in the Chemical laboratory Edited by N O R M A N V. STEERE, School of Public Health, University of Minnesota, Minneapolis, Minn., 55455
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XXII.
The Use of Biological Samples in Evaluating Toxic Exposures in the Laboratory R. G. SMITH; Ph.D. Professor-Wayne In recent years, a large measure of success has been achieved in making the chemist conscious of safety in t,he labors, tory. Regular articles in this column and elsewhere have emphasised the need for concern on the part of the chemist as he daily manipulates substances which are explosive, flammable, toxic, or ot,herwise dangerous to his person, and it is now commonplace to see safety devices, laboratory hoods, ete., being used as they should in almost any litbarntary. Nevertheless, there is one aspect of exposure to hazardous substances that must still he regarded as unsat,isfactory, and it is doubtful if one laboratory in ten is fully conscious of the nature and magnit,& of the problem. I refer to the effects of repented exposures, overa long periodof time, and usually by inhalation, to low roncent,ratians of substances which are capable of causing serious and often irreversible damage to the exposed individoals. Consider, far example, the infrared ~peetroseopist who utilizes carbon tetrachloride because it is transparent in his spectral region, and who daily dispenses small quant,ities of it to fill his cells. H e knows that the liquid is "on-flammable, and he probably han heard that excessive exposme to the vapors is hmnfrd; in fact if he reads the label on most bottles he will be amply warned of the hazards of prolonged inhalation. I t is probable, however, t,hat he relies on the nose to warn him of excessive exposure, and doesn't know that the presently accepted threshold limit value for carbon tetrachloride is 10 ppm by volume, a concentration below the odor threshold for most persons. It iq entirely possible for our spectroscopist to hreathe enough carbon tetrachloride vapors over a period of weeks or months to cause adverse health effects, even though he hardly recalls a time when the odor was objectionable. A similar condition can result from many other solvent vapors, for mercury vapor, and for eauntless other substances which a laboratory worker may use repetitively. The industrial hygienist ha3 long been dealing with exactly this kind of problem in industry, and an extensive technology has developed in regard to evaluating exposures and controlling them. The most widely used approneh has ?mdit,ionally been the measurement of air cancentrations of toxic substances, ut,ilizing a number of sampling devices and direct reading instruments which have been
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developed for this purpose. The usual procedure is to go into tho working environment a t a certain time and take a number of rnther short samples in what is hopefully termed the breathing aone of the workers. To anyone who has spent much time in industry or in chemical Inboret,ories, it is quite apparent that the breathing zone is a rat,her difficult area to define, for in relatively few cases does the exposed worker s h y in one place while breat,llin~an unvarying concentration of some contaminant. Certain measures can be adopted to improve the air nampling technique, and one of the more sophisticated is the install~tion of automat,ic air analysis equipment, which samples a t s. variety of points in the workina environment and continuously records the data. I n some instances, a further refinement is the installat,ion of an alarm device which may be activated if concentrations become excessive. Even the simplest of such systems is initially rather expensive and requires a subst,sntial annual budget for operation and maintenance, so that economic comiderations alone tend t o prevent widespread adoption of such systems. Furthermore, many situations in the laboratory simply do not lend themselves to this kind of fixed point air analysis, and could not be used even if cost were no object. I n recent times, a different sampling approach has become more popular, namely, the use of miniature devices which may be at,tached to the individual whose exposure is being studied, and which operate with lightweight pumps and battery packs. These devices are very nseful and go a long way toward better defining t,he exposure of an individual in the performance of his normal duties, inasmuch as they follow him as he moves about. No matter what met,hod of air sampling is used, the purpose is to define the inhalation exposure, with the nltimate intention of comparing the analytical results t,o some set of standard valoes. In the United States, comparisons are normally made with reference t,o the Threshold Limit Vahre (TLV) list ( I ) , put ont annually by the Threshold Limit Value Commit,tee of t,he American Conference of Governmental Indtlstrial Hygienists. This listing of chemicals, minerals, and assorted commercid substances implies that there exists a coneent,retion of any substance included in the list whieh ordi-
Ralph . Smith, I l l . rceeived )is Bachelor c~nd Master of Science legrees from Wayne State University n 1942 nnd 1949, and his Ph.D. in >hemistry there in 1953. He worked as an indwtrial chemist or two years, served in the U. S. Lrmy Air Force for four years, and ws employed from 1946 to 1955 by he Bnreau of Industrial Hygiene of he Detroit Department of Health rhere he served RS A S S O C Indu~t,rid ~R~~ Iygienist and Chief Chemist. Since 955, Dr. Smith has been associated iith Wyane State University, where te holds the position of Professor in he Ueuartment of Ocaimnt,ional and Cnvironmental Health, of tlie School f Medicine. Particularly significant among Dr. Imith's many and varied pprofessionsl ctivities are his services ns Editor of he Toxicology, Air Pollntioll, and ndustrisl Hygiene Section of Chem:a1 Abstracts, Chairman of the Comlitbee on Biochemical Assnyr of the imeriean Industrial Hygiene .4ssoeiaion, and member of the Committee n Threshold Limits of the American hnference of Governmentd Indusrid Hygienists. narily will not produce advcrse effects when the exposure period i defined as a normal work meek. The values cited in the list are intelided to be time-weighted average concentrations for a normal work day, and as such they are clearly an attempt to limit the total amount of a substance whieh an individual may breathe in any work period. (Continued on page A45)
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I n actual practice, air sampling by any of the means previously cited does not normally yield time-weighted average data, and a considerable amount of auxiliary work may he required to obtain such information. Again, in many situations it is unfortunately true that it can he nearly impossible to get redly good t,imeweighted averages, and air s a m p l i n ~ fails to achieve its intended goal. I n such situations, s. completely different approach is called for, and in many instances this approach consists of sampling the exposed individual rather than his environment. Commonly this is done by examining samples of blood or nrine, which are mt,her easily obtained, and less commonly by annlgsing breath, sweat, tissue, feces, or other materials of bialogical origin. A great many suhstances are ahsorhed after inhalation and exist essentially unchanged in the bloodstream, and:msy even be excreted unchanged ill the urine. Other mbsl.ances undergo changes after ahsorption and met&mlites may!:be present in the blood or nrine, while in still other instances a measurable change may result in the level of some naturally oecmring biochemical substance. Frequently, thes: naturally occurring snhstances are enzymes, but many non1 ensymic materials are also involved. It, is obvions that under ideal cireumtances biological sampling could he superior to air sampling, but unfortunately, t,he frequency with which ideal cirrmmstsnces occnr is not as great as might be wished. For example, certain materials when inhaled are found in the bloodstream s t varying levels which bear some wellestablished relationship to t,he total exposure of the individual, and it is possible to state that values below some level are normal, or a t least, not excessively elevated. Whenever s. certain conoentrstion is exceeded, by contrast, exce~3ive exposure or incipient health dnmege may he indicaled. I n other instances the blood levels of many substances fail t,o show any correlation with exposure, or else the correlation is so unrerI.ain as to be useless. The ehemicd laboralory is uhviously a work environment where exposure to almost any substance imagined or imsginable is possible. I n the nnnlytical labor* tory, there will tend t o be recurrent exposnrc to n number of reagents which are required by the analytical activity being performed. Thus, for example, many analytical laborat,ories will perform routine extractions with benzene or other solvents a? a first step in the isolation of some organic substances, and there may be a constant low level of benzene in the general air of the laboratory, interspersed with higher concentrations for shortor periods of time as fla-ks are filled, or samples changed. An organic labor~tttory will invariably take on s characteristic odor due to the class of chemicals with which a given research program is concerned, m d in many
(Continued a page 446)
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eases the eomhinatian of high vapor pressure and toxicity may result in exposures which are very hazardous. Almost any I&omtory c m he expected to w e mercnry, and experience has demonstrated on numerous occasioni that, exressive exposure to bhis element is rclntively cnmmonplsee. Many more examples of possible exposue of l a b ~ m t ~ o r y perrunnel to toxic substsnres mold he cited, hut. it should be elem that the lahonit,ory is a workplace where it is almost s, certainty that s ~ x hexposures will occur, and that they will very probably be non-repetit,ive, unmeasured if not unmoasurahle, and altogether fnlstratinx to a rcsporisihle health official coneenled with prevenl.ing health damage. The use of biological sample analyses i n snch rin:umstances is underst,andahly xtlmct,ive, and the remainder of this d i s e ~ ~ s s i m is concerned with some applications of this approach. I t must, be kept in mind that in no way does the use of biological sample analysis preclude the evnluat,ion of the hasards by air smrqding, and for most situations air sampling seeomparried by biological sample analyses proves to he very informative and helpful. It is not possihle l o attempt to list all of the chemical exposures which a , d d be evaluated by biological sample analysis, and the following examples were selected for several reasons. First, it is lrelieved t,hat they me representative of several classes of w~bst,anceswhir,h may be encountered; secondly, the individual snh-
stnnres chosen %rethemselves commonly enronntered in many laboratories; and finally, a considerahlo body of knowledge sxisls concerning tho interpret,stion of the results of these particular nnslyses. With these thoughts in mind, the evaluation of exposure t o mercury, benzene, carboll tetrachloride, organic nitro compnonds, and organic phosphat,es will he hriofly discussed. For many substances which are not mentioned, the chemist should he aware of soun:es of informat,ion t.o whieh he may l.urn when necessary, and severnl are deserving of note. A p u t i a l listing of informed agencies or orgxniaations will include: the industrinl ltygiene section or tho medical nr toxicology departments of the employing rrrgnniant,ion itself, when t,he employer in a large corporation, university or institution; the stat.e nr local industrial hygicne agency; and t,he United Sl.ates Puhlic Heitlth Service, Division of Oempational Health, in Cincinnati, Ohio. Oft,-t,imos iL is sntisfar:t,ory t,o r o t ~ d t . an aut,horit,ative text or journal, and sever;d whieh are particularly useful are &ed in tho reference list (24). Many other t,ext,s and journnls are available, and industrial hygiene orgsnisations can readily identify them upon reqllest.
Mercury
As stated earlier, mercury is to he found in virt~tally any labo~atory, and more often than not, it is spilled and distributed in s m h n manner that hazardous sit,ualions may arise. The entire subject ha? heen recently discussed in this column
(7), n l d remarks therein made need not. he repeated. It in sufficient to make Lhe ohservnbion that aampling and analysis of urine k uniquely useful when dealing with lahorat,ory personnel exposed to mercury, and although it is not possible to equate urinary lev& with illness, there is n clear relnt,ionship hotwoen elevated llrillary levels and exposure by inhnlation or skin d ~ s ~ r p t i o n Although . there is no hard and fast urinary mercury level which can he considered as indicating hasardous conditions, any value %hove fl.30 mg/l is usually considered exres~ive, xrrd lower villues whieh are in excess nf several hundredths of a mg/l indicate that the individual has heen exposed to mercury in some manner or ot,her. There sre a number of precsut,ims to he nhserved in the eolleetion of specimens and interpretat,ion of trilirrry mermwy values, and it is not recommended that, laborat,aries unfamiliitr with these matters al.tempt to do their own sampling and n d y s e s . Instead, the appropriat,e indostrial hygiene or medical mganisation ~ h o u l dbe consulted, and arrangements made for periodic sampling of this kind. I n the event of elevated urinary levels, of course, a. complete industrinl hygiene survey is indicated, snd consideration of !.he possibilities of health damage by L: competent, physician is wise. I t should be noted in passing that at the present time there is no reason to analyze hlood samples for their mercury cont,ent, inasmuch as most published studies hsvr not, shown any correlation between hlood
(Catinued on page 2146)
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mercury levels and exposure. To summarize, the periodic sampling and anslysir of wine specimens to determine mercury content is recommended as a means oi preventing mercorislism in laboratory workers.
Benzene The unique toxicity of benzene has been recognized by several decreases in its threshold limit value in recent years until a t present the threshold limit value is 25 ppm, with the further rewmmendation by the committee setting the limit
that a t no time should the concentration be permitted to be in excess of 25 ppm. The fact that low concentrations of this order of magnitude are either odorless or not a t all unpleasant to breathe meam that concentrations in excess of the threshold limit value are readily tolerated by most individuals, hut the effects of over-exposure to benzene are of mch serious consequence that every possible precaution should be taken to minimize exposure to it. If possible, another solvent should be used in its plaoe, but when benzene must be used, it should always be contained &bin a. good labor* tory hood or ot,her well ventilated encloswe. Absorbed benzene metabolizes rather
quickly to phenol, which is excreted in the mine in snffioient quantity to substantially increase the normal phenol concentration. For many yearn, it has been common pract,ice to messure the socalled ethereal sulfabes by a simple pracedore which distinguishes inorganic sulfates from organic sulfates in the urine. More recently, it has been shown that the determination of phenol as such is preferable to the sulfate determination (8), but it is possible to interpret results no matter which of the two tests is employed. Normally, the inorganic sulfates will account for 85% or more of the t o t d sulfates, and lesser quantities are indicative of exposure to benzene. By the same taken, normal urine will usually contain less than 200 mg of phenol per liter of urine, and the increase in cancentration is proportional to the exposure. If benzene is regularly used in any lahoratory, t,hese simple urine analyses should be made periodically as B method of detecting excessive exposure.
Carbon tetrachloride Carbon tetrachloride is wnsidered every bit as insidious in its action as benzene, as evidenced by a threshold limit value currently set a t 10 ppm, and its use should also he minimized and prevented if possible. At levels of 10 ppm carbon tetrachloride cannot be detected by odor, and hence the chemist may be qnit,e unaware of its presence in the air. Unlike benzene, carbon tetrachloride produces no simple metabolites which may be determined in urine or blood. Although some use may be made of direct measurements of carbon tetrachloride in the blood or breath, the means for performing suoh analyses are not readily available to most laboratories and int,erpretation of analyses is questionable. Carbon tetrachloride, therefore, is an example of a, substance for which direct biologicd sample analysis is restricted to the measurement of variations in the levels of some normal constituent of the blood or tissue, with the purpose of detecting organic injury. Specifically in the case of carbon tetrachloride, it is the liver which is most likely to be damaged by repeated low level exposures, and if necessity demands that carbon tetrachloride he used with any frequency then the exposed individuals should be periodically examined by a. physician who is aware of the hitzmd. Normally, the physician will supplement his examination by requesting that, liver function tests be performed by the clinical laboratory, and the actual tests chosen will be a matter of medical judgement. These remarks concerning carbon tetrachloride may be usefully applied to exposure to certain other chlorinated hydrocarbons, but care must be taken to avoid the misconception that all chlorinated hydrocarbons are toxicologically equiv* lent. For some, such as trichloroethylene, it is possible to measure metabolites in the urine to evaluate the exposure, whereas for many others reliance must be placed upon air sampling and medical examinations, while in yet other cases, breath analyses by gas phase infrared spectroscopy may be very useful. It is
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urged that the toxicological liter&.ture be consult,ed in the ease of each substance whose use is anticipated so that adequate measures may he taken to minimize the hazards of use.
Organic Nitro Compounds A large number of nitrogen-containing compounds are noted for their tendency to alter the normal composition of the blood by the iormation of met,hemoglahin, s mhstaure which, unlike hemoglobin, contains iron in the ferric state and is unsuitable for the transport of oxygen. Aniline and nitrohensene are hut two examples of commonly used chemicals with this property, and the hazards attel~dingtheir use are all the more severe because of the ease with which they can pass through the unbroken skin. When using these chemicals and others like them, great care should be taken to minimize contact with skin and inhalation of the vapors, and it is advisable to establish a program of regular medical examination if such a~bstanees are used n,ith frequency. Almost certainly the physician will wish to have periodic hematological tests performed, including methemoglobin determinations as well a s other standard hlood quality evahmtions. Although it is true that not all nitrogen-containing compounds exert t,he same toxicological action, the literature should he rhwked before significant exposures to npw compounds are permitted.
Organic Phosphate Compounds Certain organic phosphate esters have become immensely important in recent years as a result of their insectieidnl properties or their possible use as "nerve gases." I t is highly improbable that laboratories doing research with such compounds will not be fully acquainted with the hasards attending t,heir use, so these remarks will be kept very brief. Many of the organic phosphate esters are very toxic to man, and a t very low levels specifically inactivate the enzyme chalinesterxse. 811 persons who work with organic phosphates should be examined periodically. An important aspect of the examination is the determination of red hlood cell and plasma. levels of cholinesterase, for although these determinations do not specifically constitute a diagnosis of injury, they are most useful in giving evidence of exposure.
Conclusion The examples cited above should make it clear that exposure to many toxic subst,ances can he implied by the analysis of biological samples, and in some cases early diagnosis of intoxication is possible. Whenever such analyses can be performed, they have the virtue of integrating the individual's e q o s u r e to some degree, and of giving evidence of relatively brief
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high level exposures which could well go unnoticed with most air sampling programs. Furthermore, they reflect the extent to which absorption of the substance occurred, either after inhalation or by any other route of administration, and thus are potentially more inform* tive than air samples. By no means, however, should complete reliance be placed on biological sampling to the exclusion of air sampling or other hygienic measures normallv recommended for the handling of dxngeroos substances. When properly used in conjnnction with other procedures, biological sample analysis can add much to a. well conceived and executed program of health maintenance.
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References (1) 7'h~eshold Limit Values for 1966, pub. by American Conference of Governmental Industrial Hygienists, 1014 Broadway, Cincinnati, Ohio 45202. (2) PATTY, F . A,, Indust~.ialHygiene and Tozieology, Vol. I , second revised edit,ion, Interscience Pub. Co., New York (1958). (3) PATTY,F. A,, Industrial Hygiene and Tozieology, Vol. 11, second revised edition, Interscience Pub. Co., Kew York (1962). (4) ELKINS, H. B., The C h a i s l r y qf Industrial Tozieology, second edition, John Wiley and Sons, Inc., New York (1959). ( 5 ) WILLIAMS. R. T.. Detozicalion Meehnnisms, second edition, Chapman and Hall, Ltd., London (1959). (61 Hvoienie Guide Series. nub. bv Amer-
(7) STEERE, N. V., "Meroury Vapor Hazards and Control Meamres," JOURNAL OF CHEMICAL EDUCATION 42, 7: A529 (July 1965). ( 8 ) FAGNOTTO, L. D., ELKINS. H. B., BRuGscn, H. G., A N D WALKLEY, J. E., "Industrial BenzeneExposure from Petroleum Naphtha,'' Amer. Ind. Hug. Assoc. J . 2 2 : 417 (1961).
REFRIGERATOR EXPLOSIONS "Laboratory refrigeration explosions aecur with regularity. While these explosions can destroy the building, they usually are limited to the ruining of experimental materidstored in and around the refrigera tor. These accidents usually occur due to the introduction of flammable liquids into an ordinary household refrigeration unit that is unsuited far this use. This type of explosion will became ancient history when explosion-proof refrigerators are used exclusively by laboratories. The first such unit was listed by Underwriters' Laboratories, Inc. in December, 1957."
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