LITERATURE CITED (1) R. J. Pancirov and R. A. Brown, "Proceedings of the 1975 Conference on Prevention and Control of Oil Pollution", American Petroleum Institute, Washington, D.C., 1975, p 103. (2) W. W. Youngbiood and M. Blumer, Geochim. Cosmochim. Acta, 39, 1303 (1975). (3) L. R. Snyder, "Principles of Adsorption Chromatography", Marcel Dekker, New York. N.Y.. 1968. (4) T.-Doran and N: G. McTaggart, J. Chromatogr. Sci., 12, 715 (1974). (5) W. Giger and M. Blumer, Anal. Chem., 46, 1663 (1974). (8) M. Novotny, M. L. Lee, and K. D. Bartle, J. Chromatogr. Sci., 12, 606 ( 1974). (7) M. L. Lee, M. Novotny, and K. D. Bartle, Anal. Chem., 48, 405 (1976). (8) M. L. Lee, M. Novotny, and K. D. Bartle, Anal. Chem., 48, 1566 (1976). (9) J. A. Schmit, R . A. Henry, R. C. Williams, and J. F. Dieckman, J. Chromatogr. Sci., 9, 645 (1971). (IO) C. Golden and E. Sawlcki, Anal. Lett., 9, 957 (1976). (11) s. N. Chesler, B. H. Gump, H. S. Hertz, W. E. May, and S. A. Wise, manuscript in preparation. (12) W. E. May and R. G. Christensen, manuscript in preparation. (13) M. Popl, V. Dolanskq, and J. Mostecky, J. Chromtogr., 117, 117 (1976). (14) M. Popl, V. Doianskq, and J. Mostecky, J. Chromatogr., 91. 649 (1974). (15) M. Popl, V. Doianskq, and J. CGupek, J. Chromatogr., 130, 195 (1977). (16) C. A. Streuli, J. Chromatogr., 56, 219 (1971).
M. Martin, J. Loheac, and G. Guiochon, Chromtographia, 5, 33 (1972). D. C. Locke. J. Chromatogr. Sci., 12, 433 (1974). R. B. Sleight, J. Chromatogr., 83, 3 1 (1973). I. B. Berlman, "Handbook of Fluorescence Spectra of Aromatic Molecules", 2nd ed., Academic Press, New York, N.Y., 1971, p 70. (21) K. W. Bartz, T. Aczei, H. E. Lumpkin, and F. C. Stehiing, Anal. Chem., 34, 1814 (1962). (22) 8 . J. Mair, J. L. Martinez-Pico, R o c . Am. Pet. Inst., 42, 173 (1962). (17) (18) (19) (20)
RECEIVED for review August 9, 1977. ~ ~ September ~ 22, ~ 1977. The authors acknowledge partid financid support from the Office of Energy, Minerals, and Industry within the Office of Research and Development of the U.S. Environmental Protection Agency under the Interagency EnergyjEnvironmerit ~~~~~~h and ~~~~l~~~~~~program, identification of any commercial product does not imply recommendation or endorsement by the National Bureau of Standards, nor does it that the or equipment identified is neeessarily the best available for the purpose.
Determination of Some Thiourea-Containing Pesticides by Pulse Voltammetric Methods of Analysis Malcolm R. Smyth" and Janet G. Osteryoung Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523
The polarographlc behavior of thiourea, phenylthlourea, a naphthytthlourea and benzyl(kso)thlourea has been lnvestlgated In Brltton-Roblnson buffer and sodlwn hydroxlde solutlons. The waves obtained for these compounds In 1 M NaOH are recommended for analytical purposes. I n particular, dmerentlal pulse polarography has been used to resolve a mixture contalnlng thlourea, phenylthlourea or a-naphthylthlourea and benzyl(1so)thlourea. Thls technlque can be used to determlne concentratlons of thiourea and phenylthlourea down to 1 X M, a-naphthylthlourea down to 2 X lo-' M and benM under optlmum condltlons. zyl(1so)thlourea down to 5 X Slnce thlourea, phenylthlourea, and a-naphthylthlourea form Insoluble complexes wlth mercury, these compounds can also be determined by cathodlc strlpplng voltammetry at a hanglng mercury drop electrode. Thls method of analysis can determine concentratlons ot these compounds down to 1 ng mL-' and has been applled to the dlrect determlnatlon of thiourea In urlne.
During the past 20 years, thiourea TU; I) has found widespread use in a variety of industria and biological applications ( I ) . In agriculture it has b en employed as a fungicide (2) [although its use as such has been shown to be harmful in citrus growing areas ( 3 ) ] ,as an accelerator of sprouting in dormant tubers ( 2 ) ,and to decrease the content of nitrifying bacteria in the soil ( 4 ) . I t has been isolated as a urinary metabolite both of CS2 (5) and of a S-containing heterocycle (6). Its presence in urine has also been taken as a nonspecific indicator of cancer (7). Several derivatives of thiourea, i.e., phenylthiourea (PTU; 11) and a-naphthylthiourea (ANTU; 111) have also been used as pesticides and exhibit properties harmful to man. P T U exhibits both 2310
ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
herbicidal (8) and rodenticidal (9) activity and causes chronic goitrogenic and other glandular difficulties in man (10). The rodenticidal activity of ANTU was first demonstrated in 1945 (11). These thioureas appear t o exert their toxic effects by disturbing carbohydrate metabolism (12) and they have been shown to produce a tolerance to their own toxic action (13). S H2N-!-NH2
HV-g-NH,
S HY-E-NH,
There are many published methods for the determination of TU and P T U based on titrimetric procedures (14, 15) but these are not applicable for the analysis of trace quantities of these compounds in biological fluids. Colorimetric methods have been described for the determination of PTU which are based on its reaction with Folin-Ciocalteau reagent (16, 17), but these methods are usually slow (it takes 1.5 h for the color to develop), offer little selectivity, and have high limits of detection (ca. 3 X M) (16). The electrochemical methods that have most been applied to the determination of T U include microcoulometric argentimetric titration ( I @ , ion selective electrode potentiometry (19),amperometric titration (20), and hydrogen overvoltage measurements (21). The latter procedure could be used to quantitatively determine TU down to 1 X lo-' M, but it is not foreseen that this procedure could provide a convenient method for the determination of T U in biological fluids. Although polarographic methods have also been applied to the determination of T U , these have been based either on the liberation of the S atom and subsequent measurement as H2S (22) or on the catalytic wave produced by T U in the presence of Cu2+ions (23). We have therefore studied the inherent polarographic behavior of TU, PTU, and
ANTU to see if a direct method could provide the sensitivity and selectivity required for the determination of trace quantities of these compounds in situations of environmental importance. T h e polarographic behavior of T U has previously been studied by several authors ( 2 4 , 2 5 )and it is generally agreed that the mechanism involves mercury salt formation according to: Hg =+ Hg" + 2e' Hg2+t j [ T U ] =+ Hg[TU]j2+ where j = 2, 3, or 4. In strongly acidic media, T U is oxidized (on standing) t o form the disulfide, the reduction of which obscures any anodic wave obtained for T U a t the dropping mercury electrode (25). In weakly acidic or neutral solutions, the wave obtained for T U is independent of pH. Nyman and Parry (25) have calculated the formation constants for mercury-thiourea complexes for j = 2 ( k = 1.5 X loz2),j = and j = 4 ( k = 6.3 X loz6). They have 3 (k = 5 X suggested that the Hg[TU]?' form is the predominant species formed on the oxidation of T U in weakly acidic and neutral media and that i t is the uncharged form of T U which participates in this reaction. T U also exhibits an anodic wave in alkaline media (24) which has been shown t o be due t o mercury salt formation. In this case, however, the value was shown to be dependent on pH, indicating that a different process is operative in alkaline media. Similar results have been obtained for some substituted phenylthioureas in Britton-Robinson (BR) buffer and sodium hydroxide solutions (26). I t was our intention, therefore, t o further investigate the processes exhibited by T U , P T U , and ANTU in these media using direct current (DC), normal pulse ( N P P ) , and differential pulse (DPP) polarographic techniques. The oxidation of benzyl(iso)thiourea (BITU; IV) was also investigated. This compound can be regarded as a thioether and should be oxidized according to:
Since TU, PTU, and ANTU form insoluble complexes with mercury, these compounds are amenable to cathodic stripping voltammetry (CSV) a t a hanging mercury drop electrode (HMDE). This method of analysis w ill be discussed in relation t o the D P P procedure and preliminary results on its application to the direct determination of T U in urine will be reported. EXPERIMENTAL Apparatus. Polarographic curves were recorded using a PAR Model 174 Polarographic Analyzer in conjunction with a three-electrode cell system having a saturated calomel (SCE) as the reference and platinum as the counter electrode. The polarograms were recorded on a Omnigraphic Model 2000 x-Y recorder. The dropping mercury electrode used had a flow rate of 0.83 mg 5-l and a drop time of 8.05 s in 1 M NaOH and at a mercury head of 76 cm. Variation of pulse widths was obtained using the modification of the PAR 174 instrument described elsewhere (27). For the cathodic stripping voltammetric experiments, the PAR Model 9323 hanging mercury drop electrode was used in conjunction with the PAR Model 174. Drop times were controlled using a PAR Model 172 drop knocker. Reagents. Samples of thiourea, phenylthiourea, and benzyl(iso)thiourea were obtained from Pfaltz and Bauer, Inc. A sample of a-naphthylthiourea was obtained from the Quality Assurance Section, Environmental Toxicology Division of the Environmental Protection Agency. Stock solutions of these compounds M) were prepared in distilled water and stored under refrigeration. A 1 M sodium hydroxide solution was
-06.
04'
02,
TU
PTU
ANTU
BITU
- O I L
7
8
8
10
---
11
12
13
14
PH
Figure 1. Plot of
E ,,*vs. pH for
main wave, i,, exhibited by TU, PTU,
ANTU, and BITU
prepared from electrolytic grade pellets and made up in triple distilled water. A stock Britton-Robinson buffer solution (pH 1.8)composed of a mixture of boric acid, orthophosphoric acid, and glacial acetic acid, all 0.04 M, was prepared from analytical grade reagents. From this stock solution, buffer solutions of varying pH were prepared by the addition of 0.2 M sodium hydroxide solution and measuring the pH using a glass electrode. Techniques. Polarographic investigations were carried out on solutions that had previously been deaerated using oxygen-free nitrogen for 10 min. Current-potential curves were recorded in the DC, NPP, and DPP modes. The solutions were blanketed by an atmosphere of nitrogen during analysis and each solution was scanned between -0.8 V and the potential relating to the oxidation of mercury. A scan rate of 2 mV s-l, drop time of 1 s (NPP and DPP), and a modulation amplitude of 100 mV (DPP) were typically employed in most investigations. Variation of pulse width was achieved by using the PAR 1.74 modification described elsewhere (27). For pulse widths of 20, 50, and 100 ms, sample widths of 2, 5 , and 10 ms, respectively, were employed. For the cathodic stripping voltammetric experiments, reproducible hanging mercury drops we re obtained by dialing out four half divisions on the micrometer screw gauge of the PAR Model 9323 hanging mercury drop electrode. A constant rate of stirring was achieved using a Thermolyne Model S-7895 "StirMate". The solutions to be analyzed were first degassed using oxygen-free Nifor 10 min. The stirring motor was then switched on and a constant rate of stirring allowed t o build up in the cell. A fresh drop of mercury was then dialed out and dislodged. This was then repeated and the next drop used for the experiment. In most investigations, the potential was set between -0.2 V t o -0.3 V prior to plating for 2 min. With the initial potential still being exerted on the cell, the stirring motor was switched off and the solution allowed to come to rest for another 20 s. The solution was then scanned in a cathodic direction using scan rates of 2 5 1 0 0 mV s-' in the linear potential sweep cathodic stripping (LPSCSV) experiments and 2-20 mV in the differential pulse cathodic stripping (DPSCV) runs. In the latter case a "drop time" of 1 s and a modulation amplitude of 100 m V was employed in most investigations. R E S U L T S A N D DISCUSSION Effect of pH. The effect of p H on the El ( 2 values of the main wave exhibited by TU, PTU, ANTU, and BITU in DC polarography is shown in Figure 1. Whereas T U and P T U show linear portions of similar slope (67.5 and 70 mV pH-', respectively), ANTU shows two breaks from greater t o lesser slope (at pH's 8.45 and 12.0) on increasing pH. BITU on the ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
2311
E V vsSCE
E VvsSCE -0.6
-0.4
-0,s
-0.8-0.4
-03
-0.1
+O,l
0.0
I
a
b C
I
1
a pH 14
b PH 12
c PH 10 d PH 6
Figure 2. Effect of pH on NPP wave exhibited by PTU (5 X
M)
other hand shows no dependence on pH. This reflects that no protons or OH- ions are involved in the electron consuming process and supports the mechanism shown previously. I n the case of T U , only the wave related to the formation of the mercury complex (i,) is exhibited across the pH range 10-14. P T U , on the other hand, exhibits a prewave (ib)in a solution of 1 M NaOH which disappears on lowering the pH. I t also exhibits a postwave (i,) in solutions of BR buffer of p H I 8. ANTU also exhibits the postwave in solutions of pH I 8 and retains the prewave throughout the p H range 7-14. T h e effect of p H on the N P P behavior of P T U is shown in Figure 2. The maximum shown on the main wave (i,) is characteristic of reactant adsorption (28). The pre- and postwaves are indicative that the adsorption isotherms give rise to saturation coverage over a relatively narrow range of concentration (29). T U also exhibits maxima on its N P P waves in sodium hydroxide solutions but no limiting current value is reached because the wave obtained for T U is followed closely by the direct oxidation of mercury. In the case of ANTU, however, the N P P behavior shows both a maximum on the main wave (iJ and a severe depression of the limiting current. This behavior indicates that ANTU is more strongly adsorbed than either T U or PTU. The best defined waves are obtained for TU in the pH range 12-14 and for P T U and ANTU in the p H range 10--14. At lower pH values, adsorption markedly affects the shape of the DC, N P P , and D P P polarograms. In the case of ANTU, the effects of adsorption are also seen to a certain degree a t these higher p H values, b u t a t p H values below 10 this effect becomes more pronounced. This is illustrated in Figure 3 for the DC, N P P , and D P P behavior of ANTU in BR buffer p H 7. Although the DC wave is markedly distorted by adsorption, the N P P and D P P waves show well defined prewaves followed by very sharp adsorption peaks. T h e best defined waves for analytical purposes were therefore obtained in 1 M NaOH solution. Effect of Concentration. The D P P calibration curves obtained for TU, PTU, and ANTU in 1 M NaOH are shown in Figure 4. From this it can be seen that T U shows two breaks from lesser to greater slope corresponding to concentrations 5 X M and 3 X 10-s M. P T U on the other hand shows no break in its calibration curve between 0-5 X M whereas ANTU shows one break corresponding to a concentration of 3.75 X 10-5 M. T h e marked differences in peak currents obtained for these compounds using D P P is not reflected in DC polarography where the ratio of illm values for TU:PTU:ANTU:BITU at the 3 X 10-5M level is 2.4:2.1:1.7:1 (assuming BITU is oxidized in a 1 e- process). These differences in magnitude are attributed to the increasing ad2312
* ANALYTICAL CHEMISTRY, VOL.
4 9 , NO. 1 4 , DECEMBER 1977
a
DC
b NPP c DPP
Figure 3. DC, NPP, and DPP waves obtained for 7 in BR buffer DH 7
LA
X
M ANTU
/
Concentration ( x l 0 - S ~1
Figure 4. Calibration curves obtained for TU, PTU, and ANTU in 1 M NaOH using DPP
sorption in the series ANTU > P T U > TU. For all three compounds, their E , values (in D P P ) move to more negative values on increasing concentration. In the case of TU, a plot of E, vs. log [TU] in the concentration range 8 X 10-'-5 X 10-5 M proved linear with slope equal to 29.75 mV. Deviations from linearity were exhibited a t lower concentrations due to the interference caused by the direct oxidation of mercury. Assuming the slope to be equal to -RT/nF (25),a value for n of 1.988 is obtained indicating that although the D P P calibration curve shows two breaks from lesser to greater slope, these do not reflect a change in the number of electrons involved in the electrode process. ANTU also exhibits a linear plot of E , vs. log [ANTU] over the M with a slope of 39.6 concentration range lo4 M to 5 X mV. P T U on the other hand, showed no variation in its E , M. At higher concentrations the slope value up to 7 X of the E , vs. log [PTU] plot was found to be 56 mV. The waves obtained for T U and P T U in 1 M NaOH were shown to be overall diffusion-controlled since plots of i ~ p vs. p 1 / ~ (where " ~ T = pulse width) gave straight lines (of slope 17.4 and 14.2 FA ms-', respectively) which passed through the origin. The effect of varying pulse width on the N P P behavior of T U in 1 M NaOH is shown in Figure 5. With increasing pulse width, increased resolution is obtained on the maximum relating to reactant adsorption. For ANTU in 1 M NaOH, the N P P wave is nearly completely suppressed but measurements of peak heights as a function of pulse width showed the peak current to be directly proportional to 1 / with ~ a slope
n
-0.79Y
E VvsSCE -0.5
--05
-03
-03
-05
-03
a
-
b c
-=100msec
zom S ~ -=somsec
C
Figure 5. Effect of varying pulse width on NPP wave obtained for 2 X M TU in 1 M NaOH: conditions, 2 mV s-’, 1-s drop time E VvsSCE 08
-A-
-OB
-04
Figure 7. Cyclic voltammogram of 5 X
-OM
h
‘DPP 0.5 ? A
1 V
i‘
1,
a = 6 gm, ‘TU b = a - 1419m i
c = . 21 d= ,~ 2 8 , e = , 35. I
.
ANT” I
,
I
I
I
,
f = . - 4 2 , ,
Figure 6. Effect of varying concentrations of ANTU on DPP peak of TU in 1 M NaOH
of 7.26 pA ms-’ (for a concentration of 5 x M). This is attributed to the strong adsorption properties of the naphthalene moiety a t the mercury surface. I t would appear, therefore, that in the case of TU, the breaks observed in the calibration plot reflect the successive formation of different complexes of thiourea with HgZf. Since the El,* value is dependent on pH, it appears that complexes of the form Hg” (OH),[TU]j (where j = 2, 3, or 4) are being formed by the oxidation of T U in alkaline media and that increasing the concentration of T U in this medium favors the formation of the succeedingly higher complex. It is likely that P T U and ANTU will exhibit similar behavior over a wider concentration range than that examined in the present investigation. As can be seen from the plots of vs. pH in Figure I , it is possible to differentiate between T U and either P T U or ANTU. The effect of varying ANTU concentration on the D P P peak of T U in 1 M NaOH is shown in Figure 6. T h e addition of succeeding amounts of ANTU causes subsequent small decreases in the peak height of TU. Although D P P cannot be used to resolve PTU and ANTU in a 1 M NaOH solution, a degree of differentiation is obtained in BR buffer, pH 10, where although P T U gives rise to a well defined D P P peak a t -0.2 V, ANTU gives rise to a badly defined double peak (EP= -0.15 and -0.1 V, respectively). BITU can be resolved from TU, PTU, and ANTU in 1 M NaOH since its E,, value ( 4 , 7 9 V) is well removed from those of P T U or ANTU (-0.5 to -0.55 V) or T U (-0.39 V).
M PTU in 1 M NaOH
These compounds can be determined using DPP down to M for T U and PTLJ,2 X lo-’ M for ANTU, about 1 x and 5 x lo-: M for BITU. In each case the detection limit (dl) was obtained from the expression dl = 3Sb/rn where Sb is the standard deviation of the background current and rn is the slope of the calibration curve. The limits of sensitivity given above were calculated as 3 >: dl. Cathodic Stripping Voltammetry. The application of cathodic stripping voltammetry (CSV) to the determination of TU, PTU, and ANTU is based on their ability to form insoluble complexes with mercury. This is a very sensitive method of analysis as has been reported for some aryl thioamides of pharmaceutical importance (30) and for 2mercaptopyridine-1-oxide (31). I t is performed by first concentrating (“plating”) the substance of interest a t the mercury surface a t a potential where it forms an insoluble mercury complex. The complex is then “stripped” out by scanning in a negative (Le., cathodic) direction. Since D P P can be used to determine concentration of T U , PTU, and ANTU down to 1 X 10.’ M, it was decided to limit this CSV investigation to concentrations lower than 2 X lo-‘ M. Since the polarographic investigation indicated t h a t different complexes of T U (and P T U and ANTU) were being formed a t higher concentrations, we were unable to study in detail the “stripping” of trace quantities of these compounds using techniques such as chronopotentiometry which are limited to the mM range. A t these low concentrations, it was assumed that the Hg”(OH),[TU], (or Hg”(OH)2[PTU]zor Hg”(OH)2[ANTU]2) species were predominant. In the (case of PTU, which does not show a break in its D P P calibration curve up to 5 X lo-’ M, we investigated its behavior a t the HMDE in 1 M NaOH using cyclic voltammetry (Figure 7). The peak obtained on scanning anodically is due to the formation of the Hg”(OH)z[PTU]2salt which is subsequently “stripped” out on scanning cathodic. The system cannot be considered to be reversible, however, since the anodic and cathodic E l ; values are separated by over 300 mV. The effect of scan rate on the LPSCSV obtained for 12 ng mL-’ TU in 1 M NaOH solution is shown in Figure 8. At low scan rates (10-20 mV s-’) two processes are observed. At higher scan rates, however, the double wave form is replaced by a single wave and this, coupled with the resulting increase in sensitivity, makes the wave obtained a t 50 mV s-’ most suitable for the determination of T U (or PTU or ANTU, both of which exhibit similar behavior t u T U under these conditions) in the 5-20 ng mL-’ range. In the lower concentration range (1-5 ng mL-’), the double peak behavior is also manifested a t 50 mV and a scan rate of 100 mV s-’ must ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
2313
n
I,
I1
03
-05
-07
-08
E VusSCE
Figure 8. Effect of s c a n rate o n LPSCSV of 1 2 ng mL-' TU in 1 M NaOH
lOnA Y
-02
-@4
--OB
-08
-,o
-18
-,4
-1.e
E VvsSCE
Figure 10. LPSCSV of normal urine spiked with 40 ng mL-' TU
V and in the disappearance of the shoulder which accompanied this peak. Work is in progress to identify the nature of this interfering substance and to develop a convenient method of analysis for the direct determination of T U in urine. It is foreseen that such a method could prove useful in the screening of patients who suffer from defects in sulfur metabolism or who have been exposed to foreign sulfur compounds in the environment. LITERATURE CITED
P
L-'E
Figure 9. DPCSV of 1 ng mL-' TU in 1 M NaOH
be employed for these lower concentrations. A calibration graph obtained for LPSCSV of 1-5 ng mL-' T U using a scan proved linear with a slope of 26 nA ng-' rate of 100 mV SKI mL. Under these conditions the limit of sensitivity of the technique is 1 ng mL-'. Although differential pulse cathodic stripping voltammetry (DPSCV) is potentially a more sensitive technique than LPSCSV, in this case the limit of sensitivity of the DPCSV method is limited by the background interference. This is illustrated in Figure 9 for a DPCSV scan of 1 ng mL-' T U in 1 M NaOH. I t is our experience that the "blank" current obtained for DPCSV scans of 1 M NaOH solutions can change from day to day and calibration graphs must be constructed prior to running an unknown solution. Although DPCSV has the ability to measure down to 100 pg mL-', fluctuations in the "blank" measurement affect both the precision and accuracy of the results in this low concentration range. I t is suggested therefore that this method of analysis also be restricted to concentrations >1 ng mL-' for use in routine situations. Although both LPSCSV and DPCSV offer greater sensitivity over the D P P method for the determination of T U , PTU, and ANTU, these techniques are unable to differentiate between these compounds in mixtures. The choice of operating technique should therefore be made on a consideration of (i) the range of concentrations likely to be present in the sample; (ii) the constituents of the mixture; and (iii) the time required for the analysis. Application of L P S C S V t o the Direct Determination of T U i n urine. Because of the high sensitivity and rapid time of analysis offered by LPSCSV for the determination of T U , it was decided to apply this technique to the direct determination of this substance in urine. The preliminary results of this investigation are shown in Figure 10 where i t can be seen that an interfering substance in the urine is stripped a t the same potential as TU. The spiked addition of T U resulted in a n enhancement of the peak around -0.8
2314
ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
"IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man", Vol. 7, Lyon, 1974, pp 95-109. W. C. Heuper, and W. D. Conway, Ed., "Chemical Carcinogenesis and Cancer", C. C Thomas, Springfield, Ill., 1964, p 37. Plant Protection Ltd., Plant Protection Conference, 1956, "Proceedings of the 2nd International Conference at Fernhurst Research Station", Butterworth, London, England, 1957, p 199. T. I. Kolyada, Ispol'z Mikroorg. I k h . Metab. Nan. Khoz., 167 (1972). M. Pergal, N. Vukojevlc, and D. Djuric, Arch. Environ. Health. 25, 42 ( 1972). W. A. Creasey, K. C. Agrawal, R. L. Capizzi, K. H. Stinson, and A. C. Sartorelli, Cancer Res., 32, 565 (1972). K . Okazaki, M. Murakami, H. Kawada, and A. Okada, Japan Kokal 75 97, 394 (Ci. GOIN), (Aug 2, 1975); Appl. 74, 1,427; from Chem. Abstr., 83, 1751592 (1975). K. A. Nuridzhanyan, V . G. Blinova, L. D. Stonov, L. A. Bakumenko, and N. M. Usacheva, Khim. Sredstva Zashch. Rast., No. 1, 197 (1970). T. Kusano, Y. Kasahava, and Y. Kawamura, Appl. Entomol. Zoo/., 10, 19 (1975). P. E. J. Wheatcroft, and C. C. Thornburn, Nature (London) New Biol., 235,93 (1972). C. P. Richter, J . Am. Med. Assoc., 129, 927 (1945). S.N. Giri and A. B. Combs, Toxicol. Appl. Pharmacol., 16, 709 (1967). E. J. Fairchild, Arch. Environ. Health, 14, 111 (1967). A. Kurian and C. V. Suryanavayana, Analyst(London),97, 576 (1972). A. A. D. Gol'tman, Khim. Issled. Farm., 83 (1970). A. B. Combs, S. N. Giri, and S.A. Peoples, Anal. Biochem., 44. 570 (1971). E. T. Rakitzis, Anal. Chim. Acta, 7 8 , 495 (1975). J. H. Ladenson, and W. C. Purdy. Anal. Chim. Acta, 5 8 , 465 (1971). M. K. Papay, K. Toth, and E. Pungor, Anal. Chim. Acta, 56, 291 (1971). S. Ikeda, and H. Satake, BunsekiKagaku, 22, 107 (1973). P. E. Holland, J. T. Peeler, and A. J. Wehby. Anal. Chem.,41, 153 (1969). E. S. Kosmatyi and V. N. Kavetskii, Zh. Anal. Khim.. 28, 1028 (1973). H. Sohr and K. Wienhold, Anal. Chim. Acta, 83,415 (1976). M. Fedoronko. 0. Manousek, and P. Zumn, Chem. Listy, 49, 1494 (1953). C. J. Nyman and E. P. Parry, Anal. Chem., 30, 1255 (1958). W. U. Malik and R. N. Goyai, Indian J . Chem., 14A, 144 (1976). R. H. Abel, J. H. Christie, L. L. Jackson, J. G. Osteryoung, and R. A. Osteryoung, Chem. Instrum., 7 , 123 (1976). J. B. Flanagan, K. Takahashi, and F. C. Anson, J . Hecfroanal. Chem., (1977) to be published. A. M. Frumkin and B. 8. Damaskin In "Modern Aspects of Electrochemistry", Voi. 3, J. O'M Bockris and B. E. Conway, Ed., Butterworth, London, 1964, Chapter 3. I. E. Davidson and W. Franklin Smyth, Anal. Chem., 49, 1195 (1977). D. A. Csejka, S. T. Nakos, and E. W. DuBord, Anal. Chem., 47, 322 (1975).
RECEIVED for review August 5 , 1977. Accepted October 4, 1977. This paper was presented a t the 174th National Meeting, American Chemical Society, Chicago, Ill., August 29-September 2,1977. Partial financial support for this work was provided through NSF Grant Number MPS 75-00332.
