EFFECTS OF CONSTANT MAGNETIC FIELDS ON RESPIRATION OF RAINBOW TROUT ( ONCORHYNCHUS MYKISS WALB

Oxygen uptake and carbon dioxide release by rainbow tro­ ut (Oncorhynchus mykiss Walb.) embryos exposed to constant magnetic fields of 5 and 10 mT (oxygen uptake and carbon dioxide release) and 50, 150, and 300 mT (oxygen uptake) we­ re measured. The data were compared with those recorded in embryos developing under natural magnetic field (control). The magnetic fields tested were found to stimulate respi­ ratory processes in the rainbow trout embryos as shown by a si­ gnificantly increased oxygen consumption, particularly during periods of intensified morphogenesis. Exposure to 5 and 10 mT magnetic fields resulted in a sli­ ghtly higher carbon dioxide release, the oxygen consumption being observed to increase as well. The respiratory quotient of the embryos exposed to magnetic fields was slightly higher than that in the control.


INTRODUCTION
The last three decades have supplied a growing evidence that magnetic fields different from those of the natural Earth's background affect life processes of or ganisms, including fish, exposed to those fields ( Tenforde 1979;Blank 1993 ) .The effects are manifested as, i.a., acceleration or slow-down of some functions and are particularly pronounced in some locomotory functions. .Oui:_earli er studies de monstrated, i.a., significant effects of magnetic field changes on the cardiac mu-scle performance in fish embryos and larvae, and on the performance of skeletal muscles operating the juvenile fish pectoral fins (Winnicki and Formicki 1990;Formicki 1992;Winnicki et al. 1993;Formicki and Winnicki 1996).Acting on the premises that all the locomotory responses rely on energy expenditure and that the energy can be mobilised by oxygen supplied from without (as evidenced by acce leration of heart beat rate resulting in intensified circulation and acceleration of pectoral fin respiratory beat rate), it was decided to measure the amount of oxy gen taken up (and, in some cases, of carbon dioxide released) by rainbow trout (Oncorhynchus mykiss Walb.) embryos exposed to artificial constant magnetic fields and to compare the results with corresponding data obtained under the na tural geomagnetic conditions.The question was addressed whether, and how, the respiration could be altered by application of a magnetic field.The tests were run in different fields in an attempt to determine relationships between field parame ters and responses of a developing organism..

MATERIAL AND METHODS
The tests were carried out in 1994-1996 at the Department of Fish Anatomy and Embryology, Agricultural University of Szczecin on developing embryos of rainbow trout (Oncorhynchus mykiss Walb.) Materials (i.e., rainbow trout eggs and sperm), obtained from spawners kept at fish farms in Miastko and Zelkowo (District of Slupsk), were transported to the laborato ry where insemination (dry) and fertilisation were effected.Subsequently, the hydra ted eggs were distributed for incubation on Petri dishes filled with water to 6 mm he ight.Those "mini-incubators" were kept throughout the period of the development either in an insulated room or in a special water mantle (laboratory hatchery), where by the constant temperature of 10 °C ± 1 °C was maintained.
Respiration of the embryos was measured at 6 stages of the embryonic develop ment, a particular attention being paid to the following stages of intensified morpho genesis: -early gastrulation ( development of blastoderm and periblast, formation of the gastrula, division into three embryonic layers, beginning of blastopore closure): 60-80 D 0  Vernier 1967;Opuszynski 1979;Klinkhardt et al. 1987;Depeche Billard 1994).The 0 2 uptake and C0 2 release by the developing embryos were measured either with the Winkler technique (0 2 uptake) in flow-through respirometers, or the modified Warburg manometric technique (0 2 uptake and C0 2 release).In the first case, the eggs were transferred to 13.5 mm diameter, 140 mm long cylindrical flow-through plexiglass chambers, one placed between the poles of the magnetic field source and the other away from it.The water oxygen content was determined with the Winkler technique as described in "Physical and chemical methods for water and wastewater analyses" (Hermanowicz et al. 1976).The 0 2 was determined as a difference between oxygen contents in the cham ber's inlet and outlet.A single test took 240 min.during which time 6 measure ments were taken.Water temperature was maintained constant by keeping the test chambers in additional buffering containers to make sure that identical thermal conditions were prevailing in the magnetic field, control, and in the Mariott bot-Although allowing to remove metabolic products from the test chamber and ensuring stability of gas components in the incoming water, the flow-through tech nique is of low sensitivity.It was used to determine the differences in oxygen con sumption between the control and treatment embryos exposed to magnetic fields a relatively high inductances (50, 150, and 300 mT) as more conspicuous re sponses in those fields were anticipated.
To ensure a high sensitivity, the Warburg apparatus manometers were used in weaker fields (5 and 10 mT).The duration of the test was shortened to reduce ef fects of changes in gas ratio in the measuring cell which occur during the test and distort the results.A single test took 90 min.during which the manometer column height was read at 15-min.intervals.As the eggs of salmonids (including those of rainbow trout) are at some stages of their development particularly prone to mechanical damage and sensitive to vibrations (Privolnev and Razumovskij 1939;Vernier 1967;Winnicki 1967;Opuszynski 1979), it was decided against using a shaker in order to prevent additional stress to or deaths of embryos, which wo uld lead to distortion of data.
Constant magnetic fields were produced by a computer-controlled magnetic field generator for embryo-physiological tests, manufactured at the Institute of Te lecommunications and Acoustics, Wroclaw Technical University (Duchiewicz and Formicki 1994) and by an electromagnet.
A total of 5 series of tests were run.In 1995, 0 2 uptake by the rainbow trout em bryos exposed to 50, 150, and 300 mT magnetic fields was measured at various sta ges of embryonic development, from 60 D 0 to hatch.The fields were generated with Tornasz Perkowski, Krzysztof Forrnicki the electromagnet in order to produce stronger fields.In 1996, the embryos' responses to 5 and 10 mT fields at the same developmental stages were recorded.The fields were generated with the computer-controlled apparatus mentioned above, allowing a precise control and fine tuning of the weak field.An HTM hallotronic teslameter (manufactu red by the Institute of Telecommunications and Acoustics, Wrodaw Technical Univer sity) was used to measure and control field inductance.
Differences between treatments and the control were tested for significance with Stu dent's t test applied with the use of Statgraphics® v. 6.0 Manugistics ™ statistical package.RESULTS 0 2 consumption in 50, 150, and 300 mT magnetic fi elds Table 1 summarises data on 0 2 consumption by the rainbow trout embryos exposed to 50, 150, and 300 mT fields and in the control.As shown by the data, the mean 0 2 uptakes in 50 and 150 mT fields differed significantly from the control throughout the period of observations.Significant differences were found also to exist between all the treatments combined (pooled results in 50, 150, and 300 mT fields) and the control.Dif ferences in 0 2 uptake, resulting from magnetic field effects, were observed in some sta ges of the embryonic development.In the 50 and 150 mT fields, the 0 2 uptake clearly increased, relative to the control, within 160-200 D 0 (appearance of eyes), an increase being also observed within 90-100 D 0 period (formation of primordial neural disc) in the 150 and 300 mT fields.Additionally, differences in 0 2 uptake between the treatment and the control were also recorded within 60--80 D 0 (gastrulation) in the 50 mT field, while in the 150 mT field the differences occurred within 90--200 D 0 (from blastopore closure to circulatory system formation).In none of the three field inductances tested were statistically significant treatment vs. control differences recorded for the 210-290 D 0 stage (active embryo phase).A reversed trend (reduced treatment 0 2 uptake relative to the control) was observed in the 50 and 300 mT fields within 140--150 D 0 (onset of cardiac activity).0 2 consumption rn 5 and 10 mT fi elds Data on 0 2 consumption by the rainbow trout embryos exposed to 5 and 10 mT con stant magnetic fields and in the control are shown in Table 2.As those treatments involved the use of the Warburg apparatus manometers, differences in 0 2 consumption at various developmental stages, shown in Tables 1 and 2, may have resulted from different metho dologies.This finding supports observations reported by Pattee (1962) who compared re sults obtained with different techniques and concluded that results obtained under identi cal conditions may differ considerably.Statistically significant differences between 0 2 consumption in magnetic field and in the control were recorded both for the mean uptake in both the 5 mT (90 records, p = 0.001) and 10 mT (85 records, p = 0.036) fields and for all the records combined (175 records, p = 0.001).Application of 5 and 10 mT magnetic fields resulted in increased 0 2 uptake by the embryos within 60-80 D 0 ( onset of gastru lation), 110-130 D 0 ( after blastopore closure), and 140-150 D 0 ( organogenesis ), the dif ferences relative to the control being highly significant (significance levels of 0.005 to 0.014).No reversal of the trend, i.e., reduced 0 2 uptake in a magnetic field, was recor-Significant differences relative to the control were noted also in the 5 mT field wi thin 160-200 D 0 (appearance of eyes).No significant differences compared to the con trol occurred within 90-100 D 0 (formation of primordial neural disc) and within 210-290 D 0 (active embryo phase).In the 10 mT field, no statistically significant differences with the control were recorded within 90-100, 160-200, and 210-290 D 0 Noteworthy is the fact that no ne of the treatment magnetic field inductances (5, 10, 50, 150, and 300 mT) produced statistically si gnificant treatment vs. control dif ferences prior to hatch, i.e., within 210-290 D 0 (Tables 1 and 2) .
In order to visualise differen ces in rainbow trout embryos' 0 2 uptake between the treatments and the control, box-and-whisker plots were prepared with data obtained in different field inductances and with combined data for the three high-inductance treatment (50, 150, and 300 mT).The plots allow to analyse the distribution of data in the groups and to conclude that, generally, exposure to a magnetic field produces larger differences between 0 2 uptakes than does the control ( as evidenced by larger are as covered by the horizontal lines reflecting the upper and lower qu artiles).Both in high (50, 150, and 300 mT; Fig. 1) and in low (5 and 10 mT; Fig. 2) inductance fields, the median 0 2 consumption lie above the medians for the control, the quartile ranges being clearly wider.
Oxygen consumption (ml 0 2 /h/embryo) by rainbow trout (Oncorhynchus mykiss Walb.) embryos exposed to 50, 150, and 300 mT magnetic fields and in the control .Xp>XK - X p -mean oxygen consumption by treatment embryos; x K -mean oxygen consumption by control embryos; Mean number of embryos obser ved p_ er treatment was 100.Carbon dioxide release (ml C02/h/embryo) by rainbow trout (Oncorhynchus mykiss Walb.) embryos exposed to 5 and 10 mT magnetic fields and in the control     No statistically significant dif ferences in 0 2 consumption relative to the time of exposu re to magnetic field were de tected (Figs 3 and 4).Howe ver, one can notice a certain similarity between a decrease in 0 2 uptake rate and time of exposure in 5 and 10 mT fields.On the other hand, for longer periods of time in 50, 150, and 300 rnT treatments both pooled and analysed se parately, a slightly accelerated 0 2 uptake with time of expo sure could be observed.All the differences were, however, slight and not significant.
C 0 2 r e l e a s e No significant differences in C0 2 release could be detec ted both with respect to embry onic developmental stages and field inductances (Table 3).An exception is provided by the difference between mean rele ases within 160-200 D 0 for those embryos exposed to 5 mT field (0.00070 and 0.00049 ml COih/embryo in treatment and control, respec tively; p = 0.044).A slight (sta tistically non-significant) in crease in the amount of C0 2 released in the magnetic field relative to the control was re corded within 60-80 D 0 in the 5mT field and within 60-80 and 160-200 D 0 in the 10 mT one.The amount of C0 2 released did not changed significantly throughout a single test.Changes in the amount of C0 2 released in the magnetic field during the embryonic development were similar to the changes in the amount of 0 2 taken up by an embryo exposed to a magnetic field.This is particularly well visible within 160-200 D 0 in the 5 mT field when the amount of 0 2 consumed increased relative to the control and the amount of C0 2 released in creased significantly as well.Such similarities were observed to occur also, to so me extent, in the 5 mT field within 60-80 and 110-130 D 0 and in the 10 mT field within 60-80 D 0 .Re sp atory quotient 0, 9 ,--------------- --+----+----+----+---� 1 2 4 Delopmental stage I --treatment -iii-control I Oxygen uptake and carbon dioxide release was measured si multaneously in the 5 and 10 mT treatments.The results allowed to calculate the respiratory quotient (RQ).Fig. 5 shows pooled RQ values obtained for embryos exposed to both magnetic fields and the control.Generally, RQ values were higher in the control, but no statistical significance of the differences could be detected.The lowest and highest treatment RQ values in the magnetic field were 0.47 and 0.7, respectively; the respective control values were 0.56 and 0.8.

DISCUSSION
The experiment described in this paper was aimed at addressing a question if, and to what extent, a magnetic field affected embryonic respiration.The answer was sought by comparing the amounts of 0 2 consumed and C0 2 released by em bryos exposed to artificial magnetic fields and the natural one.The physiological background of magnetic field effects on respiration was investigated as well.
To elucidate the questions addressed, it was necessary to review a number of phenomena, observed in experimental studies involving exposure to magnetic affect 0 2 uptake of embryos.With respect to constant magne tic field effects on early embryonic development of salmonids, it could be assu med that magnetic field particularly affects those embryos the development of which undergoes some critical phases.The critical phases during which the em bryo is particularly sensitive to external stimuli involve intensive metabolism, re lated to different stages of cellular division and organogenesis.Studies reported by Trifonova and Popov (1937) on 0 2 deficiency effects and those by Privolnev and Razumovskij (1939) involving temperature changes allowed to distinguish three critical phases: the onset of cleavage; beginning of gastrulation; and differentia tion of an embryo.
Studies on incubation of fertilised eggs of trout (Salmo trutta L.), rainbow tro ut (0. mykiss Walb.), and Danube salmon (Hucho hucho L.) exposed to magnetic field demonstrated, too, the presence of periods of increased embryo's sensitivity to magnetic field.The embryos proved particularly sensitive to magnetic field ef fects immediately after fertilisation ( cleavage and gastrulation) (Formicki and Winnicki 1982).The periods of increased sensitivity of the embryo to magnetic field effects seem to partially overlap the so-called critical phases distinguished by other workers.
Experiments involving magnetic field effects on embryonic respiration were started at 60 D 0 , i.e., at the morula stage.Differences in 0 2 consumption between • embryos exposed to a field and the control were f ound to involve eggs from the autumn spawning (exposure to 5 and 10 mT fields).In the 50 mT field, too (the spring spawning eggs), the period of increased sensitivity coincided with the mo stage.An increased sensitivity to magnetic field effects in the 150 and 300 fields, manifested as an increased 0 2 uptake was recorded within 90-100 D 0

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The evident similarity of responses to magnetic field effects was observed also du ring organogenesis and during the circulatory system f ormation in the embryos (140-200 D 0 ).The intensified sensitivity was in that case manifested not only as an increase, but also-in two treatments (50 and 150 mT fields)-as a reduced 0 2 consumption in the magnetic field, relative to the control.
Earlier experiments demonstrated clear effects of magnetic fields on motoric activity of the cardiac muscle and pectoral fins.Application of a magnetic field was shown to produce a change in cardiac contraction rhythm (Winnicki and For nricki 1990) and to affect movements of the pectoral fins (Winnicki et al. 1993).An increase in oxygen demand could be explained by the increased heart beat ra te.It should be borne in mind, however, that the increased 0 2 consumption at the beginning of an experiment results most probably from increased response to ma gnetic stimulation at the moment the field is being applied.After this initial pe riod, the oxygen consumption decreases somewhat to stabilise at a level higher than that of the control.Thus an increase in the cardiac muscle activity rate (re verting, after several minutes, to the initial state, the one before the field was ap plied) may be one, but not the only, factor resulting in the increased 0 2 uptake.
Magnetic field affects various levels of organisation and structure of systems studied, which makes it difficult to single out a mechanism responsible for the in creased oxygen consumption.At the cellular level, magnetic field effects on biolo gical membranes may provide an explanation for the increased oxygen uptake in the magnetic field and for different responses of an organism at various developmental stages.Some layers of the cellular membrane display liquid crystal properties.Ap plication of a magnetic field results in a change in the state of liquid crystals, which alters their permeability.Moreover, particles penetrating the membranes are electri cally charged.Thus the magnetic field affects not only the membrane permeability, but also the electric charges actively transported across the membranes (Wadas 1978).Changes in transport of particles or atoms across biological membranes may result in acceleration or slow-down of cellular or organismic metabolism (Rosen 1993;Tenforde 1993).Such effects would be particularly pronounced during pe riods of intensified metabolic activity, that is during the critical phases.
On the other hand, Nossol et al. (1993) focused on low density magnetic field interacting with enzymes which catalyse cellular biochemical reactions.Operation and efficiency of intracellular metabolic pathways depend on numerous consecu tive enzymatic processes.For this reason, even a weak stimulation affecting one of the initial links may substantially affect subsequent biochemical reactions (Karlson 1971).A 300 µT constant magnetic field activates cytochrome oxydase.The enzyme is responsible for electron transport to particulate oxygen.An incre ased activity of the enzyme can be explained by magnetic field effects on either the enzyme's structure or on protons or electrons participating in the reaction (Nossol et al. 1993).On the other hand, magnetic field effects on enzymes seem to be selective, which means that not all the enzymes respond to the field and the response in those that do respond is not uniform (Haberditzl 1967).The magnetic field effects on enzymatic activity may explain the absence of significant differen ces between C0 2 release by treatment and control embryos.
It seems, however, that the following mechanism would be most consistent with the results obtained.Application of a magnetic field reduces permeability of membranes in capillaries and respiratory epithelium of fish larvae.Consequently, at the moment of field application and for some time after it, oxygen diffusion ra te slows down and the amount of oxygen taken up decreases.During this time, the larval body passes through an oxygen debt phase during which the cells use up a normal amount of oxygen, but oxygen content in the arterial blood decreases (anactic hypoxia).The larva obtains the necessary energy anaerobically from che mical substances in the body (Szabuniewicz 1964;Depeche and Billard 1994).As in other aquatic organisms, such a state cannot be prolonged in a fish larva becau se it can lead to asphyxia.Hence the observed compensatory reaction observed, involving acceleration of the heart beat rate to increase blood circulation.After a moment, another compensatory mechanism is turned on and visible as intensi fin motility, whereby oxygenated water is fed into the larval breathing system (Winnicki and Formicki 1990;Formicki and Winnicki 1996).
A magnetic field can produce stress; an embryo faces then a necessity to adapt to changed environmental conditions.Undergoing an adaptation, the embryo has to make up for its oxygen demand which is higher than that in the control.Those phe nomena occur, however, during minute-long periods of time and do not explain the that the embryos placed in a magnetic field for prolonged periods of time, at dif ferent developmental stages, take up more oxygen than the control ones, while the n1agnetic field applied throughout the embryonic period extends incubation, which most likely slows down biochemical processes (Form.icki, 1991).It would thus se em that the oxygen demand in a magnetic field should be lower, while the reverse is fact true.It seems that the paradox can be explained by progressive, hyperbiotic processes involving growth and differentiation of cells.Studies on in vitro tissue cul tures showed that embryonic cells grew faster than adult cells due to cellular growth activators.One can assume that the factors stimulating cellular growth are substanactivated in a magnetic field.This is evidenced by a a higher weight of hat ching embryos (Formicki and Winnicki 1982;Formicki 1991) and by the higher oxygen demand of growing cells and tissues.Kholodov et al. (1996) found incre ased biological oxidation in the brain as a response to application of a magnetic field.The in vitro studies of McDonald ( 1993) demonstrated constant magnetic field to have stimulated growth of cells isolated from neonatal rats.Zhang et al. (1993), when studying domestic hen embryos, found a relationship between alternated ma gnetic field effects, increased 0 2 consumption, and faster growth.
In the present study, apart from the 0 2 uptake, the C0 2 release was measured in the treatments involving 5 and 10 mT fields.The results showed a slight incre ase in the amount of C0 2 released in the magnetic field, relative to the control.The oxygen consumption by developing embryos has been rather extensively treated in the literature, while much fewer studies dealt with C0 2 release (Winnicki 1967).
In her experiments with carp embryos, Kamler (1976) found proteins and lipids to the major sources of the energy expended, the contributions of both types of substances being similar and amounting to 50 and 49%, respectively.The RQ va lues around 0.7, arrived at by Winnicki (1963) for the rainbow trout, point to li pids as the major energy source for the developing embryos of the species.In the present work; too, the RQ values were close to 0.7, while the magnetic field-affec ted embryos showed lower RQ values.
The data obtained during the experiment reported here suggest that magnetic field affects oxygen consumption much stronger than it does the C0 2 release.No-teworthy is a reference to the RQ values mentioned.Should the increase in the 0 2 uptake be identical with the amount of C0 2 release, the treatment and control RQ values would be identical.As seen in Fig. 5, the treatment value was in fact lower.Further studies are needed to explain if the mechanism responsible involves selec tive effects of magnetic field on intracellular metabolic pathways, on substrates of katabolic processes, or altered C0 2 evacuation across shell membranes.CONCLUSION 1.A constant magnetic field stimulates respiration of rainbow trout embryos, as evidenced by the statistically significant increase in the oxygen uptake.2. Respiratory stimulation in magnetic field is not uniform throughout the em -----m:-yopic development and is particularly pronounced during periods of intensi fied morphogenesis: gastrulation and blastopore closure, organogenesis, and formation and development of circulatory system.3. Rainbow trout embryos respond to magnetic field by increasing their oxygen uptake at the moment of magnetic field application.The altered level of oxy gen demand does not change significantly with time throughout the experiment.4. At the final stage of incubation (prior to hatching), the magnetic field of in ductance values used (5, 10, 50, 150, and 300 mT) did not produce any signi ficant increase in the embryonic oxygen consumption.5. Application of 5 and 10 mT magnetic fields resulted in a slightly higher car bon dioxide release (at an increased amount of oxygen consumed in the ma gnetic field), so that the respiratory quotient in those embryos exposed to the fields was slightly lower than that in the control.

Fig. 1 .Fig. 2 .
Fig. 1.Oxygen consumption by rainbow trout (On corhynchus mykiss Walb.) emb ry os exposed to 50, 150, and 300 mT magnetic fields and inthe control; all treatment observations pooled (X 1 E-3) oxygen consumption by treatment embryos; xK-mean oxygen consumption by control emb ry os; Mean number of emb ry os observed per treatment was 35.

X
p -mean carbon dioxide release by treatment embryos; :ii-mean carbon dioxide release by control embryos; Mean number of embryos observed per treatment was 35.

Fig. 3 .
Fig. 3. Changes in oxygen consumption by rainbow trout (Oncorhynchus mykiss Walb.) embryos exposed to magnetic fields and in the control throughout the experiment; observations in 50, 150, and 300 mT fields pooled; l--0xygen consumption in magnetic fields; 2--0xygen consumption in control

Fig. 4 .
Fig. 4. Changes in oxygen consumption by rainbow trout (Oncorhynchus mykiss Walb.) embryos exposed to magnetic fields and in the control throughout the experiment; observations in 5 and 10 mT fields po oled; l--0xygen consumption in magnetic fields; 2--0xygen consumption in control

Fig. 5 .
Fig. 5. Respiratory quotients of embryos exposed to 5 and 10 mT fields (pooled observations) and in the control

Table 1
-Variance Standard -Variance Standard vs. control

Table 2
Oxygen consumption (ml Oihlemb ry o) by rainbow trout (Oncorhynchus mykiss Walb.) emb ry os exposed to 5 and 10 mT magnetic fields and in the control