JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY, APRIL 1993, VOL. 8 419

Determination of Lead and Cadmium in Human Seminal Fluid by Electrothermal Atomic Absorption Spectrometry*

Jasna Jurasovic and Spomenka Te l ihan Clinical Toxicology Laboratoryf Institute for Medical Research and Occupational Health, University of Zagreb, 2 Ksaverska, 4 1000 Zagreb, Croatia

A sensitive method is described for the determination of Pb and Cd in human seminal fluid by electrothermal atomic absorption spectrometry with Zeeman-effect background correction. The method includes deproteiniz- ation of seminal fhid with nitric acid, calibration using matrix-matched standards containing a mixture of Pb and Cd and integrated absorbance measurement using a L’vov platform in a non-grooved pyrolytic graphite coated graphite tube. The detection limits (317) are 1.4 pg I - ’ of Pb and 0.05 pg I- of Cd in seminal fluid. The day-to-day precisions (relative standard deviations) of duplicate determinations in 121 samples are in the range 4-1 4% at 4-48 pg I-’ of Pb and 4-1 6% at 0.2-3.6 pg I-’ of Cd in seminal fluid. The recoveries (mean k standard deviation) are 100.6 c 3.6% of Pb and 98.8 k 4.2% of Cd and the characteristic mass values are 1 1.6 pg of Pb and 0.41 pg of Cd, indicating the absence of interferences. Data are presented on the levels of Pb in seminal fluid in normal subjects and those working with Pb and of Cd in seminal fluid in non-smokers and smokers. Corresponding ‘normal values’ (median and range) of Pb and Cd in seminal fluid are 8.6 (4.2-1 6.6) pg I-’ and 0.54 (0.1 7-1.67) pg I-I, respectively. Keywords: Lead and cadmium determination; human seminal fluid; nitric acid deproteinization; electrothermal atomic absorption spectrometry; male reproduction capacity

There is increasing interest in the possible adverse effects of environmental factors on reproduction capacity in men, which is mainly due to a decrease in sperm density noted in the general population over the past three decades, and the possibility that the human male might be more vulnerable to toxic influences than other mammals. It has been noted, however, that the human sperm output is about four times less than that of other mammals ( ie . , the number of sperm cells produced per gram of testes) and that the human ejaculate is unique when considering the relatively high number of abnormal sperm cells that are regarded as being ‘normally’ present in a fertile male.’

Lead and Cd are inevitably present in the human environment and both are known to be toxic agents which accumulate in the human body over a lifetime (including pre-natal life, particularly with Pb). Lead is mostly accumu- lated in the bones, whereas Cd is mostly accumulated in the renal cortex. The biological half-lives of Pb and Cd in the human body ( i e . , the time for clearance of half the body burden of the metal) are estimated to be ‘a number of years’ and ‘several years’, respectively,2 whereas data regarding various target organs are particularly scarce and ineonclu- sive. Although the storage of Pb in bones (up to 95O/o of the body burden of Pb) has long been considered to be a sequestration or removal of Pb from active sites in soft tissues, recent data have shown that Pb can be released from bones under certain stresses and physiological changes3 Apart from numerous sources of occupational exposure to each of the metals, the most important non- occupational sources are food, water (Pb mostly from Pb pipes in contact with soft and acidic water), air (especially Pb from petrol in dense traffic areas), smoking habits (Cd and to a lesser extent Pb from tobaccoj and alcohol consumption (Pb-contaminated alcoholic beverages). There is also some evidence of the possible effect of Pb and alcohol interaction in man, i.e., an ethanol-induced increase in the biologically active fraction of Pb accumulated in the organi~rn.~

Although experimental data for animals have indicated that both Pb and Cd can reduce male reproduction

*Presented at the XXVII Colloquium Spectroscopicum Interna- tionale (CSI), Bergen, Norway, June 9- 14, I99 1..

capacity, veiy few data are available regarding the possible reproductive effects of Pb and/or Cd in Lead and Cd have been found both in the seminal fluid and spermatozoa of patients suspected of infertility.7 A significantly higher concentration of Cd in whole semen was found in infertile subjects than in feitile subjects, whereas no significant difference in whole semen Pb concentration was observed.$ It should be mentioned that in the latter two studies7,* no information was presented regarding possible occupational exposure to Pb or Cd in the population studied. On the other hand, a significantly higher concentration of Pb in seminal fluid was found in the infertile subjects than in fertile subjects with no occupational exposure to Pb.9 In subjects with no occupational exposure to Cd a trend of increasing concentration of Cd in seminal fluid was found with respect to smoking habits, ie., cigarettes per day, although no significant correlation with the parameters of semen quality was observed. lo However, relevant human data are generally scarce and inconclusive and there is obviously a lack of published analytical methods regarding the concentrations of Pb and Cd in the human ejaculate.

A recent study of men has indicated that exposure to Pb, and to a lesser extent exposure to Cd through smoking habits, can reduce semen quality’ and can somewhat affect male reproductive endocrine function.12 During the same study a method was developed for the determination of Pb and Cd in seminal fluid, as such data are essential for establishing the location and mechanism(s) of the effects of Pb and/or Cd on reproduction capacity in men.

Experimental Instrumentation A Perkin-Elmer Zeeman/S 100 atomic absorption spectro- meter, equipped with an HCA-600 graphite furnace, an AS- 60 autosampler and a PR-310 printer, was used. The instrument was controlled and signals were processed by a Perkin-Elmer 7700 professional computer. Argon was used as a purge gas.

Solid pyrolytic graphite L’vov platforms (Perkin-Elmer, Part No. BO-1 09324) in grooved pyrolytic graphite coated graphite tubes (Perkin-Elmer, Part No. BO-109322) were used initially and were found to produce variable atomic

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420 JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY, APRIL 1993, VOL. 8

signals and analytical sensitivity. As considerably smaller variations in atomic signals were observed when using L'vov platforms in non-grooved pyrolytic graphite coated graphite tubes (Perkin-Elmer, Part No. BO-09 1 504), these were subsequently used throughout the study.

An Eppendorf 54 1 5-C centrifuge and Eppendorf 38 10 microtubes (1.5 ml volume) were also used.

Reagents De-ionized water, purified to approximately 18 MR cm-l, was used for the preparation of standards and for final washing of the laboratory ware. Working standard solu- tions, containing a mixture of Pb and Cd in 0.02 rnol 1-' HNO,, were prepared from the BDH Spectrosol solutions containing 1 g 1-1 of Pb and Cd, respectively, in nitrate form. Concentrated HN03 (BDH, Aristar grade) was used to prepare 0.02 rnol 1-1 HN03 and 1 mol I-* FINO3, which was used for dilution and deproteinization of seminal fluid.

The autosampler washing solution contained 0.2% v/v concentrated nitric acid (BDH, Aristar grade) and 0.1% v/v Triton X- 100 (BDH).

Precautions Against Contamination All the laboratory ware used (glass and plastic) was cleaned by soaking in 10°/o m/v HN03 for 24 h, rinsed with de- ionized water, soaked in 3% m/v Na2EDTA.2H20 solution for 24 h and again rinsed with de-ionized water. Such a washing procedure is essential also for the containers used for sampling of ejaculate and those for storage of seminal fluid. Because the concentrations of Pb and Cd in seminal fluid are generally very low and the sample volume is small, the impact of possible contamination of a sample is relatively great.

Sampling and Storage of Seminal Fluid The ejaculate was collected in a metal-free glass container by masturbation after 4 days of sexual abstinence. Follow- ing the semen liquefaction (approximately 15 min), sperma- tozoa were separated from seminal fluid by centrifugation at 3000 rev min-l for 10 min. Approximately 300 pl of the supernatant were transferred into an Eppendorf poly- propylene microtube and stored at -20 "C until required for analysis. Under the aforementioned storage conditions the concentrations of Pb and Cd in seminal fluid were found to be stable for more than 18 months.

A large pool (approximately 100 ml) of the seminal fluid samples collected from non-smokers, not occupationally exposed to either Pb or Cd, was applied during the experiments on optimization of the method and for a matrix-matched calibration throughout the study. The seminal fluid pool was stored in a metal-free polypropylene bottle at -20 "C and the concentrations of Pb and Cd were found to be stable for a period of 2 years (all precautions were taken to prevent contamination, e.g., aliquots were taken monthly by pouring instead of pipetting from the pool).

The human seminal fluid samples analysed for Pb and Cd in the present study were obtained from 121 healthy volunteers (aged 20-43 years): 35 subjects with no occupa- tional exposure to either Pb or Cd and 86 subjects with long-term slight to moderate occupational exposure to inorganic Pb (printing works, a factory for Pb products, a ceramics factory, a factory for Pb-based paints and a storage batteries factory). Out of the 121 subjects there were 79 cigarette smokers and 42 non-smokers (including nine former smokers).

Analytical Procedure In an Eppendorf microtube, 100 pl of a well mixed seminal fluid were added to 700 pl of 1 HN03, followed by the addition of 50 pl of 0.02 mol 1-' H N 0 3 (the 'zero' Pb and Cd working standard solution). The microtube was capped and vortex mixed, allowed to stand for 30 min, centrifuged at 14000 rev min-l for 15 min and the clear supernatant was carefully poured into a polystyrene auto- sampler cup for subsequent analyses of Pb and Cd by electrothermal atomic absorption spectrometry (ETAAS). Each sample was prepared and analysed in duplicate. (All the volumes indicated above can be halved, if necessary, i.e., the minimum sample volume required for determina- tion of Pb and Cd in seminal fluid by the present method is

The matrix-matched samples for calibration were simul- taneously prepared in the same way as the unknown sample, by using 100 pl of a pooled seminal fluid and 50 pul of a working standard solution containing a mixture of Pb and Cd in 0.02 mol 1-1 HN03. The working standard solutions (prepared from an intermediate standard contain- ing 100 mg 1-' of Pb and 2 mg 11' of Cd in 0.02 mol 1-' HN03) had concentration ranges of 25-500 pg 1-1 of Pb and 0.5-10 pg 1 - I of Cd and were found to be stable at room temperature for 6 months. (Each working standard solution was always prepared and stored in the same glass bottle which was previously only rinsed with de-ionized water.) The working standard additions correspond to concentra- tions of Pb and Cd in seminal fluid of 12.5-250 and 0.25-5 pg l-l, respectively, whereas 0.02 moll-' HNO, (the 'zero' working standard) corresponds to 0 pg 1-' of Pb and Cd.

A 'blank' sample was prepared in the same way as the unknown sample, except that 100 pul of de-ionized water instead of seminal fluid were used and centrifugation was omitted. Blanks were prepared and analysed in triplicate.

Experience has shown that all the aforementioned samples are stable for more than 48 h when stored in an autosampler cup sealed with Parafilm at 4 "C, so it is convenient that the ETAAS analyses be performed on one day for Pb and on another day for Cd.

The thermal conditioning of the graphite furnace was performed daily by running the optimum temperature programme (Table 2) without any sample injection ten times, after which a sample for calibration with the highest standard addition was injected and the drying temperature and efficacy of thermal conditioning were checked. The precision [relative standard deviation (RSD)] of six succes- sive replicate measurements of integrated absorbance was usually 4 1 Yo for Pb and 4 1.5% for Cd, ie., an RSD of <2%0 was regarded as being acceptable.

'Three replicate atomizations were run for each sample. The recalibration controls, using a sample for calibration with the highest standard addition, were performed after every 12 samples. Experience has shown that the same pyrolytic graphite coated graphite tube and L'vov platform can be used for approximately 400 firings without any notable change in sensitivity and precision.

50 pl.)

Results and Discussion Optimization of Procedure Initial experiments on the optimization of the method were performed by using a L'vov platform in a grooved graphite tube. Considerable variations were observed in the time of appearance of atomic absorbance signals (Fig. 1 ), analytical sensitivity and the characteristic mass when using combina- tions of different L'vov platforms and grooved graphite tubes, under otherwise identical experimental conditions. This can be attributed to the variable contact between the E'vov platform and the grooves, producing various extents

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JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY, APRIL 1993, VOL. 8 42 1

of electrical and conductive heating of the L'vov platform, in addition to radiative heating of the analysed sample. Smaller variations were observed when using a L'vov platform in a non-grooved graphite tube (Fig. 2), which is in agreement with the results of other worker^.*^-^^ The latter combination was therefore applied throughout the study and the observed variations in the characteristic masses of Pb and Cd during a period of 2 years were < 12%, which is regarded as satisfactory. However, a tendency towards a gradual decrease in sensitivity (i.e., a slight decrease in the slopes of the calibration lines for Pb and Cd) was observed in the same period.

Nitric acid solution was used for the dilution and deproteinization of seminal fluid to reduce the non-atomic background signals during measurements of Pb and Cd. Nitric acid also acts as a chemical modifier by reducing halide interferences. A concentration of 1 mol 1 - I HN03 was found to be optimum, i.e., dilution of 100 pl of seminal fluid in 700 pl of 1 mol 1-1 HN03 plus 50 pl of 0.02 moll-' HN03 ('zero' Pb and Cd working standard, or those containing a mixture of Pb and Cd), as it produced the lowest background absorbance signals (Fig. 3) coupled with relatively the best repeatability of the specific absorbance signals for Pb and Cd. The chosen concentration of

0.4 k I

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Fig. 1 Variation in Pb atomic signals of deproteinized seminal fluid spiked with Pb (final concentration of 263 pg 1 - I of Pb in seminal fluid) obtained by using two different combinations of L'vov platforms and grooved tubes

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Fig. 2 Variation in Cd atomic signals of deproteinized seminal fluid spiked with Cd (final concentration of 5.6 pg I - I of Cd in seminal fluid) obtained by using (a) four repeated insertions of the same L'vov platform into the same non-grooved tube, and (b) four different combinations of L'vov platforms and non-grooved tubes

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Fig. 3 Effect of nitric acid concentration on the non-atomic background signals obtained during measurement of (a) Pb and (b) Cd in seminal fluid. The concentrations shown refer to the HN03 solution used for dilution and deproteinization of seminal fluid (e.g., 1 mol I - I H N 0 3 is equivalent to a final concentration of 0.825 rnol I- ' HN03 in the analysed sample)

1 mol 1 - I HN03 is equivalent to a final concentration of 0.825 mol 1-1 HN03 in the samples analysed for Pb and Cd. Similar findings on the optimum HNO, concentration for deproteinization were reported by other workers for the determination of Cd in blood plasma16 and urine.I7

Table 1 gives the optimum instrumental conditions and Table 2 the optimum temperature programmes for the determination of Pb and Cd in deproteinized seminal fluid. Temperature programmes were optimized using a sample for calibration with the highest standard addition (equiva- lent to concentrations of 250 p g 1 - I of Pb and 5 pg 1-' of Cd in seminal fluid). Fig. 4 shows the effect of the pyrolysis temperature on the specific absorbance signals for Pb and Cd fat the chosen atomization temperature of 1600 "C for Pb and 1400 "C for Cd) and Fig. 5 shows the effect of the atomization temperature on the specific absorbance signals for Pb and Cd (at the chosen pyrolysis temperature of 600 "C for Pb and 400 "C for Cd). Although the chosen atomization temperatures are higher than the optimum regarding the size of the Pb and Cd signals when measured

Table 1 Instrumental conditions for the determination of Pb and Cd in seminal fluid using a Perkin-Elmer Zeeman/5100 spec- trometer

Lead Cadm i u rn Hollow cathode lamp current/mA Wavelengthhm Spectral bandwidthlnm Peak evaluation

Baseline offset correction timels Read delay time/s Integration time/s Injection volumelpl

8 4 283.3 228.3

0.7 0.7 Integrated Integrated

absorbance absorbance 1 1 0.9 0.9 3.0 3.5

20 20

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-0 Q, 0.20

2 4-

0.16

0.12

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Table 2 Furnace temperature programmes for the determination of Pb and Cd in seminal fluid. using a HGA-600 graphite furnace, with L'vov platform in non-grooved pyrolytic graphite coated graphite tube

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- 0.16

- - 0.12

- - 0.08

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- - 0.04

Lead Cadmium ~ I -

Temperature/ Ramp Hold Argon flow rate/ Temperature/ Ramp Hold Argon flow rate/ Step "C time/§ time/s mI min-I "C time/s time/s ml min-I

Dry 160* I 45 300 160* 1 45 300 Pyrolyse 600 10 20 300 400 10 25 300 Cool-down 20 1 20 300 20 1 20 300 Atomize 1600 0 5 0 1400 0 5 0 Clean 2500 1 3 300 2500 1 3 300 Cool 20 1 4 300 20 1 4 300

'Varied between 150 and 180 'C on different days.

as integrated absorbance, the resulting profiles of the Pb and Cd peaks are considerably better (as indicated by the peak height absorbance data). The optimum temperature programmes for Pb and Cd include a 'cool-down' step prior to atomization because this ensures faster heating of the graphite tube and more constant conditions in the tube,I8 resulting in better repeatability of the Pb and Cd signals.

The influence of non-spectral interferences on the deter- mination of Pb and Cd in seminal fluid was examined by comparing the calibration graphs for standards prepared In a different matrix, obtained by integrated absorbance measurements under identical instrumental conditions. 'Table 3 shows the relevant statistical data for the calibra- tion curves for Pb and Cd when using the standards prepared in: A, water; B, 0.825 mol 1 - I HNO,; and C, deproteinized seminal fluid containing 0.825 mol 1-' HNO,, i.e., matrix-matched standards. The standard addi- tions were equivalent to concentrations of Pb in seminal fluid of 0, 25, 50, 75 , 100, 125 and 250 pg 1-1 and concentrations of Cd of 0, 0.5, 1.0, 1.5, 2.0, 2.5 and 5.0 pg 1-l. The original concentrations of Pb and Cd in the pooled seminal fluid used for calibration were 13 and 0.6 pg l-l, respectively. All the calibration graphs were found to be linear. Although differences between the slopes A, B and C

0.5

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200 300 400 500 600 Tern perat u re/"C

Fig. 4 Effect of pyrolysis temperature on the atomic signals of (a) Pb and (b) Cd of deproteinized seminal fluid in 0.825 an01 1-1 HN03 spiked with a mixture of Pb and Cd (final concentrations of 263 pg 1-1 of Pb and 5.6 pg 1 - l of Cd in seminal fluid)

were small and were not significant between B and C for either Pb (PXl .05 ) or Cd (P>O.$O), the times of appear- ance of the peaks and the peak profiles were different (Fig. 6) and the matrix-matched calibration, C, is regarded as being optimum.

By using a pool of human seminal fluid as a matrix for calibration solutions, it was hoped that a time-consuming method of standard additions could be avoided (as proved to be so in our 10 years of experience in applying essentially the same method for determinations of blood Pb and Cd). Table 4 shows the slopes of the calibration graphs for Pb and Cd obtained by using ten different seminal fluid samples, each measured on a different day. The observed variations in the slopes (RSD) of 3.4% for Pb and 3.9% for Cd are even smaller than the day-to-day variations in the slopes obtained by using the pooled seminal fluid, viz., 5.1% for Pb and 5.8% for Cd (Table 4), indicating no need for a method of standard additions. However, a matrix-matched calibration was chosen and the values for Pb and Cd in the pooled seminal fluid were obtained each day and used for internal quality control. The results obtained on 32 differ- ent days (meantSD) were 12.6kO.63 pg 1-' for Pb and 0.64+0.035 pg 1-l for Cd in the pooled seminal fluid, corresponding to RSDs of4.9% for Pb and 5.4% for Cd. The main advantage of a matrix-matched calibration over the

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Fig. 5 Effect of atomization temperature OA the atomic signals of (a) Pb and (b) Cd of deproteinized seminal fluid in 0.825 mol I - l

HN03 spiked with a mixture of Pb and Cd (final concentrations of 263 pg I -* of Pb and 5.6 pg I - ' of Cd in seminal fluid)

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JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY, APRIL 1993, VOL. 8 423

Table 3 Statistical parameters of the calibration graphs for Pb and Cd obtained by using standards prepared in: A, water; B, 0.825 mol I - ' HNQ,; and C, deproteinized seminal fluid in 0.825 moll-' HNO, (matrixmatched standards) under identical instrumental conditions (no read delay, integration time 5 s)

Parameter A B c Lead-

Correlation coefficient 0.9988 0.9989 0.9995 Slope/s 1 pg-l 8.2 105 x 1 0-4 8.6247 x 8.8613 x SEslo& 1 K3-l 1.2922 x 1 . 0 1 6 8 ~ 7.7121 x tSl0pe 63.5 84.8 114.9 Significance of the t=2.5 19, P<0.05 t = 1.854, 0.1 O>P>O.O5

difference between slopes ( t , f) t=4.325, Pt0.01

Cadmium-- Correlation coefficient 0.9955 0.9963 0.9963 Slopels 1 pg-l 1 . 9 1 2 8 ~ 2.0295 x 2 . 0 5 4 4 ~ SEs,opds 1 Pug-' 5 . 7 2 6 0 ~ 5.4965 x 4 . 0 7 8 0 ~ ts1ope 33.4 36.9 50.4 Significance of the t= 1.470. P>O.lO t=0.364, P>0.80

difference bet ween slopes (t, P) t=2.014, O.lO>P>0.05

Table 4 Comparison of the slopes of the calibration graphs for Pb and Cd in seminal fluid obtained by using: A, ten different seminal fluids, each measured on a different day; and €3, the pooled seminal fluid (other than A) measured on 32 days

Slope/s 1 pg-l

Sample No. 1 2 3 4 5 6 7 8 9

10

Lead 9.678 x

8.879 x 8.605 x 9.052 x 9.245 x 1 0 - 4 9.176 x 8 . 7 1 4 ~ 1 0 - 4 8.828 x 1 0 - 4 8,931 x

8.963 x 1 0 - 4

A, n=10 Mean = 9.007 x 1 O-*

RSD = 3.4% (mo= 11.5 pg Pb)*

SD=3.059 x

B, n = 3 2 Mean=8.958 x SD=4.592 x

RSD=5.1% (mo= 11.6 pg Pb)*

A - B: significance of the difference in

slopes t=0.317, f 5 0 . 7 0

Cadmium 2.590 x 2.632 x 2.563 x 2.601 x 2.593 x 2.587 x 2 . 6 1 0 ~ 2.298 x 2.627 x 2.488 x

Mean=2.559 x SD= 1.002 x 10-3

RSD= 3.9% (m,=0.40 pg Cd)*

Mean=2.510x SD= 1.455 x 10-3

RSD=5.8% (m,-0.41 pg Cd)*

t=0.981, P>0.30

*Mean value of the characteristic mass (m,) of Pb and Cd corresponding to the indicated mean value of the slope.

method of standard additions is the considerably smaller volume of seminal fluid required for analyses for Pb and Cd. This is particularly important in pathological cases with reduced ejaculate volume, taking into account the fact that several characteristic parameters of semen quality (e.g., the lactate dehydrogenase isoenzymes LDH-C4 fraction, fruc- tose, zinc, acid phosphatase and citric acid) are usually also measured in the same seminal fluid specimen.

Fig. 7 shows the matrix-matched calibration graphs for Pb and Cd in seminal fluid A when using a standard procedure, i e . , an injection volume of 20 pl, and B and C when using a multiple injection (pre-concentration) proce- dure, i.e., an injection volume of B 2 x 20 pl or C 3 x 20 pl. In B and C the autosampler was programmed so as to inject an additional 20 pl of the sample following the termination of the pyrolysis step of the previous aliquot(s). The optimum temperature programme (Table 2) was then continued as usual. In this way it is possible to inject a sample three times (C) without any notable change in precision (RSD), while at the same time the detection limit of the method (expressed as the concentrations of Pb and Cd in seminal fluid) is considerably lowered. However, the

results (Fig. 7) show that the calibration graphs are linear up to seminal fluid concentrations of approximately 263 pg 1 - I

of Pb and 5.6 pg 1-1 of Cd using a standard procedure (A), 138 pg 1-1 of Pb and 3.6 pg 1-1 of Cd when using a double injection volume (B) and I 13 pg 1-1 of Pb and 2.6 pg 1-1 of Cd when using a triple injection volume (C), taking into account that the original concentrations of Pb and Cd in the pooled seminal fluid used for calibration were 13 and 0.6 pg l-l, respectively. The multiple injection procedure (€3, and particularly C) is regarded as being convenient because very low concentrations of Pb and Cd in seminal fluid can be more precisely determined by applying the same samples for calibration and the same temperature programme, whereas any additional step in the method could be a source of error.

Analytical Parameters The detection limits of the method, calculated as three times the standard deviation of ten measurements of a 'blank' sample, are equivalent to concentrations in seminal fluid of 3.7 pg 1-1 of Pb and 0.16 pg I-' of Cd when using the

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0.4 1

0 Time/s

Fig. 6 Variation in the atomic peak profiles of (a) Pb and (6) Cd obtained by using standards prepared in: A, water; B, 0.825 moll-' HNO,; and C, deproteinized seminal fluid in 0.825 mol I-' HNO, (matrix-matched standard) under identical experimental condi- tions, as shown in Tables 1 and 2

9 Addition to the concentration of Pb in seminal fluid/pg I-'

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Fig. 7 Matrix-matched calibration graphs for (a) Pb and (b: Cd in seminal fluid, obtained by using: A, single injection (20 pl); B, double injection (2 x 20 pl); and C, triple injection (3 x 20 pl) of deproteinized seminal fluid in 0.825 mol 1 - I HN03 spiked with a mixture of Pb and Cd. (The original concentrations of Pb and Cd in seminal fluid are 13 and 0.6 pg l - ' , respectively)

standard procedure (k, an injection volume of 20 pl). The corresponding absolute detection limits are 8.8 pg of Pb and 0.38 pg of Cd. However, when using an injection volume of

2 x 20 pl or 3 x 20 pl, the detection limits of the method are equivalent to concentrations in seminal fluid of 2.0 pg 1-1 of Pb and 0.09 pg 1-1 of Cd or 1.4 pg 1- I of Pb and 0.05 pg 1-1 of Cd, respectively.

The precision (RSD) of the method, calculated on the basis of seven replicate analyses of seminal fluid containing 26 pg 1-1 of Pb and 1.3 pg 1-1 of Cd, was 5% for Pb and 6% for Cd when measured on the same day (within-day precision) and 7% for Pb and 10% for Cd when measured on different days (day-to-day precision). The day-to-day precision of the method was also calculated on the basis of duplicate analyses of 12 1 samples of seminal fluid covering the concentration ranges 4-48 pg 1-1 of Pb and 0.2-3.6 pg 1-1 of Cd, and the RSDs were in the ranges 14-4% for Pb and 16-4% for Cd (i.e., at lower Pb and Cd concentrations in seminal fluid, relatively higher RSDs were obtained).

The recovery of the method was calculated on the basis of measurements carried out in 18 different seminal fluid samples. The samples originally contained 4.4-1 1.8 pg 1 - I

of Pb and 0.21-1.93 pg 1- I of Cd and were spiked with 25, 50, 75 or 100 pg 1-I of Pb and 0.5, 1.0, 1.5 or 2.0 pg 1-1 of Cd. Each measurement was performed in duplicate and the recoveries obtained (mean k SD) were 100.6 -t 3.6% (range 89-108%) for Pb and 98.8+4.2% (range 91-1 13%) for Cd in seminal fluid.

The average characteristic masses of Pb and Cd obtained during a period of 2 years were 1 1.6 pg of Pb and 0.4 1 pg of Cd, which are regarded as being in very good agreement with the results of other workers, i.e., 12 pg of Pb and 0.35 pg of Cd19 or 10.9 pg of Pb and 0.42 pg of Cd.20

Lead in Seminal Fluid of Lead Workers and Control Subjects The concentration of Pb in seminal fluid was determined in 12 1 adult male volunteers: 86 subjects occupationally exposed to Pb and 35 subjects with no occupational exposure to Pb. The results obtained, expressed as median and range because of a skewed distribution, were 15.3 (6.5-48.3) pg 1 - I for workers exposed to Pb and 8.6 (4.2-1 6.6) pg 1-1 in control subjects. The difference between the groups was highly significant according to the Mann-Whitney test (z= - 5.865, PC Our results for lead workers are considerably lower than the reported average of 148 pg 1 - I in ten Pb workers.Z1 However, in that study no mention was made of either the method used or any data on control subjects. on the other hand, our results for control subjects are similar to the reported range of 2-23 pg 1-1 in 45 adult subjects with no specified occupational exposure to Pb,' but are higher than those of Scandinavian worker^,^ viz., a reported mean 4 SD of 3.6 k 3.1 pg 1-1 in 87 adult subjects with no occupational exposure to Pb. However, as indicated by the blood Pb levels, it appears that the general population in Scandinavia is less exposed to Pb than the population in Croatia,22 which might explain the aforementioned difference in concentrations of Pb in seminal fluid.

Cadmium in Seminal Fluid of Cigarette Smokers and Non- smokers The concentration of Cd in seminal fluid was determined in the same 121 adult male volunteers who had no occupa- tional exposure to Cd: 79 cigarette smokers and 42 non- smokers. The results obtained, expressed as median and range because of a skewed distribution, were 0.86 (0.29-3.56) pg 1-I in smokers and 0.54 (0.17-1.67) pg 1-1 in non-smokers. The difference between the groups was highly significant according to the Mann-Whitney test (z= -4.683, Pc lo+). The results for both smokers and non-smokers reported in this paper are lower than the reported range of 0.15-10.00 pg 1-1 in 45 adult subjects

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JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY, APRIL 1993, VOL. 8 425

with no specified smoking habits or occupational exposure to Cd,7 but are higher than those of Scandinavian workers,'O viz., a reported meankSD of 0.28t-0.24 pug 1-1 in 27 smokers and 0.19-tO.21 pg 1 - l in 31 non-smokers. How- ever, as indicated by blood Cd levels, it appears that both smokers and non-smokers in Scandinavia are less exposed to Cd than those in Croatia,22v23 which might explain the aforementioned difference in concentrations of Cd in seminal fluid.

Conclusions The described method for the determination of Pb and Cd in seminal fluid appears to be sufficiently sensitive and reproducible for research purposes with regard to the reproductive effects of exposure to Pb and/or Cd in men. As no reference materials with certified Pb and Cd levels in seminal fluid are available, the accuracy of the method could not be adequately evaluated. However, the recoveries of Pb and Cd obtained and the characteristic mass values of 11.6 pg of Pb and 0.41 pg of Cd, which are in agreement with published value^,^^.^^ indicate the absence of interfer- ences in the method.

This study was financially supported by the International Lead Zinc Research Organization (grant LH-378/ILZRO).

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7 Pleban, P. A., and Mei, D. S., Clin. C'hirn. Acta, 1983, 133, 43. 8 Umeyama, T., Ishikawa, H., Takeshima, H., Yoshii, S., and

Koiso, K., Fertil. Steril., 1986, 46, 494. 9 Saaranen, M., Suistomaa, U., Kantola, M., Saarikoski, S., and

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T., Andrologia, 1989, 21, 140. 1 1 TeliSman, S., CvitkoviC, P., Gavella, M.. and PongraEiC, J.,

Abstracts of the International Symposium on Lead and Cad- mium Toxicology, Shenyang Association for Science and Technology, Shenyang, 1990, p. 29.

12 TeliSman, S., CvitkoviC, P., RoEiC, B., PrpiC-MajiC, D., and Pizent, A., in Heavy Metals in the Environment, ed. Farmer, J. G., CEP Consultants, Edinburgh, 1991, vol. 2, p. 13.

13 Shuttler, I. L., and Delves, €3. T., J. Anal. A t . Spectrum., 1987, 2, 171.

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I5 Shuttler, I. L., Delves, H. T., and Hutsch, B., J . Anal. At. Spectrum., 1989, 4, 137.

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17 Halls, D. J., Black, M. M., Fell, G. S., and Ottaway, J. M., J . Anal. At. Spectrum., 1987, 2, 305.

18 Falk, H., Glismann, A., Bergann, L., Minkwitz, G., Schubert, M., and Skole, J., Spectrochirn. Acta, Part B, 1985, 40, 533.

19 Slavin, W., Sci. Total Environ., 1988, 71, 17. 20 L'vov, B. V., Spectrochirn. Acta, Part B, 1990, 45, 633. 21 Chowdhury, A.. R., Chinoy, N. J., Gautam, A. K., Rao, R. V.,

Parikh, D. J., Shah, G. M., Highland, H. N., Patel, K. G., and Chatterjee, B. B., Advance.7 in Contraceptive Delivery Systems, 1986, 2, 208.

22 Assessment of Human Exposure to Lead and Cadmium Through Biological Monitoring, ed. Vahter, M., National Swedish Institute of Environmental Medicine and Department of Environmental Hygiene, Karolinska Institute, Stockholm, 1982.

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Paper 2/03 1291 Received June 15, I992

Accepted November 10, 1992

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