Effect of occupational exposure to organophosphorus insecticides on neuromuscular function.

K.-B. Effect of occupational exposure to organo phosphorus insecticides on neuromuscular funotion. Scand. j. work environ. & health 4 (1978) 255-261. Neurophysiological investigations and determinations of cholin esterase activity on plasma and erythrocytes were carried out on 11 Swedish spraymen exposed to bromophos, diazinon, dursbane, and malathion. Plasma cholinesterase activity was significantly reduced after work, while erythrocyte cholinesterase activity was unchanged. In none of the workers with a decreased plasma cholinesterase activity after work could any related acute neuromuscular disturbance be detected when the men were tested with repetirtive nerve stimula tion and with single fiber electromyography. Signs of subclinical neuropathy were present as a slight reduction in sensory conduction velocity and increased fiber density in some workers.

phorms compounds (11,15). The cholinesterase inhibition of these compounds might poss:i!bly a:fjiiect neuromuscular transmission in exposed workers. Roberts et al. (11,15) suggested that a relatively simple EMG technique was very useful and, to some extent, superior to cholinesterase measurements when the effects of exposure to organophosphorus compounds are being monitored (6,10,11,15).
In the present 11l'vestiga1ri.on neuromuscular transmission land cholinesterase activity have been studied with more sensiJtive methods than were used in eaI'llier investigations. It iJs aJl1 a-ttem:pt to determine whether a group of Swedish spraymen was eXiPOsed to organophosphorus pesticides to such a degree that EMG tests or chdliJnesteTaiSe detenminatioIl5 showed abnormal values and, if 00, to correlate the methods used. In~ddition to measuring the decremental response of the surface EMG recording and nerve conduction ve-Locity, we performed single f:i:ber EMG (SFEMG) (17). 'Drus method is particularly sensitive 'bo slight neuromuscuLar disturbances and changes in the termirnal innervation pattern. Both plasma and erythroc)'lte cholinestemse activity was measured.

SUBJECTS AND EXPOSURE
Eleven men from a company handling the commercial application of insecticides wer,e ex'amined. They were all spraymen, wOrlking daily with solutions of 4 % lindane, 0.2 % pyrethrum, 0.125 0/0 pipeI"onylbutoxide, 2.5 0J0 ma~:arthion or 2 0J0 b1"Omophos in kerosene. Intermittently they were exposed to 0.5 diJazinonand dursbane. The employment period \nari:ed between 1 and 24 yearn. 'I1hey were equipped wjlth rourtine protective dev'ices such as respiI1ators, boo,ts, overalls and gloves. The men were examined criJni,ca:l1y, neuroph)'lsiologically, and with measurements of blood cholinesterase activity. They also formed their own reference group since measurements were ,aLso made after 1-4 weeks of non-eXiposure (pveeXjposure values). The measurements following exposure were generally made within 1-24 h after a period of 'spraywovk. Exposure v,alues were taken during the sp'ring sea80n, and the preexposure values after the SUilIlmer \nacation. Bl'f)od sampLes for cholinesterase activity ana~ysis weI1e taiken immediately before each EMG investiga,tion.

DETERMINATION OF CHOLINESTERASE ACTIVITIES
The cholinesteva:se 'activjlty of plasma (BuChE) ,and erythrocytes (AChE) w,as deteI1ffiiJned with a gasometrioc technique using whole blood samples (50 ,ul app'lied 256 and dried on filter paper) and bUityryl-choLine iodide and acetyl-jJ-metykholine iodjlde as the selective substr,ates for the two activities (3,4). The activity was expressed in b 30 v,allues, Le., microliters of 'carbon dioxi:de evolved in 30 min/50 ,ul blood. These \nalues can easily be con-\nerted to nanomoles of substrate hydrolyzed ;per second per Niter of bLood (which is the new runit, Icalled 'mtal, recOIl1Jmended by the Iinternational Enzyme COIl1JmiJssion) with the follLowing £ormula:

Nerve conduction studies
Motor neI've 'conduction velocity me.alsurements were made with the standard technique with surface recording (metal discs) and stiJrnu ' 1ating electrodes (DISA, 13 K 62). 'I1he filter setting of the ampliifier w,as 2 Hz to 20 kHz. The ulnar nerve was stimulartJed on both sides. The active vecording electrode was pla,ced ave,r the belly of the abductor digiti minimi (ADM) muscle and the indifferent electrode over the 'fi:fth metaoar,pophalangea1 joint.
Sensory nerve ,conduction velocity measurements were made f110m the right suvail nerve with antidI10mic stimulation between the two heads of the gastrocnemius muscle and the recording of the sural neI'Ve 'actjlon potential at the lateral mailleolus with £ilt ,pad electmdes (DISA 13 K 62).
FurtheI1IDore the amplitude of the compound ITer\ne action poten~ial was measured with fHt pad electrodes on both sides at ,the sulcus ulnaris when Ithe 'llJlllla,r nerve was stimulated <at the level of the wrist.
Be:liore all nerve conduction studies we checked thaJt the ,eXjt1'lemiity wa's warm. The ,tempemture of the labor,atory was kept constant at 22°C ,in aH the inve.stiga-tiJons.

Muscle response
The change in the oompound muscle actton poteIl!ttal WalS measured from the abductor digiti minimi musCle with repeti-tiv€ ulnar n€rv€ stimuJl.a.tion. FOT the recoroing and stimulating ,the setting was the Slalffie 'aIS that used for the measurements 'Of the motor conduction velocity of the ulna'!" ner,ve. The amplitud€ (negative pealk) of the first response and the change in aanpHtude 'and integraJted surrace {>the whole response) between the first and fourth respons€ at repetitive 2-Hz nerve stimu}ation~decrement) w,as measured on-line with a computer (PDP 11/40). The accuracy of the amplitude measurements was 0.1 mY. On different occasions the am~iJtude may \nary up to 2 mV (,range) in ,a normal subject. The decrement measurements were made with an a{:curacy of aboUit 1 0/0, and 'a value €xceeding 5 0/0 (amplitude and surface) was considered abno1"11lial. 'I1h~e palI'ameters were measured at rest and immediately after 20 s of maximall voluntary acti'VatiolIl of the muscle (postaJCtiV'i:ty f,aci1itation). A final test was made 5 min 'after the activ,ation period {pos1lactJivity exhaustion). Caore WlaS talken that the hand muscle WaIS waI1IIl during the investigation.

Single fiber electromyography
SFEMG was performed in the extensor digitorum communis (EDC) muscle and measurem€nts were made of the j.irtJ1:er and fiJber denmty.
Jitter. Jitter recordings were made from the slightly vohmtarily aO'bivated muscle 'as previoooly described (7). The electrode was posiJtiJoned to record simultaneously activity fIiOm two or more muscle fibers belonging to ,the same motor unit. There is a va'riability in :the time iruteNai between two aotion potentials fI10m muscle fibers in the same motor unit art; cOO1ISecutive discharges, caned the jitter, which is on the order of 5-55 fls in a normal EDC muscle. This jitter is mainly due to a vadabihty in the neuromuscu[ar transmission time (17). The jitter~s increased in cases with disturbed neuromuscul'ar trarusmission even 'before impulse bl'OCking occurs, Le., before de<:re-meM ,studies or clmica'l tests show any abnormahties. The jitter analysis W1as made on a computer (PDP 11140) off-line. Exceptionally manual analysis was made from measurements of recO'l'drj,ngs on film or 'a 'storage iQsoi:lloocope. Recordings were made £rom 10-20 potential pail'S (corresponding to 20-40 motor end-plates) at each iJnVlestiga'tion.
Fiber density. Fiber density is the average number of muscle fibers £,rom one motor unit within the SFEMG elec-tI10de uptaike area (radius about 270 /.tm), obtained f;yom 20 ,recording sites. The method and normal resulrtls for different ages have been presented earlier (18).
Statistical methods. Student's t-test of paired differences wilth the one-tatled test waJS used for the statisti:Oall analysis.

Clinical investigation
None of the examined subjects repoI1ted clinioal symptoms of intoxioation indicating a reaotion to org3Jl1iophosphate exposure. Apart from ocoasional headache, sometimesI1eLated to work, the subjects had no symptQImiS or signs of any neuI1O-lOgQoaJl dilSturbance.

Cholinesterase activities
The 'cholinesterase actiVlities of ibhe 11 spraymen befoI1e and the mean difference between the values after and before exposure are presented in table 1. The values Only on€ subject had a decrease in plasma activity eX'ceeding the normail intraindividual v,ariation. In this connection it may be pertinent to report that 10 of the 11 wor'kers had a lower pilaJS1lla cholinesterase aotivity after exposure than berore. 'I1he erythrocyte cholinesterase was not affected (p > 0.2).

Nerve conduction studies
In rthe nerve conduction studies (table 1) the motor nerve conduotion velocity was above the ,lower normal limits (> 45.0 m/s) in 'all wonker,s, and no significant decrease oouLd he detected between resuilts on the two occasions (p> 0.1). The compound nerve .action potential, norma:Ily showing a great scaltter because of technical difficulties, did not show any signtficant reduction lin amplJiiude (p> 0.2). 'I1he sensory conduction velooity was sHgl11tly reduced (3 Ofo)after the eX'posure period (rp = 0.05); reduction was noted in 7 of the 11 workers. 258

Muscle response
No decrementaJ response (tabl€ 1) and 11K> postactivity £acilitation or exhaustion was observed in any of 'the wOl'kers. The decrement was less than 3 Ofo. No signs of repetitive dilScha:pges were found. The recorded compound muscle action potentiall fI10m the ADM muscle ranged from 7.6 to 14 m V and showed no significant difference befure and ·after the ,eX'posure (rp > 0.2).

Jitter
Some individual motor end-plates showed an increased jitter before or after expo-sure~table 1). No consistant difference in >the jitter with exposure was found.

Fiber density
For the group, no diff,erence was seen between pre-and postexposure meanV'.aiues of iflhe fiJber derusity (table 1). It was unchanged 'and within norma[ i1Jimits, 1.44 ± 0.15 (18) in lSeVlen of the woIikers ( fig. 1). In four cases the fiber density exceeded normal Vlailues by 2 SD before exposure. Two had increased fiber density on both the test occasions.
There was no oorrel.ation between cholinestemse activitres in p"l:aIS.lll:a or red ce1ils and .the neurophysiological parameters (conduotion velocirbies, mean jitter value, number of recoroings with increased. jitter, fiber density). Neith-er was there any com-elation between ,changes in cho[inesterase activity and changes in the neurophysidlog.ical parameters.

DISCUSSION
This investigation was performed to determine any disturbance in neuromuscular function and possible correlation with a decrease in chio1inestera,se actirvi!ty in connection with work with organophosphorus compounds. Earilier reports (11) have indicated !that neurophysioiogicail methods could reveall suoh d:il9tuI1banoes even when bLood cholJiJneste1"ase activity was normal.
The EMG surdJace respollise W1as said to show a decrement at repetItive nerve stimulation, a decrease after muscle activity, and a lower amplitude after exposure to organophosphorus compounds (11,15). The two first findings, and possibly also the "third, would indica·te disturbed neuromuscular transmission. If this is the ca'Se, ,the decrease in amplitude of the recorded action potential 'should be pro-portionaJ. to the number of ,blocked motor end-plates. Our neurophysiological investigations wil1h surface recordings showed nm:mal response amp1itudes without decrement or postactivity exhaustion. However, !these studies can be technically difficult, and there are many possibmties of artifacts giving false ipOsilj;ive or possibly negative results (e.,g., improper electrode placemeIlit, movement of the electrodes during stimul!aiion, chl3.nge in skin resistance, intramuscuiliar ,ternpemture, characteristics of the amplifier,s, e.g., filter settings). It is particularly difficult to use the absolute amplitude fur oomparison between recordings made on different occasions. For the decremerut studies it is very important, iJn situations of minor ohanges, that changes in both amp.liltude of the response and iJts integrated surfJace be compared. Whether these £actom laccount for some of the 'aJbnormal findings reported earlier (10,11,15) canrnot be determined since some of the information needed fur such an evaluation is missing. In the present study these factors were standardized and kept as CO'tl'stanrt; as possible between the investigations. By means of a computer the measurements were made with ,a high acouracy.
To determine whether any neuromuscular disturbances were present despi.te our normal sur£ace recordings, we performed SFEMG, which is a sensitive method for the study of the transmission in single motor end-plates in situ and may show abnormalities in muscles having nondecreasing surface responses. In none of the cases did the mean jitter change after a period of work. The investigation thus did not reveal any acutely disturbed neuromuscular transmission. If the surface response shows a low amplitude or decrement due to neuromuscular disturbances, there is always an increased jitter and blockings in the SFEMG recordings. The slight changes in plasma cholinesteraseactivity were not ,correlated to dis-tUl'bed neUI'omUiscular tmnsmission in the subjec,ts. In our study we found lower sensory nerve conductLon velocities after work in sev,en subj<ects. The ,aver,age reducUon for the group was very ,sma!!l (3 0/0) but statistically significant (p = 0.05).
Reduced conduction velocity is usually a sign of neuI'opathy, which thus may be present in some of our workJers. The increase lin fiber density in four subjects is interpreted as a sign of peripheral reinnerv<ltion after denervation. Such a lesion would most likely be peripherally localized since the motor nerve conducti,on velocities and 'compound nerve action potential amplitudes were normal.
The incr,eased jitter values seen in some recoI'dings most liJkely indicates an uncertain impulse tI'ansmission in reinnervation 8tructUI'es [nerv,e 'SpI'Outs, immature motor end-plates (9)] but does not relate to acute exposure. A preceding denervation due to neuropathy is a possible explanation.
V:erbeI1k and Salle (19) a,dministeI'ed mevinphos to vo~unteers and found a red blood cell cholinesterase depression of 19%. In these men a 7 % decrea,se in slowfiber mortJor nerve conduction velocity was found, but the authors :stated tha't this finding might not be found for other organophosphorus compounds.
A peI1ipheral neuropathy could cause a reduced surf,ace ,acti'on potential, e.g., due to reducedexi'tability (demy,elination) or axonal degeneration. Whether such a reduction explains the i10wered amplitude in exposed pesticide workers, reported by others, cou i ld Iliot be fumher i1Jested in our materiJal since the ampLitudes did not show any definite changes.
The slight neurophysiological abnormal-260 ities found in this investigation were unrelated to a lowered cholinesterase activity.