The Importance of Measuring Carbon Dioxide in Aquaculture

By Pete Southgate, the Fish Vet Group - While oxygen and ammonia levels are often viewed, rightly, as critical to fish health and performance, carbon dioxide tends to be ignored and very few aquaculture facilities regularly monitor CO2 levels. This is partly due to the fact that it is not easy to measure CO2 and partly due to an assumption that, if their other water quality parameters, particularly oxygen, are OK, then CO2 will not be an issue.

There are, however, circumstances when carbon dioxide levels can be relatively high and there is an increasing body of evidence to suggest that there are a number of ways in which CO2 can have an adverse effect on fish health.

Carbon dioxide levels of below 10mg/l are thought to be well tolerated by fish, although sensitivity to the gas varies between species. The level of CO2 in the water varies with the respiratory and photosynthetic activity of animals and plants in incoming water, the level of decomposition of organic material in that water (a very significant contributor to CO2 levels in some nutrient-rich waters), and the respiration of the fish themselves. CO2 can build up to significantly high levels in systems with large numbers of fish and relatively slow water turnover.

The effect of increased CO2 in water is to reduce the rate at which CO2 from the fish's own metabolism can be released from the blood through the gills, thus the CO2 in the blood also increases - this is known as hypercapnia - resulting in a drop in the blood pH, an acidosis. At the same time the oxygen-carrying ability of the haemoglobin in the blood is reduced.

So, what is the effect of this hypercapnia? In the short term the physiology of the fish can counteract the effect by balancing the acidosis with an exchange of ions such as increasing the uptake of bicarbonate and losing hydrogen and phosphate ions and little harm is done. In the long term this balancing act can have a more profound effect on the health of the fish.

Nephrocalcinosis in salmonids has long been recognised as a pathological entity related to high dissolved CO2, eventually leading to the formation of large mineralised deposits within the excretory tissue of the kidney and associated kidney pathology. The condition can result in poor condition and performance and occasional fish loss, particularly if other 'stressors' are present. The relationship between nephrocalcinosis and high CO2 is still not completely understood, but is likely to involve the excretion of minerals, particularly phosphorus and calcium, when compensating for blood acidosis.

Some of the work presented most elegantly at recent Alpharma conferences would also suggest that poor water quality in hatcheries and smolt units, particularly high CO2 and hypercapnia, may lead to an increased susceptibility of fish to pathogens which later leads to clinical disease with, for example, IPN. By paying appropriate attention to this aspect of water quality in the early rearing stages, this may reduce vulnerability to clinical disease.

Tony Wall, in a recent Fish Farming Today article (A is for Deformity) highlighted issues relating to spinal abnormalities. It has been postulated that bone demineralisation associated with hypercapnia may be playing a role in this and it is an area which needs to be rigorously investigated.

As mentioned earlier, CO2 is not the easiest of gases to monitor, standard laboratory methods are pretty complex, although there is a relatively easy titration method which is fairly accurate and could be carried out on the farm. CO2 meters tend to be costly and temperamental (insert wife joke here).

Do try to consider the effect of CO2 on your fish stocks. Remember that high levels of oxygen may not help either as the fish respiratory rate is governed by levels of oxygen, high O2 can slow the rate of elimination of CO2 from the blood, thus increasing the hypercapnia. By increasing levels of oxygen, you may just make things worse.

Source: Fish Vet Group - July 2005

Streptococcosis In Tilapia: A More Complex Problem

To improve our understanding of streptococcal disease in tilapia, Intervet/Schering-Plough Animal Health, Brian Sheehan performed extensive epidemiological studies in the major tilapia-producing countries of Asia and Latin America.

These studies have yielded almost 500 streptococcal isolates recovered from tilapia at approximately 50 sites in 13 countries over the last 8 years. The isolates were identified using standard biochemical and bacteriological identification methods and subsequently analyzed by cluster analysis (unweighted pair-group average based on percent disagreement). Interestingly, of the nearly 500 streptococcal isolates recovered from tilapia, 82 per cent were identified as Streptococcus agalactiae and 18 per cent were identified as Streptococcus iniae.

S. iniae is a significant fish pathogen causing disease and mortality in many marine and freshwater cultured fish species in tropical and sub-tropical areas. Vaccines to combat S. iniae infection in a variety of fish species including tilapia are available, and there is a large body of literature on the pathogenesis of this organism in a variety of fish species.

Considerably less information is available for fish-pathogenic S. agalactiae. Although more commonly associated with disease in human and bovine hosts, fish-pathogenic S. agalactiae has been documented from as early as 1966, when a non-hemolytic Group B Streptococcus was identified as the cause of two epizootics in golden shiners (Notemigonus crysoleucas). Today, with the intensification of aquaculture, we find S. agalactiae to be a significant cause of mortality and morbidity in both marine and freshwater cultured species and particularly in tilapia.

Table 1. Major streptococcal pathogens of tilapia and their prevalence in the Asia-Pacific region
		Global prevalence (as per cent of total streptococcal isolations)*
S. agalactiae Biotype 1		26
S. agalactiae Biotype 2		56
S. iniae		18
* Data generated by Intervet/Schering-Plough Animal Health, Singapore

Detailed analysis of our tilapia S. agalactiae isolates suggests the presence of two distinct clusters, which differ in a variety of biochemical and phenotypic characteristics. We refer to these distinct clusters as biotypes; on this basis, we differentiate between typically beta-hemolytic “classical” S. agalactiae (hereafter referred to as S. agalactiae Biotype 1) and typically non-beta-hemolytic S. agalactiae (hereafter referred to as S. agalactiae Biotype 2). These latter strains were previously classified as S. difficile /S. difficilis but have subsequently been reclassified as non-hemolytic variants of S. agalactiae.

In our epidemiological surveys to date, 26 per cent of all streptococcal isolates of tilapia were found to be S. agalactiae Biotype 1 and 56 per cent were identified as S. agalactiae Biotype 2 (Table 1). The significance of this observation and how it relates to the development of S. agalactiae vaccines for tilapia will be explored in this paper.

Materials and methods

Bacterial isolates recovered from diseased tilapia were identified as S. iniae or S. agalactiae Biotype 1 or Biotype 2 using a variety of standard biochemical and phenotypic tests. The test results were analyzed by cluster analysis (unweighted pair-group average based on percent disagreement). Alternatively, identification was made using species- and/or biotypespecific PCR.

Experimental S. agalactiae Biotype 1 or Biotype 2 vaccines were manufactured as water-in-oil emulsions containing inactivated biotype-specific bacterial antigens and metabolizable non-mineral oil. The vaccines were given as a single dose to fish weighing 15 g or more.

The efficacy of the biotype-specific vaccines was evaluated in laboratory trials following intraperitoneal vaccination of 15 g tilapia. Age-matched, unvaccinated fish from the same cohort and origin were maintained as controls.

At various time points after vaccination, vaccinated and control fish were challenged by intraperitoneal injection with virulent heterologous S. agalactiae Biotype 1 or Biotype 2 strains. Fish were observed for 14 or 15 days post-challenge and mortality was recorded daily. Post-mortem recovery of the challenge organism was performed on fish that died during the observation period and on all surviving fish at the end of the observation period.

Results

To date, we have identified the species and, for S. agalactiae, the biotype of almost 500 streptococcal isolates gathered from approximately 50 sites in 13 countries. This is not a true epidemiological analysis since some sites were visited on multiple occasions and are therefore overrepresented in the data set, but this nonetheless represents a detailed analysis of the impact of streptococcal disease in tilapia.

As mentioned above, 26 per cent of all streptococcal isolates of tilapia were found to be S. agalactiae Biotype 1 and 56 per cent were identified as S. agalactiae Biotype 2; 18 per cent were identified as S. iniae.

It is intriguing that our analysis further suggests that the S. agalactiae biotypes are present in distinct geographical zones (Figure 1).

From our sampling of tilapia worldwide, S. agalactiae Biotype 2 is the most prevalent and geographically diverse of the streptococcal pathogens. In Asia, we find S. agalactiae Biotype 2 in China, Indonesia, Vietnam and the Philippines, and in Latin America, we have found it in Ecuador, Honduras, Mexico and, most recently, in samples from Brazil.

In contrast, we find S. agalactiae Biotype 1 to be the dominant streptococcal pathogen of tilapia in Thailand, Malaysia and Singapore. S. iniae is often found in association with S. agalactiae Biotypes 1 or 2 in China, Ecuador, Honduras, Indonesia, the Philippines and Thailand. Only in the Philippines and Vietnam have we observed S. agalactiae Biotype 1, S. agalactiae Biotype 2 and S. iniae in tilapia in the same country.

It is known that S. iniae vaccines do not provide protection against S. agalactiae infection. To determine if our classification of the fish pathogenic S. agalactiae has consequences for the development of vaccines to control this devastating disease, we assessed in a laboratory challenge the ability of biotype-specific vaccines to protect against lethal challenge with S. agalactiae Biotype 1 or Biotype 2 strains. The results of representative experiments are shown in Figures 2 and 3 and are summarized in Tables 2 and 3.

Tilapia vaccinated with an experimental S. agalactiae Biotype 1 vaccine were protected against lethal challenge with a virulent S. agalactiae Biotype 1 strain (Figure 2A). However, no protection against challenge with a virulent Biotype 2 strain was observed in the Biotype 1-vaccinated fish (Figure 2B).

Figure 2A (top) and 2B (bottom). Efficacy of a S. agalactiae Biotype 1 vaccine against challenge with a heterologous S. agalactiae Biotype 1 strain (A) and a S. agalactiae Biotype 2 strain (B). Vaccinated and unvaccinated control tilapia were challenged as described at 3 weeks post-vaccination. Fish were observed for 15 days after challenge and mortality was recorded daily. Post-mortem recovery of the challenge organism was performed on fish that died during the observation period and on all surviving fish at the end of the observation period after challenge (included as “pos”).

Similarly, fish vaccinated with a S. agalactiae Biotype 2 vaccine were protected against lethal S. agalactiae Biotype 2 challenge (Figure 3A); however, there was no protection against challenge with a virulent Biotype 1 strain in the Biotype 2-vaccinated fish (Figure 3B). Thus, vaccination with biotype-specific bacterin vaccines induces biotype-specific protection against mortality caused by S. agalactiae.

Conclusions

Our data suggest that S. agalactiae, and to a lesser extent S. iniae, are the principal agents of streptococcosis in tilapia. Detailed analysis of our tilapia S. agalactiae isolates suggests the presence of two biotypes, which differ in a variety of biochemical and phenotypic characteristics.

In our experience, these two S. agalactiae biotypes cause subtly distinct disease syndromes. S. agalactiae Biotype 1 infects fish throughout the production cycle from juvenile to grow-out, while S. agalactiae Biotype 2 causes disease predominantly in larger fish.

Moreover, and most significantly from a health-management perspective, we have demonstrated that immunity is biotype-specific.

Figure 3A (top) and 3B (bottom). Efficacy of a S. agalactiae Biotype 2 vaccine against challenge with a heterologous S. agalactiae Biotype 2 strain (A) and a S. agalactiae Biotype 1 strain (B). Vaccinated and unvaccinated control tilapia were challenged as described at 3 weeks post-vaccination. Fish were observed for 14 days post-challenge and mortality was recorded daily. Post-mortem recovery of the challenge organism was performed on fish that died during the observation period and on all surviving fish at the end of the observation period after challenge (included as “pos”).

To our knowledge, there are no obvious geographical, physiological or environmental explanations for the country-to-country distribution of S. agalactiae Biotypes 1 and 2. It would be prudent, however, to consider that the distribution might change in time, probably through trade in live fish.

new Transportation of Fish with Bags

By L. Swann, Illinois-Indiana Sea Grant Program, Purdue University - Fish, shellfish, and plants often are transported in sealed plastic bags containing small quantities of water and pure oxygen.

Introduction

Bag shipment requires placing a prescribed weight of fish in 1.5 to 2 gallons of water in 3 ml polyethylene bags, 18 by 32 inches. Excess air is removed from the bag and replaced with pure oxygen. The bag is sealed, placed in an insulated container and finally into a cardboard shipping box and shipped.

Bag shipment may be the best choice for the shipper for several reasons. First, very small fish and fry could be damaged by being shipped in large tanks. Second, due to the extreme distances involved, bag shipment may offer economic advantages over standard tank transportation. This fact sheet will focus on transport of fish. With minor modifications the techniques and principals discussed also apply to shellfish.

Water quality during shipping
Fish health is affected by changes in water quality parameters while in the plastic bags during the transportation process. The parameters to be considered are temperature, dissolved oxygen, pH, carbon dioxide, ammonia and the salt balance of the fish's blood. The rate of change of each parameter is affected by the weight and size of fish to be transported and the duration of transport.

Temperature

Fish are cold-blooded, so the metabolic rate of fish is affected by the temperature of the environment. The metabolic rate of fish will double for each 18 degree F increase in temperatures and be reduced by half for each 18 degree F decrease in temperature. A reduced metabolic rate will decrease the oxygen consumption, ammonia production and carbon dioxide production. Therefore, it is essential to transport fish as low temperatures. For cool and warm water species a temperature of 55 degrees to 60 degrees F is recommended. For species such as tilapia and red drum temperatures should be nearer to 60 degrees F. Cold water fish, such as trout, inhabit colder water and should be transported at even colder temperatures, such as 45 to 50 degrees F.

To achieve the desired transport temperature, fish should be held in tanks of cool water. By holding the fish in tanks for two days, the water temperature can be gradually reduced by adding cool water. After loading the fish into bags, final decreases and maintenance of temperatures during transport can be accomplished by adding ice or (more commonly) gel packs.

Ice or gel packs often are used during transport, especially over longer transport periods that might allow increases in temperature. One-half pound of ice will reduce the temperature of one gallon of water by about 10 degrees F. Insulated Styrofoam shipping boxes also are used to prevent outside temperatures from affecting the temperature of transport water. In some instances, 20 to 40 quart coolers are used for transport.

Dissolved oxygen

The most important single factor in transporting fish is the provision of adequate concentrations of dissolved oxygen (DO). The importance of supplying adequate levels of DO cannot be overemphasized. Failure to do so results in severe stress which may contribute to fish kills two to three days after transport.

The amount of oxygen that can be dissolved in fresh water is based primarily on water temperature. The water is referred to as 100 percent saturated when the upper saturation level is reached. DO saturation is higher for cool water than for warm water. For example, at sea level DO saturation of 45 degrees F water is 12.1 parts per million (ppm) but at 60 degrees F, saturation is 10.0 ppm. Because pure oxygen is used during bag transport, DO levels in the water will be saturated and the low oxygen levels usually will not be a problem unless the bag is improperly sealed or develops holes caused by the spines of large fish. It is important to have a 75 percent volume of oxygen in the bag to ensure adequate diffusion of oxygen at the surface of the water.

The quantity of hydrogen ions (H+) in the water will determine if it is acidic or basic. The scale for measuring the degree of acidity is called the pH scale, which ranges from 1 to 14. A value of 7 is considered neutral, neither acidic nor basic; values below 7 are considered acidic; above 7 basic. The acceptable range for fish growth is between pH 6.5 and 9.0. The pH of water will be influenced by the alkalinity (buffering capacity) and the amount of free carbon dioxide. The pH of the transport water will also affect the toxicity of ammonia. Even in well-buffered transport water the pH will sometimes decrease by one pH unit.

Carbon dioxide

As fish respire they produce carbon dioxide as a byproduct. Carbon dioxide reacts with water to form a weak acid. This weak acid will in turn decrease the pH of the water. High levels of carbon dioxide (greater than 20 ppm) will interfere with the oxygen uptake in the fish's blood. High levels of carbon dioxide sometimes are found in well water. Excess carbon dioxide in well water can be reduced by mechanical aeration or by passing the water through a degassing column.

Ammonia

Ammonia build up occurs in transport water as a result of fish metabolism and, to a lesser extent, bacterial action on fish waste excreted into the water. Two forms of ammonia occur in transport water: ionized (NH4+), and un-ionized (NH3). Unlike the ionized form, the un-ionized form of ammonia is extremely toxic at concentrations as low as 0.2 ppm. In tests for ammonia, both forms are grouped together as "total ammonia nitrogen" (TAN). The percent of ammonia that is un-ionized will depend on both temperature and pH (Table 1).

Total ammonia concentrations may reach more than 14 ppm during transport. However, using Table 1, the percent of the total ammonia which is un-ionized at pH 6.5 and 55 degrees F is only 0.07 percent. Therefore, the un-ionized ammonia concentration at 14 ppm is 14 x 0.0007 = 0.0098 ppm. Un-ionized concentrations greater than 0.05 ppm should be handled with caution.

The easiest way to reduce toxic ammonia buildup in transport water is to lower the temperature of the transport water and to stop feeding several days before transporting. Fish up to eight inches long should not be fed for 48 hours before loading and transporting and those larger than eight inches should not be fed 72 hours before transporting.

Table 1.
Percent of ammonia in the un-ionized form of different temperatures (degrees F) and pH values

Chemical additives

Numerous chemical additives can be added to the transport water to alleviate several problems associated with transporting fish in bags. Since overdoses of chemical can cause death, care must be taken when measuring the dosage of each chemical. It is essential to double check every calculation and to use an accurate balance when weighing chemicals.

The most common chemical added to transport water is salt (NaCl). Salt is used to relieve stress associated with maintaining a water balance in the fish. Freshwater fish have a blood salt concentration higher than the salts of the transport water. As a result, the fish are continually losing salts to the surrounding water. Concentrations of 5,000 ppm (0.5 percent) are commonly used. A 5,000 ppm concentration can be made by adding 19 grams (one tablespoon) of salt per gallon to water used during transport. Use non-iodized salt that contain no anti-caking compounds. Canning salt is a good example.

If the alkalinity of the transport water is less than 100 ppm, some type of buffering compound should be added to the water. Properly buffered water will help remove freed carbon dioxide which causes drops in pH. Sodium bicarbonate (Na2CO3) is one of the fastest reacting buffers and should be added at a rate of 1 g per gallon. of water. Finally, the fish will suffer some stress because they are transported in crowded conditions. Sometimes a chemical anesthetic may be beneficial by producing a light sedation. The only anesthetic approved by Food and Drug Administration (FDA) for food fish is Finquel (tricaine methanesulfonate). Finquel may be used at a rate of 0.06 to 0.25 g per gallon. of water.

Carrying capacity

The maximum weight of fish that can be safely transported within a given period of time is the carrying capacity. The carrying capacity depends on the duration of haul, water temperature, fish size and fish species. If water quality conditions such as temperature, oxygen, carbon dioxide, alkalinity and ammonia are constant, then carrying capacity will depend on the fish species. In general, fewer pounds of smaller fish can be transported per gallon of water than larger fish. General carrying capacity guidelines are given in Table 2. It is important that first-time or experienced shippers handling a new species test-run a batch before undertaking a large shipment.

Table 2.
Carrying capacity in pounds of warm water fish transported in 18- x 32- inch polyethylene bags containing 2 gallons of water (about 15 pounds). Water should be moderately hard (80 to 100 ppm total hardness) and have a temperature range of 55-60 degrees F (Dupree and Hunter, 1984)

Transport procedure

Days before the fish are loaded and transported, the shipper should determine the carrier to be used, time of departure, time of arrival and shipping costs. This information needs to be communicated to the receiver well before the shipping date. All loading should be planned to allow boxes to be shipped as soon after loading as possible. With proper planning, unnecessary delays in delivery and pickup can be avoided. The receiver is responsible for contacting the shipper if any deaths occur.

Procedure for shipping fish:

• Carefully add the proper weight of fish to 1.5 to 2 gallons of clean high quality water. Water contained in the bag needs to be within two degrees of the holding water. Chemicals, if any, need to be added at this time.
  
• Deflate to remove air and then fill with pure oxygen. Approximately 75 percent of the volume in the bag should be oxygen.
  
• Twist mouth of bag tightly and secure with heavy-duty rubber bands. Castration rings or heat sealing may also be used. Place bag inside a second bag, which has a frozen gel pack, and seal the bag.
  
• Place the sealed bag inside a cardboard shipping box and seal the box. The shipping box must be clearly labeled "Live Fish," with the name and address of the shipper and receiver displayed prominently. In the case of trips which may expose the bags to extremes of heat or cold, the bags may need to be placed inside a Styrofoam cooler before being packed into the shipping box.

Procedure for unpacking fish

Unpacking is as important as packing fish in bags. Guidelines for proper unpacking are as follows.

• Float unopened bags in a shaded area of the receiving water for at least 30 minutes to allow temperature to equalize. Check water temperatures and watch for mortalities.
  
• Open bags and add 2 to 3 gallons of receiving water to the bag.
  
• Gently and slowly pour fish into the receiving water.

Suggested Readings

Dupree, H.K. and J.V. Huner, 1984. Third Report to Fish Farmers. U.S. Fish and Wildlife Service, Washington, D.C.

Piper, R. G., I.B. McElwain, L.E. Orme, J.P. McCraren, L.G. Fowler, and J.R. Leonard, 1982. Fish Hatchery Management. U.S. Fish and Wildlife Service, Washington, D.C. 517 pp.

S.K. Johnson, 1988. Transport of Fish and Crustaceans in Sealed Containers. Inland Aquaculture Handbook. Texas Aquaculture Association, College Station, TX. A1504-A1509.