Environ. Sci. Technol. 1987,2 1 , 188-193
Toxicity of Methyl Isocyanate Ernest E. McConnell," John R. Bucher, Bernard A. Schwetr, Bhola N. Gupta, Michael D. Shelby, Michael I. Luster, Arnold R. Brody, Gary A. Boorman, and Conrad Richter
National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 Mlchael A. Stevens and Bernard Adklns, Jr.
Northrop Services, Inc., Research Triangle Park, North Carolina 27709
The immediate and delayed effects of acute inhalation exposure to methyl isocyanate (MIC), the chemical reported to be the primary causative agent in the disaster in Bhopal, India, were studied in rats and mice. The acute disease was manifested as necrosis of the epithelial lining of the nasal passages, trachea, and bronchi with effusion of proteinaceous fluids and hemorrhage into the airways. Subsequently, survivors showed mural and intraluminal fibrosis of the larger airways and focal obstructive lung disease. No other target organs were identified, and there were no or minimal effects on fertility, reproduction, and immune defense mechanisms. MIC was not mutagenic in the Sulmonella reversion assay, was genotoxic in cultured mammalian cells, and produced marginal evidence of chromosomal damage in the bone marrow of mice.
Introduction Methyl isocyanate (MIC) is a highly toxic chemical that was responsible for the deaths of over 2000 people, hospitalization of over 50 000, and significant exposure of over 320000 people in Bhopal, India. The release of the gas occurred early on the morning of December 3,1984, lasted some 40 min, and over a period of 1.5-2 h blanketed an area of approximately 60 km2. The gas covered a significant portion of Bhopal, which has a population of approximately 900000 people (1-3). The disaster in Bhopal was apparently caused by the introduction of water into an MIC storage tank resulting in a severe exothermic reaction ( 4 ) . Several safety precautions in place at the chemical plant were reported to have failed, and approximately 20 tons (h18000 kg) of MIC and possibly toxic reaction products may have escaped into the atmosphere (5). MIC is used for manufacture of various pesticides, the most important of which are aldicarb, carbaryl, carbofuran, and methomyl(6). MIC, made by reacting methylamine with phosgene, has a boiling point of 39.1 "C at 1 atm, a density of 0.960 g/cm3, and a vapor pressure of 348 Torr at 20 OC (7). It is flammable and highly reactive with water, U.S. production is estimated to be between 30 and 35 million pounds per year (6). MIC is highly irritating and causes severe bronchial spasms, asthmatic breathing, and chemical pneumonia when toxic levels are inhaled. Contact with intact skin, mucous membranes, and other moist surfaces such as the eyes causes irritation and burns (7). The initial symptoms experienced by the exposed individuals in Bhopal were those of acute fulminating inflammation of the respiratory tract, with death ascribed to severe pulmonary edema and hemorrhage. In addition, the reported clinical syndrome included severe keratitis (8). In the year following exposure, reports have indicated a regression of eye lesions; however, respiratory impairment was reported in over 40 000 people 3 months after exposure and appears to still be present in many survivors more than 1 year later. 188
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Pulmonary compromise was reported to interfere with work performance and routine physical exercise. Additional complaints included sterility, spontaneous abortions, and stillbirths (9). The National Institute of Environmental Health Sciences (NIEHS) initiated a series of studies to address two broad questions arising out of the Bhopal disaster: first, to determine the nature of short- and long-term sequelae arising from a single brief (2-h) exposure to MIC and, second, to broaden the presently scant knowledge of the toxicology of MIC. Studies selected included in vitro and in vivo genetic toxicity tests, pathology and general toxicity in two species (rats and mice) with focus on the respiratory tract as well as identification of other possible target organs, correlative pulmonary function studies, effects on reproduction and fertility, effects on neonatal development, effects on the immune system and host defense, and carcinogenicity.
Experimental Procedures Materials. MIC was obtained from Union Carbide and was determined to be greater than 99% pure by gas chromatography/flame ionization analysis. The MIC was supplied in stainless steel cylinders and was stored in a ventilated Plexiglass cabinet in a secure facility. The MIC was delivered to a supply manifold (500 mL) prior to distribution to each of three 1330-L inhalation exposure chambers with dry, carrier-grade nitrogen (National Welding Supply, Raleigh, NC). A nitrogen bypass was used to purge the delivery system of residual MIC following exposures. The flow of MIC into the exposure chambers was remotely computer-controlledwith stainless steel metering valves (10). Chamber concentrations of MIC were controlled to f10% over a range of 0-30 ppm. MIC concentrations were continuously monitored in each chamber to a detection limit of 0.2 ppm by independent infrared spectrometers (Wilks Miran 80, Foxboro, Waltham, MA) operating at an analytical wavelength of 3.3 pm and a reference wavelength of 3.6 pm. MIC was monitored every 2 h to a detection limit of 20 ppb in the negative control exposure chamber, exposure room air, and in the effluent exhaust air downstream from bag-in/bag-out scrubbers (type CG whetlerized activated carbon, Barnebey and Chaney, Columbus, OH) by a high-performanceliquid chromatograph (HPLC, Waters Model 780, Milford, MA) equipped with a fluorescence detector. The HPLC analysis was performed on MIC-fluorescent adducts (fluorescamine and fluram, Hoffmann-La Roche, Inc., Nutley, NJ) following tetrahydrofuran (Fisher Scientific Co., Fair Lawn, NJ) elution of the air samples taken on XAD-2 resin (amberlite, Supelco, Bellefonte, PA) (11). Animals used for the studies were 6-10 week old F344N rats and B6C3F1 mice of both sexes (Charles River Laboratories, Inc., Kingston, NY, and Portage, MI). Also, sexually mature CD-1 mice were used for the dominant lethal test, the mating trials, and the assessment of neo-
0013-936X/87/0921-0188$01.50/0
0 1987 American Chemical Society
natal development. A total of 91 cohort mice and 50 cohort rats, representing each shipment of animals, were examined serologically and microbiologically for indigenous rodent pathogens. In addition, a total of 100 sentinel mice and 40 sentinel rats were collected from animal rooms over the course of the studies and similarly examined. Except for one group of animals inadvertently shipped from Charles River Kingston (B6C3F1,EDIM positive), no other cohort or sentinel animals were positive for any of the agents tested, which included RCV, Sendai, PVM, KRV, H-1, MHV, GDVII, EDIM, mycoplasma, enteric pathogens, and external and internal parasites. Pathology. In the general toxicology studies, rats and mice were subjected to a single 2-h exposure to 0, 3, 10, or 30 ppm of MIC. Five rats or mice of each sex and dose were killed by intraperitoneal overdose of sodium pentobarbital within 3 h follpwing exposure and at 1, 3, 7, 14, 28,49, and 91 days postexposure. Animals found dead or moribund were also necropsied (up to five/sex/dose/ species/day) and were saved in formalin. For scheduled sacrifices the following tissues were examined histologically: nasal passages (three levels), nasopharynx, trachea at the level of the thyroid and at the bifurcation, all lobes of the lung, liver, thyroid, parathyroid, esophagus, peribronchial lymph nodes, brain, eyes, kidneys, thymus, spleen, heart, and glandular and nonglandular stomach. On days 7 and 91, a more detailed microscopic examination was performed. In addition to the above tissues, pancreas, salivary gland, mandibular lymph nodes, adrenals, pituitary,larynx, six levels of intestinal tract, mesenteric lymph nodes, abdominal skin, mammary gland, urinary bladder, femur including bone marrow, and sex organs (males, testes, epididymis, seminal vesicles, prostate, and preputial glands; females, ovaries, uterus, and clitoral glands) were also examined. The head was removed at the atlanto-occipital joint, and the nasal cavity was infused (retrograde) through the nasopharynx with a few milliliters of formalin to assure adequate fixation. The lung was infused with 4-10 mL of formalin (rats) or 2-4 mL of formalin (mice) through the trachea, the trachea tied off, and the lung immersed in formalin. After fixation of the head, excess skin, fascia, and muscle were removed, and the head was placed in decalcifying solution (American Scientific Products, McGraw Park, IL). After decalcification overnight, three separate transverse sections were taken (1)a t the level of the incisor teeth, (2) midway between incisors and the first molar, and (3) at the middle of the second molar (olfactory region). Selected specimens were embedded in paraffin, sectioned 6 ym thick, and stained with hematoxylin and eosin. Animals were examined daily for moribundity and were weighed periodically. The following organs were weighed a t scheduled necropsies: liver, thymus, kidney, brain, lung, spleen, and testes. Wet and dry lung weights were determined on male rats on days 0, 1, 3, and 7. Electron microscopy (scanning and transmission) was conducted on the lungs of male rats exposed to 0,3, and 10 ppm on days 0, 1, 3, 7, and 28. Blood was collected from the hearts of rats killed on days 0, 1, 7, and 14 for hematology (complete blood count), methemoglobin levels, and clinical chemistry including creatinine, alanine aminotransferase, sorbitol dehydrogenase, aspartate aminotransferase, and blood urea nitrogen. Cholinesterase activity was determined on whole blood and on brain homogenates (for methods, see ref 12). Pulmonary Function. On days 7,14, 28,49, and 91 six male rats were tested with a battery of pulmonary function measurements that included lung volumes (vital
capacity, residual volume, total lung capacity, end expiratory volume), tidal breathing, diffusing capacity, ventilation distribution, and lung compliance. For pulmonary function measurements, pentobarbital-anesthetized rats were tracheostomized and placed in a whole body pressure-volume plethysmograph. Vital capacity was measured directly, while total lung capacity, residual volume, and single-breath diffusing capacity were assessed by gas dilution methods previously described (13). After temporary occlusion of the animal’s airway at end expiration, the end expiratory volume was calculated from an application of Boyle’s Law. Lung compliance was expressed as the slope of the deflation wing of the quasi-static pressure-volume curve, and the homogeneity of ventilation distribution was assessed by the multibreath nitrogen washout technique (14). Reproductive and Prenatal Toxicity Testing. Male and female CD-1 mice were exposed to MIC at concentrations of 0, 1, or 3 ppm for 6 h per day for 4 consecutive days. Exposed males were mated with groups of untreated females a t weekly intervals for 8 consecutive weeks and fetal resorption and neonatal survival determined. Male and female CD-1 exposed mice also underwent mating trials during the first and eighth weeks following exposure. The effects of MIC exposure on late-term pregnancy were examined by exposing pregnant female CD-1 mice to MIC at concentrations of 0, 1, or 3 ppm for 6 h per day on gestation days 14-17. These mice were permitted to deliver their offspring at which time neonatal survival and development was evaluated. Body weight data were analyzed by analysis of variance and Dunnett’s test. Fertility indices were analyzed by the Fischer exact probability test. Resorption data were analyzed by a modified Wilcoxon test. Immunotoxicity Testing. Immunotoxicity studies were conducted in female B6C3F1 mice within 5 days following a 6-h exposure to 0, 1, or 3 ppm of MIC for 4 consecutive days. Immune function tests were performed as previously described (15,16) and included the antibody plaque-forming cell (PFC) response to sheep red blood cells and lymphoproliferation to the polyclonal activators phytohemagglutinin, concanavalin A, lipopolysaccharide, and allogeneic leukocytes, as well as natural killer (NK) cell activity. In addition to immune function tests, the effects of MIC exposure on the ability of mice to resist challenge with selected infectious agents or transplantable tumor cells were examined. The infectivity models selected for examination included resistance to infection with Listeria monocytogenes, mouse malaria (Plasmodium yoelii 17XNL), influenza A2/Taiwan/64 virus, and B16F10 syngeneic tumor cells as previously described (17). Mice were monitored for mortality, parasitemia, or lung tumor foci development depending upon the challenge model employed. Immune function tests were analyzed by the Wilk-Shapiro test for normality, one-way analysis of variance, and Dunnett’s test for multiple comparisons with a control. Mortality data were analyzed by the x2 test. Genetic Toxicity. In vitro tests of genetic toxicity included the Salmonella reversion assay (Ames test), mouse lymphoma assay, and sister chromatid exchange (SCE) and chromosomal aberrations (CA) in cultured Chinese hamster ovary (CHO) cells. Salmonella tests were conducted on five strains (TA98, TA100, TA1535, TA1537, and TA97) in the presence and absence of Aroclor 1254 induced rat and hamster liver S9 and a dose range of 0.3-333.0 pg/plate (18). The mouse lymphoma assay was carried out in the absence of S9 with a dose range of 0.5-3.0 yg/mL (19). CHO cytogenetics tests were conducted in Environ. Sci. Technol., Vol. 21, No. 2, 1987
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the absence and presence of Aroclor 1254 induced rat liver S9. The dose ranges were 0.3-3.0 &nL for SCE tests and 10-25 pg/mL for aberrations tests (18). In vivo genetic toxicity tests included the Drosophiln sex-linked recessive lethal (18) and mouse bone marrow cytogenetics assays (20). For cytogenetic studies, B6C3F1 mice, between 10 and 16 weeks of age and within f 2 g of standard weight, were subcutaneously implanted with a bromodeoxyuridine tablet 1h before exposure. Animals evaluated for chromosome aberrations were killed 18 h after tablet implantation and those for SCE determinations at 24 h; all received colchicine injections 2 h prior to sacrifice. Marrow cells flushed from a femur were fixed and stained by the fluorescence plus Giemsa method. Chromosome aberrations were scored in 50 first-division metaphases from each of eight mice per dose point, and SCEs were identified in 25 second-division metaphases each from four animals. Slides were coded and scored blind. CA and SCE data were analyzed for significance at p = 0.05 by a trend test with individual animal responses as the unit of measurement (20).
Results Pathology. Clinical signs were dose-dependent in rats and mice. MIC gas was clearly irritating to the respiratory tract. Animals became restless, breathing appeared shallow, and eyes were kept closed. At lethal levels there was dyspnea, a pink frothy discharge from the nose, and excessive lacrimation. Animals were generally unresponsive to noise but were conscious throughout the exposure. Deaths were generally preceded by periods of respiratory distress. The concentration of MIC required to kill mice was greater than for rats. No deaths occurred during the 2-h exposure period, but deaths of rats exposed to 210 ppm and mice exposed to 30 ppm were observed beginning between 12 and 18h postexposwe. Most deaths occurred during the first 4 days, but a second distinct period of mortality occurred 8-10 days later and lasted about 2 weeks. In one group of 200 (100 male and 100 female) mice exposed to 30 ppm of MIC, 88 male mice and 81 female mice died within 23 days. By day 4,81 of the 88 male mice were dead whereas only 54 of 87 females had died. No female mice died on days 5-10, but 33 died during days 11-23. Similar, apparently sex-related patterns of mortality were noted following exposure of male and female rats to lethal concentrations of MIC. Occasional deaths of exposed animals occurred late during 91-day studies, with the last a male mouse exposed to 30 ppm on day 78. No control animals or animals exposed to 3 ppm died during any of the studies. A t 10 ppm, 22 of 60 rats, but no mice, died within 23 days following exposure. Under these conditions deaths of rats were delayed, and none occurred prior to day 8. Body weight gain was decreased in a dose-related manner early in this study, but survivors then gained weight a t a rate equal to controls. The only organ to show a chemically related weight effect was the lung, which was increased by up to 2-fold throughout 91day studies in animals initially exposed to overtly toxic concentrations of MIC. Wet lung weights and wet-to-dry weight ratios suggested only moderate pulmonary edema immediately following exposures, which occurred to a greater extent in males than in females. Mild increases (%fold)in activities of serum alanine aminotransferase and sorbitol dehydrogenase were observed in female rats on day 14 postexposure. No effects on these enzymes were detected at other times or in measures of BUN, creatinine, methemoglobin, serum CK, or blood and brain cholinesterase. White blood cell counts showed only increases in segmented neutrophils and decreased lymphocytes, 190
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Figure 1. Major bronchus from a male mouse that was SacrMced 3 days following exposwe to 30 ppm of methyl Isocyanate f w 2 h. The bronchial lumen (L) is free of inflammatory cells; however, the lining resplratay e p b l i u m is almost completely denuded except In a secondary bronchus. The exposed basement membrane is covered in places by a layer of fibrin (arrow).
Flgwe 2. Mahn bronchus horn a male m s e that was sacrificed91 days following exposure to 30 ppm of methyl Isocyanate for 2 h. The bronchial lumen 6) Is hee of lnfiammatay ~811s. There Is Intraluminal Rbrosis, and me Rbmus tag (arrow) Is covered by respiratay eplthelhm.
consistent with a stress response. Red cell indices were normal but showed effects of hemoconcentration in animals too sick to drink (12). Gross and histopathologic examinations showed no target organs other than the respiratory tract where dose-related changes were observed. Thymic atrophy, which was present in the high-dose animals, was considered secondary to stress and inanition. Acutely, necrosis of the epithelial lining with sloughing was present from the nasal passages, larynx, trachea, and stem bronchi (Figure 1). The alveoli were relatively unaffected. Necrosis was accompanied by transudation, exudation, and variable amounts of blood. Animals that died after 4 days showed plugging of airways with necrotic epithelial cells, proteinaceous debris, and an influx of inflammatory cells. The respiratory epithelium of the nasal passages and trachea returned t o normal by day 28 while focal defects in the olfactory epithelium persisted through 91 days in both rats and mice. In deeper airways, epithelization occurred over intraluminal projections (Figure 2). The genesis and progression of the small airway lesions were confirmed by transmission and scanning electron microscopy (Figure 3). Inflammation persisted in airways extending to the alveoli, and hyperplasia and regeneration of the respiratory epithelium remained through day 91. Mucous exudate was observed in the bronchi, bronchioles, and occasionally the alveoli. Compromises of pulmonary function paralleled the morphologic changes observed in the respiratory tract (21). By day 21 and through day 91, rats exposed to 210 ppm showed severe air trapping as evidenced by increased lung volumes (40% above control), expiratory times (1520% above control), and disrupted homogeneous ventilation. However, total alveolar diffusing capacity was minimally affected because of the increased lung volume. This evidence of obstructive disease was further substantiated by increased lung mass and decreased specific compliance (25% below control).
Figure 3. Scanning electron micrographs (SEM) of surfaces of small bronchus from the lungs of rats exposed lo 10 ppm of MIC. (a) At 72 h. after exposure to MIC. numerows rounded and s w h i n g epimeilal cells (arrowheads) as well as inflammatory cells cover a significant propwtm of the airnay surface. C d s wim shwt clla (C) are observed around a subrmcosal @andduct that m t a i n s red blood calk and deb& (arrow). (b) At 7 days after exposure, large portions of the airway surfaces exhibn dividing and spreading epIheliil cells (arrowheads) in the process of reproducing a differentiated bronchial epithelium. (c) At 1 monm postexposure. broad bands (arrows) and prmsses extend from the airway walls into the bronchial lumens. An enlargement of one of thsse processes is shown in (d). Histopattmlogic sections have confirmed lhal these changes represent intrabronchial fibroepithelial scars emanating from the bronchial interrtltium. (d) This inhabronchial fibrotic process is covered by relatively normal epithelium (Ep). but red blood cells (arrowheads) and debris (arrows) still can be found on the epithelial surface.
Reproduction and Prenatal Toxicity. Male CD-1 mice were exposed to MIC a t concentrations of 0,1, or 3 ppm for 6 h per day for 4 consecutive days, an exposure regimen that resulted in significant pulmonary pathology at the 3 ppm concentration. Subsequently, they were mated with groups of untreated females at weekly intervals for 8 consecutive weeks. There was no significant increase in fetal resorptions, which would be interpreted as a dominant-lethal effect, and there was no effect on male fertility. Male and female CD-1 mice exposed as above underwent mating trials during the first and eighth weeks following exposure. There was no effect on male or female reproductive capacity following the first mating; during the second mating, there was a slight decrease in fertility in the 3 ppm group. Because of the questionable effect, the animals were mated for a third time, and there was no effect on fertility or other reproductive parameters. Mice were exposed to MIC on gestation days 14-17 to determine the effects of exposure during late-term pregnancy. There was a marginal decrease in litter size at birth (control, 10.4; 1ppm, 8.7; 3 ppm, 8.0) and neonatal survival during lactation at the highest dose level only (mortality by 4 days of age; control, 2.0%; 1 ppm, 0.8%; 3 ppm, 11.3%) (22). Immune Function and Host Resistance. There were no marked changes in immune function of mice exposed to MIC (23). Humoral immunity, measured as the antibody plaqueforming cell response to sheep red blood cells, was not affected by chemical exposure. Results from replicate experiments revealed a slight suppression in T cell function as demonstrated by a 2&30% inhibition of in vitro lymphocyte proliferation to polyclonal activators including phytohemagglutinin and concanavalin A as well as to allogeneicleukocytes in the mixed leukocyte response (MLR). However, only suppression of the MLR in the 3
ppm treatment group was statistically significant. Four months post exposure, these immune responses in MICexposed mice were still slightly depressed compared to controls, but the differences were not statistically significant. NK cell activity, as measured by the ability of splenic lymphocytes to lyse 51Cr-labeledYAC-1 tumor cells, was not affected by MIC exposure. To determine whether MIC exposure altered host resistance, separate groups of mice exposed to MIC, as described above, were challenged with either Listeria monocytogenes, a mouse malaria parasite (Plasmodium yoelii 17xNL). influenza AZ/Taiwan/64 virus, or B16F10 syngeneic tumor cells. There were no statistically significant increases in host susceptibility in any of the challenge models as a result of MIC exposure. However, there was a trend toward increased mortality in chemically exposed mice challenged intranasally with influenza virus with death occurring in 34% of control-injected mice compared to 43% and 49% in mice exposed to 1and 3 ppm of MIC, respectively. On the basis of the lack of effect by overlapping immune functions and host resistance tests, the increased influenza mortality is likely a consequence of impaired lung clearance mechanisms as pulmonary functions were altered. Genetic Toxicity. In the Salmonella test, toxicity was evident a t higher doses, but there was no evidence of mutagenicity (18). In the mouse lymphoma assay, cultured L5178Y cells were assayed for the frequency of trifluorothymidine-resistant clones after treatment with MIC. A reproducible, dose-dependent increase in the mutant frequency was observed; the greatest increase being from 21 per IO6 in controls to 281 per lo6 at the highest dose (3.0 nL/mL) (19). SCE results from CHO cells were reproducibly positive with an increase from 8.2 in controls to 15.5 in the high dose ( 4 9 ) and from 7.7 to 12.2 SCE/cell (+S9). Reproducible, dose-related increases in chromosomal aberrations were also observed. Increases were seen in the frequencies of both breaks and rearrangements, with more than 50% of the cells showing damage at 25 fig/mL (18).
In vivo genetic toxicity tests using adult male Drosophila treated with MIC by either feeding, inhalation, or injection demonstrated no evidence of recessive lethal mutations in Drosophila male germ cells under any of the test conditions (18). B6C3F, mice were exposed to MIC by inhalation with two dose regimens (20, 24): a single 2-h exposure a t 0, 3, 10, and 30 ppm and 6-h exposures on 4 consecutive days a t 0,1,3, and, in one run, 6 ppm. Four end points were studied in the bone marrow cells-average generation time (AGT), SCE, CA, and micronuclei (MN). Only males were used in the 2-h exposures. No effects on SCE a t 3 and 10 ppm (no second-generation cells were present a t 30 ppm) or on CA and MN were observed in either single-exposure experiment. Generation time of the bone marrow cells was clearly extended at 30 ppm and to a lesser extent a t 10 ppm. Three 4-day exposure experiments a t 0.1, and 3 ppm were conducted with both male and female mice. For AGT, the results were consistent in all three experiments, i.e., dose-related increases in cell cycle time in males and no effect in females. SCE were marginally increased in males in one experiment and in females in another. For CA, all three experiments were negative in males. In females, results in the first experiment were negative, and the third could not be scored. A small but significant increase in CA in female mice was present in the second experiment with the percent of cells with aberrations increasing from 0.5 in controls to 0.75 at 1 ppm and 2.75 at Envlron. Scl. Technol., VoI. 21, No. 2, 1987
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3 ppm. Micronuclei were scored in peripheral erythrocytes in the 4-day exposures, and no effect was observed in either males or females. However, a decrease in percent polychromatic erythrocytes (PCE) was routinely observed in males and females, indicating that MIC exposure led to a depression of erythropoiesis. One additional 4-day exposure was conducted with MIC concentrations of 0, 1,3, and 6 ppm. In this experiment, MIC increased AGT in both sexes, but more dramatically in males (from 12 to 23 h for males and from 11 to 15 h for females at 6.0 ppm), caused a significant increase in CA and depressed the PCE frequency in males and females, induced a significant increase in females (5.9-13.4) at 6 ppm but not in males (because of cell cycle length, highest dose scorable in males was 3 ppm), and increased the frequency of MN in peripheral blood erythrocytes in males. Results of the in vivo cytogenetics studies were not consistent among experiments but indicate that in mice MIC is weakly genotoxic and inhibits bone marrow proliferation in vivo and that the effects are sex-dependent and occur only at highly toxic doses.
Discussion MIC is a highly toxic gas in rodents as well as humans. In agreement with other reports (25-28), acute (C4 days) lethality in rodents was likely due to the direct necrotizing action of MIC on the epithelial lining of the respiratory tract with resultant sloughing and intraluminal accummulation of fluid (edema), hemorrhage, and cellular debris. The lesions decreased in severity toward the periphery of the lung, and the alveoli were relatively unaffected a t concentrations used in this study. This may be due to the extreme reactivity of MIC, which prevented necrotoxic levels of the gas from reaching the alveoli. A second wave of deaths occurred 8-12 days postexposure. Deaths continued with decreasing frequency throughout the 91-day observation period. Although the upper respiratory tract (nasal cavity and trachea) showed epithelial repair by this time, late deaths were interpreted to be due to pulmonary insufficiency from airway blockage by fibrous tissue, mucus accumulation (rats), chronic inflammation, and cellular debris. Pulmonary function studies demonstrated significant pulmonary compromise and suggested the presence of persistent obstructive airway disease. Of equal importance and in contrast to the pulmonary damage is the lack of evidence of primary toxicity to other organs or organ systems including reproductive and immune function. Decreases in litter size from dams exposed during gestation may have been nonspecific effects related to stress. These effects were seen only in animals exposed to 3 ppm, 6 h per day for 4 days, which is approximately half of the LCs0 for mice in this exposure regimen. The signs of genetic toxicity observed in vivo are consistent with the cytogenetic effects observed in vitro (24). However, in vivo responses were generally weak and were not fully reproducible. The results are not surprising considering the extreme reactivity of MIC. In limited studies, small but significant increases in SCE frequencies were observed in lung cells but not in peripheral blood lymphocytes from mice exposed to MIC for 4 days (24). MIC is likely scavenged during initial encounters with respiratory mucus, fluids, and epithelial components, limiting its potential to produce systemic injury or genetic effects in tissues not directly exposed to the gas. In summary, if MIC was the principal chemical involved and if these animal studies are of predictive value to the survivors of the Bhopal disaster, the primary concern for health care officials should focus on chronic pulmonary 192
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disease induced by exposure to MIC. The likelihood of significant extrapulmonary effects appears low.
Acknowledgments We thank the following for assistance in conducting these studies and in preparing the manuscript: B. E. Brown, W. Caspary, D. Costa, J. Dement, S. Fitzgerald, L. D. Fowler, N. F. Gage, D. Germolec, L. B. Hall, R. C. Hamrick, M. Harris, H. L. Hong, J. Mason, S. McClung, K. S. Micor, C. R. Moorman, D. L. Myers, R. W. O’Connor, P. A. Sams, P. Y. Seabrook, M. T. Silver, R. Sloan, S. A. Stefanski, J. Tepper, M. B. Thompson, A. Tucker, L. Uriah, S. Vore, and E. Zeiger.
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(28) Fowler, E. H.; Dodd, D. E. Fundam. Appl. Toxicol. 1986, 6 , 756.
Received for review M a y 19, 1986. Revised manuscript received October 20, 1986. Accepted November 10,1986. Genetic toxicology tests were conducted under contract to the N I E H S by
the following: Salmonella, Dr. Steve Haworth, Microbiological Associates, Inc.; mouse lymphoma, Dr. Brian Myhr, Hazleton Biotechnologies Corp.; CHO cytogenetics, James Iuett, Hazleton Biotechnologies Corp.; Drosophila, Ruby Valencia, University of Wisconsin; mouse bone marrow cytogenetics, Raymond Tice, Brookhaven National Laboratory.
Hydrocarbon Distributions and Transport in an Urban Estuaryt Timothy S. Bates," Paulette P. Murphy, Herbert C. Curl, Jr., and Richard A. Feely National Oceanic and Atmospheric Administration/Pacific Marine Environmental Laboratory, Bin C 15700, Seattle, Washington 981 15
w Aliphatic and polycyclic aromatic (PAH) hydrocarbons were quantified on suspended particulates and surficial bottom sediments from the main basin of Puget Sound, WA. Total four-, five-, and six-ring PAH concentrations ranged from 0.6 to 3.2 pg/g dry weight of sediment. Concentrations of total n-alkanes and the unresolved complex mixture ranged from 3 to 35 and from 35 to 1100 pg/g, respectively. The highest hydrocarbon concentrations on particulates were found in the surface waters near Seattle. Concentrations decreased with depth in the water column and with distance from Seattle. Hydrocarbon distributions and concentration gradients show vertical flux as the major transport process. Although the hydrocarbon residence time in the water column is too short to horizontally mix the compounds, resuspension and lateral transport in the bottom nepheloid layer disperse the hydrocarbons throughout the fine-grained sediments in the center of the basin. Introduction The input of hydrocarbons to coastal and estuarine waters can have adverse effects on the quality of the environment. High concentrations of hydrocarbons in bottom sediments near urban centers, for example, have been linked to diseases in bottom dwelling fish (1). Although it is known that hydrocarbons attach to particles and accumulate in bottom sediments, the transport processes from source to sink are largely unknown (2). The transport of these compounds through the water column affects their residence time in the estuary, their availability to pelagic organisms, and their ultimate fate. If compounds remain suspended in the water column for long periods of time, they will likely be mixed (diluted) throughout the estuary and potentially carried out of the estuary. However, if these compounds, attached to particles, settle out of the water column quickly, they will likely be trapped within the estuary. An understanding of hydrocarbon transport processes in estuaries, therefore, is needed to assess and predict the effects of human activity on the marine environment. In an effort to quantify hydrocarbon transport in the main basin of Puget Sound, we have collected samples of settling particular matter and bottom sediments. Our initial study was a year long time series with five sediment-trap mooring deployments in the middle of the basin (2). These data allowed us to calculate the seasonal variations in the vertical transport of individual hydrocarbons. The concentrations and fluxes of the naturally produced organic compounds (total carbon, pristane, and the n-alContribution No. 768 from the Pacific Marine Environmental Laboratory.
kanes) showed a strong seasonal variability reflecting the increased biological production in the surface waters during summer. The anthropogenically derived hydrocarbons [polycyclic aromatic hydrocarbons (PAH) and the unresolved complex mixture (UCM)] showed no seasonal variation. During May 1981, two moorings were deployed in Commencement Bay (Figure 1)with two to three sediment traps each at nominal depths of 20,70,120, and 170 m. During February-April 1982, five moorings were deployed extending from the sill at Admiralty Inlet to Poverty Bay near Tacoma (Figure 1). Each mooring had two to five sediment traps at nominal depths of 20,50,90,150, and 200 m. These sediment traps provided data to assess hydrocarbon distributions throughout the water column over the length of the basin. In March-April 1983, sediment traps were deployed across the basin at Three Tree Point and midchannel at Poverty Bay, Dash Point, and Colvos Passage (nominal depths of 50,100,150, and 200 m) to study the cross-channel variability of hydrocarbon concentrations and fluxes and the circulation around Vashon Island (Figure l). This paper summarizes the data from 1981 to 1983. These are the first data to document both horizontal and vertical hydrocarbon gradients in a dynamically mixed urban estuary. Study Area The central basin of Puget Sound is an urban fjord-like estuary bounded on the north by a sill at Admiralty Inlet (66 m) and on the south by a sill at the Tacoma Narrows (44m). The main basin stretches 70 km from Whidbey Island in the north to Commencement Bay in the south (Figure 1). The average depth at midchannel is approximately 200 m. Circulation in the northern two-thirds of the basin is typically estuarine with surface water generally moving seaward and being replaced with more saline water at depth from the Strait of Juan de Fuca. The two-layered flow has a reversal in net motion at approximately 50 m depth and a maximum southward transport at 100 m depth (3). Circulation in the southern third of the main basin is southward below approximately 7 m in East Passage and northward at all depths in Colvos Passage (4). Strong tidal currents (150-200 cm/s) occur at both sills causing intense mixing with approximately two-thirds of the northward flowing surface water at Admiralty Inlet mixed back down into the southward flowing bottom water (5). Although tidal currents in the central basin are weaker (10 f 7 cm/s; ref 6), there is still strong mixing between the advective reaches (7). Near-bottom currents in the central basin (450 cm/s) are sufficient to resuspend bottom sediments, creating a nepheloid layer of high particle concentration in the bottom 50 m (2, 8). Particles are removed from the surface waters by gravitational settling and by downwelling at Admiralty Inlet (8). The basin
Not subject to U.S. Copyright. Published 1987 by the American Chemical Society
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