1 Evolution of analytical instrumentation: The PerkinElmer Story SDI Global 7th Edition 2002-AAS SDI Global 8th Edition 2004-AAS 2 - HAc-MIBK GFAAS ST

Transcription

1 PerkinElmer 45 AAnalyst AAnalyst AAnalyst AAnalyst AAnalyst HGA /800

2 1 Evolution of analytical instrumentation: The PerkinElmer Story SDI Global 7th Edition 2002-AAS SDI Global 8th Edition 2004-AAS 2 - HAc-MIBK GFAAS STPF NBS Pb

3 3 PbCdCr Pb 0.1NHNO_3 -NaI 4 Characterizing the Metal Adsorption Capability of a Class F Coal Fly Ash EDTA EAAS 5 Lvov AAS FCC

4 MIBK- NaCa 6 CuMnZnFeSe - Cu 2+ Ultratrace Aluminum Analysis of Nutritional Intravenous Solution Components in 1- Propanol Using GFAAS Occupational Exposure to Lead and Induction of Genetic Damage PE AA800 Zeeman

5 - SIMMA GFAAS Ag GFAAS Pb - High-Temperature, Microwave-Assisted UV Digestion: A Promising Sample Preparation Technique for Trace Element Analysis - Speciation analysis. The future of atomic absorption spectrometry Natural Organic Matter Affects Arsenic Speciation and Sorption onto Hematite On-line separation for the speciation of mercury in natural waters by FI-AAS - AAnalyst AAnalyst AAnalyst AAnalyst AAnalyst HGA /800

6 !!! ƒ!!!! ƒ ƒ 1!!!!!!!!!!! ƒ!!!! ƒ!!!! ƒ!!!!!! ƒ! 1 11 Π Π ! ! ε Π!!! ƒ

7 1! ν ƒ Π Τ Ω Β Π Β ΠΩ Α Π Α Τ Ω Χ Π Χ ΠΩ Α Π Α ))) ƒ Τ Π Τ Π ΤΠ ! ƒ!!!!!! ƒ ƒ 1!!! ƒ!!!!!!!! 1 Χ

8 Στυδψ ον Εξτραχτινγ Ρ ατεσ ανδ ετερ µινατιον οφ Χυ Μν Ζν Φε Σε ιν Ηυανγ θι ανδ Ιτσ Ωατερ εχοχτεδ Σολυτιον ±2 Αβστραχτ ƒ ƒ Κεψ ωορδσ!!!

9 !!!!!! 1!!!!!!!! 1!!!! 1 Π 1 Π 1 Π Π Π!!! ƒ

10 1! 1 1 1!!!! Π!!! ƒ Χ Π Α Π ρ Χ 11 Α Χ 1 Α Χ 1 Α Χ 1 Α Χ 1 Α ξ? Π ν 1? 1 1? 1 1? 1 1? 1 1? 1 1? 1 1? 1 1? 1 1? 1 1? 1 1? 1 1? 1 1? 1 1? 1 1? 1 1? 1 1? 1 1? 1 1? 1 1? 1 ξ? Π ν 1? 1 1? 1 ) 1? 1 1? 1 1? 1 1? 1 1? 1 1? 1 1? 1 1? 1 1? 1 ) 1? 1 1? 1 1!!!!

11 Π 1 1!!!!! ετερµινατιον οφ Ασ Πβ Χδ Χρ ανδ Ηγ ιν αχηενγθι Γρανυλεσ βψ ΑΑΣ 2 2 Αβστραχτ 2 2 Κεψ ωορδσ

12 !!!!!! 1 1!!!!! 1!!!! 1 Π Π!!! ƒ Π Π Π 1 1 1

13 ! 1 1 1!!!! Π!!! ƒ Χ Π Α ρ Χ 1 Α Χ 1 Α Χ 1 Α Χ 1 Α Χ 1 Α ξ? Π Λ ν 3 1? 1 1? 1 1? 1 1? 1 1? 1 1? 1 1? 1 1? 1 1? 1 1? 1 1? 1 1? 1 1? 1 1? 1 1? 1 3 Π 1!! 1 1!! 1 1! 1!! 1 1 1!

14 ετερµινατιον οφ Ασ Πβ Χδ Χρ ανδ Ηγ ιν Ψισηουηυιχηυν οραλ λιθυιδ βψ ΑΑΣ Αβστραχτ Κεψ ωορδσ ))) 5 6!! 5 6!!! 1 Π 5 6

15 Χηινεσε ϑουρναλ οφ Σ π εχτροσχοπψλαβορατορψ 2 ) 22 2 ε ε Λ 222!!!! )!!!! 2 ε! Λ 2 2! Α Λ

16 ! ςς ςς! ε 2!! 2 ) Β! ε ε ε ε ε 2 ε 2 2 Λ 2 Β Β Λ Λ Λ

17 2 1 ) ) 2 ) ) 3 ) ) 2 ƒ! Λ Χυ Ζν ψ ξ ψ ξ ψ ξ ψ ξ! Λ! ƒ ΛΛ 14 8) 11) 38

18 ) ƒ ƒ Ταλαντα51) Ταλαντα42) 2 2 ƒ 2 ƒ Αναλψτιχα Χηιµ ιχα Α χτα308) ) 15) ετερµ ιν ατιον οφ Χυ αν δ Ζ ν ιν αν σηεν Χηιν εσε Η ερβ ω ιτη Μ ατριξ Μ οδ ιφιερσ βψ Γ ΦΑΑΣ 2 2ƒ 2 Κ εψ Λαβορατορψ οφ Α ναλψτιχαλσχιενχε οφ Η εβειχολλεγ ε οφ Χηεµ ιστρψ ανδ Ε νϖιρονµ ενταλσχιενχε ΗεβειΥνιϖερσιτψΒαοδινγΗ εβειπρ Χηινα Αβστραχτ ƒ ε ε 2 ) Κεψωορδσ ƒ

19 . ε ƒ Ρ Λ!!!!!!!!!! 2 ε!!!!

20 Λ Ταβ. Χονδιτιονσ οφ Γ ΦΑΑΣ Ταβ. Προγραµ οφ γραπηιτε φυρναχε ε!!!! ε Λ Λ Λ Λ Β Β Λ Λ Ρ Λ ƒ Ταβ. Ρεχοϖερψ οφ µ ανγανεσε ιν σαµ πλεσ Θ Λ Λ Β

21 Λ ψ ξ(ρ Λ! Ταβ. Χοντεντσ οφ µ ανγανεσε ιν σιξ κινδσ οφ Χηινεσε ηερβ µ εδιχινε Θ Λ!!!! ετερµινατιον οφ Τραχε Μανγανεσε ιν Χηινεσε Ηερβ µ εδιχινε βψ Ζεεµ αν Γραπηιτε Φυρναχε Ατο µιχ Αβσορπτιον Σπεχτρο µ ετριχ Αβστραχτ : ε Ρ Λ 2 Κεψ ωορδσ: ƒ

22 !!!!!!!!!!! Λ ƒ !!!!!!

23 Αναλψσισ οφ Ελε µ εντσ ιν Μυλτι Εφφεχτιϖε Σκιν Ωαση Λιθυιδ ± ± Αβστραχτ : ƒ 1 ƒ 2 Κεψ ωορδσ :! 5 6 / 0 )))!

24 1 12 3! 2 Β ƒ ƒ ƒ 1 222! ƒ 111! 2 2! Ταβ11 Οπερατινγ χονδιτιονσ οφ ινστρυµ εντ Λ ƒ Ταβ12 Προγραµ οφ γραπηιτε φυρναχε ε ε ε ε ƒ

25 112 ƒ Χυ,Ζν,Φε,Μνινλεαϖεσοφπεαχη (Λγγ 1 ) ε 1 1 ƒ ? 1 1? 1? 1? 1!! 213!!! 1 Ταβ14 ετερµινατιον ρεσυλτσιν Χηινεσε τραδιτιοναλ µ εδιχινεσ (Λγγ 1 ) ƒ ƒ ƒ 1 1 Ταβ13 ετερµινατιον ρεσυλτσ ανδ στανδαρδ ϖαλυεσ οφ ε 2 2! 2 Β Σπεχτροσχοπψ ανδ Σπεχτραλ Αναλψσισ Σπεχτροσχοπψ αν δ Σπεχτραλ Αν αλψσισ 20 ετερµινατιον οφ Σιξ Νυτριτιοναλ Ελεµ εντσιν Χηινεσε Ηερβαλ Μεδιχινεσ βψ Γραπηιτε Φυρναχε Ατο µιχ Αβσορπτιον Σπεχτρο µ ετρψ Χολλε γε οφ Χηε µ ιστρψ αν δ Ενϖιρον µ ενταλ Σχιενχε, Ηεβει Υνιϖερσιτψ, Βαοδινγ,Χηινα Αβστραχτ 1 2 Β ƒ 2 ƒ ƒ 2

26 ƒ 2 Κεψωορδσ ƒ!! / ƒ 0 ± ƒ ƒ ΧΣΙ 2 ƒ 2 ƒ!! :ΗΤΤΠ/ / ΩΩΩ.ΧΣΙΞΞΞΙΙΙ.ΟΡΓ!!!! ± ƒ ) ƒ τ ƒ τ τ τ τ ƒ ƒ 2

27

28 π π 222 Τ ηε ετερµ ινατιον οφ Πβ ανδ Α σ ιν Φορσψτηια συσπερσα 2 2 Σηανδ ονγ Προϖινχιαλ Ι νστιτυτε οφ Τ ΧΜ ϑιναν Σηανδονγ Χηινα Αβστραχτ ƒ ƒ Κεψωορδσ ƒ Φορσψτηια συσπερσα! Α 2 ε ε ε ε Λ ƒ 2 ε ε ε / 0 2 Ιφ Χ Χ!!

29 !! Λ Λ [ [ [ 5 6 Β!!! Β Β Β Β Β Β

30 Λ!! 1!1!1! Α ΣΤΥ Ψ ΟΝ ΕΡΤΕΡ ΜΙΝΑΤΙΟΝ ΟΦΣΕ ΙΝ ΡΙΧΗ ΒΙΟΣΕΛΕΝΙΥ Μ ΣΑΜΠΛΕΣ. ΛΙ Ηυι. Σηενζηεν Ηψγιενε αν δ Αντι2επιδε µ ιχσ Στατιον, Σηε νζ ηεν. Αβστραχτ : ƒ 2 Λ!! Κεψ ωορδσ: ƒ 1!!! 1! 1 1 Λ 11 ε ε 18 ε ε ε 1 1 ε? ε 1

31 ε! Λ Λ Λ Π( % ) 1? Π1 ε ε ε ε 1 1! 1! 1! 2 2 1!! Λ ! 1

32 ! / 0! ±!!!!!ƒ!!!! ε!!!ƒ!!

33 ƒ!!!!!! ƒ!! ƒ!!!!!!!!!!!!!ƒ!!!!!!ƒ!!!!! ΑΣυρϖεψ οφ τηε Τραχε Ελεµ εντ ιν τηε Λεεχη Εαρτηωορ µ ιν ϑινγ µεν χιτψ ΧΗΕΝ Κε φυ,ταν Ξι ϕυν Αβστραχτ : / 0 2 / 0 Κεψ ωορδσ:

34 ΝΑΤΥΡ ΑΛ ΣΧΙΕΝΧΕ ϑουρ ΝΑΛ ΟΦ ΗΑΙΝΑΝ ΥΝΙς ΕΡΣΙΤΨ Λ ƒ!!!! ε

35 ! Κ θϖ Βθϖ Β Α!!! Α Θ Θ θϖ Βθϖ Βθϖ θϖ! ƒ ƒ

36 ƒ ρ 3 Α Χ 3 ƒ ƒ ƒ ƒ! Ενηανχεµεντ Εφφεχτ οφ Τραχε Ποτασσιυµ ετερµινεδ βψ Φλα µ ε Ατο µ ιχ Αβσορπτιον Σπεχτρο µ ετρψ Αβστραχτ : 2 ƒ 2 ƒ 2ƒ 2 2 Λ Κεψ ωορδσ : ƒ

37 ! 1! ε !!! ) ))) 113!! 3 θ (Χ ς Χς) / µ,χ,ς,χ ς,µ,

38 ,θ Τ ε θ ε θ ε θ ε !

39 !!!!! 1 1! θ θ µαξ Κ Λ Χ/ ( Κ Λ Χ) () 1,θ µαξ,κ Λ,Χ 1 1., 1 / χ / θ : / θ/θ µαξ (/ Κ Λ θ µαξ ) 3 (/ χ) () 1 / θ µαξ, / Κ Λ θ µαξ 1 1, ( ),θ χ ! χ θ

40 ΜΙΚΑΑΝΙΑΜΙ ΧΡΑΝΤΗΑ ΚΥΝΤΗ ƒ 2 ( Φοοδ ανδ Βιολογιχαλ Ενγινεερινγ Χολλεγε, Σουτη Χηινα Υνιϖερσιτψ οφ Τεχηνολογ ψ, Γυανγζηου, Χηινα) Αβστραχτ : Μικαανιαµιχραντηα Κυντη Μικαανιαµιχραντηα Κυντη Κεψ ωορδσ : Μικαανιαµιχραντηα Κυντη ; ƒ ( επαρτµεντ οφ Χηεµιστρψ, Ζηενγζηου Υνιϖερσιτψ, Ζηενγζηου, Χηινα) Αβστραχτ : ! Κεψ ωορδσ :

41 6 ƒ 1 2 Λ 1 Ζη Αναλ Κηι µ, 1 (µγγ 1 1 ) 1 ϑινδιαν Χηεµ Σοχ 1 1 ƒ Αναλψστ, ϑ Πλαναρ Χηροµατογρ2 Μοδ Τ ΛΧ ετ αλ ϑχηροµατογρ Α, ΡΣ ƒ ϑαγριχ Φοοδ Χηε µ 812 ) 813 1

42 Λ Ψ 1 Χ 1 ρ Λ 1 1 ΡΣ Λ ΡΣ 1 ν ε !! Λ Λ !

43 [ ]!! Π Π Π [ ] Χηανγεσ οφ χοντεντσ οφ υριναρψ µεταβολισµ οφ µ ονοαµινε νευροτρανσµιττερ ιν ρατσ εξποσεδ βψ αλυµινυµ. ΤΑΝΓ Ηυαν ωεν, ΩΕΙ Ξιαοµιν, ΛΙΑΝΓ Ηαιρονγ,εταλ.Σχηοολοφ Πυβλιχ Ηεαλτη, Γυανγξι Μεδιχαλ Υνιϖερσιτψ, Ναννινγ [Αβστραχτ] Οβϕεχτιϖε Μετηοδσ 2 Ρεσυλτσ Π 2 Π Π Χονχλυσιον [Κεψ ωορδσ]

44 1 2 Π!! Π Π Π!! Π ε Π Π Λ???????????? Π 3!

45 ???????????????????????? Π Π! ƒ !!!!

46 A T O M I C Ultratrace Aluminum Analysis of Nutritional Intravenous Solution Components in 1-Propanol Using GFAAS Tim Shelbourn*, Molly Chacko, Jennifer Wisser, Tammy Mortensen, Scott Ericson, Karen Nunez, Deborah Sutliff Baxter Healthcare *To whom correspondence should be addressed The ultratrace aluminum analysis of two nutritional intravenous solution components with limited water solubility can be performed by GFAAS with dissolution in 1-propanol. Use of this solvent allows aqueous aluminum stock standards and nitric acid magnesium nitrate matrix modification as in the analysis of aqueous solutions. The FDA final rule on aluminum levels (21 CFR Part 201, January 26, 2000) states that certain components of nutritional intravenous solutions, also known as total parenteral nutrition (TPN) solutions, shall contain no more than 25 ppb of aluminum. The requirements were added by FDA to characterize such solutions to address concerns from the medical community (1). Baxter Healthcare (Deerfield, IL), a major manufacturer of TPN solutions, complies with this rule by monitoring TPN final product and by screening raw materials for aluminum content using graphite furnace atomic absorption spectroscopy (GFAAS). The screening of raw materials of slight or no water solubility for aluminum content creates an analytical challenge. Two examples of such materials include the nonessential amino acid L-tyrosine, and the lipid raw material soybean oil. L-Tyrosine has a solubility in water of approximately 0.05 wt %, whereas soybean oil is practically insoluble in water (2). Wet ashing, microwave digestion, and muffler furnace combustion are widely accepted means of preparing waterinsoluble specimens for elemental analysis. However, it was desirable to develop a GFAAS method fundamentally consistent with the U.S. Pharmacopeia (USP) 206 aluminum procedure (3) for testing water-soluble raw materials. We have determined that 1-propanol acidified with nitric acid is a suitable solvent system for the analysis of aluminum in these two TPN components. EXPERIMENTAL The instrument used for these analyses was a PerkinElmer Instruments (Norwalk, CT) AAnalyst 600 graphite furnace atomic absorption spectrometer using Zeeman-effect background correction and configured with a model AS-800 autosampler running version 3.9 of PerkinElmer Instruments AAWinLab software. The testing methodology used for L-tyrosine and soybean oil was fundamentally consistent with USP 206 (3). The specific experimental parameters are listed in Table I. Three replicate injections were made of each calibration and sample solution. Unspiked samples were prepared by accurately weighing 0.5 g of L-tyrosine (Aldrich, Milwaukee, WI) or 2.5 g of soybean oil (Aldrich) into separate 25-mL nitric acid washed polymethylpentene volumetric flasks. Five separate unspiked sample replicates of each material were prepared. Approximately 10 ml of 1- propanol and 0.5 ml of nitric acid were slowly added to each flask. When adding the nitric acid to the 1-propanol in this manner, no noticeable heat was generated. (Utmost caution should always be exercised when adding strong acids to alcoholic solutions.) Each flask was swirled thoroughly to mix the contents. The flask 16 SPECTROSCOPY 17(1) JANUARY

47 A T O M I C Table I. Experimental parameters. Spectrophotometer (AAnalyst 600) containing soybean oil was diluted to the volume 25 ml with HPLC-grade 1- propanol (Aldrich). An additional 0.5 ml of nitric acid was slowly added to the flasks containing L-tyrosine before dilution to volume with 1-propanol. Each flask was capped and inverted to mix. The contents in each flask were transferred immediately into separate acid-washed 30- Graphite Furnace Element Al Step No. Temp. (ºC) Ramp (s) Hold (s) Ar (cc/min) Lamp Hollow cathode Current Recommended on lamp Wavelength (nm) Slit (nm) Signal type AA-BG Signal measurement Peak area Read time (s) Read delay (s) BOC time (s) 1 Fume extractor engaged for furnace program Tube type THGA, End Capped Sample volume (L) 20 Read step 7 Matrix modifier volume (L) 10 Calibration program Linear, calculated intercept Diluent volume (L) 5 Table II. Accuracy and precision results.* Sample Al Conc. (ng/g) % RSD % Recovery Identification (Avg. Conc. of (Five Replicates) Manual Spike Autosampler Five Replicates) (Avg. of Five Recoveries) Spike (Avg. of Three Recoveries) L-Tyrosine Soybean Oil *L-Tyrosine specimens were manually spiked with aqueous aluminum intermediate (1.00 g/ml) standard and spiked via the autosampler using the 20-ng/mL aluminum working standard. Manual and autosampler spike concentration: 5.0 ng/ml. Soybean oil specimens were manually spiked with an organometallic aluminum standard and spiked via the autosampler using the 20-ng/mL aluminum working standard. Manual spike concentration: 10 ng/ml; autosampler spike concentration: 5.0 ng/ml. Table III. Regression statistic for aluminum standard in 1-propanol. Statistical Parameter L-Tyrosine Soybean Oil Correlation coefficient (R) Slope y intercept Blank standard deviation (n 6) Method limit of detection (MLOD) 3 (ng/g) The method limit of detection was calculated from the standard deviation of six blank responses and the slope of the linear regression calibration curve as follows: 3 SD of the blank (abs units) LOD(ppb) Slope of the regression line (abs / ng / ml units) Volume (ml) Test article weight (g) ml tetrafluoroethylene bottles. L-Tyrosine was prepared at the 2 wt % level in 4% (v/v) Ultrex II grade nitric acid (J.T. Baker, Phillipsburg, NJ) in 1-propanol. Soybean oil was prepared at the 10 wt % level in 2% (v/v) nitric acid in 1-propanol. We were unable to locate analogous standard reference materials for these two samples. Therefore, accuracy and precision of the method were determined by performing a series of spiking experiments. Manually spiked L-tyrosine samples were prepared by transferring 125 L of an aqueous g/ml aluminum quality assurance standard prepared from an aqueous 1000-g/mL aluminum stock standard (High Purity Standards, Charleston, SC) to five separate L-tyrosine sample preparations before dilution to volume with 1-propanol. This resulted in a 5.0-ng/mL aluminum spike of each L-tyrosine sample preparation. Manually spiked soybean oil samples were prepared by accurately weighing approximately 0.1 g of an approximately 2.5-g/g organometallic aluminum standard (High Purity Standards) into each of five separate soybean oil preparations before dilution to volume with 1-propanol. This resulted in an approximately 10- ng/ml organometallic aluminum spike of each of the soybean oil sample preparations. In addition to the manually spiked samples, the AS-800 autosampler was used to automatically spike one of each of the unspiked L-tyrosine and soybean oil sample solutions in triplicate at the 5.0 ng/ml level using a portion of the 20-ng/mL calibration standard. Individual aluminum calibration working standards were prepared in 2% (v/v) nitric acid 1-propanol at blank, 2.0, 10, and 20 ng/ml from an aqueous g/ml aluminum stock standard (Ricca Chemical Company, Arlington, TX). The AS-800 autosampler automatically prepared a 5.0-ng/mL working calibration standard using a portion of the 20-ng/mL aluminum working standard. Quality assurance check standards were prepared from an aqueous g/ml aluminum stock standard of a different manufacturer than used to prepare the calibration standards (High Purity Standards) in 2% (v/v) nitric acid 18 SPECTROSCOPY 17(1) JANUARY

48 A T O M I C (a) Absorbance Time (s) 20 ppb 10 ppb quality control sample 5 ppb spike Tyrosine (unspiked) Figure 1. Aluminum peak profiles for (a) L-tyrosine and (b) soybean oil. 1-propanol at 10 ng/ml. The check standard was analyzed after every five autosampler locations to monitor instrument drift throughout the analysis. A matrix modifier solution of 0.5- mg/ml magnesium nitrate (Perkin- Elmer) in 10% (v/v) nitric acid was prepared in 1-propanol and stored in a 30-mL acid-washed tetrafluoroethylene bottle. An autosampler rinse solution of 1:1 1-propanol ethanol (HPLC-grade (b) Absorbance Time (s) 20 ppb standard 10 ppb quality control sample 10 ppb spike Soybean oil (unspiked) [Aldrich]) was used throughout the analyses. DISCUSSION The analytical accuracy and precision results are shown in Table II. The regression statistics for aluminum calibration standards are shown in Table III. The intermediate polarity of 1-propanol allowed complete dissolution of the two materials while allowing for the use of aqueous aluminum stock standards and magnesium nitrate nitric acid matrix modification. The solvent also has a slow rate of evaporation, is relatively nontoxic, and is free of an offensive odor. Several lots of HPLC-grade 1-propanol proved to be of sufficiently low aluminum content to be used without the requirement of further cleanup. L-Tyrosine and soybean oil sample analyses were completed within 4 h. The practical time limit for analysis of samples prepared in this manner is approximately 8 h. Table II shows the precision and accuracy results for five replicate analyses. The average of five soybean oil manual spike recoveries of 10-ng/mL organometallic aluminum standard was compared with the average of three 5.0 ng/ml spikes prepared by the autosampler with the aluminum standard. The purpose of this experiment was to demonstrate that the furnace temperature program, matrix modification, and solvent system would produce accurate results regardless of the molecular species of Circle 17 Circle SPECTROSCOPY 17(1) JANUARY

49 A T O M I C aluminum in the sample. The other consideration was to establish that the positive displacement pump of the AS-800 autosampler would meter the 1-propanolic sample preparations with the same relative accuracy as we have experienced with aqueous sample preparations. The average of five L-tyrosine manual 10-ng/mL spike recoveries of waterbased aluminum standard was compared with the average of three 5.0-ng/mL spikes prepared by the autosampler using the 20-ng/mL aluminum working standard. The percent recoveries for manually spiked and autosampler-spiked soybean oil and L-tyrosine test solutions were all within 10% of theoretical, which is considered acceptable accuracy by our laboratory. Figures 1a and 1b show aluminum peak profiles for L-tyrosine and soybean oil. A new tube was used for the L-tyrosine sample analysis (Figure 1a); the same tube was used for the analysis of the soybean samples (Figure 1b). Note that the tube had approximately 300 firings before the analysis of the soybean oil samples. The difference in peak shapes from Figure 1a and Figure 1b may be due to the relative aging of the tube. However, accuracy and precision for the soybean oil analysis was equivalent to that for the L-tyrosine analysis. In our experience, this is typical of tube lifetime when aqueous solutions are analyzed by GFAAS. CONCLUSION The use of an organic solvent of intermediate polarity proved to be a successful matrix for the GFAAS analysis of these test articles. Analytical parameters and matrix modification as suggested by the instrument manufacturer and USP 206 for the analysis of aluminum in aqueous solutions were essentially maintained. Accuracy, precision, and sensitivity of the methodology are characteristic of GFAAS for ultratrace aluminum analysis as experienced in this laboratory. Due to evaporation of the 1-propanol from the GFAAS autosampler cups, the practical time limit for conducting an analysis is more restricted than for an analysis of aqueous solutions. However, the use of an organic solvent for GFAAS analysis of these types of insoluble compounds has proven to be a practical alternative to digestion. REFERENCES (1) Federal Register 65(17), (January 26, 2000). (2) The Merck Index, 10th Edition, 1249, 1406 (Rahway, N.J., 1983). (3) United States Pharmacopeia 24. NF 19, Aluminum <206>, 1856 (Rockville, MD: U.S. Pharmacopeial Convention, 1999). Tim Shelbourn is a senior research associate in atomic spectroscopy at the Baxter Healthcare William Graham Technology Center, located at Route 120 and Wilson Road, Round Lake, IL. He may be contacted by phone at (847) , by fax at (847) , or by at tim_ shelbourn@baxter.com. Circle 19 JANUARY (1) SPECTROSCOPY 21

50 Articles Occupational Exposure to Lead and Induction of Genetic Damage Alexander Vaglenov, 1 Amadeu Creus, 2 Stoyan Laltchev, 3 Vera Petkova, 4 Sonya Pavlova, 4 and Ricardo Marcos 2 1 National Centre of Radiobiology and Radiation Protection, Sofia, Bulgaria; 2 Grup de Mutagènesi, Unitat de Genètica, Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Ceranyola del Vallès, Spain; 3 Department of Medical Genetics, 4 Clinic of Occupational Diseases, Medical University, Sofia, Bulgaria. To investigate whether occupational exposure to lead is genotoxic, we evaluated data from 103 lead-exposed workers and 78 matched controls. These data correspond to three different sampling periods, and we measured genetic damage as increases in the frequency of binucleated cells with micronuclei (BNMN) in peripheral blood lymphocytes. The levels of exposure were determined according to the lead levels in blood. Clearly significant increases in BNMN were observed in the exposed groups when compared to the control group. In addition, for the overall population (n = 181), we observed a clear relationship between lead levels in blood and BNMN (r = 0.497; p < 0.001). When we examined four exposure levels very low exposure (< 1.20 µm/l), low exposure ( µm/l), high exposure ( µm/l), and very high exposure (> 2.88 µm/l) we found significant differences in the genetic damage induction. We conclude that exposure to levels of lead higher than 1.20 µm/l may pose an increase in genetic risk. In addition, our data show that blood lead level is a good indicator of genetic damage induction. Key words: biomonitoring, genotoxicity, lead-exposed workers, lymphocytes, micronucleus assay. Environ Health Perspect 109: (2001). [Online 5 March 2001] Lead (Pb), a toxic contaminant metal used in many important industrial processes, is widely used in batteries, paint, and varnishes, as an antiknock compound in gasoline, in pipe covering, and in welding. Because of its persistence in the environment, exposure to lead has become a major public health concern. Chronic, low-level exposure to lead affects children living in old homes and/or children in families with low income (1), as well as other groups that are occupationally exposed to high lead levels. The study of these highly exposed persons provides the opportunity to establish relationships between exposure levels and different toxic end points. Although lead toxicity on different biological systems and functions has been well reported (2 4), there are conflicting data on its genotoxic and carcinogenic properties. In bacterial tests, lead seems to be generally nonmutagenic (5). Nevertheless, in eukaryotic cells this metal is usually genotoxic (6,7), through a mechanism that until now has not been well characterized and that possibly involves indirect damage to DNA, affecting the stabilization of chromatin (8) or interacting with repair processes (9). Whether lead is carcinogenic to humans is still not known. The International Agency for Research on Cancer (IARC) classified lead and inorganic lead compounds as possible human carcinogens (Group 2B) on the basis of sufficient evidence for carcinogenicity in experimental animals but inadequate evidence for carcinogenicity in humans (10). A quantitative assessment of published data, with workers heavily exposed to inorganic lead, provides some evidence to support the hypothesis of an association between stomach and lung cancer and exposure to lead (11), but this meta-analysis is limited significantly by the lack of information about potential confounding factors. To learn more about the possible relationship between lead exposure and genetic risk, we investigated genetic damage observed in a large group of Bulgarian workers exposed to lead. The data were collected in three periods (1992, 1993, and 1996) and the frequency of micronuclei (MN) was the genetic end point evaluated. MN is considered a reliable biomarker of genotoxic exposure to both physical and chemical agents (12), and increases in MN frequency indicate exposure to clastogenic and/or aneugenic agents. In addition, cytogenetic end points in peripheral blood lymphocytes have been used as biomarkers for many years and allow a reasonable epidemiological evaluation of cancer predictivity (13). Materials and Methods Studied populations. Three independent biomonitoring studies were conducted in the years 1992, 1993, and 1996, examining the genotoxic effects of lead exposure in a group of workers from a Bulgarian storage battery plant. The studies assessed cumulative exposures by monitoring concentrations of lead in blood and by cytogenetic monitoring with the micronucleus assay in peripheral blood lymphocytes. We sampled 181 men in three sessions. The first sample included 16 controls and 29 exposed, the second included 24 controls and 49 exposed, and the third, 38 controls and 25 exposed. The 103 workers exposed to lead participating in the study were employed in a storage battery plant located in Pazardzik, Bulgaria. These men were constantly exposed at their workplaces to lead concentrations in air 2 15 times the permissible exposure limit (0.05 mg/m 3 ; PEL). The control group of 78 males was divided in two subgroups as follows: an internal control group of 43 persons from the administrative and maintenance staff from the same plant and an external control group of 35 persons recruited from a noncontaminated plant in the same town. The questionnaire and clinical examination of all the individuals studied were performed the same day as blood sampling by occupational expert pathologists to select the appropriate subjects for the study. Individuals exposed to other genotoxic agents were not included in the study, and the exposed and control individuals were matched for relevant factors such as age, smoking, and drinking habits. Each donor gave written consent before the investigations, and blood samples were collected and manipulated in accordance with ethical standards. The lead concentrations in the blood of each participant were determined by using an AAS PerkinElmer 3030 device (Perkin- Elmer, Norwalk, CT, USA) after flame extraction the day after sampling. Cell cultures and MN analysis. Blood samples were obtained by venipuncture in heparinized sterile tubes, coded, and sent immediately to the laboratory where they were processed. Lymphocyte cultures were started by adding 0.5 ml of blood to 5 ml RPMI-1640 medium (Sigma, St. Louis, Address correspondence to R. Marcos, Grup de Mutagènesi, Unitat de Genètica, Departament de Genètica i de Microbiologia, Edifici Cn, Universitat Autònoma de Barcelona, Bellaterra, Cerdanyola del Vallès, Spain. Telephone: + 34 (93) Fax: + 34 (93) rmd@cc.uab.es This investigation was supported in part by the Bulgarian government (HZ-13/92) and by a contract with Elhim-Izkra Ltd. (N97/ ). We thank N. Bandev, director of the starter battery plant, for his financial support. We are grateful to the Spanish Ministry of Education and Culture, which granted a visiting research position to A. Vaglenov (DGES, SAB ). We are indebted to G. Umbert and A. Corral for their expert technical assistance. The secretarial help of M. McCarthy is very much appreciated. Received 27 July 2000; accepted 16 October Environmental Health Perspectives VOLUME 109 NUMBER 3 March

51 Articles Vaglenov et al. MO, USA), supplemented with phytohemagglutinin (PHA; Gibco, Grand Island, NY, USA), 15% fetal calf serum (Sigma), and 1% penicillin and streptomycin. The cultures were incubated at 37 C and 5% CO 2 for 72 hr. Cytokinesis was blocked with 6 µg/ml cytochalasin B (Sigma) added 44 hr after PHA stimulation. Cells were harvested by centrifugation and, after a mild hypotonic treatment with 3 ml M KCl at 4 C and another centrifugation, were fixed by adding 5 ml fixative solution (methanol:glacial acetic acid, 3:1). Then 50 µl formaldehyde were added during the next hour. We next performed two steps of centrifugation, with consequent fixation of the material as already described, without adding any more formaldehyde. Air-dried preparations were stained with 5% Giemsa (Merck, Darmstad, Germany) for 15 min. MN were scored in 1,000 binucleated lymphocytes per donor according to Fenech (12). To minimize variability, the same experts performed all the microscopic analyses during the study. All scoring was performed blind, with no knowledge of the samples origin. Statistical methods. For the investigations done in 1992, 1993, and 1996, we compared the distribution of BNMN and the distribution of blood lead concentration with the normal distribution by means of the Kolmogorov-Smirnov test of goodness of fit. Neither distribution departed significantly from normality and therefore parametric tests were adequate for the statistical analysis. For each independent study, the effects of some factors on binucleated micronuclei (BNMN) were simultaneously assessed by analysis of variance (ANOVA). These factors were occupational exposure, smoking habits, and alcohol consumption, with age of the subject considered as a covariate. To determine the relationship between lead exposure levels and BNMN, we applied a multiple regression analysis to the whole investigated population. This analysis was carried out by using the CSS:STATISTIC/W (StatSoft, Tulsa, OK, USA) statistical package. Results Tables 1 and 2 show the characteristics of the control and exposed groups, respectively. Age of individuals, years in their employment, and smoking and drinking habits are indicated. Lead levels in blood are also shown. These results are presented for each sampling period as well as for the pooled data. Lead in blood for the exposed workers was about three times higher than the values obtained for the controls, indicating that the exposure levels were high. Table 3 shows the results of the MN scoring, indicating both the average total number of MN scored for 1,000 binucleated cells, as well as the average of binucleated cells presenting one or more MN. This second measurement has been considered a good parameter for measuring genotoxic effects (14). The overall frequency of BNMN in the pooled control was ± 1.02, which is in good agreement with values usually reported for control populations. When we compared BNMN from sampling periods for controls, we found no differences between 1992 and 1993 (p = 0.67), 1992 and 1996 (p = 0.32), or 1993 and 1996 (p = 0.52). Furthermore, when all the controls were grouped as internal controls (n = 43) and external controls (n = 35), the statistical analysis showed that the differences were not statistically significant (p = 0.18). Thus, the pooled data can be considered as a good reference control. When the overall control data for BNMN were compared with the pooled exposed data, the differences were significant (p < 0.001), indicating a clear genotoxic effect of lead exposure. Figure 1 shows the values of the exposed group compared with both the internal and external control groups. These differences between exposed and controls were also observed for each of the sampling periods (p < 0.001; p = ; and p < for 1992, 1993, and 1996, respectively). The genetic effects detected in the exposed group are considered to be caused by lead exposure. The average values of lead in blood for the exposed and for the internal and external controls are indicated in Figure 2. This figure demonstrates that, despite the lack of significance for the BNMN values between internal and external controls, the values of lead in blood are higher in the internal control (p = ). Nevertheless, both control values are significantly lower than values observed in the exposed group (p < 0.001). The multiple regression analysis indicated a slight relationship between alcohol consumption and BNMN (p = 0.052). Figure 3 shows the relationship between the levels of lead in blood and genotoxic effects, as reflected by the BNMN values. This figure provides data for both the control and the exposed individuals (n = 181). A significant correlation (r = ; p < 0.001) was found between BNMN and lead in blood, indicating a direct relationship between lead levels in blood and genetic damage induction. To visualize better the relationship between lead in blood (as a measurement of exposure) and the frequency of BNMN (as a measurement of genetic damage), we categorized the donors in four groups, according to their blood lead levels. These values are indicated in Figure 4, where the four groups, corresponding to very low exposure (< 1.20 Table 1. Characteristics of the three control groups, with lead levels in blood (mean ± SE). No. of Age Years Smoking Alcohol Pb in blood Year individuals (years) employed (cigarettes/day) (g/day) (µm/l) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.06 Total ± ± ± ± ± 0.04 Table 2. Characteristics of the three exposed groups, with lead levels in blood (mean ± SE). No. of Age Years Smoking Alcohol Pb in blood Year individuals (years) employed (cigarettes/day) (g/day) (µm/l) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.16 Total ± ± ± ± ± 0.10 Table 3. Mean number (± SE) of micronuclei (MN) in binucleated cells and binucleated cells presenting one or more micronuclei (BNMN). No. of Group Year individuals MN BNMN Control ± ± ± ± ± ± 1.60 Total ± ± 1.02 Exposed ± ± ± ± ± ± 2.89 Total ± ± 1.68 We scored 1,000 binucleated cells per donor. 296 VOLUME 109 NUMBER 3 March 2001 Environmental Health Perspectives

52 Articles Occupational exposure to lead and genetic damage µm/l), moderate exposure ( µm/l), high exposure ( µm/l), and very high exposure (> 2.88 µm/l), are shown. The number of donors in each group was 71, 32, 29, and 49, respectively. As can be seen, BNMN correlate well with blood lead levels, obtaining significant differences between very low and moderate exposure levels (p = 0.05) and moderate and high exposure levels (p = 0.02), although not between the high exposure and the very high exposure levels (p = 0.16). Discussion The workers occupationally exposed to lead who were monitored in this investigation showed clear evidence of genetic damage in peripheral blood lymphocytes when evaluated by using the MN assay. These results extend and confirm our recent studies performed in another group of Bulgarian workers exposed to lead (15) with no apparent exposure to other suspicious genotoxic agents at the workplace. Once the hazardous workplace or exposures are identified, the major objectives are to establish the relationship between exposure levels and genetic risk and to define safe levels of exposure on a sound toxicological basis. In this context, biomonitoring exposed individuals is extremely important, especially in evaluating genotoxic effects, as BNMN Mean ± SD Mean ± SE Mean External Internal Exposed Groups Figure 1. BNMN values obtained for exposed and controls (internal and external). Pb in Blood (µm/l) Mean ± SD Mean ± SE Mean External Internal Exposed Groups Figure 2. Lead levels in blood from exposed and controls (internal and external). in this study. At present, large follow-up studies suggest that increases in chromosome alterations may predict an increased cancer risk (13,16). Among the different cytogenetic approaches, the MN assay in human lymphocytes using the cytokinesis-block method (17) has increasingly been accepted as a reliable biomarker of cytogenetic damage induced by genotoxic agents, both physical and chemical (12,18). Positive findings using this biomarker indicate evidence of exposure to clastogenic and/or aneugenic compounds. In particular, the MN assay has proved very reliable in assessing the genotoxic effects of metal ions in occupational exposures (15,19,20) Regarding the carcinogenic risk of lead exposure, several experiments in rats and mice showed the production of renal tumors when lead compounds were administered in food and drinking water (21). In humans, several epidemiological studies of workers exposed to lead in various occupational settings have been reported. Unfortunately, most of these studies contain some deficiencies, particularly a lack of information about cumulative past exposures, and the overall studies do not provide conclusive evidence of an association between lead exposure and increased incidence of cancer (10,21). Accordingly, lead has been classified by BNMN Regression 95% confidence interval Pb in Blood (µm/l) Figure 3. Lead in blood and BNMN relationship in the overall population. BNMN = (blood lead); r = BNMN Mean ± SD Mean ± SE Mean 5 < > 2.88 Pb in blood (µm/l) Figure 4. Lead in blood and BNMN relationship for four blood lead levels. IARC as a possible human carcinogen on the basis of sufficient evidence in rodents but inadequate evidence in humans (10). Nevertheless, a meta-analysis of published data from workers highly exposed to lead seems to support the hypothesis of an association between exposure to lead and stomach and lung cancer (11). The relationship that exists between the genotoxic and carcinogenic potential of lead has been the subject of extensive studies, although in most of them the results obtained were inconclusive. Thus, although lead seemed to be negative in an assay detecting mutation induction in bacteria (22), positive results were obtained in an assay detecting induction of λ prophage in E. coli (23). In mammalian cells, induction of mutation has been reported in the hprt locus in Chinese hamster V79 cells by lead compounds (6), although studies on the induction of chromosomal aberrations, both in vivo and in vitro, showed ambiguous results because the genotoxic response appears to depend on factors such as cell type, duration, and route of exposure and can also be influenced by synergistic effects. Thus, for instance, calcium-deficient animals exposed to lead demonstrated more severe chromosomal aberrations than nondeficient animals (24). All these variables contribute to the high variability in results. With respect to the biomonitoring studies performed in humans occupationally exposed to lead, although relatively few studies have examined the genotoxic potential of lead and they offer some equivocal results, most show increases in chromosomal aberrations (25 29). The high degree of variability in the available data represents, perhaps, different levels of exposure and makes the explanation of biomonitoring results quite complex. In view of the current inconclusive evidence of a direct relationship between in vivo lead exposure and induction of genetic damage, we think that the positive findings presented here are satisfactory evidence of a genetic risk associated with lead exposure. It must be remembered that the MN assay detects both clastogenic and aneugenic effects, covering a wider spectrum of damage than the classical chromosome aberrations test. Thus, it could be possible that lead induces genotoxicity via induction of chromosome loss, which would be interesting to confirm. Although the qualitative data obtained are important, indicating a genetic risk associated with lead exposure, more interesting is the quantitative association found between blood lead levels (as a measurement of exposure) and BNMN (as a measurement of genetic damage). The levels of genotoxins in the body fluids can be regarded as a measure Environmental Health Perspectives VOLUME 109 NUMBER 3 March

53 Articles Vaglenov et al. of the internal dose, confirming that exposure has indeed occurred. Cytogenetic alterations as measured by the MN assay are early biological effects in carcinogenesis, if we assume that lymphocytes are valid surrogate cells for the changes taking place in tissues where neoplasms may eventually develop. Nevertheless, the relationship between exposure markers and genetic changes such as chromosome alterations are not always clear (30), depending on the toxicokinetics and distribution of the compound or its metabolites in the body compartments. In our study, the large sample size and the wide variability found in the blood lead levels made it possible to obtain a good relationship between the exposure and the biomarkers. This allowed us to identify exposure levels as low as µm/l, which are associated with significant increases of genetic damage at levels where no clinical symptoms are observed. These lead levels are found in the range reported for different populations not specifically exposed to lead at their workplace (31) and exceed the biological tolerance value regulated in many countries, (e.g., in Germany this value is 3.38 µm/l) (32). In summary, this study shows a clear genotoxic effect associated with the occupational exposure to lead, indicating that this effect is dose-related to the lead levels in blood. These data are relevant and permit an estimate of the genetic risk of lead exposure by using biomarkers of exposure. REFERENCES AND NOTES 1. Pirkle JL, Kaufmann RB, Brody DJ, Hickman T, Gunter EW, Paschal DC. Exposure of the U.S. population to lead, Environ Health Perspect 106: (1998). 2. Todd AC, Wetmur JG, Moline JM, Godbold JH, Levin SM, Landrigan PJ. Unraveling the chronic toxicity of lead: an essential priority for environmental health. Environ Health Perspect 104: (1996). 3. Gerber GB, Léonard A, Jacquet P. Toxicity, mutagenicity and teratogenicity of lead. Mutat Res 76: (1980). 4. Goyer RA. Lead toxicity: current concerns. Environ Health Perspect 100: (1993). 5. Dunkel VC, Zeiger E, Brusick D, McCoy E, McGregor D, Mortelmans K, Rosenkranz HS, Simmon VF. Reproducibility of microbial mutagenicity assays: I. Tests with Salmonella typhimurium and Escherichia coli using a standardized protocol. Environ Mol Mutagen 6:1 254 (1984). 6. Zelikoff JT, Li JH, Ahartwig A, Wang XW, Costa M, Rossman TG. Genetic toxicology of lead compounds. Carcinogenesis 9: (1988). 7. Winder C, Bonin T. The genotoxicity of lead. Mutat Res 285: (1993). 8. Johansson L, Pellicciari CE. Lead induced changes in the stabilization of the mouse sperm chromatin. Toxicology 51:11 24 (1988). 9. Hartwig A, Schlepegrell R, Beyersmann D. Indirect mechanism of lead induced genotoxicity in cultured mammalian cells. Mutat Res 241:75 82 (1990). 10. IARC. Overall Evaluations of Carcinogenicity: An Updating of IARC Monographs 1 to 42. Monogr Eval Carcinog Risks Hum(suppl 7): (1987). 11. Fu H, Boffetta P. Cancer and occupational exposure to inorganic lead compounds: a meta-analysis of published data. Occup Environ Med 52:73 81 (1995). 12. Fenech M. Cytokinesis-block micronucleus techniques: a detailed description of the method and its application to genotoxicity studies in human populations. Mutat Res 161: (1993). 13. Hagmar L, Bonassi S, Strömberg U, Mikoczy Z, Lando C, Hansteen I-L, Montagud AH, Knudsen L, Norppa H, Reuterwall C, et al. Cancer predictive value of cytogenetic markers used in occupational health surveillance programs: a report from an ongoing study by the European Study Group on Cytogenetic Biomarkers and Health. Mutat Res 405: (1998). 14. Surrallés J, Antoccia A, Creus A, Degrassi F, Peris F, Tanzarella C, Xamena N, Marcos R. The effect of cytochalasin-b concentration on the frequency of micronuclei induced by four standard mutagens. Results from two laboratories. Mutagenesis 9: (1994). 15. Vaglenov A, Carbonell E, Marcos R. Biomonitoring of workers exposed to lead. Genotoxic effects, its modulation by polyvitamin treatment and evaluation of the induced radioresistance. Mutat Res 418:79 92 (1998). 16. Bonassi S, Abbondandolo A, Camurri L, Dal Prá A, De Ferrari M, Degrassi F, Forni A, Lamberti L, Lando C, Padovani P, et al. Are chromosome aberrations in circulating lymphocytes predictive of a future cancer onset in humans? Preliminary results of an Italian cohort study. Cancer Genet Cytogenet 79: (1995). 17. Fenech M, Morley AA. Measurement of micronuclei in lymphocytes. Mutat Res 147:29 36 (1985). 18. Bauchinger M, Braselmann H. Use of the micronuclei in biological dosimetry of absorbed radiation dose. In: Chromosomal Aberration, Basic and Applied Aspects (Obe G, Natarajan AT, eds). Berlin:Springer-Verlag, 1990; Bercés J, Ótos M, Szirmai S, Crane-Uruena C, Köteles GJ. Using the micronucleus assay to detect genotoxic effects of metal ions. Environ Health Perspect 101(suppl 3):11 13 (1993). 20. Vaglenov A, Nosko N, Georgieva R, Carbonell E, Creus A, Marcos R. Genotoxicity and radioresistance in electroplating workers exposed to chromium. Mutat Res 446:23 34 (1999). 21. U.S. EPA. Evaluation of the Potential Carcinogenicity of Lead and Lead Compounds. (EPA/600/8 89/045A). Washington, DC:U.S. Environmental Protection Agency, Office of Health and Environmental Assessment, Kanematsu N, Hara M, Kada T. Rec assay and mutagenicity studies on metal compounds. Mutat Res 77: (1980). 23. Rossman TG, Molina M, Mayer LW. The genetic toxicology of metal compounds. I. Induction of λ prophage in E. coli Wp22s (λ). Environ Mutagen 6:59 69 (1984). 24. De Knudt GH, Colli A, Gerber GB. Chromosomal abnormalities in lymphocytes from monkeys poisoned with lead. Mutat Res 45:77 83 (1977). 25. Forni A, Cambiaghi G, Secchi GC. Initial exposure to lead: chromosome and biochemical findings. Arch Environ Health 31:73 78 (1976). 26. Nordenson I, Beckman G, Beckman L, Northstrom S. Occupational and environmental risks in and around a smelter in Northen Sweden. IV. Chromosomal aberrations in workers exposed to lead. Hereditas 88: (1978). 27. Forni A. Chromosomal effects of lead: a critical review. Rev Environ Health 3: (1980). 28. Vaglenov AK, Laltchev SG, Nosko MS, Pavlova SP, Petkova VV, Karadjov AD. Cytogenetic monitoring of exposed workers to lead. Cent-Eur J Occup Environ Med 3: (1997). 29. Kentner M, Fischer T, Richter G. Changes in external and internal lead load in different working areas of a starter battery production plant in the period 1982 to Int Arch Occup Environ Health 66:23 31 (1994). 30. Kortenkamp A. Problems in the biological monitoring of chromium(vi) exposed individuals. Biomarkers 2:73 80 (1997). 31. Romieu I, Lacasana M, McConnell R, and the Lead Research Group of the Pan-American Health Organization. Lead exposure in Latin America and the Caribbean. Environ Health Perspect 105: (1997). 32. 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