Heavy Metals

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jhnrb

Reef enthusiast
Is it Really in the Water? A Critical Reexamination of Toxic Metals in Reef Tanks

Part I

By Richard Harker

During my recent talk at the International Marine Aquarium Conference, I outlined the evolution of modern reef keeping explaining that the hobby had evolved through a series of stages to reach its current status. I suggested that the haphazard trial and error that had helped the hobby reach its current level of understanding would carry us no further. To continue to evolve and grow, the hobby had to enter a new phase where scientific methods replaced the "voodooism" that has characterized too much of what had guided the hobby to this point.

A recent series of articles on metals in artificial seawater and reef tanks would seem to be an example of what I was advocating. (Shimek 2002a 200b) Although that is true to some degree, the series also illustrates the potential pitfalls and dangers that we face as we enter this new phase of reef keeping. The response to the articles also demonstrates that many reef hobbyists are naive regarding scientific methods and are ill prepared to interpret the validity and usefulness of seemingly significant research.

If the hobby is to benefit from scientific methods, hobbyists need to develop a greater understanding of the principles and methods of science as well as an ability to distinguish between good and bad science, The goal of this and subsequent articles examining the reef tank metals articles is to help develop a sufficient understanding within reef keeping that hobbyists can critically judge articles that appear in hobbyist literature.

What is science?

In its most basic form, science is simply systematic study of phenomena. The Oxford Dictionary defines science a little more elaborately as, "a connected body of demonstrated truths or with observed facts systematically classified and more or less comprehended by general laws, and which includes reliable methods for the discovery of new truths." There are many moving parts to the definition and each matters in deciding whether something is science. It has also been said that the history of science consists of a series of conjectures and refutations. That's because in science, most conjectures are wrong and few are right. In other words, scientists are wrong more often than they are right. So one might more accurately describe scientists as seekers of falsehoods. (This is one key difference between the psyche of the scientist and that of the reef keeper. Scientists accept error as inevitable and the uncovering of errors of others as essential elements of scientific progress. Many reef keepers seem uncomfortable with this notion.)

Scientific knowledge has grown through the centuries as scientists have built on the discoveries (and errors) of previous work. Observations led to hypotheses, that in turn led to tests and experiments. Some experiments supported hypotheses while others disproved them, but each step of the way scientists learned a little more about our world. Scientific methods refer to the use of tested and accepted methods to study phenomena. The use of accepted scientific methods lends credence to one's findings and help other scientists understand the results. It also helps others replicate the studies to confirm their findings.

The metals studies are useful in illustrating both the use and abuse of scientific methods. In the following sections, I'll examine each stage of the studies and explain what is right and wrong about the author's methods and conclusions.

Scientific work involves a series of steps, all of which determine the accuracy and usefulness of the work. A misstep at any point in the process can undermine the entire process, so each step must be closely examined. Scientific papers generally begin with an introductory section explaining the central question under study. This section will generally review previous studies and relevant literature. It will explain why the study is important, and outline the hypotheses to be tested. In the metals articles the author asserted that metal concentrations in the average reef tank were significantly higher than on natural reefs and that scientific studies had conclusively demonstrated that at the levels found in reef tanks metals were toxic to marine organisms, He proposed that metal accumulation in reef tanks might explain why some tanks deteriorate over time (old tank syndrome).

When critically examining research, one should first question the premises of the author. Are the arguments of the author logical? Given what we know, are his assertions reasonable? If they are, will the approach he proposes address the issues he raises?

Methods-and why you can't always trust the government

Scientific papers always have a methods section devoted to outlining the methods used in the study. One way to judge a study is to examine the methods used and see if the methods are a reasonable means to test the author's hypotheses. To prove that the average reef tank has high levels of metals, one has to measure metal levels in a reasonable number of tanks representing a cross section of hobbyists. Ideally, a sample of participants would be drawn reflecting all the possible variables that might affect metal levels. The age of each system, the experience of each reef keeper, the different sources of make-up water, and so on should be considered in designing a sample. In the case of this study, the author solicited the help of hobbyists who would be willing to submit tank water for evaluation and pay for the analysis. Ultimately, only 23 hobbyists submitted samples. In statistical terms, this is a self-selected sample. Rather than sample a cross section of tanks, the author simply accepted whoever had the money and inclination to participant. As a rule, self-selected samples tend to be unrepresentative of large populations. With only 23 self-selected participants, one should be quite cautious about assuming that any analyses of these tanks can be extrapolated to the hobby as a whole.

The methods section should also outline how the levels of metals will be determined. In the metals articles, the author listed the method as, "Inductively Coupled Plasma Emission Spectrometry or ICP Scan, EPA method 200.7." A long impressive name like this lends an air of authenticity to the work. Most hobbyists have probably never heard of the technique and are in no position to judge the appropriateness of the method. In a scientific paper, the methods section will often present an explanation of why a certain technique was chosen. It will also explain any limitations in the method. The series author, unfortunately, did not address limitations in the method chosen. A review of the scientific literature on the subject of measuring metals in seawater finds significant problems with the ICP method. (Crompton 1989) Saltwater is a complex soup of chemicals in widely varying concentrations. For technical reasons addressed in a recent column by Randy Holmes-Farley, an ICP scan has great difficulty differentiating metals, particularly toxic metals. (Holmes-Farley 2003) Scientists do use ICP scans to study seawater, but the metals are first concentrated using resins or other methods.

So why has the EPA approved the use of ICP scans? Because they are fast and inexpensive. The Federal government's interest is in finding a method that can provide cost effective data for monitoring sites and enforcement of environmental laws. Other methods are more sensitive, but more time consuming and costly. Consequently, an EPA endorsement of any methodology is not evidence that it is the most accurate or useful for scientific studies.

Every experiment and study has multiple potential errors, and it is important to consider how the author deals with potential error. The samples were collected by individual hobbyists and then shipped to the author who in turn shipped the samples to the lab. This means that 23 different people collected the 23 samples. No attempt was made to filter particulates out of the tested water, and it isn't clear how careful the 23 hobbyists were in collecting their samples. Under these circumstances, the risk for contamination is great. The metals of interest are in extremely small concentrations, and it would be easy for a hobbyist to inadvertently introduce foreign substances into his sample. For example, even if rinsed repeatedly, using the same cup one uses to add supplements or feed the tank would inevitably contaminate the sample water. In a study of professional marine scientists, it was found that even professionals produced wildly varying results when analyzing metals in seawater, probably because of contamination. Because contamination is so easy when testing for metals, very elaborate procedures have been developed to make sure that contamination is minimized at each stage of analysis. The author makes no mention of handling techniques, so it is unlikely that the hobbyists involved used accepted procedures for handling the water.

(CONT)
 
Part-1 (cont)

The hobby tends to confuse accuracy and precision. A measurement might be carried out to three decimal places and still be inaccurate, whereas a measurement carried out to a single decimal point may be much more accurate. ICP scans carry out most metal levels to two decimal places, but does that mean the measurements are accurate to that level? Not necessarily. Computer programs analyze the results of an ICP scan, and then make their best estimate of the levels detected. The stated detection level is the level at which a single element can be detected. However, it does not tell us how well the ICP machine can differentiate multiple metals simultaneously, and that is what a scan does. Consequently, the practical limits of detection are much less precise than the theoretical limits of the technology. Because seawater contains high levels of some metals like sodium and magnesium, ICP operators make sequential dilutions of the samples to determine the concentrations of some of the metals. Each dilution introduces another potential for contamination and error. While professional labs do their best to avoid such problems, detecting metals in seawater tests the limits of even the most conscientious lab technician.

So without going much further than the methods portion of the study, one finds serious methodological flaws that raise questions regarding the likelihood that valid data will come out of the study. The small sample of reef tanks may not be representative of most reef tanks. The method used to determine metal concentrations, the ICP scan, has serious flaws as used here, and the handling of the samples may have introduced contaminations.

Lies, lies, and statistics

Mark Twain once wrote that there were three kinds of lies: lies, damn lies, and statistics. This brings us to the analysis portion of the articles. Once one has collected the data, the next step is to aggregate it into some sort of useful summary. Since the goal of the study was to determine metal concentrations in reef tanks, a logical first step would be to average the results from all 23 tanks to determine what an "average" tank looks like. On the face of it, this seems simple and obvious, but as it turns out, one of the most serious methodological mistakes in these articles occurred at this point.

An average or mean is calculated by summing all the values and dividing by the number of values. Consider five tanks that measure as follows:

0.02 0.01 0.02 0.03 0.02

The mean for the five tanks is 0.02, which seems like a reasonable average value. We can also calculate something called the standard deviation, which tells us how much the values vary from the mean. In this case, the standard deviation is 0.01 which means that two-thirds of all values are within 0.01 of the mean and that 95% of all values are within 0.02, or two standard deviations from the mean. What happens if one tank is very different from the others? Let's say that five different tanks measure as follows:

0.02 0.01 0.02 1.00 0.02

In this case, the mean is 0.21, ten times the mean of the first example. While mathematically correct, a hobbyist should be wary of assuming that for this sample of tanks, the mean is synonymous with average. Four of the tanks were within .01, so it seems like the "average" tank should be closer to .02 but the one high value distorts the results. This points out the most serious problem in using a mean to characterize data. Means are sensitive to extreme values. In statistical terms, the 1.0 value is called an outlier. It is a data point so removed from the rest of the data that it is reasonable to suspect that it is an error or aberration.

A statistician will look at data and first determine whether the values are reasonable. If outliers exist, he may exclude them, suggest that the tests be repeated, or qualify the results by noting the outliers. Another option is to use the median of the data rather than the mean. This is particularly useful if one has a limited data set and does not want to exclude any of the data. The median is the mid-point in a distribution. It is half way from the highest and lowest values. The median for both examples is .02, which is probably closer to the average tank than the mean value.

One can calculate the median only if we have access to the original data, and the author has refused to publish the data of individual tanks. Consequently, we have no way to calculate the median metal levels. The author did, however, provide standard deviations for each of the examined metals, so we can make some inferences about the range of values. High standard deviations indicate widely varying values. For example, in the study cobalt had an average value of .037 mg/l with a standard deviation of .031. These numbers suggest that two-thirds of all tanks have cobalt levels between .006 and .068, a ten fold difference. Furthermore, it also tells us that 95% of reef tanks have cobalt levels between -.025 (let's call that zero) and nearly 0.1 mg/l. Such a wide range of possibilities raises the question of whether we can draw any conclusions about the level of cobalt in an average reef tank based on these data.

Large standard deviations should be a red flag for anyone reviewing the results of a study. In this case it means that for some metals, the concentration levels varied widely among the 23 tanks. It also means that we should be very cautious about assuming that the mean values in the articles really represent the average reef tank. This by itself is a serious problem for the study, but an even more egregious statistical error was committed in analyzing the results. Some of the tanks had metal levels below the detectable limits of the ICP scan. For example, if the level of antimony detected was less than .01 mg/l, the print-out read < .01. In other words, the machine was saying that we know the level is no higher than .0099 mg/l, but there is no way of knowing how much lower it might be.

The author chose to ignore any undetectable levels when calculating mean values. For example, the detectable limit for arsenic is .01 mg/l. One tank had a level of .02 mg/l and none of the other tanks had detectable levels. The author then claimed that the mean for arsenic was .02 mg/l. Is this reasonable? Excluding 22 of 23 tanks because the ICP scan detected no arsenic potentially creates the false impression that high levels are present in the average tank. A more reasonable method to treat undetectable levels is to use the detection level to calculate a mean. Using the detection level for the other 22 tanks, mean arsenic becomes .01, half what the author claims. And at .01 mg/l, the mean probably over estimates the level of arsenic in the average reef tank.

Once the author had calculated means for all of the metals, he then proceeded to show elevated metals by comparing the tank test results with published metal levels of natural seawater. The tank concentrations were much higher than the published NSW concentrations. Is this conclusive proof? Not necessarily. The published concentrations of metals in natural seawater is the result of elaborate studies using sophisticated equipment and procedures. None of the published studies used ICP scans to determine metal levels.

One might take the position that if levels found in reef tanks exceeds the detection limits of ICP, the method of testing is irrelevant; Levels still exceed NSW. The reality is somewhat more complex. As I pointed out earlier, the practical detection limits of ICP when testing seawater are considerably higher than the theoretical limits. Because of this, pristine natural seawater might test higher in metals using ICP scan than using the methods of published studies. Because of this possibility, the author should have tested natural seawater along with reef tank water to see if higher reef tank metal concentrations were an artifact of the chosen methodology.

In part two, we'll look at metals in natural seawater using the ICP scan, We'll also take a look at metal concentrations in several reef tanks not included in the original study to see if reef tanks are really the toxic waste dumps that the author believes they are.

References
Crompton, T.R. 1989. Analysis of Seawater. Butterworths & Company, London

Holmes-Farley, Randy. 2003. Aluminum in the Reef tank. Advanced Aquarist July 2003. http://www.advancedaquarist.com/issues/july2003/chem.htm

Shimek, R.L. 2002a. It's (in) the water. Reefkeeping.com February 2002 http://reefkeeping.com/issues/2002-02/rs/feature/index.htm

Shimek, R.L. 2002b. It's still in the water. Reefkeeping.com March 2002 http://reefkeeping.com/issues/2002-03/rs/feature/index.htm

(CONT TO PART-2)
 
Part-2

EXERT FROM FULL ARTICLE

Is it Really in the Water? A Critical Reexamination of Toxic Metals in Reef Tanks

Part 2

By Richard Harker

The data collected on 23 hobbyist tanks led the author to conclude that the average reef tank is badly polluted with heavy metals that at best probably impair some inhabitants of the typical reef tank and perhaps even ultimately kill some inhabitants.

"The water from the average reef tank is clearly dangerous to the organisms put into it." (Shimek 2000c)

He expressed the belief that levels of these metals could possibly explain unexplainable deaths in the reef tank. His conclusion was that we should periodically break down a reef tank disposing of all rock, sand, and water and begin all over again. These are certainly disconcerting conclusions and if correct cast a cloud over all of reef keeping. The assertion that virtually all reef tanks are ultimately doomed by the accumulation of toxic levels of heavy metals created considerable stir in the hobby and left many hobbyists confused and alarmed.

Has the author made a convincing case for his interpretation of the results? Are the author's conclusions supported by the data? As part one explained, there are several errors and mistakes that compromise the value of the data and since the conclusions are based on the data, one must raise into question the conclusions. We have no way of knowing whether the tanks tested are representative of an "average" reef tank. The author's conclusions ignore the fact that some of the tanks did not have detectable levels of the most toxic metals. There is also the issue of the author's interpretation of what represents toxic levels. Even if some heavy metals are found at higher concentrations in reef tanks than found in natural seawater, does that mean that the average reef tank is toxic?

Even more fundamentally, can we be sure that the data presented are accurate? As part one outlined, ICP, the method used to determine metal levels, is not the best choice for accurately determining metal concentrations in seawater. None of the studies determining the extremely low levels of metals found in natural seawater used an ICP scan to determine the levels. In a scientific paper, an author usually devotes some space to justifying the chosen methodology. The author will support his decision by providing data that confirms that the methodology can do what the author claims it can do. Unfortunately, in the case of these articles, the only justification given for using ICP was that it was cheaper than more sensitive methods. Given the realities of reef keeping research, cost is a reasonable concern, but the need for accuracy ought to outweigh the need to keep costs low. If we are to draw conclusions about the levels of metals in a reef tank, we first need to see an ICP scan of natural seawater. The seawater concentrations provide a benchmark by which we can judge reef tanks.

Given that the metals of greatest concern are found at extremely low concentrations in seawater, and given the relative insensitivity of an ICP scan in detecting these metals at very low concentrations, one might argue that measuring natural seawater is a pointless exercise. Perhaps, but it is the responsibility of the researcher to prove that it is a pointless exercise. We need to know that the coral reef waters we are emulating have undetectable levels of metals if we are to take seriously the claim that our reef tanks are heavily polluted with heavy metals. And this comparison needs to be done using the same methodology. Perhaps compared to published seawater data, reef tanks do have elevated metal levels, but what if natural coral reefs do too?

Methodology

Knowing that we needed better data on natural coral reef water to reach conclusions regarding the health of reef tanks, I collected water over reefs in Fiji and the Philippines in the Pacific, and the windward side of Bonaire in the Caribbean. At each location several liters of water were collected in pre-washed drinking water plastic bottles. The collection bottles were then refrigerated and transported back to the United States. Upon return, the water was either frozen for transport to the laboratory or preserved according to standard methods of water analysis.

"Out of whack water parameters of whatever source increases stress and makes tank inhabitants more vulnerable to other stress factors. Had the angst generated over toxic heavy metals been redirected to a more balanced reduction of a broad range of potential stress factors, many reef tanks would probably be healthier than they are today."

I also collected water from my 2000 gallon reef tank using the same collection methods used for the natural reef waters. The 2000 gallon tank is three years old and in the table is labeled as the new tank. Its design is outlined in the 2001 Reef and Saltwater Annual as well as a recent Advanced Aquarist.(Harker 2003) I also had analyzed water collected from my 300 gallon tank and frozen two years ago. The 300 gallon tank was eight years old at the time I collected the water. The tank is featured in Michael Paletta's new book, Ultimate Marine Aquariums: Saltwater Dream Systems and How They Are Created. If metals do accumulate in a reef tank, my 300 gallon tank would be a likely candidate. Both tanks have used Instant Ocean since their inception. Reverse osmosis water is used for top off, but aged tap water is used for water changes.

The waters sampled were analyzed using Inductively Coupled Plasma Emission Spectrometry (ICP scan), EPA method 200.7, the identical method used in the articles under review. The data are therefore directly comparable to the previously published data, except in one regard. The author normalized the data of each of the 23 reef tanks so that all of the tanks showed identical sodium levels. Whatever factor was used to adjust sodium levels was applied to the other constituents, so the actual concentration of a metal in each tank might be different from that quoted in the articles. This normalization is somewhat dubious. The methods used by hobbyists to maintain calcium levels in a tank such as the addition of calcium chloride and sodium bicarbonate may alter the ratio of sodium to chloride and the ratio of sodium chloride to the other constituents. The data presented here are not adjusted or normalized in any way. Metals not shown in the table were below detection levels for both reef tanks and all three natural reefs. The results from the three reefs did not differ significantly, so the three were averaged for the purposes of the table. Values shown with a less-than sign (<) are below detection levels.

(CONT)
 
Part-2 (cont)

Results

The ICP scan suggests that natural coral reefs have heavy metals levels considerably higher than those published in the scientific literature and quoted in the articles. This raises an interesting question. Is the difference between published data on seawater and the coral reef water tested here an artifact of the methodology of testing, the ICP scan, or does the water over a coral reef actually have higher metal levels? If limitations of the methodology produce higher apparent metal concentrations in natural seawater, the same is probably true for reef tank water. Consequently, we don't really know whether reef tanks contain toxic levels of metals. The only way to determine that is to test tank water using the more sensitive methods that scientists use to study seawater. Only then can we compare published metal concentrations to reef tanks. On the other hand, if coral reef water actually does have higher metal levels than open seawater tested by scientists, then coral reef inhabitants have adapted to these levels and we may not have the toxic conditions that we were told we have.

The most remarkable thing is how similar the reef tanks are to the average natural reef. Some constituents are elevated, but only slightly so. Lithium is ten times the natural level in my new tank, but only a third of natural levels in the eight year old tank. Cadmium, selenium and titanium are slightly above detection levels in my new tank, but not in my old tank. In many respects, the eight year old tank looks more similar to the natural reef than the newer tank. It is possible that the natural evolution of a reef tank gradually reduces already low concentrations of heavy metals to even lower concentrations. This suggests that periodically breaking down a reef tank and starting over may perpetuate higher metal levels, not lower them as the author suggested.

Supporters of the author may argue that I've simply proved that today's natural coral reefs are polluted, that I am comparing toxic natural reefs to toxic reef tanks. From my observation, all three areas appeared pristine with no signs of degradation or deterioration. I chose to collect water at these locations because of the healthy reefs and the lack of natural or man-made damage to the reefs. If these healthy appearing reefs are really polluted, then there may be no truly healthy reefs. I've had an opportunity to explore many outstanding looking reefs, and these are some of the best.

So where do we stand?

We have been told that the average reef tank has heavy metal concentrations many times those of natural seawater. We have been told that our tanks are toxic and dangerous to many of their inhabitants. These data suggest otherwise. Using the same methods to prove that reef tanks are toxic, I've shown that my reef tanks are no more toxic than coral reefs from around the world. Furthermore, these data suggest that heavy metal concentrations in a reef tank may actually decline over time, not increase.

So do we need to worry about heavy metals? Yes, but no more than we should worry about the accumulation of organic compounds and other potential pollutants. As a closed system, the possibility exists that any substance that is introduced into the system will accumulate, and many substances can become toxic in sufficient concentration. Based on this evidence metal accumulation is not the bogyman that we have been led to believe. Heavy metals have to be considered in the broader context of just one of the many potential sources of stress in a reef tank. The key to a successful reef is the reduction of stress factors. Out of whack water parameters of whatever source increases stress and makes tank inhabitants more vulnerable to other stress factors. Had the angst generated over toxic heavy metals been redirected to a more balanced reduction of a broad range of potential stress factors, many reef tanks would probably be healthier than they are today.

In part three we will continue our reexamination of the heavy metal controversy by turning our attention to the issue of toxicity. Are the metal levels found in this study still high enough to be of concern? A recent article in Advanced Aquarist by Habib Sekha pointed out the metals found in seawater are not necessarily in a form that make them toxic to organisms. In seawater, metals are typically bound to a number of substances including organic matter, calcium carbonate, and chemical compounds that make them far less toxic than were they in their elementary form. We will review the heavy metal toxicology literature in the context of the above findings and determine whether heavy metals impair and even kill the inhabitants of our reef tanks.

References

1.Harker, R. 2003. Featured Aquarium: The Aquarium of Richard Harker. Advanced Aquarist Volume 2 issue 8. August 2003.

2.Paletta, Michael S. 2003. Ultimate Marine Aquariums:Saltwater Dream Systems and How They Are Created. TFH Publications.

3.Sekha, H. 2003. Toxicity of trace elements: truth or myth. Advanced Aquarist Volume 2 issue 5 may 2003.

4. Shimek, R.L. 2002a. It's (in) the water. Reefkeeping.com February 2002 http://reefkeeping.com/issues/2002-02/rs/feature/index.htm

5. Shimek, R.L. 2002b. It's still in the water. Reefkeeping.com March 2002 http://reefkeeping.com/issues/2002-03/rs/feature/index.htm

6.Shimek, R.L. 2002c. Our coral reef aquaria-Our own personal experiments in the effects of trace element toxicity. Reefkeeping.com. Volume 1 number 8. August 2002.

(CONT TO PART-3)
 
Part-3

Is it Really in the Water? A Critical Reexamination of Toxic Metals in Reef Tanks

Part 3

By Richard Harker

Speaking at the 1998 Society of Integrative and Comparative Biology annual meeting, Dr. Bruce Carlson outlined the Waikiki Aquarium's success raising Acropora and other stony corals. The room full of professional coral reef scientists listened intently as Dr. Carlson explained that reef hobbyists were having the same success growing corals in their homes. After the meeting Dr. Carlson was quickly surrounded by a group of scientists who wanted to learn more about how we were able to keep corals alive for years, a feat few scientists had accomplished. The wonderment expressed by these scientists has been repeated many times since then. Invariably, coral reef scientists when seeing a reef tank for the first time are shocked and surprised by our success. They realize that we "civilians" have been accomplishing more in the area of coral husbandry than professionals in the field have. In that regard, the professionals appreciate even more then the hobby does, what we have accomplished. Clearly it doesn't take a Ph.D. to create a successful reef tank, and a Ph.D. does not guarantee success in the hobby.

One reason we don't appreciate our success as much as we should is that we seem always to be just one step ahead of disaster. Whether it is the loss of a single prized animal or a total tank crash, we all experience our own share of personal failures. As much as any other emotion, the specter of failure seems to have driven the hobby's advance over the past decade. How else can we explain the all too common act of successful hobbyists changing much of what they do because other hobbyists claimed success doing something completely different.

A reef tank is a complicated system of mechanical and biological components. There are a lot of moving parts. Disasters can generally be attributed to the failure of one or more of these parts. Whether it is the catastrophic failure of a powerhead, the inadvertent dosing of large amounts of kalkwasser, the unnoticed death of a tank inhabitant polluting the tank, or any other of a virtually limitless number of possible problems, the root cause of a problem can generally be identified. There are those events, however, that defy explanation. Inexplicable deaths where the animal is behaving normally one day and is dead the next. Old Tank Syndrome (OTS), a phenomenon observed many time whereby a consistently maintained tank thrives for years and then gradually deteriorates and perhaps even crashes. These are the most disturbing failures we face, because we have no explanation for them. We don't know what we are doing wrong, so we have no way of determining what we should do differently.

Uncertainty creates vulnerability, and too often there are those that play on our fears as a means to advance agendas. Living as hobbyists do just one step ahead of disaster, too many are all too ready to blindly accept solutions that promise to pull us just one single step back from the precipice. Anyone who proposes a possible explanation and solution to these problems finds an attentive and accepting audience. The truth is, however, that these problems may be intractable. We may never determine a root cause of problems like OTS. There may not even be a single problem but instead a combination of factors. That's why we should be wary of any simple explanation that claims to address a whole host of problems. We should critically examining the proposed explanation. What evidence supports the explanation? How strong is the evidence?

This third in a series of articles is a reexamination of one hobbyist's view that many of the inexplicable problems we experience with reef tanks are caused by an accumulation of toxic heavy metals.

"I think that the cause of many such unexplained mortalities lies hidden in our tanks, and is even abetted or accentuated by well-meaning aquarists. My hypothesis is that a primary cause of many, if not all, of these unexplained mortalities is heavy metal poisoning. I believe this metal poisoning is specifically due to excessively high concentrations of some of the trace elements that are present in tank water." (Shimek 2002)

While the author suggests that heavy metal poisoning is only a hypothesis, in an on-line exchange, he claimed that it was much more than a hypothesis. Shortly after this quote was published, the author responded to a questioner in this way:

"You may consider the idea of tank mortality due to chemical build up as a theory. It isn't. I have collected the necessary data, and I have done the necessary tests to show that it is a proven fact. Over the next month or so, additionally I will be doing some direct sea-urchin larval bioassays on various salt mixes to put the last nails in that coffin."

So even before conducting further study, the author considered the data on artificial seawater toxicity a "proven fact."

Writing in Marine Pollution Bulletin, the editors decried the decline in scientific objectivity. They wrote that:

"It is both wrong and dangerous for a scientist to become personally and inflexibly 'attached' to a theory....Becoming inflexibly attached to a theory, whether or not a scientist considers it his or her own, prevents that scientist from thinking of more useful theories." (Chapman & Giddings 1997)

As we continue this reexamination, readers should keep the words of Chapman and Giddings in mind. The hobby benefits most by viewing any proposed explanation for inexplicable phenomena with some skepticism. As we will show, despite the author's confidence, his hypothesis is far from proved, and his conclusions far from fact.

If it's in the water, is it toxic?

To prove as the author asserts that high concentrations of some heavy metals cause unexplained mortalities in a reef tank, we need to prove that 1) heavy metal levels in the average reef tank significantly exceed levels found in natural seawater, 2) that these elevated metals in the concentrations found are toxic, and 3) that the toxicity "explains" unexplained deaths. If any part of this three part hypothesis is proved false, then we have to reject the hypothesis. Unfortunately, the third element is virtually impossible to prove short of necropsies of every organism that dies in a reef tank. To prove that toxic levels of any substance killed an organism, we have to test for the suspected substances. Clearly, this is impractical. Consequently, by the rules of hypothesis testing we have to reject the hypothesis even without examining the first two elements of the hypothesis. Nevertheless, the first two elements are intriguing enough to warrant this reexamination of the author's "proof."

In part 2 of this series we showed that an ICP scan analysis of natural coral reef waters found that heavy metal concentrations in the collected water were considerably higher than published seawater data. (Harker 2004) More significantly, the analysis found levels of heavy metals in coral reef water to be very similar to the levels of metals in my own reef tanks and only slightly lower than those reported earlier for reef tanks. Perhaps natural coral reef seawater has elevated metal levels compared to the open ocean. Perhaps the complex nature of seawater prevents an ICP scan from providing an accurate view of metal content of seawater. In either case, we have insufficient evidence to conclude that reef tanks have elevated heavy metal levels. We therefore have to reject the first element of the author's proposed hypothesis, that the average reef tank has concentrations of heavy metals much higher than those found in natural seawater.

Comparing the levels of metals in coral reef water to the levels deemed toxic according to Shimek in, "Our Coral Reef Aquaria-Our Own Personal Experiment in the Effects of Trace Element Toxicity," one might conclude that natural coral reef water itself is toxic. (Shimek 2002) The author presents a litany of toxicity studies that show harmful effects of metals at levels lower than I reported for natural coral reef water. How can this be? It is because not all metal in seawater is toxic. In one toxicity study, the authors remind us that, "contaminants are only toxic if they are incorporated into an organism's tissues." (Hook & Fisher 2000) To be toxic, a metal must be bioavailable, available in a form that can be absorbed into the tissue of the organism. (Meyer 2002) The author writes, "The total concentration of a metal in the water column is not a good predictor of acute toxicity. Because of the presence of modifying factors (dissolved and particulate), the potential for the metal to interact with biotic ligands often will be less than that indicated by the total metal concentration." A recent Advanced Aquarist article explained that potentially toxic elements exist in many compounds, few of which are toxic. (Sekha 2003) Only a portion of metals found in seawater are bioavailable. Much of the metal found in seawater exists in a form that is not readily available to organisms, and is therefore not toxic.

(CONT)
 
Part-3 (cont)

The partitioning between the different forms of a metal is called speciation. Generally speaking, there are three dissolved forms of a metal. Metals can be present as a free hydrated ion, in inorganic complexes, or in organic complexes. (Valasquez, et al., 2002) The free ion form is considered bioavailable while the organic and inorganic complexes are generally considered not bioavailable. (Sundra & Guillard 1976) In addition to these dissolved forms, metals can be precipitated or sorbed onto solid surfaces, particularly carbonate surfaces. (Mwanuzi & De Smedt 1999) Since the ionic state is the only toxic form of a metal we need be concerned with, it is the ionic component that we need to measure. Unfortunately, the ICP scan cannot differentiate between the different forms of a metal. It only tells us the total of the combined ionic, inorganic and organic complexes. While there are analytical methods that can determine the speciation of heavy metals in seawater, these tests have not been done on reef tanks.

As a rule the toxic ionic proportion of a metal in seawater is generally low. Most heavy metals in seawater are organically or inorganically bound, and are therefore non-toxic. Depending on the study, ionic metals can range from 20-30% of total dissolved metals. That means that 70-80% of the concentration of heavy metals in seawater is not in a form that is toxic to reef tank inhabitants. That does not mean the metals are rendered permanently non-toxic, only that at the time of analysis, they were non-toxic. Any hobbyist who has dosed a tank with copper in an effort to rid fish of parasites has experienced this effect first hand. It is very difficult to maintain a constant level of ionic copper in a tank, particularly if the tank has sand or live rock. While the level of total copper remains unchanged, testing for copper will show that levels are declining. Hobbyist copper tests measure ionic copper. As copper complexes with organic compounds and is sorbed onto calcium carbonate, the proportion of ionic copper declines as does the toxicity of the copper. That is the case with all metals that enter a reef tank.

What about bioassays?

According to the author, the "last nail in the coffin" regarding toxicity of reef tank water was a bioassay of sea-urchins. (Shimek 2003) Inexplicably, however, the bioassay primarily evaluated "freshly mixed artificial seawater," not reef tank water, so the study ended up shedding far less light on the issue of reef tank toxicity than if water from reef tanks had been used. A bioassay subject an organism to successively higher levels of a toxin to determine the level at which the animal becomes impaired or dies. Heavy metal bioassays for seawater organisms generally use metal compounds that dissociate in seawater. Adding copper sulfide or copper chloride to seawater releases controlled amounts of ionic metals in the water. Consequently, virtually all of the metal is bioavailable. When trying to relate lethal levels of metals in a bioassay to the levels of metals in a reef tank, it is important to keep in mind that virtually all of the metal in a bioassay is toxic whereas only a portion of the measurable metal in a reef tank is toxic. This is why the findings of published bioassays are of limited value for hobbyists.

In part 1 of this reexamination, I mentioned that it is important to study the "methods" section of any article to determine whether the methods used are proper and able to accomplish the intended goal of the study. One way to do this is to read related scientific literature to see if the author followed accepted methods. Fortunately toxicity research is well funded, and the scientific literature abounds with examples. Perhaps the most interesting was a study examining the effects of copper on the larvae of a decopod. (Wong, et al., 1995). The authors used Instant Ocean artificial seawater as the control medium. The authors stated, "Artificial seawater have been used extensively in our laboratory for culturing Metapenaeus ensis larvae and postlarvae and have shown no adverse effects on growth and development." When a team of professional scientists publishing studies in a well respected journal like Marine Pollution Bulletin use Instant Ocean to raise larvae, one should be skeptical about an assertion that artificial seawater is toxic.

In another bioassay paper, the authors used Forty Fathoms Biocrystals. (Rumbold & Snedaker 1997) The authors note that the artificial seawater was aged with vigorous aeration for more than 48 hours and filtered prior to use. This provides one clue as to why professional researchers would be comfortable using artificial seawater that a hobbyist author has labeled toxic. Bioassays do not use "freshly made" artificial seawater. Scientists go to great lengths to prepare control water that can support the health of the organism. If natural seawater is used, typically the water is prefiltered and then pH and salinity adjusted before use. If artificial seawater is used, the water is aged for some time and then prefiltered before use. For decades, research scientists have emphasized the importance of aging artificial seawater. (Bidwell & Spotte 1985). The Gosse formula for artificial seawater dating from 1854 as reported in Bidwell & Spotte recommends filtering the solution through sponge and conditioning by adding, "a few clean shore pebbles and some fronds of green seaweed." Gosse recommended adding animals after one week. One hundred and fifty years ago scientists appreciated the importance of aging artificial seawater, so it is not clear why a hobbyist author would not follow well accepted methods in preparing his samples. Freshly made artificial seawater is inherently toxic, but becomes less toxic as it ages. That's why since 1854 researchers have aged their artificial seawater. Conducting a bioassay on freshly prepared artificial seawater may determine how quickly one salt dissolves compared to others, but it tells us nothing about whether any of the salt mixes are toxic after a reasonable length of time.

Fortunately the bioassay also included water from two hobbyist reef tanks, so the results from these two tanks can offer some insights. The two hobbyists both used Instant Ocean. One prepares his water with RO/DI water while the other uses well water. We have one controlled variable, the salt mix, Instant Ocean. We have one known variable, the source water, RO/DI versus well water. The bioassay found that on average five times as many larvae survived in the water of the hobbyist using RO/DI water compared to the water made from well water. Since both hobbyists used Instant Ocean and there was a statistically significant difference between the two tanks, we have to eliminate the salt mix as a possible explanation for the difference.

Both hobbyists used Instant Ocean and yet only one was highly toxic to sea-urchin larvae. Why? We don't know. Nothing in the study indicates that the waters were chemically analyzed. In the absence of metal analyses for the two tanks we cannot determine whether metal levels had anything to do with the outcome. Perhaps the well water contained higher levels of metals than the RO/DI water. Perhaps pesticides had contaminated the well water. Perhaps something unrelated to well water was the cause. In any case, we can eliminate the salt mix as the source of the problem. Despite the author's confidence, the study falls well short of being the last nail in the debate's coffin. Furthermore, since the majority of larvae in the one hobbyist's water survived, it raises additional questions about whether reef tanks are really as toxic as they have been portrayed.

(CONT)
 
Part-3 (cont)

So what is a toxic level?

For nearly all of the metals studied, levels found in typical reef tanks are significantly lower than those found to be toxic to the organisms studied. See the references cited at the end of the article for complete details.

The majority of data on toxicity are based on bioassays where virtually all of the metal in solution is bioavailable. Since much of the heavy metal in our reef tanks is not bioavailable and therefore benign, it would be incorrect to conclude that a reef tank is toxic if metal levels are found to be at levels found to be toxic in a bioassay. Quite the contrary, we know that the majority of metals in seawater are not bioavailable. As a result, if a reef tank has a total metal concentration equal to that found to be toxic, it is safe to assume that the reef tank is not toxic. Furthermore, organisms have little difficulty tolerating metal concentrations just slightly below the published numbers. Unfortunately, we have no convenient means to determine what proportion of metals in a reef tank are bioavailable, so we may want to start by assuming that 100% of the metals are bioavailable and compare these numbers to the bioassay numbers. This is a highly unlikely assumption, but it provides a tremendous margin of error.

Select findings of several toxicity studies were reported by Shimek, and present a rather dire image of reef tanks. With regard to copper, the author writes, "(copper's) effects on corals are profound and occur at concentrations below those typically found in aquaria." (Shimek 2003) Reading the complete metal toxicity scientific literature reveals quite a different picture. Natural coral reef water was found to have 14 mg/l of copper. Typically bioassay results are presented in terms of LC50, the concentration that leads to 50% mortality over a stated period of time or IC50, the concentration that causes inhibition of some physiological function. Note that at the published LC50 levels, 50% of the organisms survive. One study found that 17.4 mg/l copper inhibited Acropora millepora fertilization, and 110 mg/l inhibited larval metamorphosis. (Negri, et al., 2001) In another study looking at Acropora tenuis, the authors found that 17.3 mg/l of copper did not significantly inhibit larvae settlement. (Reichelet-Brushett 2000). "However, concentrations of 42 mg/l and 80.5 mg/l results in significantly lower mean total settlement compared with the controls." A study of Porites lutea found that 10 mg/l of copper had no effect on respiration, but 30 mg/l had a significant effect. (Alutoin, et al., 2001) In a study on gastropods, copper levels below 50 mg/l were safely tolerated by Nassarius festivus. (Cheng et al., 2002) In another study, median lethal concentrations for copper were 46mg/l for lobster larvae, 50 mg/l for spider crab larvae, and 3,304 mg/l for shrimp larvae. (Marino-Balsa, et al., 2000) Total copper in my tank measures 9.5 mg/l, the average of the 23 tanks tested was 24 mg/l, and the highest level of copper found was 38 mg/l. It is clear from these studies that the total copper levels typically found in a reef tank are quite safe for the tank's inhabitants. And considering that the bioavailable proportion of copper is a small fraction of the total level, the typical reef tank is far from toxic.

This is true for all the other potentially lethal metals. Metal levels in the average reef tank are well below toxic levels-even when we consider the total concentration of metals, not just the bioavailable portion. This should not be surprising. Copepods, amphipods, nematodes, and polychaetes readily reproduce in most mature reef tanks. Larvae are the most sensitive organisms in our tanks, and they survive quite well.

A lurking catastrophe?

It has been suggested that even if most metals in a reef tank are not generally bioavailable, that the accumulation of metals can lead to disaster if unexpected events suddenly make them bioavailable. A typical senario is a decline of pH. If the tank pH declines, a portion of the sand will dissolve and the metals adsorbed on the calcium carbonate might be released resulting in a lethal spike of metals. While this might be theoretically possible, what is the likelihood? We know that metals can be chemically bound to both organic and inorganic compounds. We also know that a relatively small proportion of ionic metals exist in seawater because of the propensity for metals to bind with organic and inorganic compounds. What is the likelihood that a high proportion of the bound metals might be released and made toxic?

We can gain some perspective by examining the laboratory procedure for reducing metals to their ionic state. To determine total metal content, the EPA requires that samples be reduced to a pH of less than 2 and then boiled in hydrochloric acid. Strong acids and an extremely low pH will release all bound metals, but these conditions are so removed from anything that a reef tank might encounter that we can safely rule out any similar release of metals in a reef tank. More likely in a reef tank with low pH is a slow release from the sand. However, a reef tank with low pH is generally one with a high organic load, so as the metals are released from the sand, they would most likely bind to organic compounds.

Conclusions

After reviewing the scientific literature and reviewing the series of hobbyist articles on metal toxicity, it is clear that evidence supporting metal poisoning as a source of inexplicable deaths in a reef tank is far from conclusive. Metal levels in the average reef tank are not significantly elevated, and the levels are below toxic levels in the scientific literature. A bioassay of reef tank water supports this conclusion. Artificial seawater made from Instant Ocean supported sea-urchin larvae survivability despite the fact that the author had previously identified Instant Ocean as one of the most heavy metal laden salt he examined.

As Chapman and Giddings wrote in their editorial, "We realize that theories and ideas require a great deal of effort, sometimes over many years. In this light, new ideas or criticisms which may not appear to be fully justified can be upsetting, and it is tempting to provide an abrupt, dismissive, or even hostile reply....Valid criticism can, at best, only temporarily be deflected. If a theory has serious flaws (or even subtle ones), they will show up eventually. Sooner or later the theory will crack under the weight of conflicting evidence. Persuasive arguments, rhetoric, and personal attacks on critics won't protect a flawed theory forever." Clearly the theory that much of what befalls a hobbyist is a result of heavy metal poisoning falls into this category. It is a flawed theory that cracks under the weight of conflicting evidence. The evidence presented in this series shows that it is possible to have heavy metal concentrations near that of coral reef water and well below levels that have been shown to be toxic.

THIS HAS BEEN AN EXCERT OF THE FULL ARTICLE.
END.

Posted jhnrb
 
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