or fascinating and worthy of protection?

The horseshoe crab - a primeval animal.

The horseshoe crab is one of the oldest creatures on earth. It has been around for 400 million years and has survived several geological catastrophes and mass extinctions, including the extinction of the dinosaurs around 66 million years ago (1). Since horseshoe crabs have changed little over these unimaginable periods of time, they are also referred to as living fossils. The up to 85 cm long primeval animals, which are not crabs at all but are probably more related to the arachnids, have a tripartite exoskeleton consisting of a front body, an abdomen, and a tail. The animal's arrow-shaped tail may look menacing but is harmless: it serves as a rudder and as a turning aid when the animal is lying on its back.

Four species of horseshoe crabs still exist today. Three of them live in the waters of Southeast Asia and one species (Limulus polyphemus) off the North American Atlantic coast. The North American horseshoe crabs mostly live hidden on the sea ground but migrate from deeper waters to their spawning grounds on the east coast of the USA in spring.

From almost worthless mass products to a valuable pharmaceutical raw material

In the mid-19th century, horseshoe crabs were so plentiful on the east coast of the United States that they were collected and used as feed for pigs and chickens. At the end of the 19th century, horseshoe crabs were processed into fertilizer on a large scale in the USA (2). This intensive use and the fact that the animals were caught on their way to mate, and thus their reproduction was prevented, led to a dramatic reduction in their numbers (2). Nevertheless, the capture of the animals for fertilizer production was only stopped in the 1970s - not because of species conservation, but because of the introduction of artificial fertilizers. The population then recovered until the 1990s, when the mass capture of the animals started again - this time to use them as bait for whelks and eels fishing (3).

Horseshoe crabs are still systematically caught and used today. Also, but not only as bait. Their blood -  of blue color due to the copper-containing molecule hemocyanin, which is responsible for transporting oxygen - can be used to detect fever-causing substances, so-called pyrogens.

Horseshoe crabs are caught on their way to spawn.

Pyrogens

Pyrogens are substances that cause fever in humans or can even lead to blood poisoning with a potentially fatal outcome. Substances that are administered to humans by bypassing the gastrointestinal tract must therefore be tested for pyrogens. Materials that need to be pyrogen-free include solutions that are injected into veins, muscles or under the skin, such as vaccines, infusion solutions and injectable drugs, but also medical materials that are implanted into the human body, such as hip prostheses or artificial heart valves. Pyrogen testing of each batch of these products is required by the European Pharmacopoeia to protect patients from harm.

Pyrogens can originate from different sources: they can either be of bacterial origin, such as the endotoxins that are released when gram-negative bacteria decay. They can also originate from gram-positive bacteria, fungi or viruses or, for example, from abrasion of rubber, plastic, and metal.

The classical method for testing for pyrogens is the so-called rabbit pyrogen test (RPT). Rabbits are confined in a small box with only their heads sticking out. The substance to be tested is then injected into an ear vein. A thermometer in the rectum of the animal is used to monitor the temperature for several hours to observe whether the animal develops a fever. The rabbits can be “used” several times in these agonizing experiments, which represent enormous stress alone by the restraint, as rabbits are flight animals. Then the rabbits are killed. While the awareness that rabbits should not be misused as measuring instruments in such a cruel way increases – also based on our campaign (4) - and more and more companies in Germany surrender the use of rabbit tests, the horseshoe crab is increasingly in the focus of pyrogen testing.

The Limulus amebocyte lysate (LAL) test

The blood cells of the horseshoe crab, the so-called amoebocytes, contain proteins that result in clotting upon contact to endotoxins. In this way, bacteria that have entered the animal's body can be rendered harmless by encapsulating them in a gel and thus preventing them from spreading. Specialized companies take advantage of this behavior of the amebocytes. For this purpose, the blood of wild-caught horseshoe crabs is drawn off, the amoebocytes are broken down and the content of the cells, the so-called lysate, is obtained. This contains coagulogen and the protein factor C. If these substances are combined with an endotoxin, the coagulogen breaks down into insoluble fragments. These fragments accumulate and form a gel.

In the so-called Limulus amebocyte lysate (LAL) test, this is used to detect contamination with endotoxins. The LAL test was introduced in the 1970s and is still one of the standard methods for detecting bacterial contamination in injectable solutions. The companies that produce LAL tests like to draw the picture of a sustainable use of the horseshoe crab: the animals are only taken from the sea for a short time and brought back unimpaired after the blood has been taken. In return, they would be protected from being overused as bait in fisheries (5).

The suffering of horseshoe crabs

With the sheer volume of IV fluids, injectable drugs, and implantable medical devices that need to be analyzed for pyrogens at batch release, it is obvious that massive amounts of amebocyte lysate are being consumed. According to a publication from 2018, around 550,000 horseshoe crabs are used for blood collection every year at the east coast of the USA alone (6). The animals are caught with trawlers and transported to the laboratories. There they are cleaned and checked for injuries. Their body is severely bent, creating a gap between the armor of the front and rear body. The horseshoe crabs are then lined up in long shelves where a thick cannula is inserted into their hearts without anesthetic (7), through which the blue blood is collected in bottles. Up to 30% of the blood of each animal is removed in this way. Then the animals are transferred back into the ocean.

Horseshoe crabs are caught en masse, many die.

Even if it is often described differently, this process is more than a short removal from their habitat, to which they are brought back after a short "milking": many animals do not survive the catch, the often lengthy exposure to the air, the transport, and the procedure of blood collection. Of the surviving animals, 13% are sold to local fishermen as bait, with the rest being released back into the sea (8). In addition, it is estimated that between 10 and 30% of the animals returned to the sea die (9). This does not include animals that are killed or injured during capture or transport, and are therefore sorted out before blood is collected. The animals surviving the procedure are also affected. To prevent repeated capture and blood withdrawal, animals are released far from where they were collected (9). It was also found that the animals are slower and more lethargic after blood collection. This restricts the animals, especially during the mating season, so that their reproductive success is endangered (10). Female horseshoe crabs particularly suffer from the blood collection. It is assumed that the production of the eggs represents a high energetic burden for the animals. Because much of the animals' capture occurs during or shortly after the mating season, the females are affected by a double burden due to egg production and blood loss. As a result, their mortality rate is higher and in some areas, an increasing overrepresentation of males is already being observed (11). It is therefore not surprising that the species Limulus polyphemus has been listed as “vulnerable” by the IUCN (International Union for the Conservation of Nature) since 2016.

The role of industry: huge profits and subterfuge

Horseshoe crabs have their blue blood drawn.

Horseshoe crab blood is traded at a price of $15,800/l (currently around €15,000/l) (12). It is therefore also referred to as "blue gold". It has been estimated that around 550,000 animals are caught each year (6), and this number is unlikely to have decreased from 2018 to the present day. It is more likely that due to the need for corona vaccines their number has increased (13). 200 to 400 ml of blood can be obtained from each animal (7). Even with a conservative estimate, at least 110,000 liters of horseshoe crab blood are collected each year, which are worth around €1.65 billion.

The following sections briefly introduce some of the companies that use horseshoe crabs and make money from their blood. A more comprehensive overview of the companies involved in this business is available online at the ERDG (Ecological Research & Development Group) (14).

Associates of Cape Cod

Associates of Cape Cod not only offers a variety of LAL products and related accessories, but also performs LAL-based pyrogen testing according to the US and European pharmacopoeias as contract work.

The company has developed a breeding program for horseshoe crabs: eggs and sperm from the animals, which are sold as bait for fishing, are collected and fertilized, and the hatched young animals are later released to the ocean. By June 2021, one million young animals had been released into the sea. In this way, the company claims to extend “their [the horseshoe crabs used as bait] genetic legacy for generations to come” (15). This seems almost cynical. Associates of Cape Cod sells the parent animals as bait. From the eggs and sperm collected from the bait animals new animals are bred, which – if they survive and grow in the sea at all – will ensure the catch and profits for the years to come. Presenting this as a project for sustainable species conservation is simply hypocritical.

Charles River

The market leader in the production of LAL is Charles River Laboratories, which is also one of the world's largest "experimental animal" breeders. The company has been bleeding horseshoe crabs for nearly 30 years and has annual sales of approximately $500 million (16).

Charles River has attempted to develop a synthetic alternative to the LAL, but the resulting test hasn't worked as well as the LAL test (16). Since then, the company has been working very actively and unfortunately also successfully against the use of synthetic LAL products, which are indeed available from other manufacturers. Charles River is publishing misleading data that cast doubt on the quality of synthetic products already approved in Europe, Japan and China, according to an investigation of The State Media Company. It is presumably due to these statements and due to Charles River's lobbying work that the synthetic alternatives are still not equated with the LAL test in the American pharmacopoeia. Charles River has also reportedly caught horseshoe crabs in protected areas (16).

The company itself states that it plays a significant role in the protection of horseshoe crabs. The needs of the biomedical industry would be the basis for better protection of the animals and corresponding laws being passed. In fact, the company helped to pass a legislation banning horseshoe crabs from being used as fishing bait in South Carolina. The company is now celebrating itself for these supposed successes (17). Of course, it is legitimate when economic interests coincide with those of species conservation. A stale aftertaste remains, however, when a company that earns millions from animal suffering eliminates fishing as a competitor for a limited resource and instead earns money from catching the endangered animals itself - and then even boasts its merits in preserving the species. In addition, Charles River´s horseshoe crab conservation efforts appear to be limited to the American species. According to the ERDG (Ecological Research & Development Group), the company also catches horseshoe crabs in Asia under the name Zhanjiang A&C Biological. In contrast to the USA, the animals are not returned to the sea after the blood collection; the death rate is 100% (14).

Use of the LAL test in Germany

Horseshoe crabs are caught on the east coast of the USA and in Southeast Asia for the Asian market. However, the product made from the blue blood of the animals is used worldwide – including in Germany. While the dimensions of industrial consumption in Germany cannot be determined, it can be assumed that it is used by almost all pharmaceutical companies, as well as by contract laboratories that offer tests for the pharmaceutical industry.

The LAL test is offered as a service by various companies:

• in Bavaria by Labor LS (Bad Bocklet) and senetics healthcare group GmbH & Co. KG (Ansbach)
• in Baden-Wurttemberg by CleanControlling Medical GmbH & Co. KG (Emmingen-Liptingen) and BBF Sterilisationsservice GmbH (Kernen im Remstal)
• in North Rhine-Westphalia by Mikrobiologischen Labor Dr. Michael Lohmeyer GmbH (Münster) and Wessling GmbH (Altenberge)
• in Berlin by the ifp Institut für Produktqualität GmbH

This short list does not claim to be complete and certainly only covers a small part of the laboratories that carry out the LAL test as commissioned work.

There is another way: Eli Lilly tests for pyrogens without animal suffering

The American pharmaceutical company Eli Lilly demonstrates that there is another way. Since 2016, the company has been applying to the US Food and Drug Administration (FDA) for approval of the use of synthetic alternatives to the LAL test for new products - the same methods that Charles River is still claiming don’t work well enough. The FDA obviously sees this differently, as in 2021 they approved an Eli Lilly antibody for COVID-19 treatment that was tested with synthetic alternatives (16).

Of course, Eli Lilly does not do this for altruistic reasons either. Jay Bolden, a biologist at Eli Lilly, states that the synthetic testing is cost-effective. He also says that a more consistent product quality can be achieved for the synthetic alternative compared to the tests derived from animals pulled from the ocean (16).

Animal-free replica of the LAL test

It might be understandable that parts of the biomedical industry would like to continue using LAL tests based on their background of decades of experience. But it is no longer necessary. Scientists from the University of Singapore developed a synthetic alternative to the LAL test back in 1997. In contrast to the LAL test, this method only requires a single protein, the recombinant factor C (rFC). In the rFC test that can be carried out with this protein, the binding of an endotoxin to rFC leads to its activation. The activated rFC then initiates a reaction that produces a dye.

The synthetically produced rFC corresponds to that from the blood of horseshoe crabs; it is only produced differently, namely from microorganisms in bioreactors that can be scaled to any size in order to be able to cover the needs of the market in a sustainable manner and independently of the horseshoe crabs, which are classified as endangered (8). Nevertheless, the rFC test, which has been commercially available since 2003, has not yet prevailed over the LAL test. Concerns are often expressed that the rFC test may be less efficient and less secure than the LAL test. But are these claims, which are also spread by Charles River, actually true?

Scientific evaluation of the synthetic rFC test

It is often claimed that the test with the rFC produced in bacteria is less reliable than the test with horseshoe crab blood. To verify this claim, a study was conducted in 2018 comparing 10 publications that examined the efficiency of the rFC test (8).

It was found that the rFC test is clearly just as suitable for the detection of endotoxins from gram-negative bacteria as the LAL test. In addition, the specificity of the rFC test is higher. This means that in the rFC test only endotoxins lead to a signal, while in the LAL test other substances such as polysaccharides, proteases or phospholipids can also produce false positive signals. In contrast, no substances are known to date that lead to false-positive results in the rFC test. In addition, the rFC test also leads to fewer false negative test results, which in the worst case could lead to endotoxins not being detected and corresponding products being placed on the market and causing serious complications for the patients. Another advantage is the better reproducibility of the rFC test, which, due to its manufacture process, has fewer batch-to-batch variations than the LAL test (8).

Legal situation

While there is ample evidence that the rFC test is a safe way to test for endotoxins, its adoption has been slow. Manufacturers and authorities are understandably cautious when it comes to changing safety-relevant processes in such a sensitive area as endotoxin testing. The manufacturers themselves can only decide to a limited extent which method they use to carry out the endotoxin test, and only methods that are accepted by the relevant competent authorities may be used. To make matters worse, different regulatory authorities (e.g. FDA in the USA and European Medicines Agency (EMA) in Europe) are responsible for vaccines and medicines that are manufactured and distributed worldwide and refer to different compendiums (e.g. US or European Pharmacopoeia).

In 2012, the FDA issued a guidance including the use of rFC as an alternative to the LAL test. In 2016, the rFC test was also included in the European Pharmacopoeia as a possible alternative. In 2018, the first drug tested with the rFC test was approved by the FDA. In the same year, the European Directorate for the Quality of Medicines (EDQM) announced that a new chapter in the European Pharmacopeia dealing with the rFC test is planned. This chapter was published in 2020 (18).

Many small steps have already been taken and it seems likely that the rFC test will be recognized as an equivalent method to the LAL test. Until then, however, it is only considered a so-called alternative method. This means that the test can be used instead of the LAL test, but a complex validation must be carried out for each individual product. An effort that many companies shy away from. We therefore demand not only the full recognition of the rFC test, the applicability of which has been sufficiently proven not least by the approval of drugs tested with it by the FDA, but also the deletion of all pyrogen tests based on animal suffering.

Monocyte Activation Test (MAT)

The pyrogen test is used to detect substances that cause fever in humans. Why is horseshoe crab blood used for this purpose at all? It seems to be more straightforward to use the mechanisms that take place in the human body during the fever reaction to detect pyrogens.

The monocyte activation test (MAT) follows this approach. Cells of the immune system, in particular certain white blood cells (monocytes), release messenger substances - the so-called interleukins - when they come into contact with fever-causing contaminations. These interleukins then initiate the fever reaction in the human body. In the MAT, the substance to be tested is therefore mixed with a small amount of blood from a healthy, voluntary donor. If pyrogens are present, the white blood cells produce interleukin-1, the amount of which can be determined. A blood donation of 500 ml is enough for 50,000 tests (19).

In contrast to the two animal-based tests using rabbits or horseshoe crabs, not only solutions but also powders, gaseous substances, blood transfusions and cell therapies can be tested with the MAT. In addition, a much wider range of pyrogens can be detected, including pyrogens from gram-positive bacteria and fungi, which are not covered by the LAL test. By using human cells, the results can be directly transferred to humans.

The MAT can be performed using standardized and commercially available test kits. There are different variants, which are equally recognized in the European Pharmacopoeia: a test with fresh human whole blood from voluntary donors, frozen blood from voluntary donors, and a human cell line (Mono Mac 6 cells).

Comparison of different methods of pyrogen testing

		Rabbit pyrogen test	LAL test	rFC test	MAT
Type of test		Pyrogen test	Endotoxin test	Endotoxin test	Pyrogen test
Approach		In-vivo	In-vitro	In-vitro	In-vitro
Animal consumption		+	+	-	-/(+)
Origin of detectable pyrogens	Gram-negative bacteria	+	+	+	+
	Gram-positive bacteria	+	-	-	+
	Viruses	+	-	-	+
	Fungi	+	-	-	+
	Others	+	-	-	+
Applicable to	Injectable pharmaceutical products	+	+	+	+
	Biologicals	+	+	+	+
	Blood products	-	-	+	+
	Cell therapies	-	-	+	+

Covid-19 and the horseshoe crabs

Due to the Covid-19 pandemic, the need for pyrogen tests has increased enormously and the majority of vaccines and antibodies against the virus have been and are tested with the LAL test (2). This increases the risk that the over-exploitation of and decline in the horseshoe crab population will be repeated (13). Horseshoe crabs do not become sexually mature until they are about 10 years old. In the short term, there will be a decrease of animals due to the increased catch. In the long term, however, the animals that have not been conceived today will not come to the beaches to spawn.

But perhaps the Covid-19 pandemic also represents an opportunity for change. The LAL test is the only medical product that is based exclusively on a single wild species, and the population of this species is declining (20). Relying on such a test in times of a pandemic seems downright daring. Dr. Jessica Ponder, toxicologist at the Physicians Committee for Responsible Medicine, believes that the pandemic underscores the urgent need to recognize synthetic alternatives to LAL (16).

How can the horseshoe crab be protected?

It seems that the horseshoe crab is not very popular. Maybe the animals are just too alien and, unlike rabbits, too far away from what is commonly considered to be cute and worthy of protection. As invertebrates, horseshoe crabs are less well protected by law than vertebrates, and even today some scientists are unsure whether our concept of pain applies to such animals (21).

Consequently, when it comes to protecting the horseshoe crab, reference is often made not to the horseshoe crab itself but to its importance to the ecosystem and to other, more pleasing species. On the mid-Atlantic coast, several migratory bird species depend directly on horseshoe crabs. Delaware Bay in particular is an important resting place for these birds, which have synchronized their arrival with the horseshoe crabs laying their eggs in the bay. The absence of horseshoe crab eggs resulted in a decrease in the number of birds and their breeding success. This includes the red knot, a bird that stops here on its long migration route to its breeding grounds in the Arctic. The red knot has been classified as endangered since 2014 (8,20).

In case of doubt, one should always assume that every animal can feel pain and suffering. Every individual should be protected from avoidable suffering, regardless of their importance to the ecosystem. Therefore, there is only one clear demand: the ban of the LAL test and immediate regulatory recognition and comprehensive use of the rFC test.

Closing remarks

In the past, the horseshoe crab's massive occurrence was fatal. Almost worthless, millions of the animals were caught and processed into fertilizer and food for so-called livestock. Today, the living fossil is enormously valuable, but that hasn’t improved its situation. Quite to the contrary: high profits lead again to the plundering of the horseshoe crab population.

The capture and exploitation of these primeval creatures can be considered a declaration of bankruptcy for the human ability to act empathetically, sustainably, and rationally, especially since cruelty-free methods for pyrogen testing have been available for years and their applicability and superiority has been proven in a number of studies. Thus, the consistent use of animal-free tests would not only benefit the horseshoe crabs and the migratory birds that depend on them, but also all of us.

And maybe a little humility would be appropriate towards a creature that has existed for 400 million years, has survived the extinction of the dinosaurs, and is now being massively exploited by us humans again and again.

Dr. rer. nat. Johanna Walter

References

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