The effects of several biocides on a simple muscle preparation of the ecologically relevant soil-organism Lumbricus terrestris L.

030&4492/88 $3.00 + 0.00 0 1988 Pergamon Press plc

Camp. &o&em. Physiof. Vol. 89C, No. 2, pp. 133-l 39, 1988 Printedin Great Britain

THE EFFECTS OF SEVERAL BIOCIDES ON A SIMPLE MUSCLE PREPARATION OF THE ECOLOGICALLY RELEVANT SOIL-ORGANISM LUA4BRICUS TERRESTRIS L.” THOMAS Department

of Neuroethology,

W. BENEKE and WOLFGANG W. SCHWIPPERT FB 19, Gh Kassel, University Land Hessen, Heinrich-Plett-Str. Kassel. FRG

40, 3500

(Received 9 February 1987) The action of different herbicides and one insecticide was studied on the oblique-st~ated muscle systems of the annelid Lumbricus terrestris L. 2. By means of a simple electrical stimulation device, preparations of the circular muscle system (CMS) were contracted in reproducible manner half-maximally. 3. From 12 tested herbicides, 7 showed a pure inhibiting and one a promoting action, two responded with both effects depending on the duration of incubation and 2 were ineffective. 4. The insecticide pirimicarb, an acetylcholinesterase blocker, enhanced the electrically evoked contractions. 5. The findings coincided in general with those obtained at a defined smooth muscle of the gastropod Helix pomatia L. Abstract-I.

lNTRODUCl’ION

The annelid Lumbricus terrestris L. inhabits networks of ducts situated directly underneath the soil-surface. In a vertical direction, their branches travel to a depth of more than 2m. The inner wall of the ducts is covered with a mucose layer making the tubes stable against erosion. The nutritive substances of the worms are dead plants which are pulled into the earth holes where residues of the detritus and excrements of the worms are finally degraded by microorga~sms. The ecological role of earthworms is strictly coupled with this mode of living while these organisms improve the ventilation and the water-capacity of the soil and support the process of humification and by it the micro-structure of soil. Also the sensitivity of plants towards disease and attacks of vermin is reduced, a fact that is referred to a better dist~bution and upgrading of relevant plant nutrition (Ghabbour, 1966; Barrett, 1976; Edwards and Lofty, 1977). Because of the ecologically prominent role of lumbricides, the question arises how far herbicides or insecticides put into the soil are able to influence the biolo~cal parameters of these animals. In previous studies, numerous side-effects of herbicides and fungicides were reported for many lower organisms such as snails, nematodes, collembola and also lumbricides (Fox, 1964; Franz and Tanke, 1974; Cook and Swait, 1975; Ulber, 1977). In particular, it was shown that for the smooth muscle preparation of the snail Helix pomatia L. herbicides, including some degradation products identified as carbamates, thio*The experimental work regarding this paper was achieved in the Department of Biology II (Zoology) of the RWTH Aachen, Kopernikusstr. 16,510O Aachen, FRG, in co-operation with Professor Fr.-W. Schlote.

carbamates, urea-derivatives, amides, phenols or anilines, more or less effectively relaxed contractions which occurred s~ntaneousiy or were evoked by suitable agonists (Schwippert, 1982, 1985, 1986; Backhaus et al., 1984a; Schwippert and Beneke, 1987). In freely moving water-snails and soil-living worms, the application of these drugs produces a diminished mobility (Backhaus et al., 1984b, c; Kosanke et al., in preparation) while the drugs are enriched presumably in the cell membranes of muscle cells, as could be demonstrated in the snail Helix as well as in the worm Lumbricus (Backhaus et a!., 1984a, c; Schwippert and Beneke, 1987). In order to evaluate the physiological effects on electrically evoked responses of the oblique-striated muscle system of Lumbricus, a simple recording device was used to measure the muscle tension. MATERIALS AND METHODS Animals All experiments were carried out with adult Lumbriws terresfris L. (source of supply: H. Miemert, 4236 Hamminkeln, FRG) which were kept in aquaria containing the breeder’s medium enriched by organic material such as roots and leaves, at 8°C and a day-night rhythm of 14: 10 hr. An artificial gradient of humidity permitted the animals to choose acceptable soil-conditions. Muscle preparation A solution with the following composition served as the preserving medium for the muscle preparations (modified according to Enders, 1952): NaCI, 126mmol; KCl, 4.7 mmol; CaCl,, 1.9 mmol; glucose, IOmmol; adjusted to pH 6.6 by 1 N HCl. The worms were anaesthetized with 5% ethanol and the 10 caudal segments were removed. The subsequent segments situated rostrally were then separated, cutting at the dissepiments with microscissors. From the 133

THOMASW. BENEKEand WOLFGANGW. SCHW~PPERT

134

obtained single segments, the remaining parts of the dissepiments, the mesenteria with blood vessels and the ventral nerve cord as well as the gut were removed totally. From the outside such a preparation consisted of the cuticule, the epidermis with sensory receptor cells and a nerve net, a muscle bilayer distinguishable in the circular muscle system (CMS) below the epidermis and a longitudinal muscle system. The latter travelled through the long axis of the body and was cut off to the length of one segment, whereas the CMS did not cross the dissepiments and was not damaged. This was used to test the contraction ability of the CMS in the presence of different herbicides and insecticides. Incubation solutions

Because most of the herbicides and insecticides applied in this study were not water-soluble exceeding a concentration higher than 10-5mol/l, ethanolic stock solutions of 2 x IO-’ mol/l were prepared. A short time before starting an experiment, the desired final concentration was adjusted by dilution with the physiological medium so that the ethanolic content of all test- and control solutions was 0.05%. The media were stirred intensively in order to get an even distribution of the test-substances. Contraction measurement

The change of the muscle tension in the CMS was measured auxotonically by electromechanical transducers. The experiments were achieved in a Plexiglas chamber suitable for a parallel study of 2 test- and 2 controlexperiments and supplied with adjustable influx and efflux tubes. Each segment of the CMS was fixed at 2 opposite platinum hooklets serving simultaneously as holder and stimulating electrodes, one connected to the electromechanical transducer, the other to an arrangement in order to adjust a reproducible back force. The recorded muscle tensions were fed on line to a pen-writer (UMR: UMP CU 003/PS 020; velocity 12-24 mm/min).

Ehrensdorfer, 8900 Augsburg, FRG; all other chemicals (pa.) were obtained from Fa.Merck, 6100 Darmstadt, RRSULTS

After the CMS-preparations were inserted into the experimental chamber, the tonus which was superimposed by phasic activities, decreased continually and exponentially. About 30min later, a constant tonus appeared and only single phasic contractions could be obtained. Eliciting phasic contractions with a 500 msec-lasting stimulus of different negative DC voltages, a half maximum tension development was obtained by applying a voltage of 2.4 V. Stimulating the preparations with this voltage, a stimulus duration of 500msec or more did not enhance the tension (Figs la and b). Thus, the stimulus experiments were achieved with a so-called standard stimulus of 2.4 V and 500 msec for every 2 min (0.~8 Hz; Fig. lc) which allowed detection of relaxing or activating effects of the studied compounds. Because of the hydrophobicity of the test-substances (Fedtke, 1982; Perkow, 1982; Jager, 1983) it was necessary to dissolve them in ethanol and to dilute the obtained stock solutions with the physiological medium. The ethanol

Course and evaluation Four CMS-preparations of 30min age were identically stretched and preserved in the physiological medium for a further 30 min. Then, they were electrically stimulated every 2 min with a DC-standard stimulus of 2.4 V for 500 msec (see also Results and Fig. 1). After the fifteenth stimulation the physiological medium was renewed. After the thirtieth stimulation 2 muscle segments were rinsed again with the physiological medium, whereas the other 2 were incubated in the contaminated medium. Both media were renewed again after stimulations 45 and 75. For recovery, the four segments were washed intensively with the physiological solution and responses to a further 30 stimuli were recorded. In each preparation, the amplitudes of 5 successive, electrically evoked contractions were averaged and regarded as the value of one single contraction (p,). Thus, during the first hour (30 stimuli), 6 so-called average contractions were obtained, of which the contraction with the largest value was defined as the maximum tension (p,,,,,). In none of the control experiments, the subsequent electrically evoked contractions exceeded pmax . The tension (F) of the nth average value delivered from 5 single contractions was calculated as

voltegc (VI

tcii

Fig. 1. Estimation of the electrical stimulus evoking a half maximum tension. (a) Dependence from the stimulus duration (negative DC-voltage, 2.4 V) (*). (b) Dependence from the stimulus voltage (durations: 0 250 msec; 0 500msec; 0 ~~rn~c). (c) Evoked contractions after applyinn the so-called standard stimuli of SOOmsec and 2.4 V (-DC) every 2 min.

F = (P,/Pm..) x 100%. Putting the averaged nth contraction of the controls to lOO%, a relative tension (F,,) was obtained:

i

F,! = 4, theprceyncc or,hc~*lF~~*~~~~,=~~ mntiOtx 100%. Age

Chemicals applied

The herbicides barbane, chloroxurone, chlorpropham, cycloate, isoproturone, linurone, MCPA, metabenzthiazurone, monalide, swep, terbutryne, triallate and the insecticide pirimicarb were obtained from Fa.Dr.S.

/CM - ~reparetiOne (RiR)

Fig. 2. The reversible relaxing action of ethanol on the electrically evoked contractions in the CMS. 0 Control = 0% ethanol; 0 0.1%; V 1%; A 2%, * 5% ethanol (in each case, N = 4). Standard deviations are not given for the sake of clearness.

Biocides

influencing

the circular

muscle

system

of _IXrtbricus

135

Table I. The relative tension F,, of the CMS-preparation in the presence of certain herbicides and the insecticide pirimicarb P

Drugs f 1) Herbicides Barbane Chlorpropham Swep ControI

Cm&l) 5 x 10-5

1

10-4 IO-’ 10-d 5 X 10-r

I 1 2

I 1 2

Cyctoate Triallate

10-4 10-4 5 X 10-J 5 X 10-s 10-4 10-d

1 2 1 2

1 2

1

Control

2 Chioroxurone Isoproturone Linurone m-Benzthiazurone

10-e 10-d 10-d lo-’ 10-a 10-4 10-d IO-‘

Control

:

1 2 :

1 2 1 2

MCPA Monalide

Terbutryne

(2) Insecticide Pirimicarb

10-a 10-a IO-’ IO-’ IO-’ 2 X 10-S 10-4 10-a

10-r 10-5 10-d IO-”

Control

0.05

f&

: 10min

I 2

1 1 2

54.6 * 14.6 + 43.3 * 5.0 f 36.3 f 100.0 + 100.0 k

6.1 3.3 9.9 1.5 7.9 1.8 5.2

4 4 4 4 4 24 4

< < > > < >

74.5 * 6.6 37.6 5 4.6 114.7 + 2.7 114.6 + 4.9 76.1 f 4.7 98.8 k 10.9 100.0 5 4.7 100.0 f 4.5 146.7+ 138.5 f 88.2 + 87.7 f 67.4 f 50.1 f 95.0 * 98.2 f 100.0 k 100.0 +

Ii.4 10.5 2.3 0.5 3.0 4.1 4.2 2.7 2.9

101.9 * 98.4 + 136.1 5 59.8 f 39.0 * 69.2 f 22.5 f 16.2 +

1.9 3.1 8.3 18.2 14.5 2.7 1.2 3.5

123.3 + 123.9 f 197.1 + 211.3 f 100.0 * 100.0 +

3.4 7.5 7.4 10.9 3.8 2.7

1.7

< < < < <

4 4 4 4 4 4 4 4 16 16

< < = = < < > > > > < < < < < <

4 4 4 4 24 16

< < < <

The mean values and SE of m appropriate contractions in physiological solution defined to 100% served as controls; c, = concentration, I = incubation time, F,, = relative tension, N = numbers of experiments, P = significance level.

content of all incubation media was adjusted to a value of 0.05%. As shown in Fig. 2, an ethanol content of less than 0.1% did not significantly affect the electrically induced contractions in the CMS. Since a 30min-lasting incubation with 5% ethanol was stopped by rinsing with the physiological solution in less than 30 min, it could be supposed that the reversibility of the 5 min-lasting anaesthesia with this concentration was guaranteed after a rinsing time of 30 min (Du~lohier and Ceorgescauld, 1979). Thus, all the effects were referred to an action of the following substances: (i) the carbamate-insecticide pirimicarb; and (ii) herbicides (a) the carbamates and thiocarbamates barbane, chlorpropham, swep, cycloate and triallate, (b) the urea derivates chloroxurone, isoproturone, linurone and metabenzthi~urone, (c) the amide derivate monalide, (d) the phenoxicarbonic acid MCPA, and (e) the triazine derivate terbutryne. With respect to the inhibition of the electrically evoked phasic contractions of the muscle segments, the carbamates were more effective than the thiocarbamates, as shown in Fig. 3 for an applied concentration of 10-4mol/l. In relation to those control

contractions which were obtained after certain durations of a herbicide application (age of the preparations 180 or 240 min, respectively) and which were defmed to lOO%, the relative tension FE, was blocked most in the case of swep (8%), followed by barbane (14%) and chlorpropham (43%), whereas the inhibitions caused by triallate (76%) and cycloate (74%) were scarcely different and smaller compared with the values of the 180 mm-old CMS-preparations (Table 1). There was evidence that the action of some herbicides failed to be linear regarding the time course and concentration dependence. For example, the second hr of incubation with chlorpropham diminished F,, about 4-times as much (1 hr: 100% --+ 43%; 2 hr: 43%+5% instead of about 20%) as the second hr of incubation with cycloate which only doubled the inhibitory effect (Table 1). Also the action of 10-4mol/l barbane or swep was about 4-times stronger than the effect of 5 x 10-5mol/l (Table 1). In contrast, triallate appeared to have a two-fold effect on the CMS-preparation, first, a slight increase of the phasic evoked contractions and, second, an inhibitory action. Applying the thio-

THOMASW. BENEKEand WOLFGANG W. SCHWIPPERT

136

70

I I

120 Ape /CM

/

I

I

180

I

240

I

300

preparations (mid

Fig. 3. The relaxing and partially reversible action of the herbicidal carbamates barbane (O), chlorpropham (A), and swep (*) as well as the herbicidal thiocarbamates cycloate (*) and triallate (0) on the CMS (concentration in each case 10e4 mol/l) in comparison with an appropriate control (-0 N = 12; --a N = 8). Every datum represents the

result of N = 4 experiments. Standard deviations are not given for the sake of clearness.

carbamate in a concentration of 5 x 10e5 mol/l led to an enhancement of the contraction amplitudes which was, however, not statistically significant, independently of an incubation time of 1 or 2 hr (Table 1). However, 10m4mol/l triallate first reduced the phasic elicited contractions during the first hr of incubation and lost this effectiveness during the second hr of incubation (Fig. 3, Table 1). The relaxing effect of the carbamates and thiocarbamates was at least partly reversible by rinsing with the physiological medium. It has to be taken into account that chlorpropham, swep, and cycloate were completely dissolved at the used concentrations, whereas about 50% of barbane and 85% of triallate were suspended in the medium taking a concentration of 10-4mol/l as a basis. Chloroxurone (10e4 mol/l), one of the four investigated derivates of urea, of which only 5% was dissolved at the tested concentration, enhanced the amplitudes of the half-maximum stimulated contractions. After an incubation time of 40 min, the increase in tension reached a maximum value of 160% compared with the lOO%-defined control value. Thereafter, Fre, remained enhanced during the following 2 hr as well as the subsequent rinsing phase, and oscillated at around 140% (each control, lOO%), indicating that the effect might be irreversible (Fig. 4, Table 1). Isoproturone (10V4mol/l) diminished the contractions during the first hr to about 90% of the control value (Table 1). The effect was rather weak and could be evaluated statistically at the 5% level with respect to the appropriate 100% control of the same animal, but not to the averaged values of all control experiments of this series (N = 16). A longerlasting incubation did not intensify the relaxing action. Any effect was lacking when incubating the worm segment with 10m5mol/l isoproturone which means that the action level ranged between 1O-4 and low5 mol/l. Also in the case of linurone, even after 2 hr, 10m5mol/l was not effective enough to relax the CMS, but 10-4mol/l diminished the electrically

evoked contractions to 50% (Fig. 4, Table 1). The relaxation set in relatively late (after 40 min) and the amplitudes were reduced to about 70% at the end of the first hr (Fig. 4, Table 1). The relaxing property of linurone and isoproturone could be more or less effectively cancelled by rinsing with the physiological solution. Metabenzthiazurone, the fourth of the tested ureaderivates, as well as the phenoxicarbonic acid MCPA showed no effect on the elicited contractions (Figs 4 and 5, Table 1). The amide monalide applied at a concentration of lo-’ mol/l and almost completely dissolved (> 95%) showed a biphasic effect. During the first 10 min after incubation the F,,, increased to a maximum value of about 140%. Subsequently, a relaxing effect set in and after 1 hr the contraction amplitudes decreased to 60% compared with the control (100%). This relaxing action continued and at the end of the second hr Fee, reached only about 40% (Fig. 5, Table 1). Within 20min this effect was abolished by rinsing with the physiological solution. The most effective herbicide also characterized by a relaxing property was the triazine compound terbutryne (10-4mol/l) which diminished the F,,, to 22.5% (1 hr) and 16.2% (2 hr), respectively (Fig. 5, Table 1). In a concentration of 2 x 10e5 mol/l (Table l), terbutryne decreased Fre, to about 70%, and even in a concentration of 10m5mol/l to about 80% significantly so that the level of effectiveness should be reached. The action of terbutryne was partly reversible by rinsing with the physiological medium. In order to demonstrate the effect of an insecticide with a chemical resemblance to carbamates and, with reservation, to triazines, the cholinesterase-inhibitor pirimicarb was tested. Because of its enzyme-blocking property, the evoked contractions in response to half-maximum, electrical stimuli were significantly enhanced which could be shown by incubating the preparations for 20 min with 10m5mol/l. The higher concentration of pirimicarb ( 10m4mol/l) even increased the response to the DC-stimuli during a

..;0

1;0 Ape 1 CM

Ii0

zio

3bo

preparations(mid

Fig. 4. The partially reversible action of the herbicidal urea derivates chloroxurone ( l), isoproturone (V), linurone (A), and metabenzthiazurone (0) on the CMS (concentration in each case 10m4mol/l) in comparison with a control (a). Every datum represents the result of N = 4 experiments (control N = 16). Standard deviations are not given for the sake of clearness.

Biocides influencing the circular muscle system of tumbricus

137

smaller than the electrically elicited standard contractions as shown in Fig. 6. This effect was obtained in the, presence of barbane (> 5 x lo-’ mol/l), chlorpropham (lo-* mol/l), cycloate ( 10e4 mol/l), triallate (> 5 x 10-smol/l), and terbutryne (low4 mol,!l>. In all cases, this effect was reversible by the rinsing procedure. DISCUSSION

_I

Aea

1CM- ero)orotions

(mid

Fig. 5. The partially reversible action of the herbicidal phenoxicarbonic acid MCPA (a), the amide derivate mon-

alide (V), and the triazine derivate terbutryne (A) as well as the in~ticid~ carbamate pirimicarb (*) on the CMS (concentration in each case 1O-4mol/l) in comparison with a control (a). Every datum represents the result of N = 4 experiments (control N = 16). Standard deviations are not given for the sake of clearness.

2 hr-lasting incubation to more than double (Fig. 5, Table I), i.e. the CMS-contractions became maximal. This effect could not be totally reversed. There was an additional action which reflected in spontaneous activities occurring between the contractions induced every 2 min. The frequency of this autonomic activity amounted to 5-20 phasic contractions per min whose amplitudes, in general, were

The toxicological property of herbicides, fungicides, insecticides, growth regulators etc., is based on numerous criteria, for example, the investigations of the acute toxicity by LD~,,studies; determination of a long-time toxicity after successive application to find cumulative effects and the “no-effect-level”; the type of metabolism and mechanisms of action within animals, plants, microorganisms etc., and more specifically, the registration of allergical, mutagenic, cancerogenic, neurotoxical and fertility-influencing reactions. In this context, mammals such as rodents, dogs, cats, agriculturally important specimens as well as other animals, e.g. fish, birds or bees serve primarily as test objects (DFG, 1976; SVR.f.U., 1978). Studies concerned with possible side-effects of such compounds on non-target organisms, in particular on soil-living invertebrates, however, are iess frequent, although these drugs are rather persistent in the soil and migrate there very slowly (DFG, 1976; Perkow, 1982). One example is the early investigation of insecticidal effects on non-target organisms such as lumbricides on account of their ecological importance (Baker, 1946; Goffard, 1949). The so-called dung worm Eisenia foptida Sav. can be kept very easily under laboratory conditions, and therefore this species has been preferred as the experimental lumbricide. But towards an application of the insecticide carbofuran, for example, Eisenia showed a noticeable insensitivity against this compound compared with Chlorpropham

Control

Triallate

Barbaee

.‘.

Cycloate

.

.

.

.

.

Terbutryee

Fig. 6. Spontaneously occurring phasic contractions in the CMS-preparation in the presence of 5 x lo- mol/l barbane as well as low4 mob1 chlorpropham, cycloate, terbutryne and triallate. Every black triangle indicates an electrical standard stimulus.

f

s P.

*9~*(1--8

138

THOMAS W. BENEKE and

the soil-living worm Lumbricus terrestris L. (Gillman and Vardanis, 1974). Analyses of the actions of herbicides and their degradation products have often been restricted to the dete~ination of the population densities or migrations whereas physiolo~cal effects within the organism have been left in the background (Fox, 1964). Thus, in the case of the soil-conditions improving species of Lumbricus it may be important to prove the injuriousness of such compounds on the physiology of their organ-systems. It was found in recent studies that the snail Hefix pmatia L. absorbs some herbicidal substances within its smooth muscles these being relaxed by the herbicides (Schwippert, 1982, 1985; Backhaus et al., 1984a, c). On account of the corresponding ability to concentrate herbicidal compounds, it was supposed that in the muscle system of soil-living worms similar physiological sideeffects regarding muscle functions take place. The use of the ion~tudinal muscle system of Lumbric~s turned out to be disadvantageous with respect to the tension studies since the damage to this tissue was extensive and the lacking pressure of the coelomic fluid allowed very strong electrically evoked contractions which disrupted the body segments at the segment borders. This did not hold for the CMSpreparation applied here. A further advantage was that numerous, almost identical test preparations could be prepared from a single individual, which could be stimulated electrically every 2 min over a long period without strong, non-linear coursing fatigue. In summary the CMS of Lumbricus has to be regarded as a very simple available, relatively longliving and low-cost test preparation. Moreover, the half-maximum stimulation of the CMS-preparation allowed us to detect inhibiting as well as promoting actions in contrast to the smooth penis retractor muscle of Helix stimulated by 10-4mol/l acetylchohne. This experimental condition allowed only the measurement of inhibiting effects (Schwip~rt, 1982, 1984, 1985, 1986). From the studied substances, the herbicides chlorpropham, swep, cycloate, triallate, isoproturone, linurone, and terbutryne induced an inhibitory action on the CMS. This agrees with the effect of these compounds on the penis retractor muscle-complex of Helix (Schwippert, 1982, 1984, 1985; Backhaus et al., 1984a, b, c). With respect to the degree of action and its reversibility, however, some differences exist. For example, it appeared that the effect of cycloate was irreversible on the CMS in contrast to the effect on the penis retractor muscle, whereas in the case of triallate the appropriate ratio was inverse. Remarkable different reactions of both muscle test-systems towards the amide monalide and the urea derivate chloroxurone could also be found. Monalide first enhanced the electrically evoked contractions but inhibited them thereafter, whereas in the penis retractor muscle monalide showed an inhibiting action from the beginning of incubation. Chloroxurone promoted contractions in the worm muscle irreversibly, but inhibited them in the snail muscle reversibly. One reason for these different actions in both muscle preparations may be due to the different architecture of the smooth and oblique-striated muscles, which should be the subject of further investigations. In particular, it must not be forgotten

WOLFGANG W. SCHWIPPERT

that the different neuronal transmitter systems in these non-denervated preparations might play a role in the action of the studied compounds. Against that possibility, however, the identified dopa~ner~c, serotonergic and chohnergic transmitter systems of the smooth penis retractor muscle were insensitive towards the herbicidal drugs (Schwippert and Beneke, 1987). However, the electrically evoked, half maximum contractions in the CMS cannot be immediately compared with the maximum contractions evoked by ACh in the snail muscle. On the other hand, it is noticeable that the herbicides terbutryne or chlorpropham which relax the electrically evoked CMS-contractions or the ACh-induced contractions in the retractor muscle, act upon KCl- or caffeine-elicited contractions in the same way, independently of the differences in the muscle systems. This may be interpreted as an indi~tion of a general mechanism. Probably, the cell membranes are involved while the molecular structure of the lipid bilayer is altered or even disturbed in the presence of the herbicidal drugs and, it may be possible that membrane-bound Ca*+-mechanisms stabilizing the Ca+-equilibrium, are in0uenced. This would explain why chemically skinned muscle ill-p~parations of both muscle systems are not influenced by these substances (Schwippert, 1984; Schwippert and Beneke, 1987). Interpretation becomes more difficult when considering the temporary double action on the CMS of barbane, chlorpropham, cycloate, triallate, and terbutryne which indeed inhibited the muscle contractions, but also evoked fast phasic contractions, often with a lower tension amplitude. One possibility for this simultaneous effect implying more than one action site may be based on the release of an excitatory transmitter from the remaining nerve endings within the CMS. The results obtained by both muscle preparations serving as test systems correspond in so far as the effective concentration levels of the substances are identical whereas the concentrations themselves are always less than the recommended solutions which are given by the manufacturers or the authorities (BBA, 1981). As proven by the example of watersnaifs, the muscle-relaxing herbicides are able to permeate the body wall of the snails and the mobility and reproduction rates of the animals are reduced (Kosanke et al., in preparation). Also lumbricides concentrate the drugs from aqueous solution (Backhaus et al., 1984a) and thus, it is possible that their mobility is also diminished. If this immobility lasts, a reduced rate of worm’s reproduction may be one consequence whereby the stability of the whole popuiation is influenced, and, with it, the quality of soil. This effect is cumulative if, in addition, the development of eggs or larvae is injured. wish to thank Professor Schlote (Department of Zoology, RWTH Aachen, 5100 Aachen, FRG) for the helpful discussions and advice and Professor Reisener and his co-workers (Department of Plant Physiology, RWTH Aachen, 5100 Aachen, FRG) for their support and efforts to make a working place available to the first author. We also thank H.-D. Tetzel for the improvements to the electronic set-up and G. Kosanke for his Acknowledgements-We

critical discussions and ideas.

Biocides influencing the circular muscle system Of Lumbricus

139

Franz J. M. and Tanke W. (1974) Nebenwirkungen von DFG-Tagung, Herbiziden auf Nutzarthropoden. Backhaus R., Beneke T. and Schwippert W. W. (1984a) Side 1l.Gct. 1974, Neustadt/WeinstraBe. effects of herbicides on the mobility of snails and earthGhabbour S. I. (1966) Earthworms in agriculture: a modern worms: uptake and site of action. Med. Fat. ~dbou~w. evaluation. Rev. Ecol. Biol. Sot. 3, 259-271. Rt&iuniv. Gent 49, 1033-1039. Gilman A. P. and Vardanis A. (1974) Carbofuran: comparaBackhaus R., Beneke T, and Schwippert W. W. (1984b) tive toxicity and metabolism in the worms Lumbrtcus Nebenwirkungen von Herbiziden auf die Muskulatur VOW terrestris and Eisenia foetida. J. Agr. Food Chem. 22, Evertebraten. Verh. Dtsch. 2001. Ges. GieBen 77, 270. 625628. Backhaus R., Beneke T. and Schwippert W. W. (1984c) Goffart H. (1949) Die Wirkung neuartiger insektizider auf Nebenwirkungen von Herbiziden auf die Muskulatur von Regenwiirmer. Am. f: Schiidlingskunde 22, 72-74. Gastropoden und Lumbriziden. Mitteilungen aus der Jiiger G. (1983) Herbicides. In Chemistry of Pesticides biologischen Bundesanst~t fur Land- und Forstwirt(Edited by Bilchel K. H.), pp. 322-392. J. Wiley & Sons, schaft Berlin-Dahlem. Heft 223, 31 I-312. New York. Baker W. L. (1946) DDT and earthworm populations. J. Perkow W. (1982) Wirksubsfanzen der Pflanzenschutz- und econ. Ent. 39, 404-405. Schiidlingsbekampfungsmittet. P. Parey, Berlin. Barrett T. J. (1976) Harnessing the Earthworm. Bookworm Schwippert W. W. (1982) Inhibition of potassium- and Pub. Co., Ontario, CA. acetylcholine-induced contractions in a molluscan BBA (1981) Pflanzenschutzmittel-Verzeichnis der biolo& smooth muscle by herbicidal carbamates. Camp. Biuchem. schen Bundesanstalt fiir Land- und Forstwirtschaft Physiol. 7X, 253-257. (BBA), Teil 1 und 2. Schwippert W. W. (1984) Uber die Wirkung von Herbiziden Cook M. E. and Swait A. A. J. (1975) Effects of some auf die glatte Muskulatur von Evertebraten. Dissertation, fungicide treatments on earthworms populations and leaf RWTH Aachen. removal in apple orchards. J. hurt. Sci. 50, 495499. Schwippert W. W. (1985) Structure-activity relationship of DFG (1976) Datensammlung zur Toxikologie der Herbiherbicides inhibiting ACh-induced contractions in a molzide. Arbeitsgruppe Toxikologie der Kommission fur luscan smooth muscle. Pest. Biochem. Physiol. 24, Pflanzenschutz, Pflanzenschutzbehandlungsund Vor174181. ratsschutzmittel der Deutschen Forschungsgemein~haft Schwippert W. W. (1986) The relaxing effect of herbicidal (Editors). Chemie, Weinheim. degradation products on a molluscan smooth muscle. Duclohier H. and Georgescould D. (1976) The effects of the Pest. B&hem. Physiol. U, 439-145. insecticide decamethrin on action potential and voltage Schwippert W. W. and Beneke T. W. (1987) Evidences that clamp currents of hfyxicola giant axon. Camp. Biochem. herbicides relaxing smooth and oblique-striated muscles Physiol. 62C, 217-223. affect the muscle cell membrane. Camp. Biochem. Physiof. Edwards C. A. and Lofty J. R. (1977) Biology of Earth(in press). worms. Chapman and Hall, London. SVR.f.U. (Sachverst~ndigenrat Wr Umweltfragen, 1978) Enders E. (1952) Die Wirkung kiirpereigener Organextrakte Umweltgutachten (1978) (Edited by Der Rat der Sachauf die Bewegungen des Regenwurmdarmes. Ann. Univ. verstlndigen fur Umweltfragen, Vorsitzender: K.-H. Saran. Natur. 1, 294308. Hansmeyer). W. Kohlhammer GmbH, Stuttgart, Mainz. Fedtke C. (1982) Biochemistry and Physiology of Herbicide Ulber B. (1977) Mobilitlt und Nahrungswahl subterraner Action. Springer, Berlin. Collembolen als Riibenschldlinge unter dem EinfluD von Fox C. J. S. (1964) The effects of five herbicides on the Herbiziden. Dissertation, Landwirtsch. Fak. Georgenumbers of certain invertebrate animals in grassland soil. August-Uni., Gottingen. .l. Plant Sci. 44, 405409. REFERENCES