Can the Common Asian Toad, Duttaphrynus melanostictus, live in saltwater?

In my previous post on tadpoles of this species I left the question of whether adult Common Asian Toads, Duttaphrynus melanostictus, can tolerate saltwater, and, if so, what strength of saltwater.

Before trying to answer that question I should point out that since the mid-1960s, the ability of a number of other amphibians to tolerate saltwater has been studied, the Green Toad (Bufotes or Bufo viridis) from Europe and the Middle East being one notable example.

In an epic of bibliographic research Gareth Hopkins and Edmund Brodie pulled together all the papers on the occurrence of amphibians in saline habitats beginning as far as I can see with Charles Darwin in 1834; their review was published in 2015. Useful though that exercise was (although I have since found at least one paper that was missed), I was less than impressed by the treatment of physiology in the review and the criteria used to extract meaningful data from individual papers, particularly those reporting observations rather than experimental evidence. I can illustrate these points by reference to a table in their review that includes Duttaphrynus melanostictus as a ‘well-studied salt-tolerant amphibian species’. Let’s look at the evidence from the papers they used to reach this conclusion.

Annandale’s paper from 1907

Quoting Nelson Annandale^†, Hopkins and Brodie show a measured salinity of 12.87g/kg for where Annandale found this species in India. Please note for this article compared with the last, I am using g/kg (parts per thousand) rather than percentages for salinity in order to use the same units as Hopkins and Brodie. 12.7g/kg is equivalent to about 40% seawater and would be well above the point of isotonicity^‡ between body fluids and the external environment. In other words, there would be an osmotic flow of water out of the body. However, there are important caveats. Annandale’s paper describes the brackish ponds at Port Canning on the Matla river, about 60 miles from the sea. The ponds were dug as part of an ill-fated scheme to build a port to rival Calcutta and Singapore in the 1860s; judging by what I can see on Google Earth, they are still there. This is how Annandale describes the water and its salinity:

     An important factor in the environment is the nature of the water. I have described the ponds as brackish, but at some time of the year the water may contain the same proportion of soluble salts as the sea, at others it may even be more strongly saline, and again at others it is much more nearly fresh. As a rule the ponds are completely isolated both from one another and from the estuary. During the cold weather they are exposed to evaporation, which becomes intensified during the hot weather. During the rainy season, on the other hand, they become filled up with fresh water and probably often coalesce. They are also liable to be placed in temporary communication with the estuary occasionally, owing to a flood bursting the embankment but this does not occur by any means every year. When it does happen, it happens owing to the estuary being swollen with fresh water, which is flowing down from up-country so that the ponds, even under these conditions, are practically cut off from the sea. 

     Stoliczka*, in 1868 or 1869, had the water of the ponds analysed; but he does not say at what time of year his samples were obtained. He found that the proportion of soluble solids was 12.87 per thousand, sea-water containing from 32 to 39 per thousand. Mr. D. Hooper, Curator of the Industrial Section of the Indian Museum, has kindly examined samples taken by myself in December and March last. Two samples came from a pond in which the Hydrozoon Irene ceylonensis, as well as the Actinian, was reproducing its species, and in which the plant Naias was abundant. A sample taken from this pond at the beginning of December, a few weeks after the end of the rainy season, was found to contain 12.13 per thousand of soluble salts, while another taken on March 17th contained 20.22 per thousand. At the latter date water from the edge of the Matla at Port Canning contained 25.46 per thousand, and that from a second pond near the first 23.16… 

     Stoliczka noted that the water in the ponds was almost fresh during the rains…All that can be said, therefore, as regards the salinity of the water of the ponds, is that it varies considerably at different times of the year…

Listing the fauna of the ponds, Annandale had this to say:

     …and the only Amphibians the equally common Rana cyanophlyctis and R. tigrina. The Indian Toad, Bufo melanostictus, is abundant at the edge of the ponds, in which it possibly breeds.

So, equating measurements of the measured salinity of the ponds at a particular season to a presumed salt tolerance of the largely terrrestrial toad (it could breed in the ponds in the rainy season when the water is ‘more nearly fresh’) is perhaps unwise. The killer blow, though, to trying to relate measured salinity to salt tolerance of this toad is the occurrence of the two species of frog. The Indian Bullfrog, Rana tigrina, now reverted to its apparently correct specific name of tigerina in the genus Hoplobatrachus, was used by Gordon, Schmidt-Nielsen and Kelly in their 1961 paper as a freshwater species to compare with F. cancrivora. Their results were clear. Adult bullfrogs could tolerate a salinities up to 9 g/kg (25% seawater). Above that physiological point of isotonicity, the frogs died in 24-48 hours, even at 11 g/kg. Therefore, the salinity quoted by Annandale cannot be equated to the presence of the amphibian species in the water at the same season.

I therefore discount the evidence taken from Annandale’s paper on D. melanostictus.

George Chakko’s paper from 1968

The only direct study on the saltwater tolerance of D. melanostictus listed by Hopkins and Brodie is that by George Chakko of Madras Christian College published in 1968. He kept four local species in various concentrations of seawater. There was a difference between species. All survived for two weeks in 25% local seawater (salinity 32g/kg), i.e. 8 g/kg. These species were Jerdon’s Bullfrog (Hoplobatrachus, then Rana, crassus), Indian Green Frog (Euphlyctis, then Rana, hexadactylus), Marbled Narrow-mouth Frog (Uperodon or Ramanella variegatus*) and, of course, D. melanostictus. Only D. melanostictus survived in 35% seawater, i.e. 11.2 g/kg. These were the maximum tolerated concentrations.

What do we now know?

Looking at the experimentally-determined salinity tolerance of a number of anurans (frogs and toads, for brevity, although there are interesting examples in caudates, the newts and salamanders) listed by Hopkins and Brodie, there are a number that show a similar tolerance to D. melanostictus. I counted 7 species in the range 10-11.2 g/kg. Above that there are only 4: Xenopus laevis at 14g/kg (reminding us salinity can be high in inland waters); the possibly aptly named Marine or Cane Toad, Rhinella marina at 16; Green Toad, Bufotes viridis, at 20-26 and then the jump to F. cancrivora at 39.

In terms of mechanisms of adaptation, it is interesting that in the four species known to tolerate higher salinities as adults, an increase in urea as well as an increase in salt concentrations in blood has been found. Is that also true of those tolerant to only slightly hypertonic saltwater like D. melanostictus, or do they rely simply on a small increase in salt concentrations in their blood?

The take-home message is that D. melanostictus just enters the physiologically interesting, in terms of adaptations needed to survive, range of salinity, in that the 35% seawater (but not the 25% seawater) would be slightly hypertonic to the body fluids of toads in freshwater. In terms of physiological adaptation, there is a question still to be answered—an ideal project for an honours year student perhaps.

On the Common Asian Toad, taking the evidence discussed in this and the previous article, I can summarise:

• Tadpoles must hatch in virtually fresh water
• Older tadpoles must live in brackish water hypotonic to body fluids
• Adults can tolerate salinities just but only just hypertonic to body fluids. The physiological mechanism has not been studied in this species.
• The evidence matches observations that these toads can live near the sea, even breed in freshwater near the sea, where they may be exposed to brackish conditions.
• D. melanostictus does not approach the capabilities of F. cancrivora in exploiting the far greater salinity of a mangrove swamp.

‡Tonicity is a measure of the effective osmotic pressure gradient, as defined by the water potential of two solutions separated by a semipermeable membrane. In other words, tonicity is the relative concentration of solutes dissolved in solution which determine the direction and extent of diffusion. It is commonly used when describing the response of cells immersed in an external solution. Unlike osmotic pressure, tonicity is influenced only by solutes that cannot cross the membrane, as only these exert an effective osmotic pressure. Solutes able to freely cross the membrane do not affect tonicity because they will always be in equal concentrations on both sides of the membrane. This Wikipedia definition is as good as any. An example of differences between isotonic and isosmotic would be the plasma composition of F. cancrivora in saltwater. Because of the impermeabilty of the membranes in question to urea, this isomotic concentration is isotonic. In a typical mammal, because urea passes rapidly across membranes, the isosmotic plasma would be hypotonic.

†Thomas Nelson Annandale (1876–1924), in 1907 Director of the Indian Museum in Calcutta.

*Ferdinand Stoliczka (1838 –1874), a Moravian (Czech) in the Geological Survey of India. An all-round palaeontology, geology and zoology, he described and named the frog Ramanella variegatus (studied by Chakko above) in 1872.

Annandale N. 1907. The fauna of brackish ponds at Port Canning, Lower Bengal. Records of the Indian Museum 1: 35–43. 

Chakko G. 1968. Salinity tolerances of some Indian anurans. Proceedings of the Indian Academy of Sciences - B 67, 233–236,

Hopkins GR, Brodie ED. 2015. Occurrence of amphibians in saline habitats: a review and evolutionary perspective. Herpetological Monographs 29, 1-27. DOI: 10.1655/HERPMONOGRAPHS-D-14-00006 

Ronald Strahan in 1950s Hong Kong: Amphibians in saltwater

I had a surprise when researching the recent article on Ronald Strahan. I discovered that Strahan had done a small piece of research in Hong Kong that we unknowingly and in part repeated ten years later.

The background is that in the mid-1960s, the story of a frog and its survival in sea water had already become a classic of the heyday of comparative physiology. Again, Knut Schmidt-Nielsen (1915-2017) was one of the two main players. He describes the background in his autobiography:

     But I was curious about amphibians, such as frogs and salamanders. Normally frogs do not live in the sea; the high permeability of their skin would cause them serious problems in sea water because their blood and body fluids contain less than 1 percent salt, whereas sea water contains about 3.5 percent. A frog in sea water, it seems, should soon resemble a pickled herring. 

     I had come across a few reports of frogs and toads that live in brackish water, and even a mention of certain frogs that swim in full-strength sea water. This sounded incredible. If frogs living in the sea really existed, their physiological mechanisms certainly deserved a careful study. 

     In the spring of 1960 I planned to search for saltwater frogs where they had been reported, on the tropical coasts of Southeast Asia. Pete Scholander told me that Malcolm Gordon, a physiologist at the University of California at Los Angeles, was planning a similar study. “Why don’t you two work together?” he asked. 

     I was more than happy to collaborate with Malcolm, and we planned to work at the Oceanographic Institute at Nha Trang, a town north of Saigon in Viet Nam. At the beginning of the summer I flew from New York to Los Angeles, my first trip in a modem jet. From there, Malcolm, a student named Hamilton Kelly^†, and I took off for Viet Nam to search for saltwater frogs. After stops in Tokyo and Hong Kong, we arrived in Saigon.

Crab-eating Frog (Fejervarya cancrivora)**

After an unwelcome introduction to discomfort of a French bed and the horrors of the tapwater, the team failed to find any frogs of the species Rana cancrivora, now known as Fejervarya cancrivora. They decamped to Thailand where they not only found saltwater frogs but discovered how they could live in such conditions. The frogs could survive in seawater. The problem, as Knut Schmidt-Nielsen explained, is of water. The concentration of the salts in seawater is much higher than that in the blood of frogs in freshwater. Water would be lost osmotically, and the frogs would die. 

The adaptation of saltwater frogs is to match the concentration of solutes in their blood to that of seawater or whatever salinity of water they are accustomed to. They do this mainly by accumulating urea. In most animal tissues urea moves rapidly in and out of cells so that a concentration gradient for osmotic water flow is not formed. By contrast, the skin of saltwater frogs is impermeable to urea. Therefore, the sum of salts plus urea concentrations inside the body equals the salt concentration on the outside and the frog does not lose water. This is the same mechanism employed by sharks and rays living in the sea

.

This is the only photograph I can find of a Crab-eating Frog
in a mangrove swamp. From here

As a result of Gordon and Schmidt-Nielsen’s research in south-east Asia, frogs that occur in coastal mangrove swamps and the mechanism by which they survive became famous overnight. An 18-minute 16 mm film, which I saw at a ZSL meeting in the 1970s, of Malcolm Gordon working in Thailand on the frogs was released in 1967*.

With that knowledge from the early 1960s you can appreciate the feeling my wife and I had when one Sunday afternoon in spring 1966, wandering along the top of a beach on Hong Kong Island, we saw an amazing sight. In an isolated pool comprised entirely of sand were tadpoles, lots of tadpoles. It seemed inconceivable that these tadpoles were not exposed to a higher degree of salinity than those in a freshwater pool. Did we have another species that, like F. cancrivora, could live in marine conditions? We had nothing to transport any tadpoles back to the lab that day. However, Alan Wright the next day was similarly enthused and we set off that afternoon in his Volkswagen Beetle to collect some of the tadpoles plus a sample of the water they were living in.

Back in the lab, the tadpoles lived perfectly happily in tap water and concentrations of salt up to 0.9%. Above that they were in obvious distress until moved again to freshwater. I cannot remember the salt concentration of the water in the pool but I think it was around 0.2%, much lower than that of seawater and lower than we had expected it to be. We could only assume that freshwater flowed down the beach after rain and was trapped.

A modern view of Big Wave Bay on Hong Kong Island. The red circle
marks the approximate site where we found the tadpoles. The buildings
and beach paraphernalia there have all been erected since 1966

Physiologists reading this will realise that 0.9% saline is the approximate concentration of salt in the blood of vertebrates and only above this concentration would the tadpoles be resembling F. cancrivora. Oh well, worth a look but not very interesting physiologically. The tadpoles were released into one of the ponds in the university compound and my notes (long gone) filed under ‘Abandoned Experiments’. Over the years, as information on the amphibians of Hong Kong accumulated, I realised that the black tadpoles with relatively short tails tadpoles were of Bufo melanostictus (now Duttaphrynus melanostictus), the Common Asian Toad.

Ronald Strahan

Getting back to the first paragraph of this article, you can imagine my surprise to discover that ten years earlier (i.e. before the experiments Schmidt-Nielsen described) Ronald Strahan had done a similar, but more extensive, study—in the same lab. His short paper, published in Copeia in 1957, began:

The ability of some am­phibians to withstand brackish water is well-known and indicated, for example, in the name, Bufo boreas halophilus. The experiments described below give some information on the extent of this ability in the tadpoles of a toad normally found in fresh water. 

In short, he collected spawn and tested salt concentrations up to 1%. The ability to withstand greater than 0.25% appeared to develop with age after hatching. At 1%, even when transferred into that concentration at 8½ days, activity was reduced and metamorphosis was retarded. Our 1966 tadpoles were probably as old or older than 8 days older and, therefore, our quick look see produced the same conclusions as those of Strahan. An important point from Strahan’s experiments was that tadpoles from spawn laid in saltwater stronger than about 0.25% will not survive.

Duttaphrynus melanostictus
Common Asian Toad, Hong Kong 1966

Notice that we, and Strahan, had studied the tadpoles of Duttaphrynus melanostictus whereas Gordon and Schmidt-Nielsen had studied adult F. cancrivora. What about the tadpoles of cancrivora? Gordon, this time with Vance Tucker from Schmidt-Nielsen’s department at Duke University, made another trip to Thailand to study that question. Tadpoles were found to be ‘abundant in brackish ponds near high-tide marks in the mangrove swamps along the north shore of the Gulf of Thailand’. 

Relatively large tadpoles coped perfectly well in seawater, but used a different method compared to the adults. The osmotic concentration of the blood increased with increasing concentrations of external salt but the increase was in salts not urea. Like salmon and other teleost fish that move to salt water, tadpoles appeared to drink the water and then get rid of excess salt, possibly through the gills. However, young tadpoles could not cope with salt concentrations higher than about 0.6%, findings reminiscent of those of Strahan in D. melanostictus.

In addition, although older tadpoles could cope with, and grow to a large size in, seawater, they did not appear to metamorphose in those high concentrations.

Gordon and Tucker concluded:

     Our observations in the laboratory indicate that the initial stages of embryonic development and metamorphosis are interfered with by salinities greater than 20% sea water [i.e. approximately 0.6% salt]. These observations, together with field data, suggest that the torrential summer rains of Thailand play an important role in the developmental biology of this frog. Spawning may occur only during or soon after heavy rains when the salinity of the spawning pools is low. We spent several nights collecting dozens of frogs with ripe gonads at the edges of the spawning pools but never observed amplexus. Spawning could have been restricted to periods of heavy rainfall, when we did not collect. The general synchrony of developmental stages in a single pond suggests that spawning could have been synchronized by a period of heavy rain.  

     Tadpoles in the laboratory usually did not metamorphose if the medium was more concentrated than 20% sea water. In the field the largest immediately pre-metamorphic tadpoles were found in the saltiest ponds. These observations suggest that metamorphosis may be delayed as long as the pond salinity is high. 

     These requirements for dilute media, and the freshwater nature of all other ranids, make it seem probable that R. cancrivora has invaded the marine environment from fresh water in relatively recent times. The high temperatures of its spawning ponds would permit rapid embryonic development and metamorphosis when torrential monsoon thunderstorms temporarily dilute the ponds. Its otherwise great salinity tolerance permits this frog and its tadpoles to enter a rich environment closed to all other amphibians. 

Studies on other species that can tolerate concentrations of seawater in the physiologically interesting range have been done since the early 1960s. However, sticking with D. melanostictus for the moment, judging by a comment made by Strahan in an autobiographical note, he was interested in this species because in Hong Kong he found it on outlying islands. 

The Zoology Department, which had been sacked during the war, was not very well equipped, so I undertook research that needed little gear. I worked for a while on the water relations of a local toad, Bufo melanostictus, and its tolerance of saline water, which could explain its prevalence on offshore islands…

In the second article of this series I will discuss what is known about adults, rather than tadpoles, of this species and its occurrence in salty water and why what is known or assumed may be misleading. However, for the moment it is quite clear, from these early studies and later ones I have not described, that the water in which the spawn hatches must be freshwater or brackish water no stronger than about 0.2% salt, i.e. less than about 5% the concentration of salt in seawater.

†Hamilton Morgan Kelly, 1936-2006, became a psychiatrist in California.

*Adaptation to a Marine Environment. 18 min, colour and b/w, sound, 1967. Distributed by McGraw-Hill Text-Films. Produced by Lamont Geological Observatory of Columbia University with a grant from the National Science Foundation. I have not been able to find a digitised online version.

**By W.A. Djatmiko (Wie146) [GFDL (http://www.gnu.org/copyleft/fdl.html), CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0/) from Wikimedia Commons

Gordon MS, Schmidt-Nielsen K, Kelly HM. 1961. Osmotic regulation in the crab-eating frog (Rana cancrivora). Journal of Experimental Biology 38, 659-678.

Gordon MS, Tucker VA. 1965. Osmotic regulation in the tadpoles of the crab-eating frog (Rana cancrivora). Journal of Experimental Biology 42, 437-445.

Gordon MS, Tucker VA. 1968. Further observations on the physiology of salinity adaptation in the crab-eating frog (Rana cancrivora). Journal of Experimental Biology 49, 185-193.

Schmidt-Nielsen K. 1998. The Camel’s Nose. Memoirs of a Curious Scientist. Washington DC: Island Press.

Strahan R. 1957. The effect of salinity on the survival of larvae of Bufo melanostictus. Copeia (1957), 146-147.