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Behavioral response of carp (Cyprinus carpio) to ammonia stress
Aquaculture 165 Ž1998. 81–93
Behavioral response of carp ž Cyprinus carpio / to ammonia stress Dorith Israeli-Weinstein, Eitan Kimmel
)
Agricultural Engineering Department, Technion IIT, Haifa 32000, Israel Accepted 7 March 1998
Abstract The behavioral responses of schools of young Koi fish Ž Cyprinus carpio . to sub-lethal ammonia concentrations were monitored using CCD cameras and computer image processing. Several geometrical parameters of the schools such as the position of the center of gravity, and the distribution of the fish were calculated continuously and plotted versus time. Swimming speed and activity were measured indirectly through a novel parameter—the Projected Mobility Picture ŽPMP.. The immediate response of the treated fish was to dive to the bottom of the tank and stay there for a time period which increased with the ammonia concentration. Later, the fish rose to depths which increased with ammonia concentration. At high ammonia concentration, the fish approached the surface. Decreased activity of the treated fish was accompanied by a smaller mean distribution. Blood glucose levels of the treated fish increased, and they became indifferent to food. In general, this remote method of continuous monitoring of alterations in fish behavior under stress has the potential to be developed into a method for detecting early signs of stress in populations of fish. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Computer vision; Ammonia stress; Fish behavior
1. Introduction Stress in fish farming systems is associated with a variety of environmental conditions such as reduced oxygen level, abrupt variations in temperature, appearance of contaminants or elevated ammonia. The effect of elevated ammonia on fish held under conditions common in intensive aquaculture is the subject of this study. When fish are stressed, stress hormones are released Žprimary response., the composition of blood and tissues is altered and variations in ventilation and heart rate frequently occur Žsecondary )
Corresponding author.
0044-8486r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 4 4 - 8 4 8 6 Ž 9 8 . 0 0 2 5 1 - 8
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response. ŽMazeaud et al., 1977.. As a result, glucose level in the blood, for instance, has become a common method for estimating the effect of stress ŽMazeaud et al., 1977; Himick and Eales, 1990; Fevolden et al., 1991; Flos et al., 1988.. In the long run, stress induces a reduction in the effectiveness of the immune system, resulting in higher susceptibility to diseases and parasites, decreased growth rate, reduced reproduction and increased mortality. All the stress-induced changes are associated with variations in the behavior of the fish ŽDonaldson et al., 1984; Passino, 1981; Pickering, 1981; Adams, 1990; Robertson et al., 1987; Maule and Schreck, 1990.. Stressed fish change their behavior. By ‘behavior’ we refer to the series of visible actions which are operated and controlled by the nervous, sensory and endocrine systems. Escape or freezing, avoidance or attraction are common behavioral responses to adverse stimuli ŽBeitinger, 1990.. Nitrogen, mostly in the form of ammonia is excreted by fish across the gills. Ammonia level in the bulk of the water is reduced by oxidization to nitrite and to nitrate Žnitrification.. In intensive aquaculture systems, where fish are reared at high densities with insufficient water exchange or filtration, excessively high levels of ammonia can arise. Typical rates of ammonia excretion by carp Ž Cyprinus carpio L. and Carassius auratus . are 2 to 5 = 10y5 mgrl kg h ŽWood, 1993.. In addition, ammonia accumulation in the water may result from bacterial decomposition of uneaten food rich in proteins ŽLang et al., 1987. or seepage of other nitrogen-containing substances such as ammonium sulfate from fertilized fields ŽSanthi et al., 1992.. Maximum ammonia levels of about 0.02 mgrl Žat pH 7.0. are allowed in fish farming ŽPillay, 1990.. Exposure to high concentrations of ammonia reduces survival, inhibits growth, and causes a variety of physiological malfunctions. Ammonia acts as a stressor and stimulates the release of corticosteroid hormones into the blood circulation ŽTomasso, 1994.. Ammonia concentrations of 0.32 to 0.60 mgrl induced mortality rates of 50% in sunshine bass, Morene chrysops, within 96 h ŽWeirich et al., 1993.. In . and the solution, ammonia exists in equilibrium between the ionized form ŽNHq 4 un-ionized form ŽNH 3 .. At pH 7.0 and 208C, only 1% of the total nitrogen is in the form of NH 3 ŽFleck, 1966; Wood, 1993.. NH 3 is more toxic to fish than NHq 4 because it easily diffuses across the gill membrane ŽRusso, 1985.. The toxic effect of elevated ammonia concentrations in the water is attributed to a reduced outward flux of ammonia excretion through the gills, so that the outward flux is blocked and a reversed inward ammonia flux occurs. As a result, ammonia levels in the fish plasma increase. Another possible mechanism for plasma ammonia build-up at high external ammonia levels, is the decreased ammonium outward flux through the NaqrNHq 4 channel and increased Ž . inward NHq 4 diffusion Wood, 1993 . Increased ammonia levels cause physiological and behavioral changes in fish. In smolts exposed to 0.08 mgrl ammonia NH 3 , increased plasma cortisol were found ŽDonaldson, 1990.. Glucose levels increased by 30% in Atlantic salmon exposed to sublethal concentrations of NH 3 Ž0.019 to 0.037 mgrl. for 8 days. The exposed fish showed an increased ventilation rate and their gill filaments were damaged ŽFivelstad et al., 1993.. Juvenile rainbow trout, exposed to 0.25 mgrl of NH 3 for 4 weeks, increased ventilation frequency ŽLang et al., 1987.. Exposure for 24 h of rainbow trout to an external ammonia ŽNH 3 . concentration of 0.72 mgrl increased the body ammonia content from 0.6 to 4 m molrg. At the same time, body Naq and Cly concentrations
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decreased by about 28% and Kq concentration decreased by 35% ŽPaley et al., 1993.. In carp Ž C. carpio L.. exposed for 48 h to sublethal concentrations of ammonia in water Ž0.32–1.04 mgrl NH 3 versus control: 0.07 mgrl., significant changes were found in concentrations of ammonia, urea, lactic acid and total protein, and in activities of hepatopancreas LDH, ADH, GDH, AST, and ALT ŽPalackova et al., 1990.. Extended exposure of carp Ž C. carpio L.. to low concentrations of 0.033 mgrl ammonia caused the appearance of signs of stress in the immune system and led to changes in the composition of blood and hemopoietic tissue Žhead kidney, spleen. after 5 weeks. However, after 10 weeks of prolonged exposure, almost all the signs of stress disappeared ŽWlasow et al., 1990.. In hybrid tilapia Ž Oreochromis mossambicus= O. niloticus ., no stress detected in concentrations of ammonia ŽNH 3 . below 0.84 mgrl. At moderate concentrations of ammonia between 1.28 to 2.13 mgrl, the fish showed signs of stress at first and gradually recovered after 24 h. At high levels of 2.97 to 4.24 mgrl NH 3 , the fish showed signs of marked excitement, followed by exhaustion and usually death within 18 h ŽCheng-Chung and Cheng Liu, 1989.. In this study, we monitored the behavioral responses to various levels of ammonia stress as reflected by the calculated changes of the school parameters. 2. Materials and methods 2.1. The fish Ornamental carp species, Koi-C. carpio, of 8 to 12 cm length, weight of about 10 g and from either sex, were used. Koi fish appear usually in schools. The dominant colors of the fish are black, orange and red. These colors improve the contrast between the fish and the white background for better image processing. The fish were obtained from a commercial fish farm, where they had been raised to the age of 6 months. All fish were from the same batch. In the laboratory, the fish were acclimatized in the experimental tanks for 3 weeks prior to the experiment. During the acclimation time, oxygen levels were kept in the range 7.5 to 8.5 mgrl, corresponding to about 90% of the saturation level. Water temperature was maintained at 19 to 218C and the ammonia level was below 0.001 mgrl, corresponding to the sensitivity level of our measuring method. The fish were fed once a day, around noon, with 3 to 4% of the total fish biomass, given in a form of dry pellets which are floated at water surface. 2.2. The experimental system The system Žschematically shown in Fig. 1. is composed of two white PVC tanks Ž70 = 90 = 90 cm., with one transparent side in each tank. Each tank is divided by a white, vertical PVC partition wall, into control and treatment compartments. The light in the room was provided by two 15 W fluorescent bulbs mounted on the ceiling, some 1.5 m above the water surface. This was the only light source in the room during the acclimation and experiment periods, and it was used only during the day. Two CCD ŽCharge Coupled Device. cameras faced the transparent side of each tank. Both cameras
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Fig. 1. The experimental system.
were 8 mm, black and white cameras ŽBurel-TC655EACX., with pixel resolution of 512 = 512 and with auto-iris lenses. The cameras were located at a distance of 160 cm from the tanks and were connected to a 486DX-PC compatible through a frame-grabber ŽScorpion-Univision, maximum resolution: 1024 = 1024.. In addition, the whole experiment was recorded by a video camera which viewed one of the tanks, through the transparent side. The image was processed using the Windows version of the Image-Pro ŽMedia Cybernetics. software package, which can support up to four cameras. The whole procedure, from grabbing a frame until it is fully processed takes about 5 s. The image monitoring and processing of the behavior parameters was continuous, one frame after another coming from the two cameras so we had a view of each tank every 10 s. Each compartment of the experimental tank was connected to a separate bio-filter ŽMagnum.. Air was pumped by an air-pump and bubbled through the water. Water was replenished with fresh water inlet, located at about the center of the tank, at a rate of 6 lrh Ž90% replacement time of about 100 h.. The temperature and the dissolved oxygen were tested twice a day, in the morning and afternoon, using the OxyGuard tester. Ammonia levels were tested three times a week, during acclimation period, and three . times a day during each experiment day. The ammonium ŽNHq 4 level in the water was measured using a color reagent reaction by an automatic method of Phenate Ž"0.1 mgrl. ŽGreenberg, 1980.. Levels of the un-ionized form of ammonia ŽNH 3 . were calculated using the measured ammonium concentrations. We found that only about 1% of the nitrogen component is in the form of ammonia. This calculation was performed by means of Table B-1 in Piper et al. Ž1982. for given water conditions of pH s 7.4, and water temperature 20 " 18C. 2.3. BehaÕioral indices The gray levels in the images varied from light gray for the background to almost black for the fish. At the beginning of the experiment, the intensity gains were calibrated
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manually for optimal contrast of the fish relative to the background. The processing cycle started by loading the image captured by the first camera, into the Image-Pro’s frame store. The fish were then counted and the features of the projected picture of the school were calculated. The same process was repeated for the image captured by the second camera. The Cartesian coordinate system Ž X,Y,Z . is shown in Fig. 1, where the origin is denoted by O and is located at the lower left hand corner of the transparent side. The image features that were used as behavioral indices were the coordinates of the center of gravity CX, and CZ ŽEq. Ž1.. of the school of fish, and the spatial standard deviations SDX, and SDZ ŽEq. Ž2.. in the directions X, and Z, respectively. Note that: n
Ý A i Xi CX s
is1 n
Ž 1.
Ý Ai is1
and similarly for CZ, SDX s
)
Ý A i Ž X i y CX . Ý Ai
2
Ž 2.
and similarly for SDZ, where n is the number of fish in the projected image, X i denotes the X coordinate of the center of gravity of the ith fish, and A i its projected area. Note that the spatial standard deviations SDX and SDZ represent the spreading value of the fish school at each sampled projection and are different from the ‘standard deviations in time’ which describes the variations over the whole monitoring phase of the parameters CX and CZ. The dimensions of CX, CZ, SDX and SDZ in Sections 3 and 4 and in the enclosed figures, are in pixels. Based on previous experiments we concluded that the parameters CY and SDY calculated from overhead projections were much less sensitive to stress conditions compared to the parameters attained from the side view. Accordingly, we prefer to use the two cameras for two side view repetitions. Both mobility and density of the school were evaluated by the ‘Projected Mobility Picture’ ŽPMP. index. The PMP was calculated from a sequence of 60 frames Fj Ž j s 1 to 60. taken at time intervals of 1 s, as follows, 1 60 PMP s Ý
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levels greater than 255 Žmaximum allowed gray level which is associated with bright white.. To conclude, the calculated PMP is an image in which the gray level of each pixel represents the average mobility in it. Clusters of white pixels appear in locations in the tank where fish prefer to reside. Behavioral indices in treatment and control were compared for statistically significant differences using the SAS software package-version 6.0 ŽSASrSTAT, 1990.. Each set of 300 values of each index, attained during a continuous monitoring, where compared by One-way ANOVA analysis Ža parametric test. and by a nonparametric estimates— Wilcoxon rank test, with other compartments Žcontrol versus treatment. at different phases of monitoring. 2.4. Experimental procedures About 30 Koi fish were put in each of the four compartments and were acclimated for 3 weeks, before each experiment. The fish were not fed during the day prior to the experiment. Ammonia levels were elevated in the treatment compartments by gradually mixing ammonium chloride ŽNH 4 Cl. into the water by the filter pump. The control water ammonia were kept at about zero level throughout the experiment. During the monitoring phase, the immersed filters and air bubbling system were removed from the tank to prevent processing errors in the vision system. A reference monitoring phase of about 50 min was recorded one day before the experiment. Blood glucose was measured every day in five fish that were captured with a net and anaesthetized by MS222 in few seconds to prevent glucose rise due to handling stress. The parameters CX, CZ, SDX, SDZ, and PMP were calculated at each of the monitoring phases. Processing time of each of the grabbed pictures during the continuous monitoring was about 5 s. The effect of the day-cycle was tested in preliminary experiments in which the fish school behavior was monitored for an hour in the morning, noon and evening. No significant differences were found in any of the above calculated indices. 2.4.1. Experiment 1: high ammonia (0.4–0.8 mg r l) Ammonia levels in the water of the treatment compartments were raised from zero to about 0.8 ppm in about 40 min, as shown in the protocol of the experiment ŽFig. 2.. Later, the ammonia level declined slowly to about 0.4 ppm after 24 h due to some ammonia evaporation from the water surface of the water and some filtration during the night. To compensate for this decline, ammonium chloride was added once every 24 h. Continuous monitoring periods of about 1 h were recorded on the first and the third days. PMP was calculated before and after each monitoring period and few times during the second day. Feeding behavior was monitored using the PMP at the end of each day, immediately after a portion of food was given to the control and treatment schools. 2.4.2. Experiment 2: medium ammonia (0.12–0.27 mg r l) The same protocol was carried out as in experiment 1, except for the ammonia levels which were 0.27 mgrl at their peak and gradually dropped to 0.12 mgrl before adding more ammonium chloride to compensate for the decline. Continuous monitoring periods
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Fig. 2. The protocol of a typical experiment. PMP Ž). was recorded 3 times a day; blood was sampled Ž@. every day; the fish were fed Žq. towards the evening each day; the monitoring phases on the first and the third day are illustrated by an heavy solid line; broken line represents the night part of the experiment.
of about 1 h were recorded on the first and the second days. No geometrical variables were monitored at the third day. 2.4.3. Experiment 3: low ammonia (0.04–0.08 mg r l) Ammonia levels were elevated in the treatment compartments to 0.08 ppm ŽNH 3 . and then gradually reduced to 0.04 ppm at the end of the first day. The protocol described in Fig. 2 was carried out during one day only. Feeding behavior was not tested. Blood glucose was measured at the end of the first day and also at the two days that followed, were the ammonia levels were almost zero.
3. Results In all the experiments, the immediate response of the treated fish to the increased ammonia level is to dive to the bottom of the tank and stay there for a period which increased with the ammonia concentration. This phenomenon is demonstrated in Fig. 3a, where a significant reduction Ž P F 0.05. of mean CZ was measured in the treated fish of experiment 2 ŽCZ s 99.8 " 20.7, 93.6 " 19.8., e.g., relative to the control mean CZ ŽCZ s 242.3 " 29.0, 250.5 " 42.2.. A more pronounced effect was observed in the high ammonia concentration Žexperiment 1. were mean CZ of the treated fish is even lower ŽCZ s 69.8 " 9.0, 72.7 " 9.9. relative to the control ŽCZ s 204.6 " 31.3, 211.3 " 44.7.. At the same time, the fish were swimming closer to one another, so that the spreading of the school reduced in both directions as shown by the smaller mean SDX and SDZ ŽFig. 3b.. The mean SDZ value of the fish stressed by higher ammonia concentration, was significantly Ž P F 0.05. smaller Žtreatment mean SDZ s 13.3 " 6.6, 23.0 " 8.4 versus
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Fig. 3. An immediate response of the fish, stressed by medium Ž0.12–0.27 mgrl. ammonia concentration in experiment 2. Variations in time of CZ Ža. and SDZ Žb. in control ŽB. and treatment Ž\., and the PMP Žc..
control mean SDZ s 57.7 " 15.2, 59.0 " 23.6, in experiment 1; and treatment mean SDZ s 25.2 " 10.3, 36.8 " 7.2 versus control mean SDZ s 79.7 " 12.9, 52.1 " 10.5 in experiment 2.. Also, the variations in the treatment PMP ŽFig. 3c. show a clear reduced activity at the bottom, relative to higher activity and wider spreading in the control. Fluctuations in CZ with time, which represent movements of the school position, reduced in frequency and amplitude in all the ammonia stress experiments as shown in, e.g., Fig. 3a, and in the lower values of the standard deviations of the CZ variable. After the immediate ‘low CZ’ response, the treated fish rose to a higher position in the tank and stayed there until the end of the experiment. The depth varied with concentration. In the high ammonia concentration of experiment 1, the fish rose almost to the water surface Žtreatment mean CZ s 424.1 " 14.7, 455.1 " 20.5 versus control mean CZ s 206.1 " 28.5, 203.5 " 61.2.. This phenomenon is demonstrated in the treatment fish by the significantly Ž P F 0.05. greater CZ ŽFig. 4a. and the upper location of the bright zone of activity in the PMP picture ŽFig. 4c.. In the medium ammonia treatment of experiment 2, the fish rose to a lower level Žmean CZ s 158.8 " 17.7,
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Fig. 4. A later response of the fish, stressed by high Ž0.4–0.8 mgrl. ammonia concentration in experiment 1. Variations in time of CZ Ža. and SDZ Žb. in control ŽB. and treatment Ž\., and the PMP Žc..
Fig. 5. Feeding behavior of the fish stressed by medium Ž0.12–0.27 mgrl. Ža. and high Ž0.4–0.8 mgrl. Žb. ammonia concentrations.
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Table 1 Blood glucose Žmean"s.d.; in mgrdl. in control and treatment fish Ž10 fish. in the experiment days Day
1 2 3
High ammonia
Medium ammonia
Low ammonia
C
T
C
T
C
T
23.6"2.7 32.3"3.9 30.6"3.4
41.0"5.6 52.5"4.9 46.8"4.8
25.9"3.1 31.3"3.4 32.6"2.9
41.4"4.0 41.6"2.2 40.4"3.4
21.0"2.3
39.4"6.8
161.0 " 24.1 versus control mean CZ s 243.2 " 36.3, 250.3 " 43.8.. An unclear response was found in the low ammonia concentrations of experiment 3, where in one of the two treatment compartments the fish rose to levels similar to the control and in the other the fish stayed at a low position near the bottom Žtreatment mean CZ s 251.0 " 38.1, 62.2 " 25.5 versus control mean CZ s 200.1 " 52.0, 180.3 " 36.5.. The ‘higher CZ’ phase ŽFig. 4a,c. in experiment 1 is also associated with an increase of mean SDZ back to values close to the control Žtreatment mean SDZ s 63.6 " 28.8, 66.4 " 40.3 versus control mean SDZ s 76.1 " 15.6, 61.2 " 26.4. ŽFig. 4b.; while SDZ stayed at low values in experiment 2 Žtreatment mean SDZ s 21.4 " 9.2, 30.4 " 12.4 versus control mean SDZ s 64.0 " 18.2, 68.2 " 22.6.. In addition, abrupt expanding and contracting of the school of the treated fish were observed. This ‘breathing’ motion of the school is illustrated in Fig. 4b by greater changes in SDZ of the treated fish. It worth noting that in spite of the breathing motion of the treated fish, their activity is much less intense as indicated by the more gray color of the treatment PMP ŽFig. 4c.. Unclear SDZ variations were detected in experiment 3 Žtreatment mean SDZ s 31.2 " 15.4, 92.6 " 23.4 versus control mean SDZ s 64.0 " 15.3, 72.7 " 20.0.. Feeding behavior was tested in experiments 1 and 2 and reduced feeding excitement in the treated fish was found at the medium and high ammonia concentrations as shown by the PMP in Fig. 5a. Table 1 summarizes the results of the blood glucose measurements in all the experiments. Blood glucose of the treated fish was significantly Ž P F 0.05. higher by 30 to 60% relative to the control in all the ammonia concentrations.
4. Discussion In general, after the ammonia stress was applied, the fish school moved down towards the bottom of the compartment and their distribution reduced significantly. Later, after a time interval which increased with water ammonia concentration, the school moved slowly upward to a very high position in the high ammonia experiment, and to a position lower than the control in the medium and low ammonia concentrations. A similar dive to the bottom was found in four salmonid species Ž Salmo salar L., S. trutta L., Oncorhychus mykiss and SalÕelinus alpinus L.. when light conditions change abruptly ŽMork and Gulbrandsen, 1994.. Diving to the bottom resembles the approach to nets and walls of tanks ŽRosenthal, 1989; Martinez Cordero et al., 1994. which can also be interpreted as an avoidance reaction. Approaching water surface was found in
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hypoxia-stressed fish ŽBirtwell and Kruzynski, 1989; Israeli and Kimmel, 1996.. In all experiments, the fish reduced their swimming activity within the school. Reduced swimming speed due to hypoxia stress was found by Israeli and Kimmel Ž1996., while Newton Ž1982. reported reduced aggression in stressed fish. When comparing the three experiments, we found that the time period of the ‘low CZ’ phase increased from about an hour, to 4 h and even to 10 h while ammonia levels were raised from low, to medium and high, respectively. Carp exposed to ammonia respond in general by reducing their activity, which might be interpreted as an exhaustion response and an attempt to save energy needed to overcome the stress condition, or could be an adaptive strategy-waiting for conditions to improve. After 2 to 3 days of moderate to high ammonia levels, the fish were so exhausted that they even ignored feeding. The physiological condition of these fish is also reflected by the relatively high blood glucose levels, probably associated with glycogen metabolism. The early accumulation of the fish near the bottom of the tank is a normal fright response and was also observed by us in fish stressed by a frightening disturbance. In high ammonia concentration, the fish slowly reached the water surface, probably due to gill damage that caused respiration malfunctions, or as a result of difficulties in gas exchange due to mucus accumulation. The same response was observed in hypoxia stress in carps ŽIsraeli and Kimmel, 1996.. Other similar behavioral responses were common to carps under ammonia and hypoxia stressors, such as the breathing-like variations in the spatial distribution of the stressed school and their reduced activity. 4.1. Feeding behaÕior The feeding excitement in the treated fish reduced during the whole experiment at the medium and high ammonia concentrations as shown by the PMP in Fig. 5a. At the high ammonia concentration, the feeding excitement reduced at the first day and vanished at the second day as shown by the PMP in Fig. 5b. Juvenile rainbow trout, exposed to 0.25 mgrl of NH 3 reduced food intake and subsequently their weight gain decreased ŽLang et al., 1987.. Also, reduced feeding in stressed fish was reported by Love Ž1981.. 4.2. Blood glucose A significant rise in blood glucose of the treated fish, by 30 to 60% relative to the control in all the ammonia concentrations is typical of stressed fish. Glucose levels in stressed fish rise, for example, from 65 to 100 mgr100 ml in rainbow trout Ž O. mykiss ., after 3 h of exposure to handling ŽFlos et al., 1988. and in Atlantic salmon Ž S. salar L.. from 40 to 60 mgr100 ml after 2 h ŽFevolden et al., 1991.. Blood glucose levels of the control fish at the end of the first day were lower relative to the other two days, probably due to starvation in the day prior to the onset of the experiment. Greatest glucose level was observed in the fish stressed by the high ammonia level. 4.3. Future applications After comparing the present work with our previous publication on carp under hypoxia stress ŽIsraeli and Kimmel, 1996., we believe that there are common responses
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of carp to different stressors. More investigation is needed. The same technique can be employed for monitoring fish behavior under different stressors and has the potential to become a routine non-invasive and non-disturbing tool for the early detection of stressful conditions in farming systems. In fish farms, where the conditions differ significantly from the laboratory, monitoring variations in fish behavior as a means for stress alarm, can be achieved by two approaches: i. analyzing images obtained by echo sound techniques, which are already used in open-sea cages and can work in the dark, in turbid water and even when the fish densities are very high, and ii. analyzing video images taken from time to time in a ‘testing’ tank where light conditions fit video applications, the fish density is much smaller than the typical fish farming density, and where the water conditions are the same as in the fish farm. At this stage of developing the technique, low ammonia concentrations, which may be encountered in fish farms, did not provide a conclusive response. Other possible uses for our video technique include: counting fish, evaluating biomass, and optimizing the geometrical design of water bodies to fit the shape of the fish school.
Acknowledgements We thank S. Hershler, S. Laiter and MagNoi of Kibbutz Hazorea. The study was partially supported by the Ministry of Agriculture and the Ministry of Science and the Arts.
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Himick, B.A., Eales, J.G., 1990. Acute correlated changes in plasma T4 and glucose in physically disturbed cannulated Rainbow trout, Oncorhynchus mykiss. Comp. Biochem. Physiol. A 97 Ž2., 165–167. Israeli, D., Kimmel, E., 1996. Monitoring the behavior of hypoxia-stressed Carassius auratus using computer vision. Aquacult. Eng. 15 Ž6., 423–440. Lang, T., Peters, G., Hoffmann, R., Meyer, E., 1987. Experimental investigations on the toxicity of ammonia: effects on ventilation frequency, growth, epidermal mucous cells, and gill structure of rainbow trout Salmo gairdneri. Dis. Aquatic Organisms 3, 159–165. Love, R.M., 1981. Stress and behavior in the culture environment. In: Bilio, M., Rosenthal, H., Sinderman, J. ŽEds.., Realism in Aquaculture—Achievements, Constraints, Perspectives. Eur. Aquacult. Soc., pp. 451–466. Martinez Cordero, F.J., Beveridge, M.C.M., Muir, J.F., 1994. A note on the adult Atlantic halibut Hippoglossus hippoglossus L. in cages. Aquacult. Fish. Manage. 25, 475–481. Maule, A.G., Schreck, C.B., 1990. Changes in numbers of leukocytes in immune organs of juvenile Coho salmon after acute stress or cortisol treatment. J. Aquatic Anim. Health 2, 298–304. Mazeaud, M.M., Mazeaud, F., Donaldson, E.M., 1977. Primary and secondary effects of stress in fish: some new data with a general review. Trans. Am. Fish. Soc. 106 Ž3., 201–212. Mork, O.I., Gulbrandsen, J., 1994. Vertical activity of four salmonid species in response to changes between darkness and two intensities of light. Aquaculture 127, 317–328. Newton, B.J., 1982. Early stress effects on growth and adult behavior in Poecilia Õeticulata. Dev. Psychobiol. 15 Ž3., 211–220. Palackova, J., Gajdusek, S., Jirasek, J., Fasaic, K., 1990. Effect of sublethal concentration of ammonia in water on changes in and correlations of some biochemical indices in carp fry Ž Cyprinus carpio L... Ichthyologia 22 Ž1., 57–67. Paley, R.K., Twitchen, I.D., Eddy, F.B., 1993. Ammonia, Naq, Kq and Cly levels in rainbow trout yolk–sac fry in response to external ammonia. J. Exp. Biol. 180, 273–284. Passino, D.R.M., 1981. Biochemical indicators of stress in fishes: an overview. In: Pickering, A.D. ŽEd.., Stress and Fish. Academic Press, pp. 38–48. Pickering, A.D., 1981. The concept of biological stress. In: Pickering, A.D. ŽEd.., Stress and Fish. Academic Press, pp. 38–48. Pillay, T.V.R., 1990. Aquaculture Principles and Practices. Cambridge Univ. Press. Piper, R.G., McElwain, I.B., Orme, L.E., McCraren, Fowler, L.G., Leonard, J.R., 1982. Fish Hatchery Management. United States Department of the Interior Fish and Wildlife Service, Washington, DC. Robertson, L.P., Thomas, P., Arnold, C.R., 1987. Plasma cortisol and secondary stress response of cultured red drum Ž Sciaenops ocellatus . to several transportation procedures. Aquaculture 68, 115–130. Rosenthal, H., 1989. Fish behavior in circular tanks: a video documentation on fish distribution and water quality. In: Lillelund, K., Rosenthal, H. ŽEds.., Fish Health Protection Strategies. pp. 61–166. Russo, R.C., 1985. Ammonia, nitrite, and nitrate. In: Rand, G.M., Petrocelli, S.R. ŽEds.., Fundamentals of Aquatic Toxicology. Hemisphere, New York. Santhi, K., Gunasekhar, K., Babu, C.S., Reddy, K.P.O., Neeraja, P., 1992. Changes in the dehydrogenase levels of fish Orechromis mossambicus under chronic ammonia stress. J. Inland Fish. Soc. India 24 Ž2., 71–73. SASrSTAT, 1990. Tomasso, J.R., 1994. Toxicity of nitrogenous wastes to aquaculture animals. Rev. Fish. Sci. 2 Ž4., 291–314. Weirich, C.R., Tomasso, J.R., Smith, T.I.J., 1993. Toxicity of ammonia and nitrite to Sunshine Bass in selected environments. J. Aquatic Anim. Health 5, 64–72. Wlasow, T., Dabrowska, H., Ziomek, E., 1990. Hematology of carp in prolonged sublethal ammonia intoxication. Pol. Arch. Hydrobiol. 37 Ž3., 429–438. Wood, C.M., 1993. Ammonia and urea metabolism and excretion. In: Evans, D.H. ŽEd.., The Physiology of Fishes. CRC Press, FL, pp. 379–402.
Behavioral response of carp ž Cyprinus carpio / to ammonia stress Dorith Israeli-Weinstein, Eitan Kimmel
)
Agricultural Engineering Department, Technion IIT, Haifa 32000, Israel Accepted 7 March 1998
Abstract The behavioral responses of schools of young Koi fish Ž Cyprinus carpio . to sub-lethal ammonia concentrations were monitored using CCD cameras and computer image processing. Several geometrical parameters of the schools such as the position of the center of gravity, and the distribution of the fish were calculated continuously and plotted versus time. Swimming speed and activity were measured indirectly through a novel parameter—the Projected Mobility Picture ŽPMP.. The immediate response of the treated fish was to dive to the bottom of the tank and stay there for a time period which increased with the ammonia concentration. Later, the fish rose to depths which increased with ammonia concentration. At high ammonia concentration, the fish approached the surface. Decreased activity of the treated fish was accompanied by a smaller mean distribution. Blood glucose levels of the treated fish increased, and they became indifferent to food. In general, this remote method of continuous monitoring of alterations in fish behavior under stress has the potential to be developed into a method for detecting early signs of stress in populations of fish. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Computer vision; Ammonia stress; Fish behavior
1. Introduction Stress in fish farming systems is associated with a variety of environmental conditions such as reduced oxygen level, abrupt variations in temperature, appearance of contaminants or elevated ammonia. The effect of elevated ammonia on fish held under conditions common in intensive aquaculture is the subject of this study. When fish are stressed, stress hormones are released Žprimary response., the composition of blood and tissues is altered and variations in ventilation and heart rate frequently occur Žsecondary )
Corresponding author.
0044-8486r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 4 4 - 8 4 8 6 Ž 9 8 . 0 0 2 5 1 - 8
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response. ŽMazeaud et al., 1977.. As a result, glucose level in the blood, for instance, has become a common method for estimating the effect of stress ŽMazeaud et al., 1977; Himick and Eales, 1990; Fevolden et al., 1991; Flos et al., 1988.. In the long run, stress induces a reduction in the effectiveness of the immune system, resulting in higher susceptibility to diseases and parasites, decreased growth rate, reduced reproduction and increased mortality. All the stress-induced changes are associated with variations in the behavior of the fish ŽDonaldson et al., 1984; Passino, 1981; Pickering, 1981; Adams, 1990; Robertson et al., 1987; Maule and Schreck, 1990.. Stressed fish change their behavior. By ‘behavior’ we refer to the series of visible actions which are operated and controlled by the nervous, sensory and endocrine systems. Escape or freezing, avoidance or attraction are common behavioral responses to adverse stimuli ŽBeitinger, 1990.. Nitrogen, mostly in the form of ammonia is excreted by fish across the gills. Ammonia level in the bulk of the water is reduced by oxidization to nitrite and to nitrate Žnitrification.. In intensive aquaculture systems, where fish are reared at high densities with insufficient water exchange or filtration, excessively high levels of ammonia can arise. Typical rates of ammonia excretion by carp Ž Cyprinus carpio L. and Carassius auratus . are 2 to 5 = 10y5 mgrl kg h ŽWood, 1993.. In addition, ammonia accumulation in the water may result from bacterial decomposition of uneaten food rich in proteins ŽLang et al., 1987. or seepage of other nitrogen-containing substances such as ammonium sulfate from fertilized fields ŽSanthi et al., 1992.. Maximum ammonia levels of about 0.02 mgrl Žat pH 7.0. are allowed in fish farming ŽPillay, 1990.. Exposure to high concentrations of ammonia reduces survival, inhibits growth, and causes a variety of physiological malfunctions. Ammonia acts as a stressor and stimulates the release of corticosteroid hormones into the blood circulation ŽTomasso, 1994.. Ammonia concentrations of 0.32 to 0.60 mgrl induced mortality rates of 50% in sunshine bass, Morene chrysops, within 96 h ŽWeirich et al., 1993.. In . and the solution, ammonia exists in equilibrium between the ionized form ŽNHq 4 un-ionized form ŽNH 3 .. At pH 7.0 and 208C, only 1% of the total nitrogen is in the form of NH 3 ŽFleck, 1966; Wood, 1993.. NH 3 is more toxic to fish than NHq 4 because it easily diffuses across the gill membrane ŽRusso, 1985.. The toxic effect of elevated ammonia concentrations in the water is attributed to a reduced outward flux of ammonia excretion through the gills, so that the outward flux is blocked and a reversed inward ammonia flux occurs. As a result, ammonia levels in the fish plasma increase. Another possible mechanism for plasma ammonia build-up at high external ammonia levels, is the decreased ammonium outward flux through the NaqrNHq 4 channel and increased Ž . inward NHq 4 diffusion Wood, 1993 . Increased ammonia levels cause physiological and behavioral changes in fish. In smolts exposed to 0.08 mgrl ammonia NH 3 , increased plasma cortisol were found ŽDonaldson, 1990.. Glucose levels increased by 30% in Atlantic salmon exposed to sublethal concentrations of NH 3 Ž0.019 to 0.037 mgrl. for 8 days. The exposed fish showed an increased ventilation rate and their gill filaments were damaged ŽFivelstad et al., 1993.. Juvenile rainbow trout, exposed to 0.25 mgrl of NH 3 for 4 weeks, increased ventilation frequency ŽLang et al., 1987.. Exposure for 24 h of rainbow trout to an external ammonia ŽNH 3 . concentration of 0.72 mgrl increased the body ammonia content from 0.6 to 4 m molrg. At the same time, body Naq and Cly concentrations
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decreased by about 28% and Kq concentration decreased by 35% ŽPaley et al., 1993.. In carp Ž C. carpio L.. exposed for 48 h to sublethal concentrations of ammonia in water Ž0.32–1.04 mgrl NH 3 versus control: 0.07 mgrl., significant changes were found in concentrations of ammonia, urea, lactic acid and total protein, and in activities of hepatopancreas LDH, ADH, GDH, AST, and ALT ŽPalackova et al., 1990.. Extended exposure of carp Ž C. carpio L.. to low concentrations of 0.033 mgrl ammonia caused the appearance of signs of stress in the immune system and led to changes in the composition of blood and hemopoietic tissue Žhead kidney, spleen. after 5 weeks. However, after 10 weeks of prolonged exposure, almost all the signs of stress disappeared ŽWlasow et al., 1990.. In hybrid tilapia Ž Oreochromis mossambicus= O. niloticus ., no stress detected in concentrations of ammonia ŽNH 3 . below 0.84 mgrl. At moderate concentrations of ammonia between 1.28 to 2.13 mgrl, the fish showed signs of stress at first and gradually recovered after 24 h. At high levels of 2.97 to 4.24 mgrl NH 3 , the fish showed signs of marked excitement, followed by exhaustion and usually death within 18 h ŽCheng-Chung and Cheng Liu, 1989.. In this study, we monitored the behavioral responses to various levels of ammonia stress as reflected by the calculated changes of the school parameters. 2. Materials and methods 2.1. The fish Ornamental carp species, Koi-C. carpio, of 8 to 12 cm length, weight of about 10 g and from either sex, were used. Koi fish appear usually in schools. The dominant colors of the fish are black, orange and red. These colors improve the contrast between the fish and the white background for better image processing. The fish were obtained from a commercial fish farm, where they had been raised to the age of 6 months. All fish were from the same batch. In the laboratory, the fish were acclimatized in the experimental tanks for 3 weeks prior to the experiment. During the acclimation time, oxygen levels were kept in the range 7.5 to 8.5 mgrl, corresponding to about 90% of the saturation level. Water temperature was maintained at 19 to 218C and the ammonia level was below 0.001 mgrl, corresponding to the sensitivity level of our measuring method. The fish were fed once a day, around noon, with 3 to 4% of the total fish biomass, given in a form of dry pellets which are floated at water surface. 2.2. The experimental system The system Žschematically shown in Fig. 1. is composed of two white PVC tanks Ž70 = 90 = 90 cm., with one transparent side in each tank. Each tank is divided by a white, vertical PVC partition wall, into control and treatment compartments. The light in the room was provided by two 15 W fluorescent bulbs mounted on the ceiling, some 1.5 m above the water surface. This was the only light source in the room during the acclimation and experiment periods, and it was used only during the day. Two CCD ŽCharge Coupled Device. cameras faced the transparent side of each tank. Both cameras
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Fig. 1. The experimental system.
were 8 mm, black and white cameras ŽBurel-TC655EACX., with pixel resolution of 512 = 512 and with auto-iris lenses. The cameras were located at a distance of 160 cm from the tanks and were connected to a 486DX-PC compatible through a frame-grabber ŽScorpion-Univision, maximum resolution: 1024 = 1024.. In addition, the whole experiment was recorded by a video camera which viewed one of the tanks, through the transparent side. The image was processed using the Windows version of the Image-Pro ŽMedia Cybernetics. software package, which can support up to four cameras. The whole procedure, from grabbing a frame until it is fully processed takes about 5 s. The image monitoring and processing of the behavior parameters was continuous, one frame after another coming from the two cameras so we had a view of each tank every 10 s. Each compartment of the experimental tank was connected to a separate bio-filter ŽMagnum.. Air was pumped by an air-pump and bubbled through the water. Water was replenished with fresh water inlet, located at about the center of the tank, at a rate of 6 lrh Ž90% replacement time of about 100 h.. The temperature and the dissolved oxygen were tested twice a day, in the morning and afternoon, using the OxyGuard tester. Ammonia levels were tested three times a week, during acclimation period, and three . times a day during each experiment day. The ammonium ŽNHq 4 level in the water was measured using a color reagent reaction by an automatic method of Phenate Ž"0.1 mgrl. ŽGreenberg, 1980.. Levels of the un-ionized form of ammonia ŽNH 3 . were calculated using the measured ammonium concentrations. We found that only about 1% of the nitrogen component is in the form of ammonia. This calculation was performed by means of Table B-1 in Piper et al. Ž1982. for given water conditions of pH s 7.4, and water temperature 20 " 18C. 2.3. BehaÕioral indices The gray levels in the images varied from light gray for the background to almost black for the fish. At the beginning of the experiment, the intensity gains were calibrated
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manually for optimal contrast of the fish relative to the background. The processing cycle started by loading the image captured by the first camera, into the Image-Pro’s frame store. The fish were then counted and the features of the projected picture of the school were calculated. The same process was repeated for the image captured by the second camera. The Cartesian coordinate system Ž X,Y,Z . is shown in Fig. 1, where the origin is denoted by O and is located at the lower left hand corner of the transparent side. The image features that were used as behavioral indices were the coordinates of the center of gravity CX, and CZ ŽEq. Ž1.. of the school of fish, and the spatial standard deviations SDX, and SDZ ŽEq. Ž2.. in the directions X, and Z, respectively. Note that: n
Ý A i Xi CX s
is1 n
Ž 1.
Ý Ai is1
and similarly for CZ, SDX s
)
Ý A i Ž X i y CX . Ý Ai
2
Ž 2.
and similarly for SDZ, where n is the number of fish in the projected image, X i denotes the X coordinate of the center of gravity of the ith fish, and A i its projected area. Note that the spatial standard deviations SDX and SDZ represent the spreading value of the fish school at each sampled projection and are different from the ‘standard deviations in time’ which describes the variations over the whole monitoring phase of the parameters CX and CZ. The dimensions of CX, CZ, SDX and SDZ in Sections 3 and 4 and in the enclosed figures, are in pixels. Based on previous experiments we concluded that the parameters CY and SDY calculated from overhead projections were much less sensitive to stress conditions compared to the parameters attained from the side view. Accordingly, we prefer to use the two cameras for two side view repetitions. Both mobility and density of the school were evaluated by the ‘Projected Mobility Picture’ ŽPMP. index. The PMP was calculated from a sequence of 60 frames Fj Ž j s 1 to 60. taken at time intervals of 1 s, as follows, 1 60 PMP s Ý
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levels greater than 255 Žmaximum allowed gray level which is associated with bright white.. To conclude, the calculated PMP is an image in which the gray level of each pixel represents the average mobility in it. Clusters of white pixels appear in locations in the tank where fish prefer to reside. Behavioral indices in treatment and control were compared for statistically significant differences using the SAS software package-version 6.0 ŽSASrSTAT, 1990.. Each set of 300 values of each index, attained during a continuous monitoring, where compared by One-way ANOVA analysis Ža parametric test. and by a nonparametric estimates— Wilcoxon rank test, with other compartments Žcontrol versus treatment. at different phases of monitoring. 2.4. Experimental procedures About 30 Koi fish were put in each of the four compartments and were acclimated for 3 weeks, before each experiment. The fish were not fed during the day prior to the experiment. Ammonia levels were elevated in the treatment compartments by gradually mixing ammonium chloride ŽNH 4 Cl. into the water by the filter pump. The control water ammonia were kept at about zero level throughout the experiment. During the monitoring phase, the immersed filters and air bubbling system were removed from the tank to prevent processing errors in the vision system. A reference monitoring phase of about 50 min was recorded one day before the experiment. Blood glucose was measured every day in five fish that were captured with a net and anaesthetized by MS222 in few seconds to prevent glucose rise due to handling stress. The parameters CX, CZ, SDX, SDZ, and PMP were calculated at each of the monitoring phases. Processing time of each of the grabbed pictures during the continuous monitoring was about 5 s. The effect of the day-cycle was tested in preliminary experiments in which the fish school behavior was monitored for an hour in the morning, noon and evening. No significant differences were found in any of the above calculated indices. 2.4.1. Experiment 1: high ammonia (0.4–0.8 mg r l) Ammonia levels in the water of the treatment compartments were raised from zero to about 0.8 ppm in about 40 min, as shown in the protocol of the experiment ŽFig. 2.. Later, the ammonia level declined slowly to about 0.4 ppm after 24 h due to some ammonia evaporation from the water surface of the water and some filtration during the night. To compensate for this decline, ammonium chloride was added once every 24 h. Continuous monitoring periods of about 1 h were recorded on the first and the third days. PMP was calculated before and after each monitoring period and few times during the second day. Feeding behavior was monitored using the PMP at the end of each day, immediately after a portion of food was given to the control and treatment schools. 2.4.2. Experiment 2: medium ammonia (0.12–0.27 mg r l) The same protocol was carried out as in experiment 1, except for the ammonia levels which were 0.27 mgrl at their peak and gradually dropped to 0.12 mgrl before adding more ammonium chloride to compensate for the decline. Continuous monitoring periods
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Fig. 2. The protocol of a typical experiment. PMP Ž). was recorded 3 times a day; blood was sampled Ž@. every day; the fish were fed Žq. towards the evening each day; the monitoring phases on the first and the third day are illustrated by an heavy solid line; broken line represents the night part of the experiment.
of about 1 h were recorded on the first and the second days. No geometrical variables were monitored at the third day. 2.4.3. Experiment 3: low ammonia (0.04–0.08 mg r l) Ammonia levels were elevated in the treatment compartments to 0.08 ppm ŽNH 3 . and then gradually reduced to 0.04 ppm at the end of the first day. The protocol described in Fig. 2 was carried out during one day only. Feeding behavior was not tested. Blood glucose was measured at the end of the first day and also at the two days that followed, were the ammonia levels were almost zero.
3. Results In all the experiments, the immediate response of the treated fish to the increased ammonia level is to dive to the bottom of the tank and stay there for a period which increased with the ammonia concentration. This phenomenon is demonstrated in Fig. 3a, where a significant reduction Ž P F 0.05. of mean CZ was measured in the treated fish of experiment 2 ŽCZ s 99.8 " 20.7, 93.6 " 19.8., e.g., relative to the control mean CZ ŽCZ s 242.3 " 29.0, 250.5 " 42.2.. A more pronounced effect was observed in the high ammonia concentration Žexperiment 1. were mean CZ of the treated fish is even lower ŽCZ s 69.8 " 9.0, 72.7 " 9.9. relative to the control ŽCZ s 204.6 " 31.3, 211.3 " 44.7.. At the same time, the fish were swimming closer to one another, so that the spreading of the school reduced in both directions as shown by the smaller mean SDX and SDZ ŽFig. 3b.. The mean SDZ value of the fish stressed by higher ammonia concentration, was significantly Ž P F 0.05. smaller Žtreatment mean SDZ s 13.3 " 6.6, 23.0 " 8.4 versus
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Fig. 3. An immediate response of the fish, stressed by medium Ž0.12–0.27 mgrl. ammonia concentration in experiment 2. Variations in time of CZ Ža. and SDZ Žb. in control ŽB. and treatment Ž\., and the PMP Žc..
control mean SDZ s 57.7 " 15.2, 59.0 " 23.6, in experiment 1; and treatment mean SDZ s 25.2 " 10.3, 36.8 " 7.2 versus control mean SDZ s 79.7 " 12.9, 52.1 " 10.5 in experiment 2.. Also, the variations in the treatment PMP ŽFig. 3c. show a clear reduced activity at the bottom, relative to higher activity and wider spreading in the control. Fluctuations in CZ with time, which represent movements of the school position, reduced in frequency and amplitude in all the ammonia stress experiments as shown in, e.g., Fig. 3a, and in the lower values of the standard deviations of the CZ variable. After the immediate ‘low CZ’ response, the treated fish rose to a higher position in the tank and stayed there until the end of the experiment. The depth varied with concentration. In the high ammonia concentration of experiment 1, the fish rose almost to the water surface Žtreatment mean CZ s 424.1 " 14.7, 455.1 " 20.5 versus control mean CZ s 206.1 " 28.5, 203.5 " 61.2.. This phenomenon is demonstrated in the treatment fish by the significantly Ž P F 0.05. greater CZ ŽFig. 4a. and the upper location of the bright zone of activity in the PMP picture ŽFig. 4c.. In the medium ammonia treatment of experiment 2, the fish rose to a lower level Žmean CZ s 158.8 " 17.7,
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89
Fig. 4. A later response of the fish, stressed by high Ž0.4–0.8 mgrl. ammonia concentration in experiment 1. Variations in time of CZ Ža. and SDZ Žb. in control ŽB. and treatment Ž\., and the PMP Žc..
Fig. 5. Feeding behavior of the fish stressed by medium Ž0.12–0.27 mgrl. Ža. and high Ž0.4–0.8 mgrl. Žb. ammonia concentrations.
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Table 1 Blood glucose Žmean"s.d.; in mgrdl. in control and treatment fish Ž10 fish. in the experiment days Day
1 2 3
High ammonia
Medium ammonia
Low ammonia
C
T
C
T
C
T
23.6"2.7 32.3"3.9 30.6"3.4
41.0"5.6 52.5"4.9 46.8"4.8
25.9"3.1 31.3"3.4 32.6"2.9
41.4"4.0 41.6"2.2 40.4"3.4
21.0"2.3
39.4"6.8
161.0 " 24.1 versus control mean CZ s 243.2 " 36.3, 250.3 " 43.8.. An unclear response was found in the low ammonia concentrations of experiment 3, where in one of the two treatment compartments the fish rose to levels similar to the control and in the other the fish stayed at a low position near the bottom Žtreatment mean CZ s 251.0 " 38.1, 62.2 " 25.5 versus control mean CZ s 200.1 " 52.0, 180.3 " 36.5.. The ‘higher CZ’ phase ŽFig. 4a,c. in experiment 1 is also associated with an increase of mean SDZ back to values close to the control Žtreatment mean SDZ s 63.6 " 28.8, 66.4 " 40.3 versus control mean SDZ s 76.1 " 15.6, 61.2 " 26.4. ŽFig. 4b.; while SDZ stayed at low values in experiment 2 Žtreatment mean SDZ s 21.4 " 9.2, 30.4 " 12.4 versus control mean SDZ s 64.0 " 18.2, 68.2 " 22.6.. In addition, abrupt expanding and contracting of the school of the treated fish were observed. This ‘breathing’ motion of the school is illustrated in Fig. 4b by greater changes in SDZ of the treated fish. It worth noting that in spite of the breathing motion of the treated fish, their activity is much less intense as indicated by the more gray color of the treatment PMP ŽFig. 4c.. Unclear SDZ variations were detected in experiment 3 Žtreatment mean SDZ s 31.2 " 15.4, 92.6 " 23.4 versus control mean SDZ s 64.0 " 15.3, 72.7 " 20.0.. Feeding behavior was tested in experiments 1 and 2 and reduced feeding excitement in the treated fish was found at the medium and high ammonia concentrations as shown by the PMP in Fig. 5a. Table 1 summarizes the results of the blood glucose measurements in all the experiments. Blood glucose of the treated fish was significantly Ž P F 0.05. higher by 30 to 60% relative to the control in all the ammonia concentrations.
4. Discussion In general, after the ammonia stress was applied, the fish school moved down towards the bottom of the compartment and their distribution reduced significantly. Later, after a time interval which increased with water ammonia concentration, the school moved slowly upward to a very high position in the high ammonia experiment, and to a position lower than the control in the medium and low ammonia concentrations. A similar dive to the bottom was found in four salmonid species Ž Salmo salar L., S. trutta L., Oncorhychus mykiss and SalÕelinus alpinus L.. when light conditions change abruptly ŽMork and Gulbrandsen, 1994.. Diving to the bottom resembles the approach to nets and walls of tanks ŽRosenthal, 1989; Martinez Cordero et al., 1994. which can also be interpreted as an avoidance reaction. Approaching water surface was found in
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hypoxia-stressed fish ŽBirtwell and Kruzynski, 1989; Israeli and Kimmel, 1996.. In all experiments, the fish reduced their swimming activity within the school. Reduced swimming speed due to hypoxia stress was found by Israeli and Kimmel Ž1996., while Newton Ž1982. reported reduced aggression in stressed fish. When comparing the three experiments, we found that the time period of the ‘low CZ’ phase increased from about an hour, to 4 h and even to 10 h while ammonia levels were raised from low, to medium and high, respectively. Carp exposed to ammonia respond in general by reducing their activity, which might be interpreted as an exhaustion response and an attempt to save energy needed to overcome the stress condition, or could be an adaptive strategy-waiting for conditions to improve. After 2 to 3 days of moderate to high ammonia levels, the fish were so exhausted that they even ignored feeding. The physiological condition of these fish is also reflected by the relatively high blood glucose levels, probably associated with glycogen metabolism. The early accumulation of the fish near the bottom of the tank is a normal fright response and was also observed by us in fish stressed by a frightening disturbance. In high ammonia concentration, the fish slowly reached the water surface, probably due to gill damage that caused respiration malfunctions, or as a result of difficulties in gas exchange due to mucus accumulation. The same response was observed in hypoxia stress in carps ŽIsraeli and Kimmel, 1996.. Other similar behavioral responses were common to carps under ammonia and hypoxia stressors, such as the breathing-like variations in the spatial distribution of the stressed school and their reduced activity. 4.1. Feeding behaÕior The feeding excitement in the treated fish reduced during the whole experiment at the medium and high ammonia concentrations as shown by the PMP in Fig. 5a. At the high ammonia concentration, the feeding excitement reduced at the first day and vanished at the second day as shown by the PMP in Fig. 5b. Juvenile rainbow trout, exposed to 0.25 mgrl of NH 3 reduced food intake and subsequently their weight gain decreased ŽLang et al., 1987.. Also, reduced feeding in stressed fish was reported by Love Ž1981.. 4.2. Blood glucose A significant rise in blood glucose of the treated fish, by 30 to 60% relative to the control in all the ammonia concentrations is typical of stressed fish. Glucose levels in stressed fish rise, for example, from 65 to 100 mgr100 ml in rainbow trout Ž O. mykiss ., after 3 h of exposure to handling ŽFlos et al., 1988. and in Atlantic salmon Ž S. salar L.. from 40 to 60 mgr100 ml after 2 h ŽFevolden et al., 1991.. Blood glucose levels of the control fish at the end of the first day were lower relative to the other two days, probably due to starvation in the day prior to the onset of the experiment. Greatest glucose level was observed in the fish stressed by the high ammonia level. 4.3. Future applications After comparing the present work with our previous publication on carp under hypoxia stress ŽIsraeli and Kimmel, 1996., we believe that there are common responses
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of carp to different stressors. More investigation is needed. The same technique can be employed for monitoring fish behavior under different stressors and has the potential to become a routine non-invasive and non-disturbing tool for the early detection of stressful conditions in farming systems. In fish farms, where the conditions differ significantly from the laboratory, monitoring variations in fish behavior as a means for stress alarm, can be achieved by two approaches: i. analyzing images obtained by echo sound techniques, which are already used in open-sea cages and can work in the dark, in turbid water and even when the fish densities are very high, and ii. analyzing video images taken from time to time in a ‘testing’ tank where light conditions fit video applications, the fish density is much smaller than the typical fish farming density, and where the water conditions are the same as in the fish farm. At this stage of developing the technique, low ammonia concentrations, which may be encountered in fish farms, did not provide a conclusive response. Other possible uses for our video technique include: counting fish, evaluating biomass, and optimizing the geometrical design of water bodies to fit the shape of the fish school.
Acknowledgements We thank S. Hershler, S. Laiter and MagNoi of Kibbutz Hazorea. The study was partially supported by the Ministry of Agriculture and the Ministry of Science and the Arts.
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