A Stealth Topic

Out of all the topics far from the minds of most reef aquarists, it would probably be difficult to find one further from consciousness than the consideration of how the animals in their tanks eliminate waste materials. If this is even considered at all, it is thought of along the lines of, "The animal poops it out, and from then on the scavengers/detritivores get rid of it." This is, of course, a very concise way of thinking about the elimination of uneaten food from the digestive tract. Unfortunately, it has nothing at all to do with what biologists consider to be waste. Not to put too fine a point on it, but fecal matter is nothing more than uneaten, partially digested and processed food. Although relatively unsightly, and probably a bit messy, this stuff is generally not overtly poisonous. In fact, many reef animals, among them many popular reef aquarium fishes, are coprophagic. "Coprophagic" is the jargon term for what might politely be termed, "excrement-eating." Ah… Euphemisms are alive and well; I long for the directness of the old Angles and Saxons. However, the point is obvious; if a lot of animals eat the stuff, it isn't toxic. It is preprocessed food.

Actual waste materials are something else altogether. Strictly speaking, to a biologist, only a couple types of materials are truly waste materials. These are the byproducts of cellular respiration and protein metabolism, which in most animals, are carbon dioxide and ammonia, respectively. Carbon dioxide removal is governed by many of the same rules that dictate how ammonia is removed, but carbon dioxide is much less toxic than ammonia, and often its removal is coupled with oxygen uptake in animals' respiratory systems, which I will discuss in a future column. Ammonia removal is somewhat more complicated and is the topic of this column.

Amino Acids

Proteins have been called "the building blocks" of animals. They are large molecules comprised of subunits called amino acids. Around 20 amino acids are found commonly in nature, and well over 100 are less common. Amino acids are small molecules with a relatively straightforward basic structure. At one end of the molecule is an organic acid group. An acid is simply a molecule that releases a proton, or hydrogen ion (H⁺), in a solution. The most common organic acids contain a -COOH group, which ionizes in water to become -COO^- + H⁺. Vinegar is one of the simplest organic acids, and it can be represented by the formula, CH₃COOH. Adding one -CH₂ group to vinegar gives CH₃CH₂COOH, which has the common name of proprionic or propanoic acid.

The basic backbone of an amino is a proprionic acid molecule with an amine, or -NH₂, group substituted for one of the hydrogen atoms on the middle carbon atom, giving the formula CH₃HCNH₂COOH. This is the formula for one of the simplest amino acids, alanine, and it has the typical amino acid structure, which is shown below.

Proteins are assembled in a cell by chemically bonding a great many amino acids, with the structure and properties of all proteins ultimately determined by their amino acid sequence and how these molecules are folded into complex shapes. In living organisms, all larger molecules have a finite "lifetime," after which they are disposed of. When proteins are broken down, enzymes slice them back down into their component amino acids. These amino acids may or may not be further broken down and their constituents harvested for new uses in the cell. If they are broken down completely to their component parts, the critical part of each amino acid is the amino group. All other parts of amino acids can be recycled and reused by animals, but the amino groups cannot be disassembled into nitrogen and hydrogen atoms; they remain together, and therein is the problem.

Ammonium Toxicity

During normal cellular metabolism, the various chemical constituents are constantly being recycled by breaking them down into their component parts and then reassembling them into something useful. However, amino groups are a dead-end in this regard. Unlike virtually all other chemical bonds, animals cannot break the nitrogen-to-hydrogen bond, and when the amino group is separated from any molecule in an animal during a catabolic reaction it immediately forms the exceedingly reactive ammonium ion, NH₄⁺. This ion is easily capable of combining with, and destroying, many vital and necessary cellular chemicals under the conditions found in cells. This extreme reactivity makes ammonium ions exceedingly toxic to many organisms, even in very low concentrations. Obviously, this toxicity is the major reason that aquarists monitor and attempt to minimize dissolved ammonia concentrations in their tanks. Dissolved ammonia gas is simply ammonium ion in water.

Ammonium ion is no less toxic to a single cell than it is to the whole aquarium, and the need to keep its concentration low is one of the driving forces of natural selection in many animal groups. The removal of ammonium ions from an organism is what biologists mean when they speak of waste elimination, or excretion, from living organisms.

Size Related Solutions

Excessive ammonium is no problem for the smallest animals, and for those acellular organisms commonly referred to as "protozoa." These organisms are so small that the ammonium ions simply diffuse out of the cell. Diffusion works in response to a concentration gradient, and as long as the ammonium concentration is lower outside the cell than in it, ammonium will diffuse out of the cell. This process is very efficient at moving materials in solution, but it works best over very small distances. At distances greater than a few small parts of a millimeter, diffusion rates begin to drop off and rapidly become too slow to prevent a rapid, and deadly, accumulation of ammonium ions.

The ammonium accumulation that occurs inside cells that are rapidly metabolizing is one of the factors limiting cells' maximum sizes. It is also of profound significance to aquarists. Many aquarium animals lack any specialized organs or structures for removing nitrogenous wastes such as ammonium ions. Although it might seem counterintuitive, this is true of many larger organisms as well as smaller ones. Most large and complicated animals have very efficient kidneys and can regulate nitrogenous wastes quite well, but many of the larger aquarium organisms, such as corals and sea anemones, have a very simple structure containing no excretory organs at all; nonetheless, they still mange to attain, apparently, a quite large size without suffering the deleterious effects of ammonium poisoning. The answer to this paradox, of course, is that looks are deceiving. While many corals appear to be large animals, their volume of living tissue relative to their entire size is quite small. Most corals are, essentially, very thin living tissue sheets layered onto a massive, non-living, calcareous skeleton. Such animals depend on their high surface area-to-volume ratio to provide enough surface area for the diffusion of materials such as ammonia and dissolved gases. Although it appears that sea anemones are large lumps of living matter, as a practical matter they also have a high ratio of surface area to volume. Most of the mass of a sea anemone is made of non-living, organic materials such as collagen and other fibrous materials located between the thin outer and inner living tissue layers.

Activity Forces Changes

Actively moving animals have higher metabolic rates than do sessile ones, and this means that animals that move are particularly size limited by their surface area-to-volume ratio. Unless they have some efficient way of performing gas exchange and removing poisons from their systems, they have to remain small. The bodily shape of the larger flatworms is thought to be a response to competing naturally selective pressures. These animals are essentially two-dimensional; although they may be quite long or wide, their thickness may approximate that of a piece of paper. There are a great many reasons why it is good for animals to be large. However, there are some significant problems to overcome before large size is attainable. One of the primary hurdles appears to be the development of a functional kidney and excretory system.

The largest free-living and active flatworms are about five feet (1.6 m) long, one inch (2.5 cm) wide and the thickness of a couple pieces of paper. These animals, like most of the larger flatworms, have an excretory system consisting of numerous internal structures variously called flame bulbs, flame cells, solenocytes or protonephridia. All of the names refer to similar structures. However, they were initially seen and described from different animals and were named independently by different biologists.

Figure 4. A diagram of a flame bulb cell. The top of the cylindrical portion to the left is continuous with an excretory tubule. The whole cell is about 1/100th of a millimeter in diameter. As the cilia in the cylindrical portion undulate or beat, water is moved through the perforations into the cavity around the cilia, and thence into the tubule and out of the animal. Wastes are carried along with the water (Modified from Kozloff, 1990).

These structures have several different designs, but basically they are blind-ending tubes with one or more ciliated or flagellated cells located at, or near, the blind end. The flagella or cilia beat inside the tube's opening. In a living animal, if viewed through a microscope, the beating of the cilia looks rather like the flickering of a candle flame, hence the name "flame bulb." The tube from the flame bulb connects eventually to the animal's exterior. Although the simplest flame bulb systems appear to be comprised of only one or two cells, in their most advanced forms, as seen in some large marine worms, the tubules may be quite long, comprised of thousands of cells and many dozens, to hundreds, of flame bulbs. The flame bulb cells are perforated near their base, allowing water to flow through them, and the flickering of the cilia causes water to move through the tubule to the pore's opening to the animal's exterior.

Generally, the interior contents of the animals possessing these protonephridia, a name meaning "first-kidneys," are more concentrated with dissolved materials than is their surrounding medium. This means that water flows into the animals through the process of osmosis. It often has been proposed that protonephridia and, indeed, other similar so-called "excretory organs," developed primarily to regulate animals' water balance. These organs may indeed be primarily osmoregulatory, but even so, much research has shown that they also function in a manner similar to vertebrate kidneys, albeit on a very small scale. Interior fluids arefiltered as they pass through the perforations in the flame bulb's cells. As the filtrate passes along the tubule, it is altered. Some materials, such as sugars, are selectively reabsorbed, while other materials, such as nitrogenous wastes, are actively secreted. What passes out of the excretory pore can be called a "urine," quite dilute compared to vertebrate urine, but a urine nonetheless.

Figure 5. The protonephridia, or flame bulbs, are often connected in complex networks, but these are often visible only with special microscopic techniques. At left above is a diagram of a freshwater planarian showing its gut and the protonephridial network; at right is a photograph, at the same scale, showing a marine planarian. Note that its gut is quite visible, but its protonephridia are not. Left image modified from Kozloff, 1990.

Figure 6. A small portion of the flame bulb network in a nemertean, modified from Kozloff, 1990. Nemerteans are among the largest animals with protonephridial networks, and these networks are very complex. They surround blood vessels, and the ducts adjoining them connect and dump into larger "kidney ducts" which have pores to the animal's exterior. These animals may have many thousands of flame bulbs imbedded in the tissues around their circulatory system.

Larger Size Brings Larger Problems

Protonephridial kidneys appear to work fine for many smaller animals, but larger animals seem to have replaced them with a different type of organ, called a metanephridium or funneled organ. As with protonephridia, metanephridia have a tubule connecting the interior business end with "The Great Out-of-Doors." Instead of a cell or small group of cells that create a flame bulb in the blind-ending tube, the metanephridium ends in an open funnel with a ciliated outer rim, called a "nephrostome," a term that means "kidney-mouth." The microscopic hair-like cilia surrounding the rim beat, thereby forcing fluid and particulate matter into the tubule. This tubule also has areas of active secretion and resorption so that the product passing through the excretory pore is a true urine. Metanephridia may also have a storage bladder. One thing that a metanephridium can do that protonephridia cannot is to sweep small particulate materials, such as bacteria, into their funnel, and thence eventually out of their body. It is, in fact, an all-purpose excretory organ, removing both solid and dissolved wastes.

Metanephridia are found in most of the larger, more complex animals. They reach their largest size in clams such as Tridacna, where they are called either kidneys or nephridia, but they are nonetheless modified metanephridia. Organs structurally identical to metanephridia but having a different name, fallopian tubules, are found in the mammalian reproductive system. Even the specialized and highly derived excretory organs of the cephalopods are thought to have been derived from metanephridia.

Bugs

Arthropods do things much differently. Beating cilia are lacking throughout the arthropods, probably as a function of both their solid exterior integument and their relatively high internal fluid pressure. As a consequence, they don't have either proto- or metanephridia; all excretion appears to be wholly glandular. The main arthropodan groups, the insects, the crustaceans, and the chelicerates, all appear to have different excretory organs. While I will discuss only the crustacean system here, the insect system is much better known and anyone who is interested in this topic as it relates to insects should consult a reference book dealing with insect physiology. The openings in the crustacean excretory system vary in position between the different groups, but are often found in the head region. Although such glands, called antennal or coxal glands, have been known for years to be osmoregulatory, and have been considered for a long time to be excretory, little consistent physiological evidence supports the latter contention. Until recently, these glandular structures have been considered to be derived from metanephridia, and their function related to them; however, given some of the recent discoveries indicating that no arthropodan animals are derived from an animal that could have possessed a metanephridium or any of its precursors, it has become increasingly clear that excretion in marine arthropods needs a lot of study before any generalizations can be made.

A Wasteful Subject

Although the study of excretory systems may seem far removed from the reef aquarium hobby, it is important to remember that one of the primary problems in our systems is the accumulation of dissolved nutrient materials. These may accumulate from a great many varied sources, but probably the major source of most of the dissolved nutrients in reef aquaria is the nitrogenous waste produced by protein metabolism in the reef's animals. Some points need to be made considering this material. In a reef with a thriving community of small organisms, the "clean-up crew," if you will, very little added food is going to be subject to direct bacterial decomposition. Almost all of it will be eaten by an animal. As a consequence of that feeding, virtually all of the dissolved nitrogenous nutrient found in the system must have passed through an animal at least once.

From this state of affairs comes several important points. First, aquarists can't rapidly or directly alter dissolved nutrient levels by changing feeding regimes. The production of nitrogenous waste is not directly related to feeding, but rather the metabolism of the animals in the system. As long as these animals are still alive, they will produce nitrogenous wastes. Food reduction will result in there being less food to eat, but protein metabolism, the major source of nitrogenous waste will not be appreciably changed until the animals start to die. While this may eventually occur due to lowered nutrient levels, it will take a relatively long time. The fastest way to lower dissolved nutrients would be water changes coupled with significant removal of animal biomass. Second, lowering nutrient levels will cause animals to starve. Starving animals first metabolize energy storage chemicals such as fats and sugars, but during this period, normal protein metabolism will continue relatively unabated. Then they start to metabolize their muscles and connective tissues. This may mean that after a prolonged period of starvation, the dissolved nitrogenous nutrient levels will actually rise. Subsequent to this, as the animals die, of course, the nutrient levels will ultimately drop. Consequently, the best way to control nutrients in animal-rich systems is to increase exports of nutrient-rich water. This is nature's way; nutrient-rich waters get flushed from the reef. The next best way, considering that aquarists don't have an unlimited water system to flush things away, is to provide some way to convert the nutrient into biomass (algae or animal) and export that, or to find some way to increase nitrogenous nutrient utilization by bacteria, thus allowing the excess nitrogen to leave the system as a gas.

References:

Kozloff, E. N. 1990. Invertebrates. Saunders College Publishing. Philadelphia. 866 pp.

Prosser, C. L. Ed. Environmental and Metabolic Animal Physiology. Wiley-Liss, Inc. New York. 578 pp.

Ruppert, E. E, R. S. Fox, and R. D. Barnes. 2003. Invertebrate Zoology, A Functional Evolutionary Approach. 7^th Ed. Brooks/Cole-Thomson Learning. Belmont, CA. xvii +963 pp.+ I1-I 26pp.