Urinary chromium as an indicator of the exposure of welders chromium.

S., KILPIO, VIRTAMO, and HAAPA, K. Urinary chromium as an indicator of the exposure of weLders to chromium. Scand. j. work environ. & healtih 3 (1977) 192-202. Five welders working with high alloy,cr-Ni steel and one working with mild steel were followed during one work week. The chromium concentration in air was measured concomitantly with urinary chromium determinations. The water-soluble chromium concentrations in air exceeded '0.05 mg/m 3 during welding with ,coated electrodes, but metal inert-gas (MIG) welding produced much lower con centrations. The proportion of water-soluble hexavalent chromium in the air was usually more than 50 010 of the total chromium concentration during welding with coated electrodes, whereas less than 10 010 of the chromium produced during MIG welding was in 'a water-soluble form. Since water-soluble chromium (hexava,lent) is the more important biologically, the determination of both water-soluble and water insoluble chromium concentrations is emphasized instead of the measurement of the total concentration. The urinary chromium concentration proved to be a good indi cator of short-term exposure to water-soluble chromium when exposure was above the current threshold limit value of 0.05 mg/m 3, ,concentrations of more than 30 ftg/g of creatinine representing an exposure level higher than the threshold limit value.

Chromium (Cr) is known to have various deleterious eff<eds upon the human organism (7,8,31). Usually sucheffeds are due to the hexavalent (VI) form of the element, but trivalent (III) chl'omium also appears to playa role, at least in connection with skin sensitization (14). Chromium has an irritative effect on skin and mucous membranes. For example chromium ulcers and nasal septal per£orations have been frequent findings in workers exposed to chromic acid or chromates (9,19,25,26). Allergic ,eczema as a result of sensitiz,ation to chI'omium is also well recognized (2, 13, 192 14,24,29,32). Semitization may also affect the respiratory organs and cause asthma (8). In connection with heavy exposure to chromium gastrointestinal symptoms have been reported (36). The most serious effect however is its ability to cause cancer in man (3,4,11,18,22). Before exposure to chromium can be effectively limited, reliable methods of measuring the exposure are needed. The determination of chromium in the workroom air is a reasonably good measur,e of chromium exposure, and threshold limit values {TLVs) for various chromium compounds provide a basis for practically evaluating the results (8,30,37). For the assessment of individual exposure a biologkal exposure test would provide additional information or even an alternative to, workroom air measurements. The concentratron of chI'omium in blood and urine can be measured, but there is a lack of well-controlled studies on the relationship between exposure and biological indicators. Moreover, the oxidation state of chromium in the air, whether tri-or hexavalent, has not been reported often. Nevertheless, maxi.mum allowable concentrations have !been proposed for urinary chromium concentrations (12,15,35,36,37). Too few dat·a exist to allow evaluatiOll1s of blood chromium measurements, but at present they seem to be of little or no value to biological exposure testing.
Borghetti et al. (6) recently published results of a longitudinal study in which they showed qualitative evidence in favor of a relationship between exposure to chromium and urinary chr-omium excretion. Shortly before we submitted this paper for publication, Gylseth et al. (1)(2)(3)(4)(5) published a study simHar to· ours in which a good correlation between chromium in air and ucr-inary chromium excretion was demonstrated. In our report we present the results of a one-week follow-up study of welders exposed to chromium during the welding of high alloy Cr-Ni steel.
The welders' regular daily worktime was 8 h, of which they either welded or did assembly work in connection with welding for some 6-7 h.
The workplace was a shop where machines for the pulJp and paper industry were manufactured. The assembly shop had an area of 5,000 m 2 and a room size of 50,000 m 3 . It was equipped with dilution ventilation with blowing and exhausting of 150,000 m 3 /h, but no local exhaust systems were used at the welding sites. All welding work was done in open -air conditions.
The metals welded were high alloy Cr-Ni steel, containing 18 % chromium, and mnd steel, in whioh chromium was presentonly as an impurity « 0.3 % Cr). The electrode wire for the MIG welding contained 20 % .chromium. The coated electrodes for theohromium steel were of the rutile type. The core wires of the coated electrodes ccmtained 18-20 Ofo chromium; and the coatings, ferrochromium corresponding to 12-20 Ofo of elemental chromium. In the welding electrodes for mild steel chromium appeared only as an impurity.

METHODS
Air and urine samples were collected during one work week. The urine samples were taken from every subject at 0700, 1100, and 1600 everyday of the week plus the previous Friday at 1600 and the following Monday at 0700. Air samples w~re collected each morning and afternoon on Monday through Friday except for the control subject, for whom they were taken on Tuesday and Thursday only. Blood samples were also collected, but only on Monday at 0700 and Tuesday at 0700 and 1600.

Welding fume measurements
Sampling. The welding fume samples were oollected on cellulose ester membrane filters (Millipore Filter AAWP ,,37 mm, mean por,e size 0. All samples were taken from the breath-ing~one, the filter holder being placed inside of the welder's faoe mask. Each sampling pump and membrane filter combination was calibrated against a wet gas meter. Flow rates averaged 1.7 IImin for the MSA samplers and 2.5 IImi:l). for the ranged between 180 and 210 min a1nd sample air volumes between 300 and 500 1.
Analysis. The total fume concentrations were determined from their weight and calculated in milligrams per cubic meter.
According to some earlier studies (13,16), and also to our own observations, a part of metallic chromium oxidizes in the welding arc to hexavalent chromium compounds, some of which are watersoluble. Part of theohromium in the fumes is water-insoluble and probably oonsists of trivalent chromiu,m compounds and metallic ,chromium (33). Therefore, the ohromium oOOltents in fume samples were de-194 termined separately for the water-soluble and the water-tnsoluble fraction.
For the determination of water-soluble chromium the samples were treated for 30 min at 80 D e with 30 ml of water. The mixture was filtered through a membrane filter and the solution was diluted with water to 50 ml. The water-soluble fraction was analyzed by atomic absorption spectrophotometry (AAS) and in addition with s~diphenylcarbazide(DPC) for hexavalent chromium.
The AAS determinabons were made with a Perkin Elmer Model 403 spectrophotometer with an air-acetylene flame a,ccording to the manufacturer's manual.
The detection limit was about 0.01 pg Cr/ ml, which corresponds to 1-2 pg Cr/m 3 in air samples of 300--500 1.
Hexavalent chromium forms a pink complex with DPC, -and this reaction is very specific and sensitive {20, 30}. For the colorimetric determinations, with DPC, 20ml portions were taken from the filtered water solutions, 1.25 ml of sulfuric acid (1 + I) and 1 mg of DPC 1n acetone were added, and the mixture was diluted to 50 ml with water. The absorbances were measured at 540 nm. Potassium dicl1rornate solutions were used as the colorimetric standards. The detection limit of the DPC method was 'about 0.05 pg Cr (VI}/ml . em, which corresponds to about 5 fig Cr (VI)/ m 3 in air samples of 300-500 I.
The results of the chromium determinations made fr-om the water-soluble fraction with AAS and the colorimetric DPC methods appear in table 1. The results of the two agree well Nearly all of the water-soluble chromium was hexavalent.
For the determination of the acid-soluble chromium compounds the water-insoluble residue from the fume samples was treated with a mixture of 10 ml of concentrated nitric acid and 5 ml of concentrated hydrochloric acid and evaporated carefully to dryness. The rest was diluted to 25 ml with 5 % nitric acid. The chromium concentrations were determined with AAS. The results are underestimates of the true chromium concentration because not all water-insoluble chromium compounds dissolve in a nitric acid/hydrochloric acid mixture. No corrections have been made in the present results since the undissolved proportion was unknoW'll.
Interferences with the determination of hexavalent chromium with the DPC method. The reduction of hexavalent chromium to trivalent chromium that is caused by some agents, e.g., organic material, is possible during the collection and storage of fume samples and during the preparation of samples for analysis.
Some authors have reported on the reductive effect of cellulose ester membrane filters on hexavalent chromium (1). However, we did not find any significant reduction when we determined hexavalent chromium from the same fume samples immediately 'after sample collection and again after storage for two weeks. It is possible that -a wet aer-osol such as chromiumacid mist may be reduced on filters, but not a dry aerosol such as welding fume.
In studying the possible reduction of hexavalent chromium during sample preparation, we treated and analyzed known amounts of potassium dichromate 0Il1 cellulose ester membrane fi:lters using the same procedure as for the fume samples. No reduction was found to be caused by the dissolving, heating and filtedng operations.
Iron, nickel and molybdenum (VI) in high concentrations may interfere when hexavalent chromium is determined with the DPC method. Iron produces significant interference if the ratio Cr : Fe = 1: 5 is exceeded. Nickel interferes only in over a thousandfold excess (20). In the sample solutions the concentrations of iron aIlld nickel, determined with AAS, were low and under the levels of interference. Electrode wires for acid-resistant steel contain 2.3-2.5 Ufo molybdenum. Molybdenum was not analyzed in the welding fume samples, but the possible interference was studied by the addition of ammonium molybdate to the potassium dichromate standard solutions. A hundredfold ratio of molybdenum to chromium produced no interference in the absorbance.

Biological samples
Sampling. Polyethylene bottles were used for the collection of the urine samples. All bottles were allowed to stand~n a 10 Ufo Deconex sOllution overnight and were thereafter rinsed several times, first with tap water and finally with distilled water.
Special emphasis was placed on the avoidance of contamination. Before the sampling the subjects took off their work clothes ·and washed their hands. In addition, before the last sample of the day (at 1600) the subj-ects took a shower. The morning urine samples were collected at the workplace, and the subjects had been advised to urinate after waking up. Thus, this sample represented excretion after the night, not during it. The 5-ml venous blood samples were drawn into carefully washed, hepariniz'ed tubes.
AnaLysis. From the urine samples we first measured the specific gravity with a TS-meter (American Opticals) and took an aliquot for creatinine measurement. The cr,eatinine concentrations were determined by the Jaffe reaction with the Technicon Auto Analyzer.
In the urine and blood chromium analyses we used electrothermal AAS (Perkin-Elmer 400, HGA 74). We made a 0.5 ml + 4.5 ml dilution of the samples with Hamilton's Digital Diluter, and 20 ,ul of these solutions were injected into the graphite tube by an auto sampler (Perkin-Elmer AS-I). The measuring conditions for the atomic absoI1ption spectrophotometer were as follows: A 358 nm, slit 0.7 nm, hollow cathode lamp Cathodeon 7 mAo Those for the heated graphite atomizer were: sample 20 ,ul, purge gas argon; dry 1, 30 s, 115 0 C; dry 2, 30 s, 135 0 C, the temperature program from 135 to 980 0 C in 45 s, ash 20 s, 980 0 C; atomize 11 s, 2,500°C.
The calibration of the method was performed with aqueous calibration standards of 2, 5, 10, and 20 ,ug Cr/l as potassium chromate prepared daily from a stock solution of 1,000 ,ug/ml. Our calibration studies showed that a 1:10 diluted urine matrix had no effect on the absorption of chromium; thus simple aqueous calibretkm could be used.
The l'eliability of the method was checked with urine samples spiked with 20 ,ug Cr/l and 100 ,ug Cr/l. The urine samples of each welder formed one series of ,analyses, and we repeated the measurement of th'ese spiked samples three or four 196 times within each series. The results are presented in table 2.
The precision of the method was a'lso checked by the ,analysis of duplicate samples. The coefficient of variation of the results of duplicate determinations was 4.9 0/0 at the level> 100 flg Cr/l and 6.5 0/0 at < 50 ,ug Crtl. These imprecisions agree well with those obtained for the spiked urine samples. The practical detection limit of the method was 2 fig Cr/l.

RESULTS
The results of the ,air measurements are shown in talble 1. It can be seen that the total chromium component consists ofa water-soluble and an acid-soluble (waterinsoluble) fracnon. The total chromium concentration is probably an underestimate, however, because not all add-soluble chromium compounds dissolve in the nitric acid/hydrochloric acid mixture used. The water-soluble fraction is mainly hexav,alent and the acid-soluble fracti-on is probably trivalent, but part of it may also be other forms -of chromium, e.g., metallic chromium.
The concentration of water-soluble chromium (hexavalent) in air exc-eeded the TLV of 0.05 mg/m 3 almost daily for subjects A, B, and C. It exceeded the TLV on one day for subject D but was constantly below the TLV for subject E and control subject F.
From the total chr-omium concentration in air, the proportion of heX!avalent chromium vari'ed between 3 and 91 % and was higher for the more exposed subjects. In regard to subjects A, Band C the percentage of hexavalent chromium was almost constantly higher than 50 0/0. Sub-Ject D welded only occasionally on Monday; consequently, h'is exposure was very low. His exposure was also quite low on other workdays, the highest value being an the order of the TLV. The low values of subject E can be explained by the fact that he did metal inert-gas~IG) welding only. This mode of work seems to be associated with much lower exposu,re than welding with coated electrodes. Moreover, the proportion of water-soluble chromium seems to be lower in this type of welding.   The results of the measurements of chromium in the urine and a'ir are shown in fig. 1 for each subject separately. Chromium in air in this figure means water-solu'ble chromium as measur·ed by AAS. The chromiumconcemtraUon iill air that exceeded the TLV resulted tn a rapid increase in urinary chromium excretion (subjects A-C). In subjects D and E the exposure was lower, and only when the concentration of ·chromium in air exceeded the TLV did an increase in the urinary excretion occur. The urin,ary chromium excretion of the control subject (F) remained constant during the study period. This level was higher however than the concentration found in occupationally nonexposed subjects such as laboratory personnel.
The increase in the ex·cretion of chromium for the heavily exposed subjects (Ã C) was observed already after some 3 h of weIding, i.e., in the second urine sample of the day. The highest values were usually measured from the ,afternoon samples taken at the end of the worlkday. The  In the calculation of the Pearson correlation coefficients all -the values of each subject were considered as different points. This method has the drawback that exact levels of statistical significanoe c,annot be determined for the ,coefficients because the calculations have been performed with data involving successive readings from the same subjects.
A good oorrelation was observed between water-soluble chromium in the air and the creatinine corr,ected urine afternoon values {fig. 3). The relationShip improved when the morning values were subtracted from the afternoon ones, i.e.,  morning values were low md suggested a rapid excretion during the night. The excretion pattern for the weekend and for two successive workdays is shown is fig.  2. In spite of the rapid excretion, a slight accumulation seems to have occurred during the week for the most exposed subjects since the mOl'lling values were a little higher towards the weekend than on MQIIlday morning ( fig. 1). In order to study the relationship between the water soluble and insoluble chromium on one hand and the urinary excretion of chromium on the other, we compared the different air measurements (total chromium, water-soluble chromium and acid-soluble chromium) to urine concentrations (as measured values, values corrected to 'a specific gravity of 0.018, and as values calculated per gram of excreted creatinine). Sinoe the air sampling covered the total work time and its duration was about equal during the morning and afternoon, the arithmetic means of the morning and aHernoon concentrations were considered to represent the time-weighted average 'concentration of the day ac,curately enough. The length of the sampling periods for welding fumes differed by a few minutes, 'but the difference was not oonsidered to have any practical significance in the interpretation of the data. Table 3. Relationship between various cromium (Cr) measurements in workroom air and urine.

Relationship
Water soluble Cr in air, daily mean (mg/m 3 ), vs. urine Cr (p,g/g creatinine) at 1600 ( fig. 3) Water soluble Cr in air, daily mean (mg/m 3 ) , vs. f:, urine Cr (p,g/g creatinine) ( fig. 4) Water soluble Cr in air, daily mean (mg/m 3 ), vs. urine Cr (p,g/l) at 1600 Water soluble Cr in air, daily mean (mg/m 3 ), vs. t, urine Cr (p,/l) Water soluble Cr in air, daily mean (mg/m:!) , vs. D. urine Cr (p,g/l), corrected to specific gravity Total Cr in air, daily mean (mg/m3), vs. /:;, urine Cr (p,g/g creatinine) Water insoluble Cr in air, daily mean (mg/m 3 ), vs. D. urine Cr (p,g/g creatinine) the increase in urina,ry chromium excretion was compared with the chromium concentration in the ,air ( fig. 4). From the regression lines it can be estimated that the 'afternoon urinary concentration corresponding to a mean daily chr(}mium concentration of 0.05 mg/m 3 (the present TLV) was about 33 flg/g creatinine aIl1d the corresponding mcrease in the urinary ccmcentration during the wOI"kday was about 7 flg/g creatinine. The measured, uncorrected afternoon urinary concentration corI"espondimg to 0.05 mg/m 3 was ab(}ut 38 flg/l. For the mutual comparison of the uncorrected and corrected urinary concentrations fue increase in the excretion of chromium was compared to the watersoluble chromiUiIll concentration in the air (table 3). The corI"elation was best for the values corrected to creatinine, but it was also quite acceptable for the uncorrected values and the values corrected to specific gravity. The scatter ar,oUlIld the regression hne was the greatest for the uncorrected v,alues, and the smallest for the values corrected to creatiJDine.
We  (table 3.). Water-soluble chromium showed the best correlation, followed by total chromium, while the concentration of water-iJDsoluble chromium failed to show any signifka.nt correlati<m to the urine coneentration.
The results of the blood chromium measurements are shown in table 4. Those of the most heavily exposed subjects point toward a possible dose-effect relationship, b.~t no conclusions can be drawn because of the small num'ber of observations. Nevertheless, all the values were above the normal concentration of less than 0.5 fl'g/ 100 ml 1Jhat we have found in nonexposed workers in our laboratory.

DISCUSSION
In -our study welding high alloy Cr-Ni steel caused unacceptably high exposure to chromium for some of the subjects. The level of exposure was comparable to t1hat recently reported by Vorpahl et al. (35) in connection with the welding of highly alloyed Ni-Cr steel. The symptoms of the welders of our study were relatively milnor. Generally the irritation symptoms associated with welding ,can be attributed to fluoride exposure, which is <:ommon when basi<: coated electrodes are used. Our subjects used rutilecoated electrodes however, and, except for the control subject, none of them were exposed to fluoride. Thus the chromium exposure may have caused their symptoms.
It should be emphasized that high levels of exposur-e to chromium were measured only during welding with coated electrodes. The exposure level during MIG welding was muoh lower (subject E). Since the exposure of welders using coated electrodes was found to be unacceptably hig(h, the enterprise for whioh the welders woriked has already taken measures to diminish exposure and is installing a more effective local exhaust system. The ultimate solution of the problem would be a new building with facilities for MIG welding as the main type of welding method.
The MIG welding caused the lowest total exposure and, moreover, the proportion of hexavalent chromium was remarkably low. Thus the health hazard caused by exposure to chromium during welding oam be considerably reduced if welding with coated electrodes is replaced by MIG welding.
The analysis of the welding fumes showed that only part of the chromium to which welders are exposed is in the biologically more active hexavalent form. The large variation in the proportion of hexavalent chl'omium of the total chromium indicates that the latter cannot. be reliably used for the assessmen t of exposure to hexav.alent chromium and the analysis of the water-soluble fraction is essential. Otherwise completely misleading conclusions may be drawn.
The urinary concentration of chromium reflected the exposure to water-soluble chromium (hexavalent) well, but no cor-200 relation seemed to exist for the waterinsoluble (trivalent) form. This finding agrees with experimental results (5) imdi.cating that very little trivalent chromium is absorbed from the lungs to the blood stream. Korallus et al. (21) reported an increased excretion of chromium in workers exposed to trival-ent chl'omium as compared to nonexposed controls, but he did not report any correlation to air chromium concentration.
Thus, provided that the exposure is indeed to hexavalent chromium, measuring the urin.ary chromium concentration is a good method for estimating individual short-term exposure. Unfortunately, this method does not seem to be applicable to low levels of exposure. Below the current TLV there was no concomitant elevation in the excretion of chromium. Nevertheless, the baseline concentratiiOlIls of all the welders, including the oontrol subject, were higher than the levels measured in occupationally nonexposed persons, possibly because of chronic exposure. The excretion pattern during high short-term exposure indicates that excretion occurs rapidly ( fig. 2). In animal experiments a three-phase excretion pattern has been demonstrated for trivalent chromium (27) with half-times of 0.5, 5.9 and 83.4 days, respectively. Whether this ,applies to man is not known, but it is probable that the excretion pattern found in our present study represents a rapid compartment, the increased baseline excretion being an indication 'of increased chromium body burden, whioh wouLd have a longer half-time.
When urine samples are used, their general limitations shouLd be kept in mind (10,28). Our results agree with the opinion of Lauwerys (23) that correction to creatinine should be preferred to correction to specific weight.
The increase in chromium excretion duriJngthe workday seems to reflect exposure better than the measurement of afternoon urine concentration only. Nevertheless, the difference is so small that, for practioal purposes, measurement of chromium excretion from spot urine samples at the end of the workday can be recommended for routine testing.
As early as 1955 Vigliani and Zurlo (34) suggested that a urinary concer:tration <:,f 50 p,g/l would correspond to an all' chromiC acid concentration of 0.05 mg/m 3 . Franzen et a1. (12) pl'oposed a urine concentration of 25 j.tg/l as an indication of a need for further studies. In the guidelines for the periodical medical examinations of the Federal German Republic a urine concentration range of 20-30 j.tg/l is given as tolerable (37). Recently, Gylseth et a1. (15) suggested that a concentration of 40-50 j.tg/l would ,correspond to the TLV of 0.05 mg/m 3 . Their conclusion was based on measurements from a few subjects only, and they used uncorrected urinary concen trations.
According to our data, a concentration of about 38 j.tg/l (uncorrected value) corresponded to the air concentration of 0.05 mg/m 3 • When correction to creatinine was used, ,a concentration of about 33 j.tg/g creatinine corresponded to 0.05 mg/m 3 of chromium in air ( fig. 3). Thus our results agree well with the ear.li,er mentioned figures. It seems that, if the urine concentration is below about 30 j.lg/g of creatinine, the air concentration of 0.05 mg/m 3 is not exceeded. It should be emphasized, however, that this 'observ,ation applies to short-term exposure only. The relationship between chronic exposure and urinary chromium excretion is more complex and relatively unknown.
Under optimal conditions, the relationship should be known between a biological exposure test and both exposure and effect. This situation is, unfortunately, very rare. Strictly speaking, biological monitoring in connection with exposure to lead is the only pattern which meets this criterion (17).
The symptoms in connection w~th exposure to chromium have been extensively studied. However, their relation to the chromium concentl'ation in air needs further investigation (8,32). Even more obscure from the point of view of a doseeffect or dose-respo,nse relationship is the connection between exposure to chromium and lung cancer (8,32). Thus the relationship between exposure and the effect ,of chl'omium is far from solved today and, consequently, so is the relationship between urine concentrations and biological effects.
Basing biological limit values on air TLVs is always a preposterous process.
For chromium it is even more ambiguous since the air TLV is uncertain. Thus, the limitations of this ,approach should be kept in mind when the suggested biological limits are considered. If exposure to chromium is allowed at all, it should be kept so low that the urinary excretion of chromium does not increase. The suggested concentration of 30 j.tg/g creatimine seems to meet thi's criterion for short-term exposure. Should the TLV be lowered for "carcinogenic ohromium," as suggested by the National Institute for Occupational Safety and HealVh in the United States (30), urinary determinations would probably be too insensitive for the surveillance of exposure.