Live Shipment of Flounder: Developing Methods to Enhance Survival, Improve Product Quality, and Increase Value. Mike Frinsko North Carolina Cooperative Extension Service, Pitt County Extension Center, Greenville, NC 27834 James E. Willis Harkers Island, North Carolina, 28531

Abstract

Live fish fisheries are an important and still growing component of the domestic and international commercial fishing industry. One challenge inherent to this fishery is the ability for suppliers to dependably deliver fish alive and in excellent condition to retailers after many hours (over 48 hrs.) in transit. This experiment was designed to address that challenge for summer flounder, an attractive candidate for inclusion in this fishery by area suppliers. We compared survival, changes in water quality, and muscle degradation among summer flounder, Paralichthys dentatus, held at three temperatures and with three water treatments for 60 hours. Three groups of 12 flounder, averaging 0.97 kg, were acclimated for 24 hours at either 5⁰C, 10⁰C, or 18⁰C. Individual fish were then packaged in individual bags with one of four water treatments. Three fish at each temperature were held in either water (control), Amguard (an amine polymer), AQUI-S (an anaesthetic), or Amguard + AQUI-S. A variety of environmental parameters (dissolved oxygen, nitrate levels, etc.) were measured before each bag was closed. Finally, each bag was placed into a styrofoam/cardboard transport box and sealed. After a 60 hour holding period, the boxes were opened and the contents examined. We noted survival of each fish, took a sample of axial muscle to measure pH, and measured changes in the environmental parameters. Analysis of the survival data indicated that mortalities were significantly higher for fish held at 18⁰C than at either 5⁰C or 10⁰C. Water treatment had no impact on survival. Of special interest, muscle pH was not significantly different among treatments at any temperature after 60 hours. These results indicate that flounder shipped at either 5⁰C or 10⁰C would likely survive most domestic or international travel and without significant degradation in muscle quality.

Introduction

The commercial harvest of flounder in North Carolina is an integral part of the state's overall commercial fishing industry. In 2000, landings of over 6,600,000 lbs were reported with a dock-side value in excess of $11,600,000 (North Carolina Division of Marine Fisheries, 2001). For commercial fisherman, however, environmental and regulatory trends over the past decade have led to a general climate of uncertainty in the industry.

Recent regulations, including a quota system, have limited the size and volume of flounder that can be caught and have led to increased competition among fishermen. Fishermen with larger vessels have an advantage because they are capable of large harvests early within the season. Historically, more traditional family operations have been successful while operating one or a few, smaller and less efficient vessels. This new competitive environment is negatively impacting these family businesses and many are being forced into other occupations. For family operators to stay in business they now must increase their level of efficiency. This includes investigation of non-traditional markets where enhanced product value can be developed.

One potential market for enhance product value is live fish. The targeted markets focus primarily in those national and international metropolitan areas that have large ethnic Asian communities. Historically, Asian communities, especially of Chinese, Japanese and Korean heritage, have placed a high value for fish sold alive. Live products are considered a guarantee of freshness and quality. In addition, consumers feel a heightened sense of confidence when they are able to examine a live product prior to purchase. Depending on the season, live flounder can command a premium price that is two to eight times the value of their fresh counterparts. This added revenue can be a critical economic boost to the earned income of smaller family fishing enterprises.

While live flounder markets have existed overseas for many years, sales of this type are a relatively recent phenomena in the U.S. Traditional methods of packing flounder for live shipment have often resulted in high mortality, especially when shipped great distances. Metabolic waste buildup, oxygen depletion, and other stressors associated with handling and transport are all considered deleterious to the final condition and ultimate product quality at the marketplace.

The rationale for this study was to expand marketing opportunities for area commercial fisherman. Our goal was to investigate methods and procedures that would increase the survival of live-shipped market-sized flounder. A primary focus was to minimize physiological factors known to negatively impact the health and condition of fish during transport. A major consideration targeted reduction of overall metabolic activity. This was manipulated by modifying simulated shipping temperature and by the addition of chemical sedatives. Reductions in metabolism would not only relieve potential oxygen stress and lactic acid buildup in muscle, but also reduce the accumulation of toxic waste products during transport. Regarding the latter, we also explored the impact of chemical additives that would reduce the accumulation of toxic ammonia. If manipulation of temperature and additives to the shipping water would maintain high water quality during the prolonged shipping period, increased survival and maintenance of product quality would result. This would lead to enhanced product marketability and value, resulting in greater profit potential for both fishermen and wholesalers interested in this unique and growing area.

Materials and Methods

Market sized flounder were caught using pound nets by commercial fisherman, Mr. J. E. Willis, during the fall 2000 fishing season. These nets were set in Core Sound near Harkers Island, North Carolina. Batches of approximately 30 fish each were selected using long handled dip nets from the pound nets on two days, approximately one week apart in mid November. The fish, which averaged 0.97 kg, had been held in the nets for no more than 24 hours. Only flounder in good condition were selected.

The flounder were transported from the pound nets to shore using a small 5.5 m motor boat. The vessel was equipped with an oval polycarbonate holding tank with a capacity of approximately 379 L. Transport water was flushed frequently using an electric pump to bring clean water in and force used water overboard via a drain line. Oxygen levels were kept near saturation using pure bottled oxygen and silica diffusers. The trip to shore lasted approximately 20 minutes. Once at dock, fish were kept on board and trailered with the boat for an additional 45 minutes to the holding facility at Carteret Community College.

At the community college, fish were unloaded into a 4.6 m diameter PVC lined outdoor pool. Water from the sound was pumped through a sand filter into the pool to a depth of 1.1 m. An exchange of new water was made every three days, depending on visual inspection for fecal contamination or accumulation of other debris. Aeration was provided using an 2.4 m length of partially submerged 7.62 cm PVC pipe and an air hose. The hose was placed through the length of the pipe, with the open end at the low end of the pipe at the pool bottom. Air bubbled to the water surface through the pipe creating a simple air-lift type pump; this destratified the holding water and simultaneously provided aeration. Fish were not fed during the holding period nor was the holding water treated in any way.

In a building adjacent to the holding pool, two 530 L circular fiberglass tanks were set up for temperature-acclimating the fish. The tanks received the same water as the holding pool. Ambient water temperature was approximately 10-12⁰C. Due to the limitations of having two tanks and three temperature treatments, the experiment was divided into two parts.

The 18⁰C temperature group, was tested first. A digitally controlled, 115 volt, 1800 watt submersible electric titanium heater (model QP20-T, Cleveland Process Corp.) maintained water temperature at 18⁰C for the duration of the experiment. For the 5^oC experiment, the holding water in one of the fiberglass tanks was chilled to 5⁰C with a one HP electric water chiller (model D1-100, Frigid Units, Inc.). The last group, at 10^oC, used fish maintained at ambient temperature.

For each temperature group, 20 flounder were randomly netted and carried to the indoor holding tanks. For the 18 ^oC and 5^oC groups the holding water was either heated or chilled gradually over an approximately four hour period. The fish were then held at their experimental temperatures for approximately 24 hours. Fish in poor condition were removed during this time. Oxygen levels were maintained at over 80% saturation. All oxygen measurements for this study were made with a polarographic oxygen meter (Model 55, Yellow Springs Instruments, Inc.). In addition, initial levels of CO₂, and pH in the holding water were determined spectrophotometrically (Model #2000, LaMotte Co.).

After 24 hours of temperature acclimation, individual flounder were netted and placed in a combination styrofoam/ cardboard flatfish transport box, 53.3 x 27.9 x 17.8 cm (Arjay National Inc.) containing a 40.6 x 55.9 x 20.3 cm, square bottom, poly transport bags (model # SB82, Aquatic Ecosystems, Inc.). Individual fish were weighed in their tarred boxes. Enough of the appropriate acclimation water was added to each bag to cover the gills of each fish. This was determined to be 6.3 kg of water per kg of fish. There were a total of 12 fish in each temperature group.

The 12 fish in each temperature group were randomly assigned to one of four water treatment groups. In the first group (controls) three fish were packaged just with water from the temperature acclimation tank. A second group of three (Amguard treatment) received the amine polymer, Amguard (Seachem, Inc.); this reacts with ammonia to form a stable, non toxic compound. 1.6 grams of Amguard power was dissolved in 1 L of transport water and the solution added to the transport bag. The third group (Aqui-S treatment) received the clove oil based anaesthetic Aqui-S (AQUI-S New Zealand, Ltd.) before individual packaging. Aqui-S was added to the holding tank water at a concentration of 10 ml/1000 L. After light sedation was observed, the fish were weighed and bagged as in the other groups, but with 10 ppm Aqui-S. In the fourth group (Aqui-S + Amguard), three anaesthetized fish were bagged and boxed with holding water that contained Aqui-S; in addition, Amguard was added to each bag as described above.

After the appropriate water treatments were established, pure oxygen was added to the water of each bag, to saturation. The remaining void space in each bag was filled with oxygen. The bag tops twisted and wrapped closed using rubber castration rings (model # FBT1, Aquatic Ecosystems, Inc.) Lids were added to each styrofoam transport box and then the cardboard outer box sealed with adhesive tape. Notations were made of the time sealing occurred and which experimental group (temperature and water treatment) to which the box belonged.

The boxes were held in an insulated laboratory space at Carteret Community College at a constant room temperature of 24.5⁰C for 60 hours. This simulated a typical transport duration and temperature conditions encountered during shipping.

After the 60 hour holding period, boxes were opened in the order of their sealing and examined. As each transport bag was opened, the concentration of dissolved oxygen was measured. Survival of each fish was then determined. The surviving fish were then sacrificed by pithing. A dorsal axial muscle sample was removed from all fish, survivors and mortalities. These tissue samples were then frozen and stored at -70^oC. The samples were later thawed and sliced with a scalpel along the sagittal plane to expose uncontaminated tissue. Muscle pH was directly recorded using a flat surface pH probe (model 136411163, Fisher Scientific Co.). Finally, the temperature, pH, and CO₂ levels in the holding water after 60 hours were measured.

Results

Key to drawing inferences among temperature or water treatments is ensuring that the fish were randomly assigned to treatments at the start of the experiment and that there were no biases in the distribution of fish among groups. We evaluated this with a two-way analysis of variance (ANOVA) with temperature treatment and water quality treatment as independent variables and fish mass as the dependent variable. There was no significant difference among temperature treatments or water quality treatments in the size of flounder assigned to each treatment (see Table 1). The flounder in this experiment averaged 0.97 kg. Similarly, there were no differences in the amount of water added to each container (see Table 2). Water volume averaged 6.3 kg water per kg of fish. Thus, the only initial conditions that varied among treatments were those that were part of the design, temperature and water treatment.

Because there were insufficient replicates to address the impact of the two factors together on survival, survival of flounder was analyzed separately for the temperature and water treatment groups. All flounder that were acclimated and initially packed at 10oC or at 5oC survived the 60 hour holding regime; however only 33% of the fish in the 18oC group survived (Table 3). This difference in survival among temperature treatment groups was highly significant (X2 = 20.57, P < 0.001).

Survival was also compared among water treatment groups, ignoring temperature differences. Survival among the four treatments ranged from 78%-89%; in fact, the control fish without any water treatment had the highest survival (Table 4). A chi-square test comparing survival among the four water treatment groups indicated that there was no significant difference among the four chemical treatments (X2 = 0.53, P>0.75).

A key indicator of fish quality is muscle pH. We initially analyzed variation in muscle pH values among temperature and water treatment groups via a two-way analysis of variance. This analysis revealed no differences among groups in muscle pH (Table 5); the average muscle pH was 6.5. Focusing just on live fish, there were no significant differences among temperature or water treatment groups, also (Table 6).

There were no dramatic differences in water quality variables among temperature and water treatments. Analysis of CO2 concentrations demonstrated that there was no significant different between transport containers that held fish that survived versus those that held fish that died (Table 7). Other physical variables (pH, and O2 concentration) also did not demonstrate significant differences at a specific temperature.

Total ammonia was to have been measured for each fish at the beginning and again at the duration of the experiment to determine the impact of the anaesthetic and amine polymer, separately and in combination, in reducing metabolism at each temperature. This did not occur as the test reagents were unavailable at the time of the experiment.

Table 1.   Analysis of Variance: dependent variable is fish weight; independent variables are temperature (50C, 100C and 180C) and water additive (Amguard, Aqui-S, Amguard + Aqui-S, or water).
 
 

Source	Sum-of-Square	df	Mean-Square	F-ratio	P
Temperature	0.093	2	0.047	0.344	0.71
Additive	0.154	3	0.051	0.378	0.77
Interaction	1.024	6	0.171	1.256	0.031
Error	3.260	24	0.136

Table 2. Analysis of Variance: dependent variable is water weight; independent variables are temperature (50C, 100C and 180C) and water additive (Amguard, Aqui-S, Amguard + Aqui-S, or water).
 

Source	Sum-of-Square	df	Mean-Square	F-ratio	P
Temperature	3.977	2	1.989	0.371	0.69
Additive	6.803	3	2.268	0.423	0.74
Interaction	40.257	6	6.709	1.250	0.32
Error	128.779	24	5.366

Table 3. Survival of flounder among temperatures and water additive treatments
 

a) Temperature (0C)	Additive	#dead	#alive
5	water	0	3
5	Amguard	0	3
5	AQUI-S	0	3
5	A + A	0	3
10	water	0	3
10	Amguard	0	3
10	AQUI-S	0	3
10	A + A	0	3
18	water	2	1
18	Amguard	1	2
18	AQUI-S	3	0
18	A + A	2	1

b) Observed Survival At Each Temperature:
 

Temperature (0C)	Dead	Alive	Total	% Total
5	0	12	12	100%
10	0	12	12	100%
18	8	4	12	33%
total	8	28	36	78%

X2 = 20.5714 with 2 degrees of freedom. P<0.001

Table 4. Survival Among Water Treatments

a) Observed Survival at Each Treatment:
 

Treatment	Dead	Alive	Total	%Total
Water	1	8	9	89%
Amguard	2	7	9	78%
Aqui-S	2	7	9	78%
Amguard + Aqui-S	2	7	9	78%
Total	7	29	36	81%

X2 = 0.53202 with 3 degrees of freedom. P>0.75

X2 at P = 0.05 is 7.815.

There is no significant difference among the 4 treatments

Table 5.   Analysis of Variance: dependent variable is muscle pH; independent variables are temperature (50C, 100C and 180C) and water additive (Amguard, Aqui-S, Amguard + Aqui-S, or water).
 

Source	Sum-of-Square	df	Mean-Square	F-ratio	P
Temperature	0.019	2	0.010	0.453	0.64
Additive	0.049	3	0.016	0.757	0.53
Interaction	0.045	6	0.007	0.348	0.90
Error	0.514	24	0.021

Table 6. Analysis of Variance: dependent variable is muscle pH from surviving specimens; independent variables, temperature (50C, 100C and 180C) and water additive (Amguard, Aqui-S, Amguard + Aqui-S, or water), are combined in the overall model.
 

Source	Sum-of-Square	df	Mean-Square	F-ratio	P
Model	0.109	10	0.011	0.426	0.91
Error	0.436	17	0.026

Table 7. Analysis of Variance: dependent variable is CO2 concentration; the independent variable is fate (alive vs. dead) at 18oC .
 

Source	Sum-of-Square	df	Mean-Square	F-ratio	P
Fate	2926.042	1	2926.042	3.614	0.09
Error	8096.875	10	809.688

Discussion

The main goal of the experiment was to develop insight into methods that might increase survival of flounder during transportation. Specifically, we examined the role of temperature and of various possible additives that might reduce stress and mortality of flounder. Our experiments demonstrated that the only factor that had a significant impact on survival was water temperature. Two thirds of the fish held at 18oC failed to survive 60 hours in the transport boxes, while no fish in the 5oC or 10oC treatments died. Our recommendation to the industry is that flounder destined for the live markets should be packaged at temperatures no higher than 10oC.

Surprisingly, there were no observed differences in survival among the water treatments. At 5oC and 10oC, all fish survived regardless of treatment. In addition, at 18oC, more control fish survived than did those in the three additive groups, albeit by one fish in each case. Clearly, however, there was inadequate replication to address the impacts of the additives. These preliminary experiments would indicate that under the conditions of our simulation, additives are not necessary to ensure good survival at lower temperatures; they would add material and handling costs without any advantage. However, there may be a combination of additives that would improve survival at higher temperatures. Only additional studies with various concentrations and combinations of additives can answer this question.

The lack of a difference among treatments in muscular pH indicates that this shipping simulation induced no negative impacts on the quality of the flesh. This consideration is critically important in light of the discriminating nature of the end-use consumers. Of course, our simulation lacked the disturbances due to transfer of shipping containers or movement of vehicles that might induce activity by these fish. Essentially, these fish were in "resting" metabolism for 60 hours. One might expect that the physiological challenges might be greater
if transport induced more "active" metabolism in these fish. Future simulations should address this issue.

In retrospect, the study may have been better designed to examine the impact of the chemical amendments at a single temperature using a greater number of replicates. The initial protocol called for the use of four fish per treatment. Material constraints of fewer holding boxes resulted in the use of three specimens for each treatment at a given temperature. In either case, this would not have allowed for sufficient comparison among each test category.

Acknowledgements

A project of this type could only be possible through the interest and support of others. The authors are indebted to the following people who provided assistance from conception to conclusion. Dr. Ron Hodson, University of North Carolina Sea Grant College Program, assisted with the grant protocol and initial project development. Mr. Skip Kemp, University of North Carolina Sea Grant College Program, assisted with specimen collections and facility maintenance. Dr. David Green and Mr. Greg Bolton North Carolina State University, Seafood Laboratory, Center for Marine Sciences and Technology, Morehead City, provided advice and technical assistance with muscle sample analysis. Dr. Stephen F. Norton Department of Biology, East Carolina University, Greenville, provided assistance with data analysis and final report review. Dr. Mary Kirk and Mrs. Penny Hooper, Carteret Community College, Morehead City, facilitated our use of the mariculture "wet-lab". To all those mentioned above, we offer out sincere thanks.
 

Last updated on March 26, 2003.