Hydrology of Natural and Manmade Lakes (Proceedings of the Vienna Symposium, August T991). IAHS Publ. no. 206,1991.
Biodégradation and anticholinesterase activity of methyl isocyanate in the aquatic environment of Bhopal
R. SEN GUPTA, A. SARKAR National Institute of Oceanography, Dona Paula, Goa 403 004,India T. W. KUREISHY Department of Marine Sciences, University of Qatar, Doha, State of Qatar
ABSTRACT A comprehensive study with a monitoring programme on the estimation of biodégradation product of methyl isocyanate (MIC) and its anticholinesterase activities in human blood and different organs of various species of fishes collected from the Lakes of Bhopal was carried out for a period of six months with bimonthly observations in the aftermonth of the leakage of MIC from the Union Carbide Factory in December, 1984. The concentration of the bio-degraded product of MIC (i.e. monomethylamine) in water of both the Lakes of Bhopal was estimated on each occasion, which was found to indicate a decreasing trend. However, a depth-wise increase of concentration of monomethylamine was observed in the Lower Lake. Bio-accumulation of monomethylamine was estimated in different tissues of various species of fishes, collected from the Lower and Upper Lakes of Bhopal and from Aligarh. Among all the species of fishes available in the Lower Lake of Bhopal, Puntius ticto was found to be the most susceptible to MIC-poisoning. There is a remarkable change in the values of pH, ammonia-nitrogen and urea- nitrogen concentration in the Lower Lake water. The anticholinesterase activities of MIC in human blood and in different tissues of the fishes were found to be well below the normal range of values in these samples.
INTRODUCTION
The leakage of a large quantity of the highly toxic gas, methyl isocyanate, from the Union Carbide Plant at Bhopal on the night of 2/3 December, 1984 led to the tragic loss of life and to serious damage and disabilities of thousands of people. This is the worst tragedy of its kind in the history of chemical industry
125 R. Sen Gupta et al 126 anywhere in the World. Methyl isocyanate has long been used as the most sophisticated technology (Bhalerao, 1978) for the largescale production of carbamate pesticides throughout the world. All the chemical components (e.g phosgene, monomethylamine, carbon monoxide, chlorine) used in the synthetic route to the carbaryl pesticides are highly toxic. But the toxicity of the deadly poisonous gas, MIC, towards living beings is so instantaneous that it leads to excessive tear formation, congestion in the windpipe, pulmonary edema and increased movements of the intestines and urinary blodder, characteristics of certain pathological hyperactivity in the body system. Methyl isocyanate undergoes a series of exothermic reactions and gets polymerised in the presence of a trace amount of catalyst and water to produce various bi-products such as N-methyl carbamic acid, N,N'- dimethyl-uric acid, N,N'N'-trimethyl iso-cyanouric acid, etc which on subsequent degradation in the aquatic environment leads to the end product, monomethylamine. Moreover, Methyl isocyanate is known to inhibit the acetylcholinesterase activity (AChE) in human blood and possibly in various species of aquatic organisms. A considerable amount of work has, already, been carried out by different research organisations on the exothermic reactions of methyl isocyanate in the storage tank of the Union Carbide establishment and on its environmental contamination. However, as no organisation was examining its effects on aquatic environments we were advised to look into this. Our study was aimed at the bio-degradation product of MIC. As MIC is known to inhibit the acetylchlinesterase activity (AChE) in living beings we thought it will be, perhaps, a good starting point to eventually examine the problems relating to neurological disorder. There are two lakes at Bhopal, one of them, the Upper Lake, is the source of water supply to the city and is quite far away from the Union Carbide factory, the source of pollution. It is also the recipient of sewage from the city. The other lake, the Lower Lake, is very close to the factory, smaller in area and is completely eutrophicated.The present communication deals with the following aspects of MIC pollution.
1) Preliminary studies on the determination of pH, ammonia- nitrogen and urea-nitrogen concentrations in water of both the Lakes of Bhopal. 2) Evaluation of the concentration of the bio- degradation product of MIC, monomethylamine, in water of both the Lakes and also in the different tissues of several species of fishes. 3) Anticholinesterase activities of MIC on human blood and different tissues of various species of fishes collected from both the Lakes. The 127 Biodégradation of methyl isocyanate in Bhopal
results have already been presented as a technical report (NIO, 1985).
MATERIALS AND METHODS
The period of collection of the first set of samples was from 23 to 30 December, 1984 i.e., three weaks after the leakage of MIC to the environment. Water samples were collected, using a Niskin PVC water sampler, from various depths (surface, 5m and 10m) of the Lower Lake. Due to technical problems, collections were restricted only to the surface water of the central part of the Upper Lake. All the samples were examined for their pH values on the spot using a field pH-meter. The second and third set of samples were collected from 25 to 26 February and 1 to 2 May 1985 respectively. Ammonia-nitrogen and Urea-nitrogen concentrations in water were estimated by applying the indophenol blue method (Koroleff - Cited in Grasshoff et a_l 1984), and the diacetyl monoxime method (Koroleff - Cited in Grasshoff at a_l 1984) respectively using a field spectrophotometer (spectronic mini 20). The extraction of the bio-degradation product of MIC (i.e. monomethylamine) was carried out according to the adsorption and desorption method (Glasstone and Lewis,1964) using activated charcoal. On each occasion 10 litres of water samples from the surface, 5m and 10m depth of the Lower Lake and from the surface of the Upper Lake were shaken continuously for 6 hours with the activated charcoal and the charcoal was allowed to settle. The clear supernatant was decanted off and stored in PVC bottles for further analysis. For the estimation of the bio-degradation product of MIC in water, the activated charcoal extract of the water samples were brought to acidic pH by adding 4N HC1 to convert the amine into hydrochloride. This was done as the hydrochloride is quite stable for analysis. The adsorbed amines were desorbed by shaking for 6 hours with 0.IN HCl (Glasstone, 1964). In case of aquatic organisms different tissues of fishes were digested with 4N HCl overnight to extract the amine. The digested extract were then neutralised by ION NaOH in a reaction flask. The methylamine gas, thus released, was taken into 0.001N HCl using nitrogen gas as carrier. All the aqueous solutions were treated similarly. Samples were concentrated to 5 ml and analysed spectrophotometrically applying the lactose method (Snell and Snell, 1962). The red complex was measured at 540 nm. The detection limit of the method, calculated by replicates of several standard solutions, was 0.2 ug/ml of the test solutions. Several varieties of common edible fishes were collected from the Upper Lake. Their identification, length, sex and stages of maturity were noted down as R. Sen Gupta et al. 128
far as possible. They were dissected and representative portions of different body tissues (mussels, gills,- liver, swim blodder, intestine) were collected and deep frozen for further analysis. The Lower Lake was completely eutrophicated and was devoid of the same varieties of fish as in the Upper Lake. Whatever small fishes could be collected from this Lake were frozen immediately for further analysis. However all the species of fish were not available on all the occasions. During the first visit to Bhopal a few blood samples, 2 ml each were collected from a few MIC- poisoned male patients, who were still present at the Hamidia Hospital. Blood samples were centrifuged and the plasma were collected. The AChE activity of these samples was determined spectrophotometrically, using acetylcholine bromide as the substrate and bromothymol blue as the indicator (Zweig, 1982 and Das et aj^, 1979). The absorbance was measured at 620 nra. Acetylcholinesterase enzymes were extracted from different tissues of fishes by grinding them with saline solution (containing 8.6g sodium chloride and 8.12g manganous chloride in 1 litre of water) and phosphate buffer (pH-8, 0.08 M) followed by centrifuging at 3000 rpm under refrigerated condition in order to carry out the anticholinesterase activity of MIC. The extracts were then analysed as above.
RESULTS AND DISCUSSIONS The results of the measurements of the bio-degradation product and the anticholinesterase activity are presented in Tables 1 through 4.
(i) pH, ammonia and urea in water
Table I indicates the values of pH, ammonia-nitrogen and urea-nitrogen concentrations of water at different
TABLE 1 pH, ammonia and urea in lake and tap water of Bhopal.
Locations Depth pH NH+ N Urea-1M
(mg/1) mg/1
A B C A B C A B C
Upper Lake 0 7.91 9.52 5.1 9.6 4.7 7.04 Tap water 0 7.70 2.75 4.25 at RRL Lower Lake 0 8.39 9.28 9.19 5.49 2.83 6.98 5.50 1.43 2.81 Lower Lake 5 8.31 7.82 8.78 5.05 2.51 12.2 5.17 2.14 8.05 Lower Lake 10 8.18 7.82 7.89 21.07 10.8 24.5 5.25 3.57 11.7
A - after 20 days of MIC accident B - after 80 days of MIC accident C - after 140 days of the accident 129 Biodégradation of methyl isocyanate in Bhopal
TABLE 2 Methylamine concentrations in the Lake waters of Bhopal. Location Depth Monômethylamine (ug/1) (ra) A B C
Upper Lake 0 Nil Nil Nil Lower Lake 0 0.03 0.005 Nil Lower Lake 5 0.05 0.009 Nil Lower Lake 10 Nil
A - after 20 days B - after 80 days C - after 140 days depths of the two Lakes on the three sampling occasions. It was noticed during some of the samplings that effluents from the city of Bhopal were being discharged into the Upper Lake. These effluents can be expected to contain high amounts of nitrogen and phosphorus, the effects of which could be seen in the coastal areas of the Lake in the growth of copious amounts of weeds and algae. As stated earlier, the Lower Lake was fully eutrophicated which was evident from the large quantities of algae in water samples from all depths. The presence of these algae will certainly add to the concentrations of nitrogen and phosphorus compounds in water, perhaps influenced by the instant toxic effect of MIC, before its breakdown in water. Values of pH, ammonia-nitrogen and urea-nitrogen in the Lower Lake, in general, decreased from the first to the second occasion while on the third occasion they increased. It is presumed that due to the instant toxic effect of MIC some of the organisms died and sank to the bottom, which was evident from the high concentrations of ammonia-nitrogen and urea- nitrogen in the bottom waters. A considerable contribution must have also come from the degradation of MIC which resulted in very low concentration of methylamine in water (Table II). Values during the third occasion would indicate eutrophicated condition. High values at the surface waters of the Upper Lake was due to the effluent discharges, as methylamine was absent on all the three occasions (Table -II). For comparison, pH, ammonia -and urea- nitrogen concentrations in the tap weater of RRL, Bhopal was also estimated during the first occasion. These values appear to be fairly reasonable. We made an attempt to compute the mole-percentage of free ammonia, by applying literature data. In the Lower Lake, these were 13, 11 and 9% of ammonia nitrogen during the first occasion, while during the subsequent occasions it increased from 60 to 63% at the surface, 5 to 42% at 5 m, and 5 to 26% at 10 m. The toxic limit of free ammonia for aquatic R. Sen Gupta et al 130
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£0 3 O I I I I •H Sj m co in n > co o^ en en H HM WCNCO I ÎCNI i en en en \ o w . en I I I I I I M en • H "H * % CO I ! I I I 1 I I I I 4-> HOI CO CO CO c COCO U3 0) Ij r--ococN-^-co-^ Tko n 4) CO f «3* f co CO co i rococo I «H "° o o a. co -3* ^f in I M 0) •H 4J •H co in o > I «fin I •H r-t co r-i r^ ^r in o co « co CN co co +J "H « \o in « y?vo^) ^oinin^ I ^D^i^^o^o oj e 0! O ffl *H to • 3 CO OJ »J 3 10 C c0 4J 0) rt! C organisms, particularly fishes, is 1.2 mg/L. All the values in the Lower Lake were higher than this limit. No wonder, there is hardly any aquatic life there. In the Upper Lake the mole percentage of free ammonia at the surface increased from 4 to 78% from the first to the third occasion. Care has to be taken to reduce free ammonia there to enable the aquatic life to sustain. (ii) Methylamine in water Table II presents the monomethylamine concentrations in water of both the Lakes. The concentrations in the Lower Lake decreased from the first occasion (0.03 ug/L at the surface and 0.05 ug/L at 5m) to the second (0.005 ug/L at the surface and 0.009 ug/L at 5m). An increase of concentration with depth could be noted. Monomethylamine was, however' not detectable during the third occasion. It was also not detectable at the surface waters of the Upper Lake on all the occasions. This indicates that the Lower Lake, being nearer to the factory than the Upper Lake, was comparatively more affected by MIC. (iii)Methylamine in fishes Methylamine concentrations from fish samples of both the Lakes and from Aligarh are presented in Table III. Some bio-accumulation of methylamine can be seen in the muscles of Labio gonius, Puntius serrana and Mestacemphalus sp.; in the swim bladder of Labio calbasu; and in the gills of Labio calbasu and Puntius serrana from the Upper Lake on the first occasion. The gall bladder of a Labio calbasu was observed to be highly distended. This was due to some bacterial or viral infection. Methylamine concentrations in those species of fishes, which could be collected during subsequent occasions, were non-detectable. This would indicate that in course of time the metabolism of the fishes converted the methylamine to some other nitrogenous compounds. Methylamine concentration in Puntius ticto from the Lower Lake was fairly high (9.2 ug/Kg wet weight) during the first occasion. This reduced to 2.23 ug/Kg during the second occasion to non- detectable during the third. Absence of methylamine in other species of fishes from the Lower lake on all the occasions would suggest that Puntius ticto was very susceptible to MIC poisoning and took longer time to convert methylamine to other nitrogenous compounds. Fishes, collected at Aligarh, did not have methylamine in them. This leads us to conclude that methylamine is not naturally present in fishes. Whatever quantities were observed in fishes from the Lakes of Bhopal were certainly the end-product of degradation of MIC. However in the absence of definite information, it is not sure if these quantities were harmful to the 133 Biodégradation of methyl isocyanate in Bhopal fishes themselves or to human beings after their consumption. (iv) Anticholinesterase activity in fishes The data on the anticholinesterase activities of MIC in different tissues of various species of fishes are reproduced in Table-IV. On the first occasion, AChE activities of different tissues of fishes from the Lower Lake were markedly lower than those from the Upper Lake. In course of time the values in muscle, gill and liver of Labio calbasu of the Upper Lake increased. In fishes of the Lower Lake the values are definitely ' very low indicating, however, a relative increase with time. The AChE activity in Glassogobius qiurus showed a significant decrease from the first to the second occasion. The AChE activities in different tissues of a few species of fishes from Aligarh were also estimated. These were assumed to be normal values for those species of fishes. Comparing the two sets of data, from Bhopal and from Aligarh, the following could be observed. a) Muscles, gills, liver of Labio calbasu from Bhopal, though affected, appeared to be recovering with time, excepting in the intestines; b) Muscles and gills of Labio gonius from Bhopal were unaffected, while liver and intestines appear to be affected somewhat; and c> Compared to Puntius stigma from Aligarh, Puntius ticto from Bhopal appear to be badly affected. Similar trends could be expected for the other species of fishes from Bhopal, at least at a first approximation. TABLE 5 Acetylcholinesterase activities of MlC-ooisoned human blood 20 days after the acident. Age of the Patients Acetylcholinesterase activities all male umoles/ml blood plasma (Years) 45 54.54 35 57.14 25 68.18 40 75.0 ( iv)Anticholinesterase activity in human blood The AChE activities in blood plasma of the MIC-poisoned patients were highly significant. All the values (Table- V) were well below the expected range of 90 to 150 umoles/ml of blood plasma (Nath, 1976) in normal human beings with good health. The normal process of splitting R. Sen Gupta et al. 134 of acetylcholine by the acetylcholinesterase within the nervous system of the body gets stopped due to the blockage of the active sites of the enzyme. It was observed that excepting for the 40 year old patient, the other three values indicate an inverse age-AChE relationship. This is clearly an indication of some sort of neurological disorder in the nervous system. CONCLUSIONS Based on our observations in the aquatic environments of Bhopal the following conclusions have been drawn: I) The waters of the Upper Lake appear to have recovered from the effects of MIC, but contain an excessive amount of free ammonia as a result of effluent discharges. II) The Lower Lake is completely eutrophicated and after the effects of MIC can be considered as dead for all practical purposes. III)Fishes in the Upper Lake seemed to have recovered from methylamine, but had low AChE activities in some of their body tissues. Low AChE activities in some of the fishes from the lakes of Bhopal would indicate that these too have been affected due to the effects of MIC on their environments. Could this effect have been transferred from the fishes to the human beings consuming them? Acknowledgements We express our deep gratitude to Dr. S.Z. Qasim, former Secretary, Dept. of Ocean Development, Govt. of India and presently Vice chancellor, Jamia Millia Islamia, New Delhi for advising us to investigate in the aquatic environments of Bhopal after MIC leakage. We are grateful to Dr. V.V.R. Varadachari, Late Dr. H.N. Siddiquie and Dr. B.N. Desai for kindly providing all the material help for the investigations, processing of data and drafting of the report. We deeply appreciate the valued help of Dr. M. Patel and his colleagues of the Chemistry Department of Regional Research Laboratory,Bhopal for rendering all help during the collection in the field and processing of the samples. REFERENCES Bhalerao, V.T. (1978) Indegeneous know-how development and raw material manufacture for some insecticides. Pesticides, vol.XII,7,pp 40-43 Das,B.K., Sarkar.A., and Chaudhuri,P. (1979) Investigation of some organophosphorus compounds 135 Biodégradation of methyl isocyanate in Bhopal having pesticidal activity: II synthesis, insecticidal activity, toxicity, anticholinesterase activity and hydrolysis of O-(2-ethoxy)ethyl 0,0- diparanitrophenyl phosphorothionate Pesticides, Vol. XIII, 1, 34-37. Glasstone, S. and Lewis,D. (1964).Elememnts of Physical chemistry. Mcmillan & Co. Ltd. London. 560pp. Grasshoff ,K. , M. Ehrhardt & Kremling K.Q984) Methods of seawater analysis, Verlag Chemie. Weinheim 419 pp. Nath,R.L. (1976) Practice of Biochemistry in clinical medicines. Oxford and IBH Publishing Co., New Delhi. 122 pp National Institute of Oceanography (1985) On biodegradational and anticholinesterase activities of methyl isocyanate \r± the aquatic environments of Bhopal. Technical report NIO/TR-4/85,16pp(mimeo). Snell, D.F.& Snell,C.T. (1962) Colorimetric methods of analysis j_ Vol.IV. Van Nostrand, New Jersey. Zweig, G. (1982) Analytical methods for pesticides, plant growth regulators and food additives vol.1, p.392, Academic Press, Canada.