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UNCLASSIFIEDSECURITY CLASSIFICATION OF THIS PAGE (ten 0 Date Entered)

REPORT DOCUMENTATION PAGE READ INSTRUCTIONSBEFORE COMPLETING FORM

I. REPORT NUMBER 2.GOVT ACCESSION NO. 3. RECIPIENT'S CATALOG NUMBER

4. TITLE (and Subtlle) S. TYPE OF REPORT & PERIOD COVEREDLn' CELLULAR EFFECTS OF ELECTROMAGNETIC RADIATION FINAL TECHNICAL

02/01/82 - 07/31/85.0 S. PERFORMING ORG. REPORT NUMBER

7"O 7. AUTHOR(,) S. CONTRACT OR GRANT NUMNER(s)

William F. Pickard N00014-82-K-0261

s. PERFORMING ORGANIZATION MAME AND ADDRESS 10. PROGRAM ELEMENT. PROJECT. TASKWashi ngton University AREA A WORK UNIT NUMBERS

Department of Electrical EngineeringSaint Louis, Missouri 63130II. CONTROLLING OFFICE NAME AND ADDRESS 12. REPORT DATE

* Life Sciences Programs / Biological Sciences Div. August 16, 1985* Office of Naval Research (Code 441CB) IS. NUMBEROFPAGES

800 North Ouincy St. / Arlington. VA 22217 1014. MONITORING AGENCY NAME I ADDRESS(i 41lifoRnil hm Cmonttoling Office) IS. SECURITY CLASS. (of tls relpea)

. Director, Office of Naval Research Detachment, UnclassifiedChicago ___________________DONGRDIN

Room 286, 536 South Clark St. ,s. OECLASSIFICATOONU I WNGRADIN

Chicago, Illinois 60605 _ _ __D__._

IS. DISTRIBUTION STATEMENT (of thle Report)

Scienti 1 0ffi cer ,.(T Blk.1 1 LSThIBUTION STATEMENTA calAdmi Oraf ra ,ing 6 C r ',______ _ __ " ) 1Cenr '

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IS. SUPPLEMENTARY NOTES DE CF E

'S. KEY WORDS (Continue an teerse side iI nocewy end Identify by black numbme)

Nonionizing radiation) ,.-Biological effects of electromagnetic fields,

20. ABSTRACT (Contimee an revers side II neceesmv aiId identify by Wek numbe")

, .P-Giant cells of characean algae were examined for electrophysiologicalsequelae to acute electromagnetic field irradiation at 10 mW/cm Carrierfrequencies ranged from 50 MHz to 12.4 GHz and a variety o-fTmodulationprotocols were employed. No effects were detected at this power level.

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REPORT NUMBER:

CELLULAR EFFECTS OF ELECTROMAGNETIC RADIATION

Final Technical Report for the period02/01/82 - 07/31/85 onContract N00014-82-K-0261 of theOffice of Naval Research800 North Quincy StreetArlington, Virginia 22217

Submitted 08/16/85 byWilliam F. PickardDepartment of Electrical EngineeringWashington UniversitySaint Louis, Missouri 63130

CLASSIFICATION: Unclassified

DISTRIBUTION: Scientific Officer (see 81k. 11 DD2222) [1 copyj

Administrative ContractOfficer (see Bik. 8 DD2222) [1 copy]

Director, Naval Research Laboratory (1 copy]ATTN: Code 2627Washington, DC 20375

Defense Technical Information Center [12 copies]Building 5, Cameron StationAlexandria, VA 22314

10 pages

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SYNOPSIS

SUMMARY OF ALL WORK ACCOMPLISHED:3

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This is provided in the sections following.

INDEX OF ALL TECHNICAL REPORTS:

KThis final technical report is the only technicalreport produced on this contract.

INDEX OF ALL PUBLICATIONS:

Ell K. M. Brunkard and W. F. Pickard, The membrane potentialof characean cells exposed to amplitude-modulated,low-power 147-MHz radiation. Bioelectromagnetics 5,353-356 (1984).

E21 A. V. Gokhale, K. M. Brunkard, and W. F. Pickard,Vacuolar hyperpolarizing offsets in characean cellsexposed to mono- and bichromatic CW and tosquarewave-modulated electromagnetic radiation in theband 200-19000 MHz. Bioelectromagnetics 29 357-360(1984).

[3] A. V. Gokhale, K. M. Brunkard, and W. F. Pickard, Theabsence of significant short-term electromagneticbioeffects in giant algal cells exposed to CW. andpulse-modulated X-band bursts. IEEE Trans. MicrowaveTheory Techniques MTT-32, 795-797 (1984).

E41 A. V. Gokhale and W. F. Pickard, Evidence that characeanmembrane transport is not significantly altered byincident electromagnetic radiation. Radiation Res. ,.Qj,300-306 (1985.

(5] A. V. Gokhale, W. F. Pickard, and K. M. Brunkard,Low-power 2.45-GHz microwave radiation affects neitherthe vacuolar potential nor the low frequency excessnoise in single cells of characean algae. J. MicrowavePower _Z, 43-46 (1985).

E63 A. V. Gokhale and W. F. Pickard, Extremely low frequencyresistance fluctuations of Chara membrane. PlantPhysiol. To be published.

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E7] W. F. Pickard, The electrical noise of a cell:Correlations between resistance noise and voltage noise.. Submitted to Math. Biosci.

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INTRODUCTION

The aims of this contract (NO0014-82-K-0261) were (i) to usean extremely sensitive electrophysiological technique of W. F.Pickard and Y. H. Barsoum [J. Membrane Biol. fi, 39-54 (1981)] toscreen for short-term electromagnetic bioeffects over frequencyranges of interest to the Office of Naval Research and (ii) toinvestigate irradiation modalities (e.g., mixed frequencies orpulse modulated fields) more characteristic of the shipboardenvironment than the single-frequency continuous-wave fieldscommonly employed in bioelectromagnetics research.

Not all of the seven papers listed above are of direct andobvious relevance to the these aims; and, in fact, some areperipheral. Hence, a brief guide the publications under thecontract will be given before any details of the fulfillment ofaims (i) and (ii) are provided. Paper [1] is listed only becauseit was carried out using equipment in part purchased under thecontract and because it served as a test bed for a noveltechnique later employed in [4]; it will not be discussedfurther. Paper [2] employed power densities well above thosewhich could reasonably be anticipated in a naval environment butis listed because it served to train A. V. Gokhale, who actuallydid most of the critical measurements under the contract, andbecause it employed novel frequency combining techniques; it willbe discussed at slightly greater length later. Papers E3], [4],and [5] describe the studies intended to fulfill the aims of thecontract; their contents will be excerpted later. Papers [6] and[7] arose from some surprising observations made in passingduring work on paper [5]; they are of considerable relevance tomembrane ion transport but will not be discussed further becausetheir completion required very little time and because they arenot directly related to the electromagnetic field sensitivity ofan organism.

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EXPERIMENTAL TECHNI QUES

Experimental technique has been exhaustively documented inpapers (1] - [5]. Hence the description here will be limited tothe briefest of overviews with emphasis placed upon thephilosophy of the method.

* If an electromagnetic field is to have an effect on a cell

other than the directly thermal or the classicallyelectrophysiological, then it must have a locus of action. Oneobvious locus is the cell membrane. Any action at this locusshould exert some effect on the upon the electrophysiologicalproperties of the cell, in particular upon the electricalpotential of its interior but also upon its membrane resistance.There are many ways of searching for such effects, and severalwere tried during the course of this contract. What is ofimportance is that most are unambiguously quantifiable, severaloffer extremely high resolving power, and those of high resolvingpower tend in addition to be fast.

The exposure apparatus was a simple microstrip with a ceramicslab of dielectric constant 75 separating the strip from itssubjacent ground plane; a channel, down which test solution wasflowed, had been cut into the ceramic perpendicular to the strip.A giant cell of a characean alga (normally about 15 mm long and300 .um in diameter) would be placed in the channel downsteamfrom the strip, impaled in its downstream end by a glassmicropipette electrode (two electrodes for resistancemeasurements), and then slid upstream until its umimpaled end wasunder the strip. In this configuration, one end of the cellcould be irradiated by the known quasi-TEM mode of the microstripwhile the electrophysiological response of the cell was sensednonperturbingly by the micropipette in its field-free downstreamregion. Moreover, since the cell impedance-matched the channelsolution which in turn impedance-matched the ceramic slab, theelectromagnetic field in the cell was known.

In the most common experiment, the cell was irradiated by aseries of widely spaced bursts of radiation and the responses ofits internal potential accumulated by a signal averager.Typically this internal potential was on the order of 50 mV ,

while a response on the order of 5 V was detectable by signalaveraging; this is a resolution of roughly 1 part in 10,000and 5 V is below the normal stochastic fluctuation of thecell's potential. In various experiments, there were employedbursts of CW, bursts of pulses, and bursts of swept frequency.Less commonly used techniques will be described briefly in theresults sections below.

5

MIXED FREQUENCY STUDIES

The deck of a naval vessel is not a simple fixed-frequencyelectromagnetic environment. Many frequencies may be present atonce, and some sources may hop their frequencies. The hypothesisthat, at or below 10 mW/cm2 , a potpourri of frequencies couldhave a relevant biological effect. even though any single onewould not is unfalsifiable: for, however many combinations areshown to be of no effect, there will always be others which havenot been tested. Hence, evaluation of'this hypothesis can neverbe more than inadequate. Yet, if the envisioned effects of mixedfrequencies are robust, then a random (exploratory) selection offrequency combinations might just reveal something positive.Acting on this possibility, three such combinations were assayed.

First [2], it was asked whether, at field levels well withinthe range where thermal effects are noted, the admixture of twodiscrete frequencies would reveal a nonlinear interaction thatmight be indicative of an athermal phenomenon. For this it wasassumed that power windows were not a problem and that anypositive findings would become monotonically more positive withincreasing power levels. This possibility was examined using theburst irradiation technique at power levels roughly 10,000-foldthe nominal 10 mW/cm safe exposure level; at these powerlevels, clear thermal sequelae are detectable. Using mixtures of245 & 810 MHz, of 366 & 705 MHz, or of 547 & 1019 MHz, nononlinear interactions were detected.

Second [4], it was asked whether discrete bursts of X-bandirradiation of non-constant frequency would have an effect. Forthis, the frequency was swept from 8.0 to 12.4 GHz either by a50 kHz triangle wave or by a 20-50,000 Hz white n ise source.At a variety of power levels from 0.01 to 500 mW/cm , no offsetof cellular potential was detected by the burst irradiationtechnique.

Third [unpublished], preliminary measurements in which theirradiation was swept from 10-1,000 MHz by a audio-frequencytriangle wave, revealed no effect at 10 mW/cm

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.STUDIES OF EXPOSURE TO AMPLITUDE-MODULATED FIELDS

The electromagnetic fields to which naval personnel areexposed tend to more pulsed than CW. Therefore it is desirableto examine the hypothesis that, even though continuous waveirradiation might have no athermal sequelae, non-constantirradiation might. Once again, this hypothesis is notdefinitively falsifiable. However, if careful study of a varietyof different non-constant fields turns up no athermal effects,then one can express guarded optimism that such effects are atworst subtle.

Fjrst [2J, at the distinctly thermal power level of 145,000mW/cm , the effect of bursts of CW was compared to the effect ofbursts of the same carrier frequency 100% squarewave modulatedat 100, 1000, or 10000 Hz . No statistically significantdifference was observed although nine different carrierfrequencies from 200 to 1000 MHz were tried.

2Second (3J, at 10 mW/cm and nine different carrier

frequecies from 8.2 to 12.4 GHz , three different burstirradiation protocols were employed: CW, 1.0 us pulses 1 msapart, and 0.1 us pulses 0.1 ms apart. In no instance was aneffect seen on the cellular potential despite the 1:10000resolving power of the technique.

T~ird [3J, at 9.09 GHz and power levels of 10 and 100mW/cm , twenty different combinations of pulse interval andpulse width were employed. Again, the burst irradiation protocolrevealed nothing.

Fourth [3), at 9.09 GHz, bursts of CW were compared withbursts of 1 us pulses separated by 1 ms . Neither burstprotocol revealed an effect at 0.1, 0.2, 0.5, 1, 2, 5, or 10mW/cm

Fifth (4], twenty different combinations of pulse period a~dpulse length were tried at power densities of 10 or 100 mW/cmThe burst protocol revealed no effect at carrier frequencies of50, 147, 450, or 917 MHz

Sixth [4], a 450 MHz carrier was modulated by 60 us widepulses. A lock-in amplifier examined the cellular potential forshifts correlated with the pulses. At a variety of pulserepetition frequfncies from 4 to 64 Hz and power levels from0.2 to 100 mW/cm , no effects were detected over 2 min

*. averaging periods despite the fact that any offset above 250 nVshould have been obvious.

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STUDIES OF LONGER TERM IRRADIATION

Because the burst protocol is specialized for the examination

of effects whose rise times are a second or less, it could well

miss effects which require longer term irradiation to become

manifest. Therefore, additional studies were undertaken

employing 2 min or 1 h irradiation times.

First [4], the internal potential of the cell was dc-nulled

and amplified 100-fold. This gave a trace in which

irradiation-correlated offsets on the order of 250 V would

have been apparent if they occurred during a two minute period of

irradiation or within a tw minute control period following

irradiation. AL 10 mW/cm and at frquencies of 50, 147, 450,

and 917 MHz , continuous-wave irradiation had no effect.

Second (4], the amplified and nulled cellular potential was

integrated to give a slope in which irradiation-correlated kinks

would have bee apparent if they had occurred on a time scale of

ten to sixty minutes and had arisen from long-term cell potentialshifts of 5 mV or more. After a 1 h 2 control period,continuous-wave irradiation at 10 mW/cm was applied for 1 h

and was followed by a further 1 h control period. There was no

detectable effect at frequencies of 50, 147, 450, and 917 MHz

Third [4], the amplified and nulled cellular potential was

band-pass filtered (0.2 - 2.0 Hz), was RMS-to-dc converted, andwas integrated to yield a slope in which irradiation-correlated

kinks would have been apparent if they had occurred on a time

scale of ten to sixty minutes and had arisen from shifts of 25%or more in the low frequency excess (voltage) noise of the cell.

Under the same conditions of irradiation as in the previous test,no effect of irradiation was noted.

4

STUDIES AT 2450 MHZ

Because 2450 MHz is by a wide margin the most popularfrequency for performing bioelectromagnetics screeningexperiments, it was singled out for special study [5].

At incident power densities of 10 mW/cm2 , no effect wasdiscovered in studies with the burst irradiation protocol or withthe 2 min or 1 h protocols of the previous section.

To explore whether environmental stress might potentiatemicrowave sensitivity, selected cells were examined in the abovethree fashions while being stressed by (i) the chloride channelblocker ethacrynic acid at a concentration of 100 nM, or (ii) atemperature of 15 OC (standard was 25 0 C ), or a temperatureof 10 0 . The stressed cells showed no microwave response.

Finally, it was asked whether cells irradiated for 1 h byI. an incident fields of either 10 or 100 mW/cm would show a

significant change in membrane resistivity. (These experimentswere carried out at 15 0 C using the two pipette method of

resistivity measurement.) No statistically significant changesin membrane resistivity were seen.

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DISCUSSION AND CONCLUSIONS

Because neither naval personnel nor the general public areapt to encounter average p wer densities in excess of the oldANSI standard of 10 mW/cm- , screening experiments which employ

this level of incident irradiation are probably sufficient, atleast in a first search for bioelectromagnetic phenomena. Oursearch for effects at this power density turned up nothingdespite the large number of different frequencies, irradiationprotocols, and electrophysiological end points used. If, in thegiant cells of the Characeae, there is a cellular effect of acute(i.e., one hour or less) exposures, it.must be presumed to besubtle -- unless of course the experimental studies were insome way flawed. Three possible flaws will be discussed.

First, the experimental technique may have been flawed. Itis always possible that one or another of the several techniquesused may have been flawed, either in design or execution; but itdoes seem rather unlikely that all were flawed. Moreover, theburst irradiation protocol did yield positive results at

distinctly thermal levels of irradiation [2]. Because many otherindependently performed studies in this laboratory on the sameexperimental preparation likewise revealed no athermal effectdespite the many other frequencies used (For a summary, seeReference [4].), it is doubtful that flawed technique cantotally account for the absence of observed effect: probablyeither (i) there is no significant effect to observe or (ii) whateffect there is is subtle.

Second, the experiments were carried out at known incidentpower densities rather than at known specific absorption rates.This is not the place to debate the philosophical question ofwhat the most appropriate descriptor of electromagnetic exposuremight be. Indeed, the answer to that question may be somewhatirrelevant because, despite the fact that by any reasonablemeasure a goodly amount of exposure was taking pla2e (See [4] forSAR estimates.), no effects were seen at 10 mW/cm

Third, parameter space is vast, and the experiments actuallyperformed covered only an infinitesimal fraction of it. Thehypotheses that there are sharp frequency resonances, or thatthere are sharp power resonances, or that the cell's environmentwas unsuitable can never be falsified. The experiments areflawed by inadequate coverage of parameter space, and the flaw isirremedial. But, any practical importance of this flaw onlyserves to substantiate the assertion that effects missed areindeed subtle because a large number of parameter combinationswere tried in the unsuccessful search for athermal bioeffects.

In summation, the experiments performed under this contractrevealed no biological effects of acute low-level electromagneticirradiation of the experimental preparation used. They offer no

evidence that such irradiation may be biologically deleterious.

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