Daniel sinclair

Ammonia Production of Commonly Used Aquatic Fish Foods Through Decomposition *D.J., Sinclair & C., Baldwin, 2011 *daniel.sinclair077@live.cornwall.ac.uk

Ammonia is a deadly nitrogen compound which can devastate live stock involved within aquaculture; however, very little information is available on the subject of ammonia levels produced by decomposing fish feed. This study uses a simple, cost effective method to determine the concentration of ammonia produced by 2g of a variety of feeds over a 36 hour period. The results show that ammonia production does not correlate with protein content, but this study suggests that protein type may be more influential. Lowest ammonia concentration- 0.45mg/l; highest ammonia concentration5.00mg/l.

Key words: ammonification, ammonia production, decomposition, fish feed, nitrification.

INTRODUCTION Development of pollutants, ammonia (NH3 / NH4+) in particular, from uneaten aquatic fish feed should be considered in all aquatic systems. Over feeding is known to result in heavy water fouling, which can cause severe stress to fish stocks as well as the surrounding ecosystems where applicable.

As a rule of thumb, hobby-aquarists will feed ornamental fish an amount which can be consumed by the fish within a few minutes. Any uneaten food is then removed to avoid decomposition to take place, and pollutants to accumulate. This is an ideal method to control the amount of potentially harmful products entering the systems in situations where such observations are possible. However, in areas of aquaculture where feeding observations can not occur, calculations are required to assist in determining the weight of food which is fed to a number of fish with a stock; a crucial equation is known as the feed conversion ratio (FCR) and symbolises the amount of feed required to produce 1kg of stocked-fish. In ideal situations, a well managed salmon or trout farm will produce a FCR of ~1, creating no losses of food and no mortalities of fish stock.

Unfortunately, this isn't realistic so monitoring of the stock numbers must still occur regularly (Ali & Salim, 2004; Sahzadi, et al., 2006).

Ammonia production is the first stage of nitrification, so it is important to understand that this nitrogen compound is the key to establishing a system, as well as being the most harmful to fauna, triggering illness. High ammonia concentrations seriously weaken a fish, leaving an individual vulnerable to almost any disease. In home-aquaria, ammonia levels from over feeding can be small and easily over come. Across a fish farm on the other hand, stocks are considerably denser, increasing the spreading rates of disease resulting in a much more costly loss.

As matter (plant/fish/feed material) is decomposed via saprobic bacteria, proteins and nucleic acids are released into the water; a variety of bacteria are present at this stage. Sepers (1981) isolated 68 different species of bacteria when monitoring this ammonification process and often refers to decomposition as remineralisation as it is the recycling of materials through biological activity (Sepers, 1981; Sarmiento & Gruber, 2006). These chemicals are then broken down (fixed) even further by the bacteria abundance, producing ammonia (NH3 / NH4+) (Sawyer, 2008). The nitrification cycle follows this process. Ammonia will occur at extremely low levels within a mature system as a healthy colony of nitrofying bacteria will be present, fixing the nitrogen compounds, however the desired ammonia concentration from a standard test kit is 0.00mg/l.

Within home-aquaria, fish flakes, pellets, bloodworm and daphnia are the most common foods used to sustain fish. Fish farms and public aquariums tend to follow a bulkier approach using mackerel and squid. A variety of widely used foods have been chosen to be tested, allowing the ammonia production to be quantified over a 36 hour period.

It is important to understand that most ammonia analysis techniques test for Total Ammonia [Nitrogen] (TAN). This is a combination of both ammonia and ammonium. Ammonium (NH4+) is an ionized form of ammonia and occurs more over in water with a lower pH. Ammonia (NH 3) concentrations increase as the pH levels increase (Sawyer, 2008). [ Low pH ] NH4+ + OH- ↔ NH3 + H2O [ High pH ] As the pH of a body of water increases, more hydroxyl ions are present, causing any ammonia species to bond with them, resulting with NH4+ being the product. As the pH of a body of water decreases, less hydroxyl ions are present, restricting ammonia bonding. This results in NH3 being the product.

Therefore, it is vital to understand that temperature and pH FIG. 1: The dependence of both have an effect on NH3 and NH4+ concentrations;

+ ammonia/ammonium (NH3/NH4 ) ratio as a function of pH

represented in FIG. 1.

Amazingly, there is very little evidence of any experiments which evaluate the ammonia production of decomposing fish feeds, and Merlin Entertainments Group realised this when trying to determine how much feed to give to live stock in their world wide public aquariums. Blue Reef Aquarium, Newquay, Cornwall, have also required such information. The National Marine Aquarium, Plymouth, expressed keen interest in the potential results and suggested feeds which are used within the aquariums at the establishment.

MATERIALS AND METHODS Exactly 2g of any chosen feed is used per test, and each test repeated 6 times. An initial pH and ammonia test is taken, using the adequate Tetratest kits, and recorded. Temperature is recorded using a thermometer. The feed is placed in a 600ml glass beaker containing 400ml of distilled water which is then placed under a light source. The water is again tested, using the correct Tetratest kits and a thermometer, every 3 hours over a 36 hour period.

Overall, 19 foods were tested 6 times: Aquarian Algae Wafers & Tropical Flakes; Hikari Algae Wafers & Frozen Daphnia; King British Algae Wafers & Tropical Flakes; Mackerel; New Era Marine Pellets, Marine Flakes, Tropical Pellets & Tropical Flakes; Nutrafin Max Tropical Flakes; Ocean Nutrition Frozen Bloodworm; Sanfrancisco Bay Frozen Daphnia & Bloodworm; Squid; Tetra Pro Tropical Crisp Food, Pleco Wafers & Prima Pellet Food. These foods were chosen through personal communications and suggestions- specifically Craig Baldwin. The amount of feed used (2g) was chosen as it is the highest amount of food which would be placed into a volume of 400ml (Jabeen et.al., 2004).

Tetratest kits were used because they are inexpensive and atleast 3 kits will need to be used for the entire experiment. Using this test also allows it to be easily repeated within home aquaria as well as across other areas of the industry.

Distilled water was chosen as it provides no ‘micro’ nutrients which may benefit bacteria, influencing decomposition and ammonia metabolism. The volume of water was chosen to correlate with the FCR and maximum stocking densities of aquaculture.

RESULTS TABLE I. The results show the accumulation of ammonia (mg/l), represented as Am, from the decomposing fish feed over the 36 hour period, so the final result of each test is expected to show the highest ammonia concentration. Results were recorded as the following:

The feed which produced the lowest concentration of ammonia is Hikari Algae Wafers. The feed which produced the highest ammonia concentration is mackerel; represented in the following tables:

Unpaired t-test is taken to statistically analyse the lowest and highest feed-ammonia production concentrations. The final measurements (at 36 hours) are compared in the following ttest.

H0 There is no difference between the concentration of ammonia produced (via decomposition) by Hikari algae wafers and Mackerel over a 36 hour period.

H1 There is a difference between the concentration of ammonia produced (via decomposition) by Hikari algae wafers and Mackerel over a 36 hour period.

Pooled Standard Deviation:

Test statistic:

Sp = 4.42 √ (1/6) + (1/6) = 2.55

T = 8.11

A 2 tail P value with a statistical significance of 0.05 and degrees of freedom of 10 = 2.228.

As 8.11 is greater than 2.228, the null hypothesis (H0) is rejected; there is a significant difference between the concentration of ammonia produced by Hikari algae wafers and Mackerel over a 36 hour period.

DISCUSSION To allow the resulted to be discussed more easily, the term average maximum (avg. max.) is used to describe the mean of each result at 36 hours. An average maximum is taken per food used and represents mg/l as in ; shown in TABLE II.

The results show that overall, algae wafers produce the least ammonia through decomposition over a 36 hour period. Mackerel, on the other hand, produced a significantly higher ammonia concentration. As proteins are a vital stage of ammonia metabolism, the food content of each food is analysed.

TABLE III. The major content of feeds which were used, as a percentage (%).

If the theory of higher protein content results in higher ammonia production is correct, Tetra Prima Pellets would show the highest levels of ammonia as a result due it containing the highest protein content (48%); this was not the case (avg. max. of 1.50mg/l where as highest avg. max. is produced by mackerel- 5.00mg/l). Also, mackerel produced the highest level of ammonia over 36 hours, but consists of only ~30% protein- almost 20% less protein content than Tetra Prima Pellets, however this pellet brand shows a significantly higher result when compared to the lowest- Hikari algae wafers (avg. max. 0.45mg/l) so it can still be considered that the protein content has influenced the resulting ammonia levels. Also, bloodworm and daphnia feeds contain less that 10% protein but produce results (bloodworm- avg. max. 1.79; daphnia- 0.76) which can be considered similar to flakes and pellets, so it is likely that protein types also have an effect on rates of decomposition. Algae wafers may contain higher amounts of plant/vegetable proteins which are not classed as “essential ammino acids” (Antonio, 2003). Algae wafers are the lowest ammonia producing feed, and mackerel (which could be classed as a 'raw' feed containing a high abundance of essential amino acids) the highest ammonia producing feed. Pellets may also contain high levels of essential amino acids as the results show a high concentration of ammonia at 36 hours.

Conversely, Hikari frozen daphnia produce the second lowest ammonia concentration, and squid produces results (avg. max. 1.29) which can be compared to that of flake feeds (avg. max 1.19). In production, the proteins contained may become severely denatured, leaving few amino acids to be processed by bacteria, limiting decomposition rates.

Surface area of feed can also be considered when determining results. Flakes, for example, could show higher ammonia production as each flake provides a large surface area for decomposition processes to occur- however, this isn't the case. This suggestion would show mackerel as a low ammonia producer within this experiment, which clearly isn't the case.

Additives which are added to the feeds also allow boasting of “promotes water quality” (Aquarian flake product quotation) so, from this experiment, it is plausible to suggest that decomposition rates may be limited as a result. Flakes, pellets and algae wafers contain additives which “promote” water quality. More 'raw' foods (i.e. daphnia, bloodworm, squid and mackerel) wouldn't [naturally] contain additives, but it is known that companies may produce products (bloodworm and daphnia in particular) which have fed on similar additives. This would explain the significantly high result observed with mackerel but not squid.

In conclusion, this study shows that protein content does not correlate with ammonia production via decomposition, but decomposition is suggested to be influenced by additives within the fish foods.

ACKNOWLEDGEMENTS Thank you to Craig Baldwin for presenting the need and providing the equipment for this investigation; Colin Smith and Tel Eneber for helping with the countless problems encountered while using computer programmes (Microsoft Excel in particular) to create this journal article; Blue Reef Aquarium and National Marine Aquarium for their interest in the potential of this study; and the National Marine Aquarium for suggesting and providing feeds to be tested.

REFERENCES Ali, T. & Salim, M., 2004. Studies on the growth response and feed conversion ratio (FCR) of Labeo rohita fingerlings fed on rice polish, fish meal and sunflower meal. International Journal of Agricultural Biology. 6 (5): 914-917.

Antonio, J., 2003. Essential Amino Acids. Strength and Conditioning Journal. 25 (3): 48-49.

Jabeen, S., Salim, M., & Akhtar, P., 2004. Feed Conversion Ratio of Major Carp Cirrhinus mrigala Fingerlings Fed on Cotton Seed Meal, Fish Meal and Barley. Pakistan Veterinarian Journal. 24 (1): 42-45.

Meisinger, J.J., & W.E., Jokela, 2000. Ammonia volatization from dairy and poultry manure.

Managing Nutrients and Pathogens from Animal Agriculture. 334-354. Ithaca: New York.

Sahzadi, T., Salim, M., Kalsoom, U.M-E., & Shahzad, K., 2006. 'Growth Performance and Feed Conversion Ratio (FCR) of Hybrid Fingerlings (Catla catla X Labeo rohita) Fed on Cottonseed Meal, Sunflower Meal and Bone Meal' Pakistan Vetinary Journal. 23 (4): 163-166.

Sarmiento, J.L. & Gruber, N., 2006. Ocean Biogeochemical Dynamics. Princeton University Press: New Jersey, USA. Sawyer, J. 2008. Surface Waters: Ammonium is Not Ammonia [online]. Available at: http://www.extension.iastate.edu/CropNews/2008/0521JohnSawyerMattHelmers.htm [10.11.2010].

Sepers, A.B.J., 1981. 'Diversity of ammonifying bacteria' Hydrobiologia. 83 (2), 342-350.

FIG. 1: Figure 4 of Meisinger, J.J., & W.E., Jokela, 2000. Ammonia volatization from dairy and poultry manure. Managing Nutrients and Pathogens from Animal Agriculture. 334-354. Ithaca: New York.

APPENDICES Results placed by brand with colour to highlight which foods produce a higher concentration of ammonia through decomposition over a 36 hour period.