Pestic. Sci. 1977, 8, 345-353

The Uptake of the Molluscicide, 4'-Chloronicotinanilide into Biomphalaria glabrata (Say) in a Flowing Water System

John Duncan, Nicolette Brown and Robert W. Dunlop

Centre for Overseas Pest Research, College House, Wriglits Lane, London W8 5SJ

(Manuscript received I November 1976)

A system is described for the exposure of freshwater snails to pesticides in flowing water. The method allows groups of seven snails to be confined in about 20 ml of water under constant conditions of pesticide concentration, oxygen tension and temperature for up to 5 days. The uptake rate of 4'-chloronicotinanilide into the planorbid snail, Biomphuluriu glubraru (Say), has been measured using this method with liquid scintillation counting techniques. The necessity for an acclimatisation period prior to exposure and the possible influence of activity on uptake is demonstrated. The advantages of the flowing system over exposure in static solutions are discussed and the use of the apparatus in deciding whether relative uptake rates can account for interspecific differences in susceptibility or contribute to the improvement of mollus- cicide specificity, is indicated.

1. Introduction

Preliminary work in this laboratory evolved a method for rearing the planorbid snail, Biornphularia glubruta (Say) in order to produce uniform specimens for experiments on the mode of action of molluscicides (Duncan and Brown, unpublished). It was also shown that the amounts of trifenmorph (N-triphenylmethylmorpholine) taken up by this snail in a given time interval bore an exponential relationship to shell diameter. Subsequent work gave variable results for uptake rates in experiments carried out at different times. In all these experiments, the snails were exposed singly in the mollus- cicide solution (25 ml) in glass beakers. This method had the disadvantage that, even during expo- sures for a few hours, the snails crawled out of the medium and had to be returned to it frequently.

This also occurred in the absence of molluscicide and was tentatively attributed to falling oxygen tension or progressive fouling of the water. It seemed that the increased activity induced by these conditions might contribute to the variability in results; a possibility given substance in this paper. It was decided to develop a system without these factors which would then allow the accurate comparison of uptake rates of various chemicals. The present paper describes a flowing water system for the purpose.

2. Materials and methods 2.1. Synthesis of the molluscicide 4'-Chloronicotinanilide, was prepared' with a tritium label by an acid catalysed exchange reaction between tritiated water and 4-chloroaniline. This was followed by acylation with nicotinyl chloride. The specific activity used was 1.14mCilg and the compound had chemical and radiochemical purities of more than 99%.

2.2. The flow-cell apparatus The apparatus for exposing snails to solutions of molluscicide consisted of a cylindrical glass cell, 5 cm high x 2.5 cm diam. (Figure 1). A glass tube with a bell-shaped end [Figure 1(A)] was clamped so as to stand about 1 mm away from the bottom and sides of the cell. Another glass tube [Figure

345

346 J. Duncan ct a/.

Polythene- collar

I

Figure 1. The exposure cell.

1 (B)] whose end had been pulled out and turned through 90°, was held within the first tube by a polythene collar. Its end pointed away from a 2 mm diameter hole in the wall of the first tube (latterly, these glass tubes have been replaced with 1 mm bore stainless steel tubing which is more durable). The exposure cell was held level with the tops of two, 2.5 litre, brown-glass, Winchester bottles (Figure 2). A peristaltic pump was used to drive air in to displace water from one of the bottles [Figure 3 (A)] into the cell. The air line passed almost to the bottom of the Winchester thus

Figure 2. A single cell unit with inflow and outflow connections.

Uptake of 4’-chloronlcotinanillde by snails 347

Figure 3. Schematic drawing of the apparatus operating a single flow cell.

helping to keep the water aerated. After passing downwards under the bell, the water was evacuated through a tube [Figure 1 (B)] by means of a second peristaltic pump set to work slightly faster than the first. This arrangement maintained the water levels steady as shown in Figure 1. It also tended to cause a constant cross-sectional flow to pass downwards through the cell and any excreted prod- ucts to be removed from the liquid surrounding the snails. A three-way tap could be employed to redirect the air flow to the second Winchester bottle [Figure 3 (B)] which usually contained the pesticide solution and this solution then passed into the cell. The exposure cell was replicated 10 times and the entire apparatus was contained in a 135 litre bath (Figure 4) which was itself in a constant-temperature room at 28°C.

Figure 4. Replicated cell arrangement housed in water bath.

348 J. Duncan ct al.

2.3. Liquid scintillation counting methods Usually seven snails, with shell diameters in the range 4-11 mm, were held in each cell. After a period of exposure to labelled molluscicide, the snails were removed from the cell with a small spatula, carefully blotted dry externally and within the shell opening, wrapped in a Combustocone" and immediately burnt in a model 305 Oxidisera set to wash the condenser with distilled water (1 ml) and add "Instagel"" scintillant (14 ml) to a scintillation vial. Any snail which lost haemolymph, whether through a haemal pore2 or by rupture of the external membrane, was rejected. The samples were counted at 13.5 k 1 "C in a model 2425" liquid scintillation spectrometer using the automatic external standard to correct for quenching. Counting efficiencies of about 40% were obtained.

3. Results

3.1. Experimental conditions Trials were made with the flow-cell apparatus to find peristaltic pumping rates which would effect a rapid change of water in the cell for a solution containing molluscicide. Tritiated water was used in this case to represent molluscicide and samples (50 pl) were removed from near the bottom of the cell using a Hamilton syringe at 1 min intervals after influx of labelled material had started. Each sample was added to "Instagel" scintillant (14 ml) and counted as described above. At a pumping rate of 10 ml/min, the level of the radioactivity in the cell approached 90% of that of the replacing medium after 1 min (Figure 5) . At about 7 ml/min, the same level was achieved after 3 min. In subsequent experiments, the change-over has been made at 10 ml/min for 10 min which is judged from Figure 5 to be the conditions for virtually complete exchange of the cell contents. It has been found that the cell system may be operated up to 5 days at a pumping rate of 0.3 ml/min and that a group of 7 snails will remain in the water during this time even when they are not fed. The usual experimental regime is to allow a 2 h acclimatisation period at 1 ml/min after the animals are placed in the cell followed by the 10 min change-over at 10 ml/min and then exposure to the molluscicide solution again at 1 ml/min. Experiments using time-lapse photography indicate that the snails are not disturbed by the changing of the medium (Asim A/R Dafalla, personal communication).

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Figure 5. Time required to exchange water in the flow-cell with incoming medium at different pumping speeds. A, 10.36 ml/min; 0, 6.83 ml/min; x , 3.47 ml/min; A , activity of replacing medium with 95% confidence limits.

Products of Packard Instrument Ltd, Caversham, Berks.

Uptake of 4‘shloronicotinanilide by snails

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3.2. The acclimatisation period To find a suitable acclimatisation period, the uptake of molluscicide by snails was measured after a 15 min exposure following various lengths of time in an untreated medium. Groups of 7 snails were exposed at 24°C in the flow-cell apparatus to 0.03 WM trifenmorph ([U3H]-labelled in the morpholino ring; specific activity 23.45 mCi/g) dispersed as a 1 % solution in dimethyl sulphoxide into the appropriate volume of a 5 mM Sorensen’s phosphate buffer containing CaCln plus MgS04.7H~0 (10.4+26.0 mg/litre) and which had a pH of 7.80. It was seen that the amount of chemical taken up (expressed here as disintegrations/min) in the 15 min exposure period fell to a steady value once the acclimatisation period exceeded 2 h (Figure 6). Each point on the graph represents the amount of chemical taken up by a 7.5 mm snail, this value being calculated from the regression of log (amount of chemical taken up) on log (shell diameter) for the group of 7 snails in the cell. A shell diameter of 7.5 m is about the middle of the size range employed. On the basis of this result, 2 h acclimatisation periods have been used in subsequent experiments.

Acclirnatisation time ( h 1

Figure 6. Uptake of trifenmorph by 7.5 mm Biomphuluria glubratu after various acclimatisation times in the flow-cell. Vertical bars represent 95 % confidence limits.

3.3. Uptake of molluscicide into B. glabrara The rate at which 4’-chloronicotinanilide is taken up by B. glubrutu has been measured over periods of 18 and 32 h. The molluscicide was freshly formulated as a 1 % solution in dimethyl sulphoxide and dispersed in an artificial hard water medium (AHW; CaClz plus MgS04.7HzO (104 mg+ 260 mg/litre distilled water; pH 5.50) to give a 0.1 p~ solution which was applied to the snails. This concentration compares with a 24 h, LC1 value of 0.26 PM against B. glubrutu of 9.41 50.73 mm shell diameter (Duncan, unpublished) and it was hoped, therefore, that the concentration was sufficiently sublethal to avoid any toxic effect on the snail during the period of the experiment. The concentration applied yields statistically acceptable count rates for each snail in a reasonable time without giving rise to errors due to gross contamination of the exterior of the animal or memory effects in the subsequent Oxidiser sample preparation for liquid scintillation counting.

350 J. Duncan ei a/.

The exposures were made at 24°C and each point on the uptake curves (Figure 7) represents the amount (At pg) of chemical taken up by a 7.5 mm snail derived from the original data as described above. For each of the groups of snails the slopes of the linear regressions of log (chemical taken up) on log (shell diameter) are variable and do not show any recognisable trend with time (Table 1). A common slope has, therefore, been calculated with a value of 2.0076 (Sheila Green, personal communication) but, in fact, values for the amount of chemical taken up by 7.5 mm snails do not differ much if either this common slope or the actual slope are used. This is due to the mean size of snails used being near 7.5 mm.

Table 1. Statistical analyses on uptake of4'-chloronicotinanilide into 7.5 mm Biomphalaria glubrata (7 snails/cell at each time interval)

Regression of log (pg chemical taken up) on log (shell diameter), for each cell

~ ~ ~ 1 7 . 5 IIWI Corr. coefT pg/7.5 mm snail calculated

Time (h) ( R ) Slope snail from common slope -

(a) Exposure for 18 h 2 4 6 8

10 12 14 16 18

+0.91 +0 .92 +0.41 +0.95

+0.91 +0.96 +0 .94 +0 .96

+0 .86

2.969 3.022 0.602 2.058 1.485 2.490 1.426 2 . I29 2.038

0.110 0.193 0.292 0.333 0.407 0.406 0.494 0.549 0.545

0.107 0.196 0.292 0.333 0.409 0.401 0.505 0.547 0.545

(b) Exposure for 32 h 2 +0.61 I . 355 0.092 0.087 4 +0 .95 3.207 0.163 0.168 6 +0.91 I ,970 0.227 0.227 8 +0 .86 I ,428 0.269 0.269

10.5 +0.91 2.047 0.364 0.364 23 +0.88 2.602 0.623 0.658 24.75 +0 .85 I . 755 0.662 0.646 27 +0 .84 1.800 0.641 0.633 29 +0.95 2.520 0.710 0.750 32 +0 .94 2.125 0.674 0.681

When fitting lines to the data for the uptake curves, a quadratic curve can be shown to be a significantly better fit than a straight line (F test at the 1 % level). The equations for the two curves, where r = time, are:

18 h exposure At = 0.0475t - 0.00099 1 f + 0.01 95 32 h exposure At=0.0391t-0.000545t2 + 0.0123

This gives two values for the rate of uptake (dAt/dt at t=O) of 0.0475 and 0.0391 pg/h. Analysis shows that the shapes of the two curves are not significantly different but that their positions are significantly different (F test at P=O.Ol) and with this constraint it is not possible to represent the points by one line. A further analysis treated the data assuming first order rate kinetics expressed by the equation,

At= Am- Am exp ( - k r )

where Am=amount (pg) of molluscicide taken up after an infinite time ( t ) assuming an equilibrium value to be reached. At = amount of molluscicide contained by a snail at time, t. A, was calculated

Uptake of 4thloronicotinanilide by snails 35 1

by a non-linear least squares analysis which allowed the determination of k, and hence the rate of uptake of molluscicide.

dAt Rate of uptake=-= -kAm at t = O dt

Values of 0.0541 pg/h and 0.0456 pg/h were obtained for the 18 and 32 h exposures respectively. A mean value of 0.047 pg/h with 95% confidence limits of kO.009 pg/h is obtained from the four values for uptake into a 7.5 mm B. glabrata. Since the wet tissue weight of such a snail can be calculated as 51.95 mg, (Duncan, unpublished) the uptake rate may be alternatively expressed as 0.905 pg/g wet weight of snail tissue/h.

The concentration factor (C.F.) of 4’-chloronicotinanilide in snail tissue may be calculated as radioactivity (disintegrations/min)/g of wet weight of tissue divided by radioactivity (disintegrations/ min)/ml of exposure medium. For a 7.5 mm snail, values for C.F. at 18 h from the two uptake curves (Figure 7) are 421.8 and 449.1 and that for 32 h exposure, 544.8. The highest concentration achieved in tissue was of the order of 0.7 pg/7.5 mm snail or 13.47 p g / g wet weight.

0.0 c 0.7 L T i-

V I I I I I I I I

4 8 12 16 20 24 28 32

ExDosure time ( h 1

Figure 7. Uptake of 4-chloronicotinanilide by 7.5 mm Bioniplialoriu g/abrura. Vertical bars represent 95 y; confidence limits.

4. Discussion

The advantages of using an apparatus for the exposure of aquatic snails to pesticides under flowing water conditions over using small, unchanged volumes are that a constant concentration of oxygen and pesticide are maintained in the cell and that soluble excretory or secreted products are not allowed to accumulate. Faeces collect under the bell-shaped tube [Figure 1 (A)]. Knowledge of the stability in water of the chemical under study is essential. Exposure times were based on the time for 50% decomposition of 4-chloronicotinanilide (23.27 pg/litre) in distilled water at pH 5.50 being 10.2 days.’ Similarly, the phosphate buffer medium used in the exposures involving trifenmorph had

352 J. Duncan et al.

a pH of 7.80 which prevented any significant hydrolysis of the c ~ m p o u n d . ~ The cell has been designed so that the molluscicide solution is only in contact with the glass surface while it passes through the cell since at least certain molluscicides may be shown to readily adsorb onto polythene (Duncan, unpublished).

It has been shown that uptake rate is at a constant value after 2 h acclimatisation prior toexposure. During acclimatisation, the snail will be initially active in gaining a foothold on the sides of the cell. It will respond to the presence of other snails which impinge on its “active space”,4 an activity which decreases with time and it will follow mucus trails which in the case of Physu ucuta (Muller) at least, are capable of influencing behaviour for up to 30 min after their deposition.5 The animal will also be adjusting to differences between the aquarium water in which it was reared and that of the cell. These differences include changes in salt concentration and a fall in temperature from 26 to 24°C. We believe that uptake is influenced by the activity of the animals and that activity decreases during acclimatisation. Preliminary results from a study of activity using time-lapse photography seem to support this latter view (Asim A/R Dafalla, personal communication). In a number of papers the relationship between the product of concentration and time of applica-

tion of molluscicides and the mortality of snail populations has been discussed. I t has been shown! for example, that phenylsalicylanilide is proportionately more effective against B. glubrata in 1 h exposures when compared with those of 6 and 24 h. This might be explicable in terms of the present results in that snails would be expected to be active and taking up more of the toxicant in the first hour of exposure when placed directly into a molluscicide solution. Thus extrapolation of such lab- oratory data in the choice of optimum field dosage regimes may not always be a reliable guide.

Variable slopes have been obtained for the regression of log (chemical taken up) on log (shell size) from the various groups of snails used in the construction of the uptake curves. In view of this, a common slope of 2.0076 was calculated. Shell diameter (S) and the amount of molluscicide taken up ( A t ) are related by an exponential equation (Duncan and Brown, unpublished), which in this case is, At=uS2. The relation between shell size (S) and wet tissue weight ( W ) of B. glubruta has been calculated (Duncan, unpublished) as

and therefore,

Thus, it would appear that the concentration of 4-chloronicotinanilide (At/ W ) expressed as pg/g of snail tissue, is inversely related to the weight of the snail body (At/W=a”W-0.26). In other words, as the body weight of the snail increases, the concentration of molluscicide in tissue decreases. A common regression slope of 0.77 and weight-specific slope of -0.23 have been reported7 for the content of a variety of trace elements in certain species of estuarine molluscs. These values, like ours, are similar to that describing the relationship between the metabolic activity of poikilotherms and body weight.*

The positions of the uptake curves (Figure 7) are significantly different. Before proceeding to comparisons of the uptake rates of two or more chemicals, it seems, therefore, that it will be neces- sary to carry out a number of runs of the same treatment to find out how much variability there is inherent in the method.

I t is proposed that the cell system will be used in future to compare uptake and loss rates of both molluscicidal and non-molluscicidal compounds to see whether these rates influence molluscicidal activity and also whether they may be the basis of interspecific differences in susceptibility. Should resistant strains of snails appear, then the possibility could be examined that a genetically controlled decreased penetration may be operating as has been described for insecticides entering houseflies.9 Other factors possibly affecting uptake and loss rates such as behaviour, temperature, age, infection with trematode parasites, ingestion, starvation and endogenous rhythms might be investigated.

S= a’ W0.37

At=a”W0.74

5. Conclusions 1. The flow-cell apparatus has been found to work accurately to its design specifications and to provide a system in which uptake rates of chemicals into B. glabrutu can be measured under more

Uptake of 4’-chloroaicotinanilide by snails 353

constant conditions than with static exposures. It has eliminated the influence of acclimatisation to a new environment. We think that a method is provided which can be used to make comparisons between uptake rates of various chemicals though some further investigation of variability inherent in the method may still be required.

2. The mean uptake rate with 95% confidence limits of 4’-chloronicotinanilide into a standard size of 7.5 mm shell diameter, B. glubrutu has been determined as 0.047 k 0.009 pg/h or more gener- ally as 0.905 pg/g wet weight of snail tissue/h.

Acknowledgements This work has been supported with the aid of a grant from the World Health Organisation. The authors thank Miss Sheila Green for advice on statistical methods, Mrs Glenda Colquhoun for the photographs, Mrs Audrey Walpole for the text figures, Dr R. F. Chapman for reviewing the manu- script and the Workshops of the Tropical Products Institute for help with construction of the apparatus. Dr C. B. C. Boyce, Shell Research Limited, Sittingbourne, Kent, kindly supplied the sample of radioactively-labelled trifenmorph.

References 1. Dunlop, R. W. Ph.D. thesis, University of London, 1976. 2. Lever, J.; Bekius, R. Experientia 1965, 21, 1. 3. Beynon, K. 1.; Crossland, N. 0.; Wright, A. N. Bull. Wld Hlth Org. 1967, 37, 53. 4. Simpson, A. W.; Thomas, J. D.; Townsend, C. R. Behav. Biol. 1973, 7 , 731. 5. Wells, M. J.; Buckley, S. K. L. Anim. Behav. 1972, 20, 345. 6. Ritchie, L. S.; Fox, I. Bull. Wld Hlrh Org. 1968, 39, 312. I. Boyden, C. R. Nature, Lond. 1974, 251, 311. 8. Schmidt-Nielsen, L. Fedn Proc. Fedn Am. Sors exp. Biol. 1970, 29, 1524. 9. Sawicki, R. M. Pestic. Sci. 1970, 2, 84.

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