424 NOTES candidate molluscicide on all stages and sizes of snails were more susceptible than older egg stages vector snails. Approximate concentrations of and in effect initiated a new toxicity range that NaPCP that will kill all snails have been determined tended to rise with the size of the snail. These for representative stages and sizes of Australorbis observations suggest that eggs in different stages of glabratus, and LC50 and LCgo values, and their incubation cannot necessarily be equated with 95% confidence limits were computed in order to motile snails of various sizes in establishing dilution permit statistical comparisons with other mollus- series for testing of molluscicides. cicides. The results obtained in this study revealed For economical employment of manpower, 6-hour the difficulty in obtaining a satisfactory 100% molluscicidal applications are preferable to 24-hour mortality end-point even with a twofold dilution applications. For this reason it is important to series. determine the slope of the time concentration curve The capacity of a molluscicide to kill an entire in order to find out whether the molluscicide in stage-size array of snails within a narrow range of question is effective at reasonable concentrations for concentrations is a distinct advantage. NaPCP has short periods of exposure. The data from the this attribute with 24-hour application, the range of present experiments indicate that when the exposure concentrations being only 1.0 p.p.m. to 4.0 p.p.m. time was reduced from 24 to 6 hours, about nine Within this limited range two overlapping ones oc- times as much NaPCP was required to give an LC90 curred that involved stages of egg incubation and for mature snails. The disproportionate increase in hatched snails. All eggs were relatively susceptible; molluscicide requirement as time of exposure was however, they exhibited increased tolerance to reduced was also characteristic of Bayluscide and NaPCP as incubation progressed. Newly hatched to about the same degree.a

Structure and Activity in Molluscicides: the Phenacyl Halides, a Group of Potentialiy Useful Molluscicides by J. P. DE VILLIERS, National Chemical Research Laboratory, and J. GRANT MACKENZIE, National Institute for Water Research, Councilfor Scientific and Industrial Research, Pretoria, South Africa

The importance of molluscicides as a means of organic derivatives of tin, lead, and mercury belong- controlling the snail vectors of bilharziasis is well are all compounds known to poison sulfhydryl, established. The basis, however, on which com- containing enzyme systems. They are thus expected to pounds are selected for testing as molluscicides kill snails by interfering with such enzyme systems. remains largely empirical and any approach that Most of the common organic molluscicides, with enables selection to be made with a better under- the exception of acrolein, belong to the first group of standing of molluscicidal activity would be an compounds. As a novel approach it was decided to advance. investigate compounds that fall into the second A study of the nature and mode of action of class, but do not rely on a heavy metal atom, such molluscicides shows that most of them fall into one as copper, lead, and mercury, for activity. of two groups. The one group is characterized by, In connexion with lachrymatory activity, Mack- among many other effects, interference with the worth a and Dixon b made a detailed study of simple osmoregulatory system of the snail when dosed at organic compounds that are specific for sulfhydryl low rates. A wide variety of compounds such as groups. Dixon showed that these compounds, Bayluscide, pentachlorophenol, dinitrocyclohexyl though normally rather unreactive, react rapidly and phenol and ICI 24223 belong to this group. There is at present no mechanism postulated as to how any a Mackworth, J. F. (1948) Biochem. J., 42, 82. member of this group actually kills the snail. b Dixon, M. (1948) Reactions of lachrymators with enzymes andproteins. In: Williams, R. T., ed., The biochemical The other group of molluscicides, to which copper reactions of chemical warfare agents (Biochemical Society sulfate, arsenic salts, acrolein and the very toxic Symposia No. 2), Cambridge, University Press, p. 39. 1310D STRUCTURE AND ACTIVITY OF PHENACYL HALIDE MOLLUSCICIDES 425 irreversibly with the sulfhydryl groupings of both TABLE 2 cysteine and proteins; and Mackworth has shown MOLLUSCICIDAL ACTIVITY OF SOME that many enzymes are strongly inhibited by such PHENACYL HALIDES compounds, but that many other enzymes are totally . Minimum LD5o LD5o Ini concentra- unaffected. Compound in millimoles tion for (P.p.m,l The molluscicidal activity of a number of such P.p.m. mol.weight) 100 % kill compounds has been tested and the results are given in p.p.m. in Table 1, together with the values found for pentachlorophenol and Bayluscide for comparison. Phenacyl chloride 1.5 0.01 2.5 Phenacyl bromide 1.0 0.005 2.5 TABLE 1 p-Bromphenacyl bromide t.0 0.003 2.5 MOLLUSCICIDAL ACTIVITY OF SOME ORGANIC SULFHYDRYL REAGENTS p-lodophenacyl chloride 1.5 0.006 2.5 p-lodophenacyl bromide 0.7 0.002 1.5 Compound C LDsop.p.m.in 1 (p.p.m./mol.LD5o in millimolesweight) m-Nitrophenacyl chloride 1.0 0.005 5 m-Nitrophenacyl bromide 0.7 0.003 2 Brombenzyl cyanide 2 0.01 p-Nitrophenacyl chloride 0.9 0.0045 1.5 N-ethyl maleimide 0.5 0.004 p-Nitrophenacyl bromide 1.0 0.004 10 Acrolein 0.1 0.002 o-Hydroxyphenacyl chloride > 10 - - Phenacyl chloride 1.5 0.01 p-Hydroxyphenacyl chloride 2.5 0.02 5 Allyl isothiocyanate 3 0.03 Bayluscide 0.1 0.0003 the two p-iodo-phenacyl compounds and the two Pentachlorophenol 2 0.004 m-nitrophenacyl derivatives. Theory would predict that with the nitrophenacyl chlorides, where the nitro group further activates From the compounds in Table 1, phenacyl the chlorine atom, p-nitrophenacyl chloride would be chloride, co-chloracetophenone (I), and its derivatives more toxic than the meta-isomer, and the meta- were chosen for further study. compound would be more toxic than phenacyl

0 0 0 chloride itself. This has been found to be the case. In 11 the hydroxyphenacyl chlorides, the hydroxyl group, C-CH2-Cl C-CH2X C-CH2-Cl while increasing the hydrophilic nature of the compound, and thus probably facilitating its trans- R port within the snail, deactivates the compounds as a whole. o-Hydroxyphenacyl chloride, where hydrogen bonding further decreases the reactivity of NOa the chlorine atom, is the least active of all the phenacyl compounds tested. I II III Thus, increasing the "positive" nature of the halogen atom, either by going from chlorine to Phenacyl halides bromine or by the correct substitution on the phenyl Dixon states that the greater the electron with- ring, increases the molluscicidal activity. drawal from the halogen atom, X, in compounds of The members of this group of compounds with type II above, the more active the system is as a low molecular weight, such as phenacyl chloride, are lachrymator and thus the more reactive it is towards lachrymators. But, besides reactivity with sulfhydryl sulfhydryl groupings. This same argument should groups, a reasonable vapour pressure at ambient apply to the molluscicidal activity of these com- temperature is necessary for lachrymatory activity. pounds, and from Table 2 it can be seen that on both This highly undesirable side-effect can be sup- a p.p.m. basis and on the more relevant mole pressed in compounds intended as molluscicides equivalent basis, phenacyl bromide is more active by increasing the molecular weight of the compound than phenacyl chloride. This is also the case with and thus lowering its vapour pressure. The vapour 426 NOTES pressure of the phenacyl bromides and the sub- Acid-soluble sulfhydryl content stituted phenacyl chlorides are all too low for these The presence of acid-soluble (non protein-bound) compounds to show any lachrymatory activity. As sulfhydryl groups, presumably as glutathione in the phenacyl bromides are inherently more expensive solution, is postulated in animals as functioning as a than the phenacyl chlorides, and in certain cases on a protection for the sulfhydryl groups of their enzymes. p.p.m. basis no more active, probably the most This acid-soluble sulfhydryl content is thus a satisfactory molluscicide tested in this series is measure of the animals' resistance to sulfhydryl- p-nitrophenacyl chloride (III). inhibiting reagents. This has been demonstrated by The anomalous minimum concentration for 100%Y Whitehead c for arsenic poisoning in the blue tick, kill of p-nitrophenacyl bromide, which is less toxic where the arsenic-resistant strain has a greater free than the corresponding chloride, is probably due to sulfhydryl content than the susceptible strain. the reactivity of the bromide being so high as to Treating the resistant strain with sulfhydryl-inhibit- cause it to react with other non-sulfhydryl groupings ing reagents increases their susceptibility to arsenic and thus to be deactivated. This would imply that poisoning. p-nitrophenacyl chloride may represent the most The acid-soluble sulfhydryl contents of Bulinus active molluscicide possible in this series. tropicus and of Physopsis africana were determined before and after poisoning with p-nitrophenacyl Molluscicide trials chloride and also Bayluscide. The method of Sakai & Dan d1 was used and the results are given in Table 3. The snails used for LD60 and other determinations The determinations were done on deshelled snails. were taken from a laboratory-reared colony of The values obtained for the acid-soluble sulfhydryl Bulinus tropicus which was fully acclimatized to content of the hepatopancreas of Bulinus was these conditions. The colony was maintained in identical with that obtained for the total deshelled tap water with a hardness of 75-110 p.p.m. CaCO3 snail. equivalent. The compounds to be tested were dissolved in the TABLE 3 minimum aqueous acetone and diluted to 400 ml ACID-SOLUBLE SULFHYDRYL CONTENT with tap water buffered to pH 7 with phosphate OF ANIMAL TISSUE IN MICROMOLES buffer. PER GRAM WET WEIGHT Twenty randomly selected snails were used and Poisoned each test bottle was placed in a constant temperature Un- with p-nitro- Poisoned Material poisoned~ phenacyl with bath at 25°C for 1000 minutes. The bottles were chloride Bayluscide then removed and the snails washed four times in tap water and then left in 400 ml tap water to allow for recovery. After 24 hours the percentage mortality Deshelled Bulinus 0.76 0.63 0.76 was determined for each bottle. Deshelled Physopsis 0.58 0.44 - Each compound was tested in three stages: Tilapia livers 6.6 3.2 5.7 Stage I at 10 p.p.m. for 100% mortality. Stage II at 10 p.p.m., 5 p.p.m., and 1 p.p.m. to estab- lish the mortality range. As is expected, Bayluscide causes little or no Stage III at smaller concentration ranges to establish reduction in the acid-soluble sulfhydryl content of an accurate LD50 and the complete range of concentra- the snail and its mode of action is thus not by tions for 0-100% mortality. affecting the sulfhydryl-containing enzyme systems. From Table 3 it can be seen that the acid-soluble Duplicate tests were run at stage III, each duplicate sulfhydryl content of both species of snail prior to test solution being made up independently. Controls poisoning is remarkably low compared with that of were run at all stages of testing. more active animals, and that only a small reduction Besides those on Bulinus tropicus, tte values for in the acid-soluble sulfhydryl values leads to death. p-nitrophenacyl chloride were also determined on It has not been confirmed whether the enzyme Physopsis africana, where an LD50 of 1 p.p.m. and a minimum concentration for 100% kill of 5 p.p.m. c Whitehead, G. B. (1961) J. Insect Physiol., 7, 177. were obtained. cl Sakai, H. & Dan, K. (1959) Exp. Cell Res., 16, 24. STRUCTURE AND ACTIVITY OF PHENACYL HALIDE MOLLUSCICIDES 427 systems, the inactivation of which causes death, are selectivity in the action of the phenacyl halide inhibited simultaneously with the disappearance of molluscicides, and it has been found possible, in the acid-soluble sulfhydryl groups, or whether the tank trials with p-nitrophenacyl chloride, selectively acid-soluble sulfhydryl groups have to be reduced to kill Bulinus in the presence of Tilapia at a molius- to a certain limiting value before the enzyme cicide concentration of 2.5 p.p.m. sulfhydryl groups are affected. As snails and other animals seem to recover rapidly and totally from a ** * non-lethal dose of the molluscicide, it would appear that the limiting value mechanism operates. for the acid-soluble sutfhydryl content The authors wish to thank Dr J. Dijkstra, of the The value Biochemical Division of the National Chemical Research of the livers of Tilapia mossambica are included in Laboratory, for many stimulating discussions and much Table 3 and show that this fish, for example, has a helpful advice. They would also like to thank Mr P. J. higher initial content and can also tolerate a greater Swart of the Veterinary Research Laboratory, Onder- reduction of its acid-soluble sulfhydryl groups stepoort, for the gift of Bulinus and advice on their before dying. This would imply a measure of maintenance.