Recent findings that di (2-ethylhexyl) phthalate (DEHP) caused reproductive toxicity and carcinogenicity in animals have again raised a concern over the human health implications of the widespread use of phthalate esters (PAEs) as plasticizers in poly vinyl chloride (PVC) products. Topics included in this review are 1) environmental toxicological aspects, 2) comparative metabolisms and pharmacokinetics, 3) feto/embryo toxicity and teratogenicity, 4) gonad toxicity to produce testicular atrophy, 5) genotoxic potency ; mutagenic and other genotoxic effects, 6) hepatocarcinogenicity which may be associated with peroxisome proliferation and tumor promoting activity. The results of our toxicological study on PAEs are also included.

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A method for the determination of phthalic anhydride (PA) in poly (vinyl chloride) (PVC) products by reversed-phase high performance liquid chromatography (HPLC) is described. By the reaction of PA with ethanol, PA is converted to monoethyl phthalate (MoEP), which is further methylated to methyl ethyl phthalate (MEP) with diazomethane. PA could be successfully determined by quantitating MEP. The method is as follows : one gram of chopped PVC sample and 10 mg of Ca, Zn stearate are dissolved in 10 ml of dehydrated tetrahydrofuran. Ten milliliter of ethanol is added to the solution, and the solution is refluxed for 20 min (PA→MoEP). PVC polymer is precipitated by the addition of methanol, and removed by filtration. After shaking the filtrate with cation-exchange resin, MoEP is methylated with diazomethane (MoEP→MEP). The reaction mixture is loaded on a silica gel column, and washed with 50 ml of diethyl ether-hexane (5 : 95) mixture, and eluted with 50 ml of diethyl ether-hexane (10 : 90) mixture. The eluate is evaporated to dryness, and the residue is redissolved in 1 ml of methanol. Five microliter of this solution is injected to HPLC apparatus. HPLC conditions : column, Nucleosil 5 C₈ (250 mm×4.6 mm i. d.); mobile phase, acetonitrile-water (60 : 40); flow rate, 1.0ml/min ; detector, ultraviolet detector ; detection wavelength, 254 nm. Calibration curve based on the peak height of MEP was found to be linear in a wide range of 2-1000 μg of PA. However, the amounts of PA found in PVC products were only less than 2.6 μg/g.

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Since fishes in fresh-water and seawater are well-known to have different physiological mechanisms for the regulation of their osmotic pressure, the accumulation of environmental, chemical pollutants in fish might differ between fresh-and seawater areas. To see this difference, the intakes of chlordane and pentachlorophenol (PCP) were investigated by utilizing seawater-acclimated tilapia (Tilapia nilotica), an originally fresh-water fish. When tilapia was exposed to 50 ppb chlordane-containing water for 24 h, the amount of chlordane recovered from the several organs and tissues was higher in fresh-water tilapia than in seawater-acclimated. Particularly, hepatic chlordane in fresh-water tilapia was as much as about five-fold higher than in seawater-acclimated. The same tendency was observed when chlordane-containing food was dosed. The intake of PCP was also higher, especially in the liver, in fresh-water tilapia than in seawater-acclimated, when the fishes were exposed to PCP-Na (100 ppb) for 2 and 24 h. An acute toxicity test indicated that the LC₅₀ value of PCP on seawater tilapia was approximately twice that on fresh-water tilapia. These differences between fresh-and seawater fishes in the intake and lethal toxicity of chemicals may give a new viewpoint in the ecological evaluation of chemical pollutants.

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Rapid and sensitive colorimetry of paraquat adsorbed on silica gel or florisil minicolumn (7 cm×1.2 mm) is described. The blood containing trace amounts of paraquat is deproteinized by the addition of a mixture of chloroform and methanol (3 : 2, v/v). After applying the supernatant to the silica gel minicolumn, the color reaction of paraquat is achieved by passing color-producing reagent (6% alkaline dithionite solution) through the minicolumn. A blue color due to paraquat radical (≨0.36 μg) is easily observed. After the adjustment of pH by the addition of activated alumina to pH=8.2-8.3, paraquat in the urine can be also detected by a similar method to silica gel minicolumn. For the semi-quantitative analysis of paraquat adsorbed on the minicolumn, paraquat is eluted with 1 N sodium hydroxide solution and the color-producing reagent is added to the eluate. Paraquat ion (0.72 μg in the blood 1 ml) can be easily determined by colorimetry. A similar method by using florisil minicolumn can be used for the qualitative analysis of paraquat in the blood and urine with pH above 13. These micromethods are suitable for the emergency detection of paraquat in the blood and urine of patients poisoned with paraquat.

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An improved method for extracting barbiturates and benzodiazepines was examined. These drugs were reacted with α-chloro-p-xylene to form p-methylbenzyl derivatives, which were extracted with n-hexane. The experimental procedures were as follows ; Add 3 ml of 3% sodium carbonate solution, 0.3 ml of α-chloro-p-xylene and 10 ml of acetone to 2 ml of blood sample in 30 ml Elenmyer's flask. Warm it on a water bath for 15 min keeping the original volume by adding acetone, and for 15 min without adding acetone at 80±3°C. After cooling, add 2 ml of n-hexane. Shake it for 1 min and place it into a 15 ml test tube. Centrifuge the content of the test tube for 2 min at 3000 rpm. Take 10 μl of the n-hexane layer, and analyze it by HPLC. For the extraction of barbital or nitrazepam from blood sample no interference could be observed with the compounds, such as chloropromazine hydrochloride, perazine maleate, prochloroperazine maleate, glycine, phenyl alanine, glucose, and urea.

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This report describes the trace analytical method of n-alkanes, based on the inclusion ability of urea, from a mixture of iso-alkanes, cycloalkanes, poly aromatic hydrocarbons and organic sulfur compounds. n-Alkane-urea inclusion complexes were formed by adding ethanol 2 ml of saturated with urea to 0.5 ml of a mixed solution of acetone and n-hexane (3 : 2) containing the above described compounds. The inclusion compounds formed in the hydrophobic medium were precipitated by adding ice-cold ethyl ether and then cooling for 5-10 min in an ice-bath. The precipitates were separated with centrifuge (3000 rpm, 6 min) at 5°C from organic reaction medium. The precipitates were dissolved in 30 ml of 4% Na₂SO₄ and extracted with n-hexane (total volume, 20 ml), and also the supernatant by the addition of 30 ml of 4% Na₂SO₄ was treated with n-hexane (20 ml). n-Alkanes were measured in the former n-hexane solution while iso-alkanes and/or others in the latter by each gas chromato-graphic analysis. The recoveries of n-alkanes of C₁₂ and C₁₄ were 67% and 80%, respectively, being recoverd in the past reports with yield of under 40%. The yields of n-alkanes of C₁₅-C₂₈, 2, 2, 4, 4, 6, 8, 8-heptamethylnonane, heptylbenzene, pristane, dibenzothiophene and 3, 4-benzpyrene were 91% on the average, 77%, 79%, 80%, 83% and 84%, respectively. This analytical method was applied to the solvent extracts of a bait of cultured fish and the sea bottom sediment.

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The distribution and effects of vanadium on the lipid peroxide level in the mouse lung, kidney and liver were investigated by short term inhalation of vanadium pentoxide aerosol (80 mg/m³ as V₂O₅, 1 h), a single oral and a single intraperitoneal (i. p.) administration of sodium vanadate (170 μmol/kg body weight as NaVO₃). The results obtained are as follows. 1. The highest distribution of vanadium was found in the lung in inhalation and in the kidney in case of oral and i. p. administration. 2. Accumulation and disappearance patterns of vanadium in the lung, kidney and liver were observed to have a similar tendency among 3 kinds of route of administration except lung of inhalation. That is, vanadium concentration in these tissues reached maximum value at 3 h after administration and decreased rapidly within 18 h, and then decreased slowly. 3. In the liver after i. p. administration, the highest distribution of vanadium was found in the 105000×g supernatant fraction at 3 h (64% of total fraction) and in the 10000×g pellet at 18 h (68% of total fraction). 4. Thiobarbituric acid (TBA) value, an index of lipid peroxidation, increased in the liver after inhalation and in the kidney and liver after i. p. administration at 0.5-3 h, 0.5-18 h and 7-18 h respectively. 5. The elevation of TBA value in the liver was recognized when hepatic glutathione concentrations was as high as 60-70% of control values.

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By determining methamphetamine levels in rat femora marrow by gas chromatography-mass spectrometry (GC-MS), an investigation was undertaken to estimate its serum concentration. The concentration was attained to its maximum at 5 min in the serum and at 60 min in bone marrow after administration. The ratio of serum to marrow levels at 45 to 120 min after administration was 0.140±0.021. At doses between 1-10 mg/kg the average ratio of serum to marrow methamphetamine 60 min after administration was 0.111±0.044 with a corelationship coefficient of 0.837.

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A method for the determination of mercaptobenzothiazole-type accelerators (MBTs) was studied by reversed-phase high performance liquid chromatography (HPLC) with Nucleosil 5 C 18 column by using methanol-water (95 : 5) as a mobile phase. After extraction of rubber samples by shaking with an acetone-chloroform (1 : 1) mixture at room temperature, MBTs were analyzed by HPLC with ultraviolet (UV) detector at 275 nm before and after methylating with diazomethane. Two kinds of rubber sheets for medical use and 9 kinds of commercial rubber footwears (2 kinds of rubber boots and 7 kinds of shoes with rubber soles) were analyzed by the proposed method. Consequently, only 2-mercaptobenzothiazole (MBT) and/or dibenzothiazyl disulfide (MBTS) were found in all rubber samples. The ranges of contents were as follows : MBT, 21.7-1190 μg/g ; MBTS, 27.3-392 μg/g. Patch testing to MBTs was also studied in 40 patients with allergic contact dermatitis from rubber or poly vinyl chloride (PVC) materials (rubber group or PVC group), who were the same subjects in the previous papers. Generally speaking, rubber group reacted to MBTs in higher incidence than PVC group.

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Hydrogen sulfide (H₂S) in fluid and tissue samples of rat or human was determined by gas-liquid chromatography (GLC) with a flame photometric detector (FPD). By using a stationary phase of 25% TCEP on Chromosorb W (AW-DMCS), suitable conditions were obtained and the H₂S peak appeared as a retention time of 0.7 min. The minimum detectable concentration by this method was 1 μg/ml. For the determination of fluid sample, to 1 ml (or 1 g) of sample (In the case of tissue samples : 1 g of sample was homogenized within 4 ml of cold water. After centrifugation, 1 ml of supernatant was used in the same way as fluid sample.) was added 5 ml of acetone and 0.5 ml of 20% hydrochloric acid. After mixing for 2 min and centrifuging for 5 min at 1000 g, the supernatant was used for GLC-FPD. The recoveries of H₂S from fluid and tissue samples in rat or human were about from 50 to 100%. The GLC-FPD method for the determination of H₂S is very useful in its simplicity, rapidity, efficiency and reproducibility.

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Since the available thiobarbituric acid (TBA) methods, the index of fats deterioration, have poor linearity and reproducibility in spite of high sensitivity, a new method with good linearity and reproducibility was developed and compared with the conventional methods. This new method (water extraction-TBA method) consists of extraction of only TBA reactive materials such as malondialdehyde in sample with distilled water and the reaction of the extracts with TBA. The results obtained were as follows ; 1) Much better linearity, reproducibility and lower blank values than various TBA methods were observed. 2) The recovery of malondialdehyde added to the sample was over 80% after extraction for three times. 3) A good correlation was observed between this new method and other non-TBA index of fats deterioration. These results demonstrated that this new method was useful for the index of fats deterioration.

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We investigated an analysis of trace amounts of bromide ion in water by gas chromatography. The recommended procedure is as follows ; a sample (containing more than 0.3 μg of bromide ion) was evaporated to dryness. The remaining bromide reacted with citric acid, potassium permanganate and manganse dioxide in sulfuric acid to form pentabromoacetone, which then turned into bromoform for hydrolysis in haloform reaction. The bromoform was measured by head space gas chromatography (ECD). By this method, bromide ion of 0.3-5.0 μg was determined. The coefficient of variation was 4.3% for 2.0 μg of bromide ion. It had no effect on the determination of bromide ion in the presence of coexisting substances such as chloride. The bromide contained in environmental water samples (sea, river, lake, ground, tap, and rain water) was determined successfully by the proposed method.

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