Tetraethyllead Is a Deadly Toxic Chemical Substance Giving Rise to Severe Psychotic Manifestations. for Its Excellent Properties

Home , Antiknock agent, Lead compounds, Tetraethyllead

Industrial Health, 1986, 24, 139-150.

Determination of Triethyllead, Diethyllead and Inorganic Lead in Urine by Atomic Absorption Spectrometry

Fumio ARAI

Department of Public Health St. Marianna University School of Medicine 2095 Sugao, Miyamae-ku, Kawasaki 213, Japan

(Received March 10, 1986 and in revised form May 21, 1986)

Abstract : A method was developed for the sequential extraction of tetraethyllead

(Et4Pb), triethyllead (Et3Pb+), diethyllead (Et2Pb2+) and inorganic lead (Pb2+) from one urine sample with methyl isobutyl ketone and the subsequent sequential

determination of the respective species of lead by flame and flameless atomic ab-

sorption spectrometry.

When 40 ml of a urine sample to which 2 ƒÊg of Pb of each of Et4Pb, Et3Pb+,

Et2Pb2+ or Pb2+ had been experimentally added was assayed for the respective

species of lead by flame atomic absorption spectrometry, ten repetitions of the

assay gave a mean recovery rate of 98% for each of Et4Pb, Et3Pb+, and Et2Pb2+,

and 99% for Pb2+, with a coefficient of variation of 2.0% for Et4Pb, 0.7% for

Et3Pb+ and Pb2+, 2.6% for Et2Pb2+, and a detection limit of 4 ƒÊg of Pb/L for

Et4Pb, 3 ƒÊg of Pb/L for Et3Pb+, and 5 ƒÊg of Pb/L for each of Et2Pb2+ and Pb2+.

Examination of urine samples from a patient with tetraethyllead poisoning 22

days after exposure to the lead revealed that the total lead output was made up of

about 51% Pb2+, about 43% Et2Pb2+, and about 6% Et3Pb+ but no Et4Pb. Ad-

ministration of calcium ethylenediaminetetraacetic acid (Ca-EDTA) was followed

by no increased urinary excretion of Et3Pb+ or Et2Pb2+.

Key words : Tetraethyllead poisoning—-Urinary lead—Triethyllead-Diethyllead- Inorganic lead

INTRODUCTION

Tetraethyllead is a deadly toxic chemical substance giving rise to severe psychotic manifestations. For its excellent properties as an antiknock agent for gasoline engines, however, its use as an additive to gasoline is widespread on a worldwide scale, and consequently it has been responsible for a number of tetraethyllead poisoning cases. Tetraethyllead (Et4Pb), when administered to experimental animals, is metaboli- cally degraded in vivo to triethyllead (Et3Pb+), then to diethyllead (Et2Pb2±) and finally to inorganic lead (Pb2+) before it is excreted from the body.1-4) Henderson and Snyder, 5) Bolanowska2) and Imura et al.6) have reported colorimetric methods 140 F. ARAI for the determination of Et3Pb+, one of the metabolites of tetraethyllead, and in addition, a microassay by gas chromatography (GC),7) hydride generation-atomic absorption spectrometry (AAS),8) and GC-AAS9) have been established. For the determination of Et2Pb2+, colorimetric methods,5, 10) hydride generation-AAS,8) anodic stripping voltammetry,11) GC,12) GC-AAS,9) and flameless AAS13) have been established. From the colorimetric assay of urine in cases of acute tetraethyllead poisoning, Bolanowska et al.14) reported that Et3Pb+ accounted for a small percentage of total lead in the urine samples. Chiesura15) determined by colorimetry the benzene-extractable lead (Et3Pb+), precipitable lead (Pb2+) and unprecipitable lead (organic lead) in the urine of workers exposed to tetraethyllead. He further showed that precipitable lead (Pb2+) accounted for 20% ; benzene-extractable lead (Et3Pb+), about 10% , and unprecipitable lead (organic lead), about 80-90% of total lead in the urine of patients with Et4Pb poisoning ; that the difference between the amount of this unprecipitable lead and that of the benzene-extractable lead represented the amount of Et2Pb2+, and that this species (Et2Pb2+) was the main metabolite of tetraethyllead. In a previous paper16) on the determination of Et3Pb+, Et2Pb2+ and Pb2+ in the urine of a patient with tetraethyllead poisoning by hydride generation-AAS, the author and his collaborators reported that Et2Pb2+ accounted for about 50% of total lead in the urine 21 days after exposure to tetraethyllead. As a result of assay by flameless AAS of urine samples from tetraethyllead poisoning patients hospitalized after drinking 10-100 ml of tetraethyllead, Turlakie- wicz et al.13) reported that about one half of the total lead in the urine was made up of Et2Pb2+. In another of urine samples from 26 workers exposed to tetraethyllead at work, Turlakiewicz and Chmielnicka17) revealed that the tetraethyllead concentration in the air was intimately correlated with that in the urine, and that Et2Pb2+ could be a specific indicator of occupational tetraethyllead exposure. In the present study, the author developed a method for sequentially extracting Et4Pb, Et3Pb+, Et2Pb2+ and Pb2+ from one urine sample with an organic solvent, and determining the respective species of lead sequentially by flame and flameless AAS, and performed a trial determination of the respective species of lead in the urine of a patient with tetraethyllead poisoning.

MATERIALS AND METHODS

1. Reagents Et4Pb standard solution : A sample of tetraethyllead (Strem Chemicals Inc., U.S.A.) was washed with a 1 : 4 mixture of redistilled water and ether 3 times, and the ether was evaporated before use. The lead content of the washed tetraethyllead sample was found to be 98% of the theoretical value. This purified DETERMINATION OF ETHYLLEAD COMPOUNDS BY A. A. S. 141 compound contained 0.06% Et3Pb+, but neither Et2Pb2+ (not more than 0.004%) nor Pb2+ (not more than 0.006%). After the washing, 0.166 g of the tetraethyllead sample was diluted with 25 ml of benzene, and then with ethanol. Et3Pb+ standard solution : A sample of triethyllead chloride (Alfa Division, Ventron Corp., U.S.A.) was purified by recrystallization from a 7 : 3 mixture of redistilled water and ethanol. The lead content of this triethyllead chloride sample was determined to be 98% of the theoretical value. This compound contained neither Et2Pb2+ (not more than 0.1%) nor Pb2+ (not more than 0.1%). After the purification, 3.30 mg of the sample was dissolved in 100 ml of water. Et2Pb2+ standard solution : A sample of diethyllead dibenzoate was synthesized from tetraethyllead by the method of Heap et al.18 This sample was mixed with gaseous hydrogen chloride to synthesize diethyllead dichloride, and the product recrystallized from a 1 : 9 mixture of redistilled water and ethanol was purified. The lead content of this sample was 99% of the theoretical value. The compound contained neither Et3Pb+ (not more than 0.06%) nor Pb2+ (not more than 0.1%). After the purification, 3.35 mg of the diethyllead dichloride sample was dissolved in 100 ml of water. Pb2+ standard solution : The Pb2+ standard solution was prepared by dissolving 100 mg of metal lead (Soekawa Chemical Co., Ltd., Tokyo) in 100 ml of 1 N HNO3. These 3 standard lead (Et3Pb+, Et2Pb2+ and Pb2+) solutions were diluted with redistilled water as necessary before use. Glyoxal-bis(2-hydroxyanil) solution10) (GHA) : In 100 ml of methanol was dissolved 0.24 g of dotite GHA (Dojindo Co., Ltd., Kumamoto), and the solution was allowed to stand in a brown bottle at room temperature for 24 hours before use. The nitric acid, perchloric acid, ammonium hydroxide, sodium diethyldithio- carbamate (SDDC) and methyl isobutyl ketone (MIBK) were of super special grade, and the other reagents were of special grade.

2. Procedures for determination

For the experimental determination of Et4Pb, Et3Pb+ and Et2Pb2+ in the urine,

40 ml of a urine sample was transferred to a separating funnel and the respective

species of lead were sequentially extracted with an organic solvent (MIBK), and

determined by flame and flameless AAS. For the determination of Pb2+ in the urine, this species was isolated as a

precipitable lead from the urine sample by the method of Bolanowska.2) The

precipitated Pb2+ was transferred to a Uniseal decomposition vessel (Uniseal Decomposition Vessels, Ltd., Israel), and decomposed with heat and pressure in

5 ml of concentrated nitric acid (130•Ž, 90 minutes), then extracted with an

organic solvent (SDDC-MIBK), and determined by AAS.

For the determination of total lead in the urine, 10 ml of a urine sample was 142 F. ARAI transferred to a 70-ml Uniseal decomposition vessel, and after degradation with heat and pressure in 5 ml of concentrated nitric acid, the lead was extracted with SDDC-MIBK and determined by AAS.

3. Procedures for extraction Figure 1 illustrates the flowchart of the procedures for sequentially extracting Et4Pb, Et3Pb+ and Et2Pb2+ from a urine sample with an organic solvent, and at the same time extracting Pb2+ with an organic solvent following its isolation as a precipitable lead. The respective procedures are described below. Et4Pb : Transfer 40 ml of the urine sample to a 200-ml separating funnel, add 1 ml of 1M dibasic ammonium citrate and 10 ml of MIBK, shake vigorously for 10 minutes, and allow the mixture to stand at room temperature for 15 minutes. Then transfer the top MIBK layer to another 200-ml separating funnel, wash with 20 and then 10 ml of 1 HNO3 by shaking gently for 1 minute, combine the washings, and allow them to stand at room temperature for 15 minutes. Use this MIBK layer as the sample for the determination of Et4Pb. Transfer washing (b) (see Fig. 1) to another 200-ml separating funnel for later use as the sample for the determination of Et3Pb+.

Fig. 1. Procedure for extraction of lead. DETERMINATION OF ETHYLLEAD COMPOUNDS BY A. A. S. 143

Pb2+ : Transfer the bottom water layer (a) to a 200-ml conical flask, and after the addition of 3.5 ml of 60% HClO4, allow the mixture to stand at room temperature for 1 hour, and adjust it with ammonia water to a pH of 4.5. Then add 5 ml of saturated ammonium oxalate solution and 0.5 ml of 10% calcium chloride solution, to produce a white precipitate, and allow the mixture to stand at a low temperature (+4•Ž) for 1 hour. Transfer this suspension into a 60-ml centrifuge tube, centrifuge it at 2500 g for 15 minutes, and transfer the supernatant

(c) to another 200-ml separating funnel. Use the solution as the sample for the determination of Et3Pb+ and Et2Pb2+. Dissolve the residue in the centrifuge tube in 5 ml of concentrated HNO3, transfer it to a Uniseal decomposition vessel, and decompose it with heat in a drying oven at 130•Ž for 90 minutes. Transfer this decomposed product into a 200-ml conical flask with about 50 ml of redistilled water, add 1 ml of 1 M dibasic ammonium citrate, adjust mixture with ammonia water to a pH of 7, add 10 ml of MIBK and 2 ml of 3% SDDC, and shake it vigorously for 10 minutes to extract Pb2+ into the MIBK layer. Use the

MIBK layer as the sample for the determination of Pb2+.

Et3Pb+ : To supernatant (c) after centrifugation, add 18 g of NaCl and 10 ml of MIBK, and shake vigorously for 10 minutes to extract the residual Et3Pb+ into the MIBK layer (the bottom water layer is later used as the sample for the determination of Et2Pb2+). Back-extract Et3Pb+ from this MIBK layer into 20 and 10 ml of 1 HNO3. Combine the extracts and add them to the washing of Et4Pb (b), and after the addition of 18 g of NaCl and 5 ml of MIBK, shake the mixture vigorously for 10 minutes to extract Et3Pb+ into the MIBK layer.

Use this MIBK layer as the sample for the determination of Et3Pb+.

Et2Pb2+ : Adjust the bottom water layer (d) from which Et3Pb+ has been removed to a pH of 7 with ammonia water, extract Et2Pb2+ with 2 aliquots of 2 ml of

3% SDDC and then 10 ml of MIBK into the MIBK layer by shaking vigorously for 10 minutes, combine the extracts, and back-extract with 40 and then 20 ml of

1% HNO3 by shaking gently for 1 minute. Combine the extracts, add 1 ml of

1 M dibasic ammonium citrate, adjust the mixture with 40% NaOH to a pH of

9.8, add 4 ml of GHA and 5 ml of MIBK, and shake vigorously for 10 minutes to extract Et2Pb2+ into the MIBK layer. Use this MIBK layer as the sample for the determination of Et2Pb2+.

4. Apparatus The determination of the species of lead was performed with a Shimadzu atomic absorption/flame emission spectrometer model AA-640-01 (Shimadzu Sei- sakusho, Kyoto) at a wavelength of 283.3 nm. For flame AAS, a 10-cm slit burner was attached to the spectrometer, to produce an acetylene-air flame. For flameless AAS, a Shimadzu peak catcher model PCA-1 was connected with the spectrometer, and a Shimadzu graphite furnace atomizer model GFA-4A was attached to the spectrometer, using argon 144 F. ARAI

Table 1. Analytical conditions of flameless AAS for urinary leads.

5. Urine samples

Urine specimens from a 48-year-old man with tetraethyllead poisoning19) were used as samples. The patient was exposed to tetraethyllead while cleaning an aviation fuel tank on December 9, 1974. He recovered 2 months after admission, when he was discharged. The author determined Et4Pb, Et3Pb+, Et2Pb2+, Pb2+ and total lead in the urine samples collected 22, 24, 27 and 29 days after the exposure, and also determined Et4Pb, Et3Pb+, Et2Pb2+ and total lead in the urine collected after intravenous drip of 1000 mg of Ca-EDTA per day (34 days after the exposure).

All urine samples were preserved frozen (at -20•Ž) until they were assayed.

RESULTS

1. Recovery rate and coefficient of variation

Table 2 shows the mean recovery rates of the 4 species of lead and the coefficients of variation in 10 repetitions of flame AAS and also 10 repetitions of flameless AAS of 40 ml of a normal human urine sample to which 2 ƒÊg of Pb and 20 ƒÊg of Pb each of Et4Pb, Et3Pb+, Et2Pb2+ and Pb2+ had been added

(the addition of 2 ƒÊg of Pb making a Pb concentration of 50 ƒÊg of Pb per liter of urine, and that of 20 ƒÊg of Pb, 500 ƒÊg of Pb per liter of urine). The recovery rates of the respective species of lead were as high as 95.4•`99.8% , associated with high reproducibility, the coefficient of variation being 0.4•`3.6 % . The flame AAS gave a slightly higher recovery rate and accuracy than the flameless

AAS.

2. Detection limit and specificity

The detection limits of the flame and flameless AAS's for the species of lead were 3•`5 ƒÊg of Pb/L (Table 3). Fig. 2 depicts the calibration curve for 2, 4,

10, 14 and 20 ƒÊg of Pb of Et2Pb2+ added to 40 ml of a urine sample. There was a good linear relation for not more than 20 ƒÊg of Pb. In the determination of Et4Pb, Et3Pb+, Et2Pb2+ and Pb2+ in the urine, only the target species of lead DETERMINATION OF ETHYLLEAD COMPOUNDS BY A. A. S. 145

Table 2. Recovery rates and coefficients of variation of lead from urine by Came AAS or flameless AAS.

Table 3. Limits of detection of lead from urine by flame AAS and flameless AAS.

3. Urine of a patient with tetraethyllead poisoning Table 4 shows the concentrations of Et4Pb, Et3Pb+, Et2Pb2+, Pb2+ and total lead in the urine of a patient with tetraethyllead poisoning at 22, 24, 27 and 29 days after the exposure to the lead as determined by flame AAS. The total lead in the urine 22 days after the exposure was composed of about 51 Pb2+, about 43% Et2Pb2+, and about 6% Et3Pb+ (total lead = Et3Pb+ +Et2Pb2+ + Pb2+). The total lead in the urine 24 days after the exposure was made up of about 63% Pb2+, about 28% Et2Pb2+, and about 9% Et3Pb+. The total lead in the urine 29 days after the exposure consisted of about 60% Pb2+, about 29% Et2Pb2+, and about 11 Et3Pb+. No Et4Pb was thus detected in the urine at any stage. Table 5 shows the assays of the sample shown in Table 4 but by AAS with the attachment of the flameless atomizer. There was no great difference between 146 F. ARAI

Fig. 2. Calibration curve for urinary Et2Pb2+.

Table 4. Concentrations of Et4Pb, Et3Pb+, Et2Pb2+, Pb2+ and total lead in the urine of a patient with Et4Pb poisoning as determined by flame AAS.

Table 5. Concentrations of Et4Pb, Et3Pb+, Et2Pb2+, Pb2+ and total lead in the urine of a patient with Et4Pb poisoning as determined by nameless AAS. DETERMINATION OF ETHYLLEAD COMPOUNDS BY A. A. S. 147 the concentrations of any species of lead by flameless and flame AAS. Table 6 shows the concentrations of Et4Pb, Et3Pb+, Et2Pb2+ and total lead in the urine after the administration of Ca-EDTA. On the 34th day after the exposure, intravenous drip of 1000 mg of Ca-EDTA per day was begun at noon, and each urine specimen voided thereafter was collected. The total lead in the urine collected at 12 : 30 hours on the 34th day was made up of about 5% Et2Pb2+ and about Et3Pb+, while that in the urine collected at 02: 30 hours on the following2% day was composed of about 12% Et2Pb2+ and about 1 Et3Pb+. The concentration of total lead in the urine collected at 12 : 30 hours on the 34th day and that in the urine collected at 02: 30 hours on the following day was two or more times as high as that in the urine collected on the 29th day after the exposure, while the concentrations of Et2Pb2+ and Et3Pb+ did not increase. Table 7 shows that assays of the samples shown in Table 6 by AAS using the flameless atomizer. There was practically no difference between the assays by flameless and flame AAS.

Table 6. Concentrations of Et4Pb, Et3Pb+, Et2Pb2+ and total lead in the urine of a patient with Et4Pb poisoning after intravenous drip of Ca-EDTA as determined by flame AAS.

Table 7. Concentrations of Et4Pb, Et3Pb+, Et2Pb2+ and total lead in the urine of a patient with Et4Pb poisoning after intravenous drip of Ca-EDTA as determined by flameless AAS. 148 F. ARAI

DISCUSSION

In a previous study, the author and his collaborators developed the hydride generation-AAS method for the determination of Et3Pb+, Et2Pb2+ and Pb2+ in the urine.8) This method, however, requires a special apparatus and reagents for the generation of the hydride. The apparatus is composed of a glass reaction bottle, a cooling glass tube and a quartz U-letter tube, with the quartz cell fitted to the atomizing section. An acid and sodium borohydride (in 0.2N NaOH solution) are required to generate lead hydride, and liquid nitrogen is used to fix the lead hydride, using helium gas as the carrier. This method, converting Et3Pb+, Et2Pb2+ and Pb2+ into the respective hydrides and isolating the respective species by the difference between their boiling points, is characterized by its ability to detect the respective lead ions directly. The hydride-generating reaction is complicated, and requires skill. The method developed in the present paper is based on the same procedures for extraction as used in the hydride-generating method by which the respective species of lead in a urine sample are extracted sequentially into an organic solvent and then assayed by flame or flameless AAS. For the flame AAS, a 10 cm slit burner was used as the lead atomizing apparatus, and for the flameless AAS, a graphite furnace atomizer was used. This method allows the samples extracted from a urine sample to be led directly into an atomizer for the rapid determination of the species of lead. The author used this method in the determination of the species of lead in the urine from a patient with tetraethyllead poisoning. The assays of the urine specimens from this patient at 24, 27 and 29 days after the exposure to tetraethyllead by the hydride generation-AAS method are reported in a previous paper.16) The urine samples from the same patient at 22 and 34 days after the exposure were assayed for the first time in the present study. The total lead in the urine taken 22 days after the exposure contained about 43% Et2Pb2+. The ratio of Et2Pb2+ was slightly low in the later samples, and it was about 29 % in the urine taken 29 days after the exposure. The ratios of Et2Pb2+ to total lead as determined in the present study were similar to those found in the previous study. In the previous study, the ratio of Et2Pb2+ to total lead was about 50 % in the urine at 21 days, and about 51 in the urine taken at 23 days after the exposure. The urine voided after the intravenous drip of Ca-EDTA was then assayed. No Pb2+ was detected in this urine. This appears to be the result of the lack of formation of precipitable lead due to chelation of Pb2+ with EDTA.1,15) The concentration of total lead in the urine after the administration of Ca-EDTA was about twice as high as that in the urine taken 29 days after the exposure or before the administration, while neither Et2Pb2+ nor Et3Pb+ increased. These find- ings suggest that the administration of Ca-EDTA may not be effective for elimi- nating organic lead. DETERMINATION OF ETHYLLEAD COMPOUNDS BY A. A. S. 149

ACKNOWLEDGEMENT

The author would like to acknowledge the continuing guidance of Prof. Y. Yamamura, Department of Public Health, School of Medicine, St. Marianna Uni- versity.

REFERENCES

1) Cremer, J. E. (1959). Biochemical studies on the toxicity of tetraethyl lead and other organo-lead compounds, Br. J. Ind. Med., 16, 191. 2) Bolanowska, W. (1968). Distribution and excretion of triethyllead in rats, Br. J. Ind. Med., 25, 203. 3) Arai. F.. Yamamura. Y. and Yoshida, M. (1981). Excretion of triethyl lead, diethyl lead and inorganic lead after injection of tetraethyl lead in rabbits, Jpn. J. Ind. Health, 23, 496 (In Japanese). 4) Arai, F., Yamamura, Y., Yamauchi, H. and Yoshida, M. (1983). Biliary excretion of diethyl lead after injection of tetraethyl lead in rabbits, Jpn. J. Ind. Health, 25, 175 (In Japanese). 5) Henderson, S. R. and Snyder, L. J. (1961). Rapid spectrophotometric determination of triethyllead, diethyllead, and inorganic lead ions, and application to the determination of tetraorganolead compounds, Anal. Chem., 33, 1172. 6) Imura, S., Fukutaka, K., Aoki, H. and Sakai, T. (1971). Spectrophotometric determination of triethyllead in aqueous solution, Japan Analyst, 20, 704 (In Japanese). 7) Hayakawa, K. (1971). Microdetermination and dynamic aspects of in vivo alkyl lead compounds, Part I. Analytical methods, Jpn. J. Hyg., 26, 377. 8) Yamauchi, H., Arai, F. and Yamamura, Y. (1981). Determination of triethyllead, diethyllead and inorganic lead ions in urine by hydride generation-flameless atomic absorption spectrometry. Ind. Health, 19, 115. 9) Chau, Y. K., Wong, P.T.S. and Kramar, 0. (1983). The determination of dialkyllead, trialkyllead, tetraalkyllead and lead (II) ions in water by chelation/extraction and gas chromatography/atomic absorption spectrometry, Analytica Chimica Acta., 146, 211. 10) Imura, S., Fukutaka, K. and Kawaguchi, T. (1969). Spectrophotometric determination of dialkyllead ion in aqueous solution with glyoxal-bis (2-hydroxyanil), Japan Analyst, 18. 1008 (In Japanese). 11) Birnie, S. E. and Hodges, D. J. (1981). Determination of ionic alkyl lead species in marine fauna, Environ. Technol. Lett., 2, 433. 12) Forsyth, D. S. and Marshall, W. D. (1983). Determination of alkyllead salts in water and whole eggs by capillary column gas chromatography with electron capture detection, Anal. Chem., 55, 2132. 13) Turlakiewicz, Z., Jakubowski, M. and Chmielnicka, J. (1985). Determination of diethyllead in the urine by flameless atomic absorption spectrometry, Br. J. Ind. Med., 42, 63. 14) Bolanowska, W., Piotrowski, J. and Garczynski, H. (1967). Triethyllead in the biologi- cal material in cases of acute tetraethyllead poisoning, Archiv far Toxikologie, 22, 278. 15) Chiesura, P. (1970). Urinary excretion of lead tetraethyl catabolites in man, Med. Lavoro, 61, 437 (In Italian). 16) Yamamura, Y., Arai, F. and Yamauchi, H. (1981). Urinary excretion pattern of triethyllead, diethyllead and inorganic lead in the tetraethyllead poisoning, Ind. Health, 19, 125. 17) Turlakiewicz, Z. and Chmielnicka, J. (1985). Diethyllead as a specific indicator of 150 F. ARAI

occupational exposure to tetraethvllead. Br. J. Ind. Med.. 42. 682 18) Heap, R., Saunders, B. C. and Stacey, G. J. (1951). Organo-lead compounds. Part IV. (a) A new method for preparing diethyl-lead salts. (b) Derivatives of mixed plum- banes, J. Chem. Soc., 658. 19) Yamamura, Y., Takakura, J. Hirayama, F., Yamauchi, H. and Yoshida, M. (1975). Tetraethyl lead poisoning caused by cleaning work in the aviation fuel tank, Jpn. Ind. Health, 17, 223 (In Japanese).