Ozone has been used in reef aquaria for many years. It is claimed to have many benefits, ranging from increased water clarity to decreased algae. It has never, however, risen in popularity to the point where a seeming majority of reef aquarists use it. Many reasons likely prevent its widespread use, including its cost, complexity and safety concerns for both the aquarist and the aquarium's inhabitants. Speaking only for myself, my reasons for never having used it in my first ten years of maintaining reef aquaria were driven primarily by concern over ozone byproducts' toxicity in the aquarium, and the lack of a perceived need.

Back in the early to mid 1990s there was a fair amount of emphasis on ozone and other oxidizers as a way to raise the water's ORP (the oxidation reduction potential). The ORP, in turn, was incorrectly described as a good way to measure the water's "cleanliness." So aquarists raised ORP. Then ozone and other oxidizers (such as permanganate) fell out of favor for a variety of reasons, not the least of which was the overall trend toward less technological approaches to reef maintenance.

It appears, however, that the use of ozone may be on the upswing. In a recent (December 2005) survey I did of experienced reef aquarists, the results were equally split between those who had never tried it, and those who were presently using it or who had in the past and would do so again in an appropriate aquarium. For most people who had used it, the emphasis is now on water clarity, not ORP as some surrogate of something that was vaguely defined but that was supposed to be beneficial.

This article is the first in a series that addresses the myriad issues around the use of ozone in reef aquaria. The articles should help aquarists understand why ozone is used and what molecular level processes take place when using ozone. Together, they should help aquarists determine for themselves if ozone is something they want to use, and if so, how to do so.

The articles are:

Ozone and the Reef Aquarium, Part 1: Chemistry and Biochemistry
Ozone and the Reef Aquarium, Part 2: Equipment and Safety
Ozone and the Reef Aquarium, Part 3: Changes in a Reef Aquarium upon Initiating Ozone

After a brief introduction to how ozone is used and some of its claimed benefits, this first article proceeds to describe what ozone is and how it reacts with seawater. It also relates ozone's perceived benefits to the actual chemical and biochemical changes that it can cause. In a sense, it provides the mechanistic framework for understanding why ozone does what it does, helps aquarists understand its limitations and details the changes in the aquarium water that ozone will cause, whether they are apparent to most aquarists or not (and, in fact, many are not).

The subsequent articles in this series will address the types of equipment necessary to effectively and safely use ozone, and the benefits that accrue upon initiating ozone in an aquarium system (mine) that had been operating for many years without it.

The sections of this first article are:

• What is Ozone Supposed to Accomplish in a Reef Aquarium?
• How is Ozone Used in Reef Aquaria?
• What is Ozone?
• Ozone and ORP
• What Happens to Natural Ozone in Natural Seawater?
• What Happens When Ozone is Applied to Seawater?
• Halogens
• Ammonia
• Iron
• Oxidation of Organics by Ozone: Decoloration
• Oxidation of Organics by Ozone: Skimming and Nutrients
• Ozone and Problem Algae
• Ozone Reduction of Organic Toxins in the Water
• Reducing Bacteria When Using Ozone
• Reducing Other Pathogens When Using Ozone
• Toxicity of Ozone Produced Oxidants (OPOs)
• Toxicity of Bromate
• The Effect of Activated Carbon on Ozone Produced Oxidants
• Removal of Bromate by Biological Means
• Conclusion
• References

What is Ozone Supposed to Accomplish in a Reef Aquarium?

I've asked many aquarists what they believed dosing ozone accomplished in their aquaria. The list is always headed by increasing water clarity, but also includes other possibilities. Below, in no particular order, are the sorts of claims that are made:

1. Increased water clarity (even if it had been very clear before ozone)
2. Increased light penetration
3. Decreased yellowness
4. Decreased algae
5. Decreased cyanobacteria
6. Decreased skimmate production
7. Increased skimmate production
8. Increased ORP
9. Reduced nitrate
10. Decreased pathogenic bacteria
11. Reduced circulating toxins
12. Cleaner (more pure) water

Some of these make perfect sense, and the chemical and biochemical mechanisms that cause them through ozone's use will be detailed in the subsequent sections of this article. Others may not be correct assertions (decreased pathogenic bacteria, for example) and these issues are also discussed.

Some instances of apparent problems and perhaps underlying issues with the use of ozone are subtle enough that most aquarists never notice them. Bleached corals, for example, are obvious and have been reported. Perhaps the bleaching that has been experienced is related to a rapid increase in light penetration. But suppose that some small invertebrates in the aquarium were less prone to successfully reproduce due to residual bromate in the water. Or that the incidence of fish cancers from bromate (a suspected carcinogen) increased from, say, 1% to 2% for some particular fish species. How many reef aquarists would notice those changes, or attribute it to the ozone, even if it were true?

On the other hand, many aquarists might not particularly care about such subtle issues, and want the water to be clearer regardless of hypothesized problems. In any case, the data such as they are will be presented and aquarists can decide for themselves if ozone use is a practice they want to pursue or not. At the end of the last article in the series, where I present the results in my aquarium, I'll comment on whether I think it is desirable to continue using it or not in my system.

How is Ozone Used in Reef Aquaria?

How ozone is used will be the primary topic of the second article in this series, but in order to understand many of the issues presented in this article, it is necessary to have a rudimentary understanding of how ozone is used.

The pathway for ozone entering an aquarium starts with an ordinary aquarium air pump. The air travels out of the pump and often into an air dryer. The air's moisture is removed as it is absorbed by very hygroscopic solids. Not all aquarists perform this step, but removing the air's moisture has at least two benefits as the air passes into the next stage of the process. The next stage is a small device that generates ozone. The method used by most ozone generators is to pass the air through a high voltage electric discharge that breaks apart some of the oxygen (O2) molecules, and when they recombine, some ozone (O3) is formed (a second, less effective method uses UV light to accomplish the same process, either by passing air or the water itself past a UV light source). Moisture in the air reduces the amount of ozone formed in the generator, and it also results in the formation of nitric acid (HNO3; from water and nitrogen gas in the air). This nitric acid can reduce pH and alkalinity, and provides nitrate to the aquarium (which will be discussed in further detail next month).

After the ozone-containing air passes out of the ozone generator, it usually is sent into some sort of mixing chamber where aquarium water and the gas are mixed well, and are kept in contact for at least a few seconds. Aquarists often use skimmers or specially made ozone reactors for this purpose, and selection of suitable materials is a concern as the ozone can degrade some types of plastic, rubber and tubing. The amount of ozone delivered varies widely. Many manufacturers recommend on the order of 0.3 to 0.5 mg/hour per gallon of aquarium water, but many aquarists use less, or do not use it all of the time. They believe that using less ozone achieves their need for clearer water, reduces the need for more expensive equipment and air dryers, reduces concerns about toxicity due to byproducts and reduces its negative impact on skimming.

Inside the contact chamber, the ozone reacts with many different chemicals in the seawater including organics, ammonia, iron and other metals, bromide and iodide. It may also interact with viruses, bacteria and other organisms drawn into the chamber. The ozone itself survives for only a few seconds in seawater, but it leaves other reactive oxidizers (called ozone produced oxidants, OPO; for example, hypobromous acid, BrOH) in its wake. These can further react with organics and other materials and are also potentially toxic, so they should be removed before the water is released to the aquarium. Much of ozone's benefits happen in this chamber, where, for example, the water is made "clearer" as certain pigments in dissolved and particulate organic molecules are destroyed.

Water leaving the reactor is optimally passed over an amount of activated carbon sufficient to remove the remaining ozone produced oxidants. The carbon catalytically (and also noncatalytically) breaks down these oxidants before they enter the aquarium. The air passing out of the reactor also contains ozone, and is also best passed over activated carbon to reduce the aquarist's concern for airborne ozone's toxicity.

In order to ensure that not too much ozone enters the aquarium, aquarists should monitor ORP (the oxidation reduction potential) in the aquarium's water. For those aquarists using a small amount of ozone, monitoring may be adequate. For those aquarists using large amounts of ozone, an ORP controller is important. It can be used to shut off the ozone if the ORP rises above a set point (that point being either an emergency shut-off point that is rarely, if ever achieved, or a target ORP where the generator is actually running only part of the time, and only when the ORP controller says that ORP needs to be raised to the set point).

For comparison to other studies reported in this article, reef aquarists typically use up to about 0.3 ppm ozone in the "contact chamber" and have contact times on the order of a few seconds before the water passes into the aquarium. This value of 0.3 ppm ozone is based on adding ozone at a rate of 100 mg/hour (a typical addition rate suggested by ozone generator manufacturers for a tank of about 200 gallons) to a contact chamber (like a skimmer) that has a flow of 333 L/h; 100 mg/h / 333 L/h = 0.3 mg/L). Higher flow rates, lower ozone addition rates or incomplete transfer of the ozone into the water will give lower ozone concentrations in the contact chamber or skimmer.

All of these aspects of ozone use in reef aquaria will be explored in more detail next month.

What is Ozone?

Ozone (O3) is a gas at room temperature, but is not stable enough to be stored in a bottle. Because it is unstable, aquarists always generate it on site just before use. The mechanisms for generating ozone will be detailed in this series' next article, but in short, ozone is generated by splitting apart oxygen molecules (O2) from the air and letting them recombine into ozone.

At low temperatures (below -180° C), ozone can be condensed to a dark blue liquid. It has a pleasant, sweet odor which permits aquarists to detect when it is being formed or released, although it is also potentially toxic. When added to seawater, it has a very short half life of only a few seconds before it breaks down.

Ozone consists of three oxygen atoms connected in a bent line (with an O-O-O angle of ~117° ), while regular diatomic oxygen consists of two oxygen atoms connected (O2). Diatomic oxygen is much more chemically stable than ozone. That is, in part, why ozone is such a strong oxidizing agent. O2 comprises about 21% (210,000 ppm) of the atmosphere at sea level, while ozone comprises only a very tiny fraction (typically about 0.05 ppm).

High in the atmosphere (above about 30 kilometers), light from the sun breaks apart diatomic oxygen molecules into monatomic oxygen atoms (O), and that form predominates at all altitudes above about 150 kilometers. At altitudes between 30 and 90 kilometers, when O is formed, it often collides with an O2 molecule and produces ozone (O3). That is the atmosphere's "ozone layer." For a variety of reasons, the actual ozone concentration peaks at about 50 km. It is a strong absorber of UV light with wavelengths between 200 and 310 nm. It is a far stronger absorber of UV light than are other gases in the atmosphere. Consequently, it helps shield the lower atmosphere and the earth's surface from UV radiation.

Ozone also can be formed in the lower atmosphere and is generally considered a part of "smog." In this case, much of the ozone is produced when nitrogen oxides (NO and NO2) from fossil fuel combustion break down to release monatomic oxygen (O). As at higher levels in the atmosphere, this O reacts with O2 to form ozone. Unfortunately, ozone is much less desirable at lower elevations, where people and other organisms that breathe it can experience lung damage. When I was a boy growing up in California's San Fernando Valley, the sky was often fringed in a brown haze of smog. After vigorous exercise, my lungs would often hurt when breathing deeply. That effect is one of elevated ozone's undesirable attributes to humans.

The second article in this series will deal with ozone's health effects in more detail, but it is worthwhile to show some basic information on ozone concentrations here. For many, the potential undesirable human health effects may be sufficient to choose to not use ozone in the home for that reason alone:

Ozone and ORP

One of the first things that all aquarists learn about ozone is that it raises the water's oxidation reduction potential (ORP). But what does that really mean? In fact, natural seawater's ORP is a very complex issue, and it is not well established what has actually changed in seawater when its ORP rises or falls by a small amount. It may be that the exact ratio of the more reduced forms of iron and manganese (those being Fe⁺⁺ and Mn⁺⁺) decreases as ORP is raised, and more oxidized forms (those being Fe⁺⁺⁺, MnO₂, etc) increase.¹ Is that something that aquarists care about? Is it beneficial?

A previous article has detailed the issues around ORP's measurement and what it means in seawater and reef aquarium water:

ORP and the Reef Aquarium
http://www.reefkeeping.com/issues/2003-12/rhf/feature/index.php

Aside from water's exact chemical properties that lead to ORP, ORP is an indicator of the balance of oxidation and reduction reactions taking place in seawater. Many of those reactions will be strongly influenced by adding a strong oxidizer such as ozone and its chemical byproducts (bromate, hypobromite, etc.). In that sense, determining the aquarium's ORP level is useful to aquarists using ozone to ensure that they do not overdose the ozone.

With sufficient ozone addition, the water will be filled with highly oxidizing chemical species and the aquarium's inhabitants themselves will begin to be oxidized by these species in the water. At high enough levels, these processes will kill organisms, and it has done so in significant overdoses. Many aquarists choose to use a particular ORP value as a target for the amount of ozone to add. In my opinion, the most important way to use ORP is to stop the ozone addition if the ORP rises too much. In part this opinion is based on the lack of a direct relationship between the water's "quality" and the ORP itself when using a chemical oxidizer. There is, however, a clear relationship between excessive ORP (say, above 500 mV) and harm to organisms.

Fortunately for aquarists, many of ozone's benefits, such as increased water clarity and decreased yellowness, can be attained without the ORP reaching excessive values. Often the water can become visibly clearer (to the point where the aquarist simply no longer notices the water in a normal sized aquarium) with the ORP hardly above 300 mV. On the other hand, whether undesirably high levels of certain ozone byproducts are in the water at those acceptable ORP levels has not typically been studied. What information exists will be detailed in subsequent sections of this article. The next article in this series will expand significantly on how to use ORP with ozone in reef aquaria.

What Happens to Natural Ozone in Natural Seawater?

Ozone is not significantly generated in the ocean, but it does get deposited into the ocean from the air. At the low concentrations of ozone that get deposited that way, and at the natural concentrations of iodide usually present in seawater (much higher than ozone), the ozone can react with the iodide present with a half time of less than a tenth of a second.² In this example, the iodide is oxidized to hypoiodate (IO^-) and hypoiodous acid (HOI):

O₃  + I^-  à  IO^- + O₂

IO^- + H₂O  à  IOH + OH^-

Because hypoiodous acid's pKa (in freshwater) is 10.4, it is largely in the protonated (uncharged form) in seawater.³ The hypoiodous acid is itself a strong oxidizer and can go on to react with other organic or inorganic materials.⁴ It has also been suggested that very low levels of molecular iodine (I2) may be generated in this way in a thin layer on the ocean's surface (0.0002 ppm, or 0.3% of the total iodine).⁵

One of this reaction's implications is that the use of ozone will skew a reef aquarium's iodine speciation, and this is detailed in the next section.

What Happens When Ozone is Applied to Seawater?

Halogens

When ozone is applied in seawater in concentrations higher than are naturally present, a larger variety of chemical reactions take place. Chief among these is oxidation of bromide to hypobromite:^6,7

O₃  +  Br^-  à  BrO^-  +  O₂   

BrO^-  +  H₂O  à  BrOH +  OH^-

The first reaction is very fast, and the half life of unreacted ozone in water with a lot of bromide (such as seawater) is on the order of a few seconds.⁸ Because hypobromous acid's pKa (in freshwater) is about 9, it is primarily in the protonated (uncharged form) in seawater, but a significant amount of BrO^- is also present.³ The hypobromous acid is itself a strong oxidizer and can rapidly oxidize other organic or inorganic materials.⁴

The hypobromous acid can also react in a variety of ways (including disproportionation and additional oxidation with ozone) to form bromate:

BrOH  à  à  à  BrO₃^-

The hypobromous acid can also be catalytically broken down by ozone to return to bromide:

BrOH + O₃ à 2O₂ + Br^- + H⁺

About extensive ozonation of seawater, one group concluded:

"Ozonization of seawater oxidizes bromide ion to Br (hypobromous acid and hypobromite ion) and then to bromate. If seawater is ozonized for >60 min, essentially all bromide is converted to bromate."⁹

That level of ozonation, however, is far more than would take place in a reef aquarium. The various reactions leading to bromine-containing byproducts of water's ozonation have been extensively studied (especially in the context of disinfecting fresh drinking water that contains bromide). Nevertheless, it is a complex problem. One recent review³ stated:

"Because bromate formation during ozonation of bromide-containing waters is a highly non-linear process, kinetic modeling has been applied to improve mechanistic understanding and to predict bromate formation. The full model consists of more than 50 coupled kinetic equations which can be solved simultaneously with a computer code…"

and then went on to say,

"the predictive capabilities of such models for the ozonation of any water should not be overestimated."

Well, we won't try to calculate what happens in reef aquaria, but we will conclude that bromate and hypobromite may be significant.

Bromate is typically the longest lived after ozonation of bromide-containing water. It is, in fact, one of the biggest concerns with ozonation as a purification method for drinking water, because bromate is a suspected carcinogen. For this reason, the US EPA limits it to only 10 ppb in drinking water. So in considering the properties of the treated seawater in aquaria, both BrOH/BrO^- and BrO3^- must be considered.

There is at least one study in the literature of bromate in a seawater aquarium.¹⁰ Here the ozone was used for disinfection, so the doses used may be higher than many aquarists employ. I also do not know whether or how effectively they treated the post ozone water with activated carbon. Nevertheless, the bromate levels in the Living Seas exhibit at Walt Disney World's Epcot Center were tracked. The researchers studying this display found that bromate had risen to about 0.6 ppm (with nitrate at about 600 ppm). After adding a batch denitrifying system, the bromate and nitrate concentrations began to drop, suggesting a sink for bromate that might well exist in many reef aquaria as well (that is, in systems or locations where denitrification takes place).

The same reactive pathways that lead hypobromous acid to bromate will take hypoiodous acid to iodate.

IOH  à  à  à  IO₃^-

In the ocean, iodine's predominate form is iodate (IO3^-) with a smaller but significant fraction of iodide (I^-). These two forms' bioavailability to macroalgae and other organisms varies from species to species, but iodide is often more bioavailable than iodate. Regardless, the use of ozone will likely skew the fraction of total iodine toward iodate and away from iodide. That may or may not be important for reef aquarists, because the importance of iodine's availability from the water column to organisms kept in reef aquaria is undemonstrated, but it may have strong implications if test kits are used detect some species and not others.

This concern was studied by one group in the Smithsonian National Zoological Park's Department of Animal Health.¹¹ It claimed that fish need iodide in the water column in the form of iodide to make the hormone thyroxine. Regardless of whether that is true or not (that is, whether fish need iodine in the water or whether they can get it from food), they showed that seawater's ozonation to an ORP of 400 mV (equivalent, they claim, to the level attained by skimmer driven use of ozone) reduced the iodide concentration by more than half. Ozonation also decreased the concentration of organoiodine compounds, and raised iodate levels. In the aquarium itself, iodide and organoiodine compounds were not detectable when using ozone. They go on to suggest that iodide supplements might be beneficial in cases when ozone is used. Therefore the conclusion that "iodine is an unnecessary additive for reef aquaria," when that conclusion is based on success in aquaria not using ozone, may not extend to aquaria that heavily employ ozone.

As long as bromide remains in the seawater, the equivalent reaction of ozone with chloride

O₃  +  Cl^-   à  ClO^-  +  O₂

is unlikely to be significant as it is much slower than reaction with bromide. The small amount of ClO^- that may form can react with bromide to form BrO^-.^3,6,8

Ammonia

Another of ozone's potential reactions and its byproducts with inorganic compounds in seawater is with ammonia. In fact, ozone is quite effective at converting ammonia into nitrate. The reaction is fast enough that if sufficient ammonia is present in seawater, it will happen preferentially to reactions that lead to bromate.^3,12,13 An intermediate species in the process is bromamine (the bromine equivalent of chloramine), but fortunately (because it is toxic) it usually is further oxidized to bromide and nitrate.

BrOH + NH₃  à  NH₂Br 

NH₂Br  +  O₃  + 2OH^-  à  NO₃^-  +  Br^-  +  2H₂O

Presumably it is not harmful, and may be beneficial to reduce the ammonia to nitrate more rapidly. It may lead to higher nitrate concentrations in the aquarium, however, and may also lead to a different ratio of nitrogen export via different mechanisms because some methods (such as growing some species of macroalgae) prefer ammonia over nitrate.

Iron

Iron can be present in two primary forms in seawater: ferric ion (Fe⁺⁺⁺) and ferrous ion (Fe⁺⁺). Ferric ion is the more stable form in oxygenated seawater, but ferrous forms may remain for a substantial period before being oxidized to ferric ion. The ferrous form is more readily taken up by many organisms (including people), partly because it is more soluble and partly due to biological membrane transport mechanisms. But many organisms can convert ferric ion into ferrous ion on their surfaces just as they are taking it up, so the importance of the exact form is not entirely clear. I dose ferrous ion when adding iron to my aquarium.

Ozone can readily convert ferrous ion into ferric ion.^14-16 That oxidation may, in fact, be part of what is actually measured in seawater's ORP changes. The conversion may be even faster for complexed ferrous ion than for free ferrous ions in seawater, and the complexing to organics may be able to keep the ferric iron in solution even after oxidation.¹⁷

Finally, ozone may serve to break iron free from very strong complexes in which it is not readily bioavailable. Iron EDTA complexes, for example, may require photolytic cleavage to become bioavailable in aquaria without ozone, and oxidation of the complex with ozone may serve a similar purpose.

Oxidation of Organics by Ozone: Decoloration

The oxidation of organics is, it turns out, the primary reason that reef aquarists use ozone because it is the organic material in seawater that causes clarity and color issues. Its impact on organic materials is also why ozonation impacts skimming. While most organic compounds that are exposed to enough ozone for a long enough period will be oxidized in some way, some are very much more sensitive than others. In fact, at the levels of ozone attained in a typical reef aquarium contact chamber (less than about 0.3 ppm ozone) or even disinfection applications where the doses are much higher, the total dissolved carbon does not appreciably change during the ozone exposure (although it may later if bacteria find the newly oxidized organics more bioavailable; see below).

In a marine mammal pool,¹⁸ for example, it was found that disinfection with 4 ppm ozone with a 30 minute contact time (a disinfection level much higher than is typically used in reef aquaria) did not reduce the pool's total organic carbon (TOC) (~13 ppm TOC), while the use of granular activated carbon (GAC) did reduce it by 37%. Interestingly, the combination of ozone and GAC was even more effective, removing 60-78% of the TOC, suggesting that the ozonation may have altered some of the molecules in a way that made them bind more strongly (or more rapidly) to GAC. An alternative explanation that cannot be ruled out involves biological transformations of the organic compounds taking place on the GAC surface as it became colonized with bacteria).

One research group¹⁹ studying the reaction between a variety of organic compounds and ozone concluded:

"…comparisons of rate constants with chemical structures of the reacting groups show that all reactions of O₃ are highly selective…"

Fortunately, many of the organic compounds that are most reactive with ozone coincidently are those that aquarists want to eliminate from aquaria. As seawater ages in marine aquaria, the water often becomes yellow as a wide variety of different organic pigments build up. Because of the ozone's reaction with many natural pigments, it is often used in drinking water purification for the purpose of "decoloration;" not organic removal per se, but decoloration.²⁰

In order to understand this effect, it is first instructive to understand which organic molecules lead to coloration, because not all of them do. In fact, most organic molecules are not colored. That is, they do not absorb visible light. Looking through bottles of purified organic compounds, the vast majority are white powders. Organisms, however, have a significant need to absorb light, for example, to photosynthesize or to see.

In order to generate molecules that absorb visible light, natural systems often turn to conjugated carbon-carbon double bonds. Figures 1 and 2, for example, show the structures of chlorophyll and b-carotene. Both of these molecules are widespread in organisms, and both contain conjugated double bonds that lead to the absorption of visible light. These figures do not show the hydrogen atoms (there are dozens of them), but all of the other atoms are shown, and there is a carbon at each intersection of two or more lines. This is how chemists often show structures, allowing the important features to stand out and not get lost in a clutter of atomic letters. What is important here is each segment with a C═C (shown in red). Without going into ridiculous chemical detail for a reef article, having a bunch of C═C bonds arranged together with a single C─C bond between them can lead to the absorption of visible light. That is why organisms have developed such chemical structures for the absorption of light despite their instability toward oxidation (see below).

It is just that instability, however, that aquarists take advantage of when employing ozone. Figure 3 shows, for example, where ozone first attacks oleic acid (a dietary fatty acid).^21,22 It is attacked at its double bond, breaking it apart into smaller fragments that no longer have a C═C bond. Consequently, while a huge dose of ozone lasting a very long time will break down these bits even more, even a small dose will remove the C═C bond.

Translating that reactivity to the pigments shown in Figures 1 and 2 makes it apparent why ozone is so good at reducing seawater's coloration and increasing its clarity: it reasonably selectively targets many of the structures that nature uses to absorb light, and converts them to nonabsorbing chemical structures.

A second type of colored organic compound that accumulates in seawater (in both the ocean and aquaria) is one of the functional groups in humic and fulvic acids (the compounds often identified as the yellowing agents in aquaria).²⁰ These "compounds" are complex mixtures of many compounds, but among them is the phenol functional group (Figure 4). Phenol can be attacked by ozone,^23,26 with breakdown products shown in Figure 4. It is the Ring-OH group that is colored when in the Ring-O^- ionized form, and many of these breakdown products lack such a functional group. Hence the oxidation of such phenolates in humic acids with ozone will reduce color in aquarium water.

The various chemical products described in this section are, of course, not the only reaction products of ozone, hypobromous acid and hypobromite with organic compounds. Other products include brominated organic compounds and many other chemical structures. These have not been fully elucidated, a fact which is not surprising since even in the absence of ozone, the nature of all of the organics in natural seawater or reef aquarium water remains poorly defined.

Oxidation of Organics by Ozone: Skimming and Nutrients

Another result of breaking some organics into smaller, more hydrophilic bits (Figure 3 and 4) is that it often increases their bacterial biodegradability.^27-29 Therefore, the ozone may need only to start the degradation process, and bacteria in the aquarium can finish off the organics by uptake and metabolism. Large humic acid molecules, for example, are converted by ozonation into smaller fragments that are more readily taken up and metabolized.²⁹ This process may, in fact, be why some aquarists report drops in nutrient levels after initiating ozone. It is not because ozone directly impacts either nitrate or phosphate (it does not react directly with either), but the newly bioavailable organics may drive bacterial growth, just as adding ethanol (e.g., vodka) or sugar might. The growing bacteria need nitrogen and phosphate, and if they satisfy those needs by taking up nitrate and phosphate, the levels of those nutrients in the water may drop. That effect, however, may be only temporary as the initial burst of new bioavailable organics winds down, and a new stable state is reached with lower levels of organic material and similar levels of inorganic nutrients.

Skimming is a complex process that has many subtleties. The previous sections have discussed how ozonation modifies organic molecules , and we can then extrapolate how those processes impact skimming. Years ago it was widely claimed that ozone use increased skimming, and I claimed then that I didn't see how that could happen directly. Most organic compounds likely to be found in significant quantities in a reef aquarium will become more polar and likely less skimmable after it reacts with ozone. Figure 3, for example, shows how oleic acid (readily skimmed) gets converted into more polar compounds that will not be so readily skimmed as they will not be as strongly attracted to an air water interface.

A small portion of organic molecules in reef aquarium water may become more skimmable if, for example, they become more hydrophobic after reaction with ozone. They may also become more skimmable if they were totally hydrophobic before ozone and were transformed into molecules with polar and nonpolar parts (called amphiphilic) which more readily absorb onto an air water interface and are skimmed out.

Are there other ways that skimming might be increased besides these two processes? I hypothesized in a previous article that it was due to the growth of bacteria (either in the water itself, or bound to surfaces), and possibly also the release of new organic molecules as they grew, that caused the effects some aquarists observed.

It seems as if the tide of opinion has turned, however, and most aquarists now claim that the amount of skimmate is reduced significantly when using ozone. Many claim that the collection of skimmate has nearly stopped in their aquaria when starting ozone. Why the difference compared to past opinion? That's hard to say, and may depend on the types and qualities of the skimmers available now compared to years ago, as well as changes in other husbandry practices. In any case, the overriding experience of many aquarists today is that skimming is reduced, and the presumed reason is that the organics are being made chemically less skimmable by ozone. The remaining organics would then be removed more by bacterial processes than before the initiation of ozone in the same aquarium.

Ozone and Problem Algae

Many aquarists report a reduction in problem algae when initiating ozone, although it is not universally observed. Whether it happens in my aquarium is one of the observations that I will report in the third article in this series. However, more people report it than I would expect if it were a simple placebo effect, where new users might be looking for a decrease in algae, so they "see" it. How might algae be decreased? The answer is not clear at all. No clear explanations were provided to me when I asked very experienced chemists who have used ozone in aquaria for many years. Nevertheless, this section provides some potential causes.

As described above, ozone breaks large organic molecules down into more bioavailable fragments. Perhaps using ozone to drive that process increases the rate of bacteria growth in the aquarium, and the bacterial growth consumes nutrients just as happens when aquarists dose organic carbon sources to aquaria to drive bacteria. This process would be related to the decrease in skimming, where organic molecules are no longer as effectively skimmed out. Where would they go? Into the hungry mouths of bacteria that then multiply faster, and consume nitrate and phosphate in order to produce the biomolecules of life (proteins, DNA, RNA, phospholipids, etc.).

Another, vaguer, explanation has to do with the ORP itself. It has been suggested that increased ORP hampers the growth of microalgae relative to macroalgae and other organisms that aquarists maintain. There may be a bit of the chicken vs. the egg argument here, where it is not clear if the lower ORP drives the algae (by altering the availability of metals such as iron, for example), or if the algae drives a lower ORP (by releasing large amounts of organic molecules, for example). In any case, raising the ORP may well alter the bioavailability of important metals such as iron. In fact, even without raising ORP, ozone may break down strong metal/organic complexes, increasing the bioavailability of the metal. In either case, ozone may tip the delicate balance of nutrient flow away from microalgae and toward other organisms (macroalgae, bacteria, corals, etc).

Ozone Reduction of Organic Toxins in the Water

In addition to the water's decoloration, another potential benefit of the ozone's reaction with aquarium water is the destruction of organic toxins. Many marine creatures secrete toxins that are designed to be harmful to other organisms. If these are allowed to build up in aquaria, they might become stressful for certain organisms. In addition to using activated carbon and skimming to remove them, ozone may also play a useful role.

As discussed above, ozone's reaction with organic molecules involves fairly specific types of reactions, and it does not remove all organic materials from the water passing through the contact chamber. However, many toxins have very specific structures, being toxic specifically because they fit exactly into or onto some important biomolecule in a living organism, thereby interfering with its normal activity. Even a small chemical change will likely reduce the toxicity of even a very potent natural toxin..

As an application of this principle, ozone has been used to remove toxins from water,^30-33 including natural marine toxins.³⁴ Ozone has been shown, for example, to detoxify botulinum toxin in freshwater at concentrations of 0.01 ppm ozone and a contact time of less than a minute.³²

Does ozone's reaction with organic toxins impact reef aquaria? Unfortunately, it isn't possible to answer that. It isn't even known if such toxins ever become significant in reef aquarium water. If they do, the answer will depend on the exact structures of the particular toxin(s) involved. Ozone may be beneficial from this standpoint, and it is very unlikely to make such problems worse, but using activated carbon may be a more effective method than ozone for toxin removal.

Reducing Bacteria When Using Ozone

Bacteria and other organisms suspended in water can be killed by adequate exposure to ozone. That process is widely used to disinfect drinking water and wastewater in a variety of applications. The doses and exposures of ozone required for disinfection, however, are quite high. They are higher than are used in reef aquarium applications, where typical doses of ozone range up to about 0.3 ppm in typical contact chambers, and last for only a few seconds. Consequently, aquarists must be careful when translating disinfection literature to reef aquarium effects.

In a recent study of a recirculating seawater system,³⁵ the dosing of 0.52 ppm of ozone was tested for its ability to decrease the system's bacterial load. That dose is similar to a 300 mg/hr ozone unit applied to a typical small skimmer flow rate of 150 gallons per hour (568 L/h). In this experiment, the levels of suspended bacteria (both Vibrio and coliform) were analyzed in a variety of locations (intake, pre-ozone, post-ozone, pre-tank, and post-tank). In no case was there a statistically significant reduction in bacteria. Even the addition of a venturi injector to the contact chamber did not adequately help (although it trended toward fewer bacteria, the result was not statistically significant). For comparison purposes, at higher ozone concentrations and contact times (5.3 ppm ozone for 240 minutes), Vibrio vulnificus is easily killed, with fewer than one in a hundred million of the initial bacteria remaining.³⁶

How much ozone, and for how long, is required to kill suspended organisms in seawater? In one study of a suspended dinoflagellate algae (Amphidinium sp. isolated from Australia's Great Barrier Reef), it was found that 5-11 ppm ozone for six hours of exposure was required to kill 99.99% of the organisms.³⁷ While that kill rate is impressive, that exposure is far higher than is ever achieved in a reef aquarium application. Lower doses and shorter contact times had smaller effects. A dose of 2 ppm and a short contact time (with the time not stated in the paper) showed a reduction in bacteria of abut 98% (which is still quite significant, but would not be referred to as disinfection).

Similar results were found for the spores of the bacterium Bacillus subtilis.³⁸ In this case, doses of 14 ppm ozone for 24 hours were required to kill 99.99 percent of the spores. In another study 99.9% of fecal coliforms, fecal streptococci and total coliforms were killed with 10 ppm ozone and a contact time of 10 minutes.³⁹ The exposure of Vibrio species and Fusarium solani (bacteria that are pathogenic to shrimp) to 3 ppm ozone for five minutes killed 99.9% of the bacteria.⁴⁰ Water from a seawater swimming pool was effectively sterilized using 0.5-1.0 ppm ozone in a contact tower.41

The data for the disinfection of freshwater systems are much more extensive, and so include more data at lower contact times and concentrations. In one experiment at a Rainbow trout hatchery, the addition of 1-1.3 ppm of ozone with a contact time of 35 seconds reduced heterotrophic bacteria in the aquarium water itself by about 40-90%.⁴²

Does the ozone used in a typical reef aquarium application reduce bacteria? Maybe, but certainly not to the extent required for disinfection. Still, a reduction of 50% of the living bacteria could have significant effects. The above study in the trout hatchery showed that the use of ozone at several times the typical reef aquarium rate and for about five to ten times the typical contact time results in such a drop. While the data are unavailable, I expect that the bacteria in the water exiting a normal reef aquarium's ozone application are not decreased by as much as 50%.

It seem reasonable to conclude from such literature studies that most bacteria that enter the ozone reaction chamber in a typical reef aquarium application will not be killed by ozone or its byproducts. If killing bacteria in the water column is a goal, then a UV (ultraviolet) sterilizer may be more useful.

Reducing Other Pathogens When Using Ozone

There has been extensive analysis of the amount of ozone needed to kill the human pathogen Cryptosporidia parvum in freshwater. Most such studies are looking for significant disinfection, but some data points show the effects at lower doses and contact times, and some researchers have developed models that suggest the amount of killing at any dose/time combination.⁴³ For example, at 22° C approximately 63% of the organisms would be expected to be killed at 1 ppm ozone with a contact time of one minute. The contact times and concentrations are inversely related, so at a contact time of six seconds, the required dose to kill 63% is on the order of 10 ppm ozone. At 0.3 ppm ozone and a six second contact time, typical for the high end of reef ozone applications, less than 5% of the organisms would be expected to be killed.

Many viruses are much easier to inactivate with ozone than are other pathogens.⁴⁴ Enteric adenovirus, for example, is inactivated to the extent of 99.8% after exposure to 0.5 ppm for 15 seconds.⁴⁴ Feline calicivirus is inactivated to the extent of 98.6% after exposure to 0.06 ppm for 15 seconds.⁴⁴ Poliovirus type 1 was inactivated to 99% within 30 seconds of contact time at 0.15 ppm ozone.⁴⁵ Hepatitis A virus was inactivated to the extent of 99.999% within one minute at 1 ppm ozone.⁴⁶ Norwalk virus was inactivated by 99.9% in 10 seconds of contact at 0.37 ppm ozone.⁴⁷ Adenovirus type 2 was inactivated by 99.99% by 0.2 ppm ozone with a contact time of about one minute.⁴⁸

The eggs of a pathogenic helminth (Ascaris suum) were killed to the extent of 90% by exposure to 3.5-4.7 ppm ozone for one hour. One additional hour of exposure killed the remainder.⁴⁹

It seems reasonable to conclude from such literature studies that many viruses that enter the ozone reaction chamber in a typical reef aquarium application may be killed by ozone or its byproducts. Larger pathogens, however, are likely much more resistant to ozone, and are unlikely to be killed. For such ends, a UV sterilizer may be more useful, but still may not be completely effective.

Toxicity of Ozone Produced Oxidants (OPOs)

Two sorts of toxicity studies of ozone produced oxidants (OPOs, such as bromate, hypobromous acid, etc.) are relevant to reef aquarists. The first involves the testing of seawater that has been exposed to ozone, and the second involves the testing of specific compounds dissolved in seawater that are known to form when using ozone. Most of the OPOs are unstable, and so have few or no specific toxicity studies. Bromate (BrO3^-) is the notable exception, and its toxicity is examined in the next section.

Much of the study of OPOs stems from applications slightly different from aquaria, and such studies must be viewed in that light. Often they relate to aquaculture facilities, where ozone is used at high doses to sterilize the water. Other studies are done on the disinfection of wastewater using ozone, another high dose application. Bear in mind that OPOs in reef aquarium applications will be at a maximum of about 0.3 ppm in typical reaction chambers, and will be lower (hopefully, much lower) once the water passes over activated carbon (assuming it does) and finally enters the aquarium. The concentration of OPO is always given in terms of the weight of ozone that produces that amount of oxidant.

In terms of the toxicity of ozonated seawater itself, one group concluded that fish were relatively insensitive to OPOs:

"Ozonation of estuarine or marine waters can produce significant amount of bromate…Toxicity studies showed that the concentrations of bromate which theoretically could be formed in an ozonated discharge were not toxic to the early life stages of striped bass (Morone saxatilis) and juvenile spot (Leiostomus xanthurus)."⁵⁰

Larvae are, in general, more sensitive to OPOs than are eggs,⁵¹ adults or juveniles.⁵² Japanese flounder eggs were found to be impacted by OPOs to the extent that 50% did not hatch after one minute of exposure to 2.2 ppm OPO. Larvae aged 3-15 days were killed to the extent of 50% in 24 hours at 0.02-0.05 ppm OPO. Larvae aged 44 days were killed to the extent of 50% in 24 hours at 0.15 ppm OPO. In this case, the larvae were shown to have damage to their branchial tissues.⁵³

The eggs and larvae of Japanese whiting (Silago japonica) also have been tested for toxicity by OPOs. In this case, half of the eggs and larvae died in about 24 hours when exposed to 0.18 and 0.23 ppm OPOs, respectively.⁵⁴

Certain microalgae are also relatively insensitive to OPOs (perhaps to the disappointment of many aquarists). The growth of the microalgae Tetraselmis chuii was found to be unaffected at levels up to 0.7 ppm.⁵⁵ At 1 ppm, growth was impacted negatively.

Toxicity tests of OPOs on shrimp show them to be less sensitive than fish. Penaeus chinensis and Paralichthys olivaceus were found to live up to 48 hours at OPO concentrations of more than 1 ppm, while Bastard halibut (fish) in the same study lived only three hours at 1 ppm and 48 hours at 0.13 ppm.⁵⁶

As for other organisms, the damage to the American oyster (Crassostrea virginica) by OPOs varied with their age. Even for adults, fecal matter accumulation was reduced at OPO levels as low as 0.05 ppm.⁵⁷

The effect of OPOs on rotifers (Brachionus plicatilis) has also been determined.⁵⁸ No effect on survival was seen at less than 0.22 ppm OPO, but effects became significant above that level. The authors point out that bacteria and other pathogens can be killed at that level, so rotifer cultures can be used with that amount of continuous ozone to reduce bacterial contamination.

Are these levels of OPO toxicity important to reef aquarists? That is difficult to answer without knowing the levels that are attained in reef aquaria. In a typical ozone application in reef aquaria that might produce 0.1-0.3 ppm OPO in a reaction chamber, the levels are quite significant compared to potential toxicity to fish larvae and other organisms at as little as 0.02-0.05 ppm. After passing the reactor's effluent over activated carbon, the OPO concentrations should be much lower, but exactly how low is unclear and will vary considerably in different setups.

Toxicity of Bromate

The toxicity of ozone and bromate at "natural" levels in the ocean has been assessed and usually found to be minimal.⁵⁹ Few studies have examined the toxicity of excess bromate itself to marine organisms.⁶⁰ One review article concluded:

"Bromate toxicity tests on marine animals indicate the levels of bromate produced by chlorination or ozonation of power plant cooling waters are not acutely toxic. The LC50 ranged from 30 ppm bromate for Pacific oyster, Crassostrea gigas, larva to several hundred ppm for fish, shrimp and clams."⁹

One individual study showed that Pacific oysters (Crassostrea gigas) had abnormal larval development at bromate levels of 30-300 ppm.^61,62 Fertilized eggs of the oyster Crassostrea virginica were killed at 1 ppm.⁶³ The clams Protothaca staminea (littleneck) and Macoma inquinata (bent-nosed) were killed by 880 ppm.⁶⁴ The marine dinoflagellate Glenodinium halli showed changes in population growth at 16 ppm.⁶⁵ The marine microalgae Isochrysis galbana showed changes in population growth at 8 ppm.⁶⁵ The marine diatom (Skeletonema costatum) showed changes in population growth at 0.125 to 16 ppm.⁶⁵ The marine diatom Thalassiosira pseudonana showed changes in population growth at 16 ppm.⁶⁵ The salmon Oncorhynchus keta was killed at 500 ppm and the perch Cymatogaster aggregata at 880 ppm.⁶⁴ Two shrimp (Pandalus danae and Neomysis awatschensis) were killed at 880 and 176 ppm, respectively.⁶⁴

Are these levels of toxicity important to reef aquarists? That is difficult to answer without knowing the levels that are attained in typical reef aquaria. The one study in the literature of bromate in a seawater aquarium, described above, showed the accumulation of up to 0.6 ppm bromate, although that was an application in which ozone was used for disinfection, so it was used at high doses. That level is high enough, however, to cause toxicity to certain organisms, but not others. In a typical reef aquarium ozone application, the bromate in the aquarium water is likely to be much lower. How much lower will likely depend on the way it is used, especially the dose and whether it is passed over activated carbon before entering the aquarium. It may also depend on the other husbandry practices used in the aquarium, because some procedures (such as denitrification) may reduce bromate levels. In any case, the potential toxicity data for bromate support the practice of using activated carbon after ozone exposure.

The Effect of Activated Carbon on Ozone Produced Oxidants

In order to reduce ozone's potential toxicity, aquarists typically try to reduce the OPOs in the effluent coming from the ozone reaction chamber. There are a variety of ways to accomplish that, but by far the most commonly used is passing the water over activated carbon (GAC).

In a previous article on how reverse osmosis/deionizing water purification systems work on tap water, Reverse Osmosis/Deionization Systems to Purify Tap Water for Reef Aquaria, I showed how hypochlorite reacted with activated carbon. Bromate and hypobromite are expected to react similarly. The reactions within the activated carbon that break down these compounds rely on having enough active surface area and time for these catalytic reactions to take place. How effective that is in a high flow application such as a skimmer's effluent is unclear. It is effective in reverse osmosis/deionization (RO/DI) applications because the flow is low and the carbon's surface area is very high.

When bromate and hypobromite interact with the activated carbon's surface, they are broken down into bromide ion (Br^-) and oxygen as shown below for bromate, where C* stands for the activated carbon and CO* stands for the activated carbon with an attached oxygen atom.

BrOH + C*  à  Br^-  +  CO* +  H⁺

Some of the oxidized activated carbon remains, and some breaks down to produce oxygen (O2):

2CO*  à  2C*  +  O₂

Some of the CO* can also break down to CO2 (carbon dioxide) in a noncatalytic breakdown of the OPO, but that is typically a small fraction of the total. None of these products of reactions are of significant concern to reef aquarists.

The big question for each aquarist is how effective is the GAC that is being used? As is true for many things examined in this field, the studies often have been done at high OPO concentrations relating to disinfection, and are usually in freshwater. In one patent application, a GAC bed was used to reduce the OPO in the water passing through it from 1.1 ppm to less than 0.2 ppm.⁶⁶ Another group showed that completely removing the bromate required a contact time with the activated carbon of more than 15 minutes.⁶⁷ In this test and in many others that have been published, older activated carbon was less effective than new activated carbon. The reason is that organics occupy portions of the GAC's surface where bromate and other OPOs are broken down.

A second group studying bromate in drinking water showed that GAC could remove 78-96% of bromate.⁶⁸ They found that contact time and age of the carbon were important parameters affecting the removal percentage.

Besides activated carbon, there are other potential ways to remove OPO's. In one patent application, researchers have shown that the water used in aquaculture applications can be treated with ozone, and then with reducing agents that react with and destroy these agents, thereby reducing its toxicity.⁶⁹ They recommend sulfite, bisulfite, metabisulfite or thiosulfate for that purpose, but it clearly is not simple to accomplish this automatically in a reef aquarium.

Does GAC or any other of these methods work well enough for reef aquarists to use ozone without undesirable side effects? The answer likely depends on the care which is used in the GAC treatment, and the aquarist's tolerance for OPOs to pass into the aquarium. The answer is likely not well enough when using the highest doses typically used by aquarists and the lowest tolerance for OPOs (that is, the lowest levels likely to cause ANY undesirable effects). Because it is not easy for most aquarists to measure low concentrations of OPOs, the most prudent course of action (aside from not using ozone) is to pass the ozonated aquarium water over as much GAC as possible before letting it re-enter the aquarium.

Removal of Bromate by Biological Means

In addition to the methods described above for removing bromate and other OPOs before they get to the aquarium, they can also removed by biological processes taking place in aquaria. In this situation bromate is apparently the one that builds up in aquarium water. Many studies have shown that biological filters (bacteria on surfaces) can break down bromate in ozonized drinking water.^70-72

Bacteria living under denitrifying conditions can also reduce bromate. As was mentioned earlier in the article, there is at least one study in the literature of bromate in a seawater aquarium.⁴² Here the ozone was used for disinfection, so its doses were high. Nevertheless, the bromate levels in the Living Seas exhibit at Walt Disney World's Epcot Center were found to have risen to about 0.6 ppm. Upon adding a batch denitrifying system, the bromate and nitrate concentrations began to drop.

Several conclusions can be drawn from this information:

1. When using ozone it may be prudent to have some denitrification taking place in the aquarium, either in live rock, live sand or in special denitrification systems.

2. Conclusions about ozone's safety or suitability, even if directed at exactly the same organisms in two different aquaria, may depend on the nature of the other husbandry practices in the two aquaria. For example, using ozone without GAC may be fine for 653 particular organisms living in tank A that also happens to have a large amount of live rock that can provide denitrification, but that same amount of ozone dosed to tank B containing the same 653 organisms without as much live rock may show more toxicity.

Conclusion

Ozone has many effects when used in a reef aquarium. The most useful of these is the degradation of organic materials. Most importantly, and quite coincidently and fortunately for aquarists, the colored organic pigments in marine aquaria are very sensitive to ozone. For this reason, ozone can remove seawater's color quite readily, and much more effectively than it removes the overall load of organic material. Its effects on water clarity described by most aquarists range from minimal to very dramatic, with most aquarists reporting significant beneficial effects.

Another big effect of ozone is the bioavailability of the organics in the water. Many organics in the aquarium are not readily metabolized by bacteria, and such materials may last for hundreds or thousands of years in the ocean. Ozone, however, has the ability to make many organic materials more readily absorbed and metabolized by bacteria. So in a sense, ozone triggers a bacterial attack that can reduce the load of circulating organic materials. This reduction in organic materials may also usefully apply to circulating toxins released by the aquarium inhabitants in an effort to kill each other with chemicals.

Ozone and its byproducts can, in high enough doses, kill many pathogens. The levels of ozone encountered in reef aquaria, however, may be inadequate to have any significant effect on total bacterial populations. Viruses are more susceptible than bacteria to ozone, and they may be effectively inactivated by typical use. Larger pathogens and parasites are much harder to kill and are not likely to be killed by ozone in reef aquaria.

Ozone also has a dark side. When reacted with seawater, ozone produces a variety of highly oxidized halogens such as BrOH and BrO3^-. If the ozone produced oxidants are not largely removed with activated carbon, they may enter the aquarium and be hazards to the most sensitive organisms in the aquarium (which are likely eggs or early stage larvae).

Finally, ozone alters a variety of other inorganic materials in ways that may or may not be important. It alters the aquarium's redox balance, raising the ORP (which may mean as little as altering the ratios of different forms of manganese in solution). It may permit more rapid conversion of ferrous ion to ferric ion, and may increase its bioavailability, but perhaps decrease the lifetime of strongly complexed iron such as EDTA iron. Ozone also oxidizes ammonia to nitrate. While that is likely beneficial, it may alter the relative effectiveness of different nitrogen export pathways (macroalgae vs. denitrification, for example). It may drive the speciation of iodine toward iodate and away from iodide. Is that good or bad? I expect neither, although others have different opinions, but it is a good poster child for the many things that happen in reef aquaria when using ozone that normally take place without any notice or recognition of them by the aquarist.

So with all things considered, is the use of ozone in a reef aquarium worthwhile? Many aquarists answer with a resounding, "Yes!" I'll leave that question unanswered until additional information is detailed in the next two articles discussing what equipment and methods are most useful for applying ozone to aquaria, and reporting on what impact it had in my aquarium.

Until then,

Happy Reefing!

References:

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