Sea Salts, Part Two: The M.A.R.S.H. Salt Study
by Eric Borneman and Kim Lowe

Artificial sea salts are the basic ingredients of any marine aquarium whose owner does not have access to natural seawater. Myriad brands and formulations exist, and opinions based largely on personal preference and a few limited studies (see Borneman, 2006 for a review of these studies) continue to fuel debates about which brand is best. Much discussion has occurred regarding the elemental composition of salts, and although impressive longevity for some marine species has been accomplished by using these artificial salts, we have little idea how artificial sea salts compare to each other and to natural seawater. It is a question that should be answered, if possible.

The original idea to study the practical effects of artificial sea salts in a reef aquarium was initiated by the Marine and Reef Society of Houston (M.A.R.S.H.) who enlisted the voluntarily services of Eric Borneman as principle investigator and to design an appropriate experiment. The generous M.A.R.S.H. membership and several other contributors (see acknowledgements) deemed the project worthy, and we embarked on what has turned into an extensive study of the differences between various commonly available artificial sea salt mixes' effects on organisms common to reef aquaria. Our hypotheses were that there would be differences in the survival, growth and reproduction of species in equivalent aquaria utilizing different sea salt mixes with a null hypothesis that there would be no differences. Natural seawater controls were provided consisting of samples from three oceanic sources.

Proposed Study: Artificial Salts' Effects on Growth and Reproduction in Reef Aquaria


Ten 10-gallon tanks are set up on a long bench and illuminated with two sets of 6' long high output T5 (one white and one blue) fluorescent bulbs (Innovative Lighting). A single new Maxi-Jet 1200 powerhead (Marineland) is mounted in the center of each tank's short end, facing lengthwise just below the water's surface. An automatic feeder is the only other equipment used on the tanks, and it is filled with 0.1g of a homogenized mix of multiple dry foods (Formula One™, Formula Two™ (Ocean Nutrition), Green 2000, Cyclop-Eeze® (Argent Labs) and beta glucan (SeaVive)).

Each tank houses clonally derived ramets of various coral species, including stony corals, soft corals, zoanthids and corallimorphs. We chose Montipora digitata, Cladacora caespitosa and Porites cylindrica as representative scleractinians. In addition, a corallimorpharian, Sinularia grayi, Zoanthus sociatus and Plexaura flexuosa of the same genotype were used as non-scleractinian coral species. We also included two algae of the same genotype, Chaetomorpha sp. and Gracilaria sp. Other organisms included Astraea tecta, Nassarius sp., Cerith sp. snails, Clibanarius tricolor hermit crabs, Asterina sea stars, Leucetta sp. sponges and Amphiprion percula clownfish from the same brood to minimize genetic variability. Our idea was to provide a representative sample of the numerous phyla encountered in reef aquaria including corals, gastropods, echinoderms, crustaceans, algae, sponges and vertebrates to each of the tanks.

No live rock was used in the tanks because of potential variation in the diversity of life introduced into the tanks by live rock, but we added a layer of autoclaved crushed coral as a substrate for benthic flora and fauna.

We acquired 11 batches of each artificial salt brand separated as far in time and space as was practical from various vendors across the country to ensure both variation in batches and that the products were those normally available to aquarists. In no case were any samples provided directly by their manufacturer, in order to ensure that samples were representative of those available commercially. Each bag was new and unopened, and approximately 1500g of salt was removed directly from the bag or container into new, clean Ziploc bags and sealed immediately. We recorded the donor, the place and date of purchase and the size of the container. Each group of salt samples were then marked as Samples A-J, rather than by manufacturer, in order to ensure the study was conducted blindly.

Sea salt mixes are prepared twice. In the first case, 35g of each salt are added to 1 liter of double-distilled water and stirred for 30 minutes on a stir plate fitted with a stir bar. At the end of this period, we qualify the resultant solution in terms of its clarity and amount of undissolved and suspended material. We also take immediate readings of pH and salinity using lab quality pH meters and refractometers calibrated before each reading. These data are recorded to calculate the volume of salt needed to fill each tank water container and to determine variation within salt brands. Then, deionized water is used to fill triple-washed containers with water and enough salt to bring the solution to 35 psu salinity, and the solution is stirred using a clean powerhead for at least one hour or until all salts are dissolved. This water is prepared once each month for a total of 10 months, accounting for 10 separate batches of each salt mix to be added with a nearly 100% water change to each tank each month.

Natural seawater was used as a control. For the first three months water was utilized from the National Cephalopod Research Station at the University of Texas Medical Branch in Galveston, Texas. Its water is pulled from approximately 30 miles offshore to the facility. The water was then sequentially filtered to 0.22 microns through Millipore filters for use as control water. During the next four months seawater controls were obtained from surface waters above the Flower Garden Banks coral reefs, approximately 100 miles off the Texas coast. For the final three months water was provided from Stetson Bank, a mixed coral and hardbottom community 70 miles off the Texas coast. In all cases, the seawater was filtered to 0.22 microns.

The salts used in the study were as follows:

  • Instant Ocean® (Aquarium Systems/Marineland)
  • Reef Crystals® (Aquarium Systems/Marineland)
  • Kent Sea Salt™ (Kent Marine)
  • Crystal Seas Bioassay Formula™ (Marine Enterprises)
  • Red Sea (Red Sea Fish Pharm)
  • hW Marinemix (HW Weigandt)
  • Tropic Marin® (Tropic Marin USA)
  • Oceanic™ (Oceanic Systems)
  • Coralife® (Energy Savers Unlimited)
  • Natural seawater

Initially, all tanks were maintained in a climate-controlled room with HEPA filtration and were covered with acrylic to reduce evaporation and limit the input of airborne contaminants. After filling each tank with the seawater, the tanks were cycled with the addition of a known amount of homogenized flake food (once) and the addition of reagent grade ammonium chloride (Sigma). The cycling period lasted for six weeks before the autoclaved substratum was added to the tanks. The following week, the species were added to each tank with positions determined randomly but with each species in the same position within each tank to minimize microhabitat variation. Recently, the tanks have been moved to the main lab to ensure security because the climate controlled room could not be locked and some concern arose over potential incidents that could occur without the added security.


All tanks have the following water quality parameters measured at the start of the experiment, weekly (within batches), monthly (old and new between batches) and at the end of the experiment:

Temperature: measured using a thermometer (Fisher Scientific)
Salinity: measured using a calibrated lab quality refractometer (Reichert)
pH: measured using a calibrated pH pen (Milwaukee Instruments)
Oxygen: measured using an oxygen probe (YSI, Inc.)
PAR: measured using a lab quality PAR meter (LiCor Industries)
Ammonia: measured using colorimetric test kit (Salifert)
Nitrite: measured using colorimetric test kit (Salifert)
Nitrate: measured using colorimetric test kit (Salifert)
Phosphate: measured using colorimetric test kit (Salifert)
Calcium: measured using titration based test kit (Salifert)
Alkalinity: measured using titration based test kit (Salifert)

Each volunteer responsible for water quality testing must read the tanks for the entire duration of a test period (= one month) to ensure reproducibility of results. All instruments were calibrated prior to each test.

Following testing, the following actions may be taken. Salinity is corrected through the addition of the same batch of salt if hyposaline, or through the addition of Class I deionized water to maintain 35psu. Calcium will not be maintained but will be allowed to decline over the course of the month-long periods because calcium is generally not limiting to calcification. Alkalinity will be maintained weekly using a mixture of reagent-grade sodium bicarbonate (VWR) and sodium carbonate (Sigma) brought to a pH of 8.2 to maintain an alkalinity of 3.0meq/l and any additions are recorded as to the volume of carbonates added. pH will be not be adjusted during each month-long period.

Every other day, 0.1g of homogenized food is added to each tank through the automatic feeders. Initially, food was added manually through 0.1g aliquots prepared and stored in closed microcentrifuge tubes.

All tanks receive nearly a 100% water change (siphoning to the gravel level) each month for 10 months, with a new batch of the same salt brand being used for each water change. Between batches, species are counted, photographed, measured and weighed as described above. Any mortalities that occur are recorded and replaced immediately or as soon as possible after losses are recorded. Soft corals, because of the water contained in their tissues, are recorded qualitatively in terms of their appearance and assigned a ranking. The gorgonian, being rigid, is measured by length, width and height, using calipers and qualitatively by description. Cladocora and zoanthids are measured by counting mature and incipient polyps. Montipora and Porites are weighed, measured with calipers or described by percent coral tissue alive according to the Atlantic and Gulf Rapid Reef Assessment (AGRRA) protocol. Algae are cleaned of detrital material or other algae, patted dry and weighed. Fish are weighed using the buoyant weight technique. Gastropods, crustaceans and echinoderms are counted as absent or present. Sponges are accounted for by their presence or absence and the number of incipient sponges being produced on their surface or found within the tank. Each month, any filamentous algae or cyanobacteria are removed from the species to limit the observed effects on species to salts, rather than algal overgrowth, as responsible for any partial or total mortality. The tanks' front pane was cleaned with a razor weekly so that photographic documentation of each tank could be recorded, and the tanks' sides are wiped clear of nuisance algae before siphoning old water prior to the water change. Coralline algae and other sessile invertebrates, such as bryozoans, were left intact where present. The powerhead filters were cleaned and strands of attached Chaetomorpha and Gracilaria were removed, dried and weighed. Also, qualitative descriptions of each tank were performed monthly and occasionally weekly, as needed. This process was repeated for a total of 10 salt batches (= 10 months).

Our argument and contention is that all salt mixes are supposed to be reasonable substitutions for seawater, and that a 100% water change shouldn't stress the organisms unless some sort of significant acclimatization to large shifts in water chemistry occurs over the interim period. In these tanks, the test period is only one month. While we concur that 100% water changes do not seem well-tolerated in established tanks, theoretically, the total water change should be, if anything, beneficial. It will also highlight any results of salts that had particularly notable deleterious effects. A 100% change will likely show salt-based effects more strongly than would smaller water changes.

At the end of the experiment, all species will be reweighed as described above. All reproductive events will be counted. All mortalities will be counted. All algae will be scraped from the glass and weighed. The calcareous species will be soaked in bleach to dissolve tissues, then the remaining calcareous crusts will be weighed. All other organisms, such as amphipods, polychaetes and other flora and fauna will be removed, filtered and counted or weighed.


Inter- and intra-tank variation of all data will be determined using repeated measures (ANOVA). Additional pairwise comparisons may be required using Student's t-tests. Nonparametric tests will be performed on ranked data.


  • The microbiota will play similar roles in all systems and will exert relatively small effects on the species' growth and survival.
  • No other species introductions will occur through the use of living organisms that will affect the species' growth and survival.
  • All species are healthy and free of pre-existing conditions that would affect their growth and survival.
  • Each salt tested is representative of a "batch" of salt. Each salt mix (n=10) will be purchased from different sources at different times. Each purchase of salt results from a separate "batch."
  • Conditions in each of the aquaria are sufficiently similar that they will not affect the species' growth and survival.
  • All additions to tanks in terms of buffer, water and food are sufficiently similar or of such small effects that they do not affect the species' growth and survival.
  • All populations are assumed to be normally distributed in terms of growth and survival in their original systems; in the ocean or with aged water using many salts. An assumption exists that species are alive and growing, some reproduction is occurring and that mortality and recession exist.
  • The data collected may not be valid for comparison with wild reefs (true control).


The data we have collected to date have been extensive and surprising. Not only will the targeted results be interesting, but so are many of the unexpected results we have encountered over the course of the experiment to date. This experiment ends on the weekend of MACNA (Sept. 23/24, 2006). The preliminary results and highlights will be presented at that meeting, where we hope to see a strong attendance. The data analysis, however, due to the overwhelmingly large amount of data, will take time to perform and present in a usable and digestible manner. These results will first be presented for consideration in a peer-reviewed journal and then written as a series of articles for aquarium magazines around the world. We very much look forward to presenting the results of our work to the aquarium hobby and hope this is a step toward understanding more about the intrinsic properties of various artificial seawater mixes available to the trade. Furthermore, thousands of articles have been written in peer-reviewed journals using synthetic sea salts predominantly from two brands - and under the assumption that they perform equivalently to natural seawater. The results of our work will further determine if such assumptions in the literature can be considered valid.


Foremost, we would like to thank our spouses, Brandee and Mark, for tolerating our time away to complete this project, as well as helping us out in our time of need. This work was supported by various dedicated members of the Marine Aquarium and Reef Society of Houston (M.A.R.S.H.), the Dallas-Fort Worth Marine Aquarium Society (D.F.W.M.A.S.) and the Marine Aquarist Association of South Texas (M.A.A.S.T.). Donations of equipment and livestock were provided by The Atlanta Reef Club, the Orlando Reef Caretakers Association, Exotic Aquatic and Pets, Innovative Lighting, Marine Depot, Maroon Lagoon, Premium Aquatics, Salifert, Village Tropical and various members of M.A.R.S.H. We would also like to thank all others not mentioned above who advised and supported us in this project.

If you have any questions about this article, please visit my author forum on Reef Central.


Borneman, EH. 2006. Sea Salts, Part One: A review and a new study to determine their
effects on reef aquarium inhabitants
. Reef Hobbyist Online 2(3).

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Sea Salts, Part Two: The M.A.R.S.H. Salt Study by Eric Borneman and Kim Lowe -