Quantification of acetochlor degradation in the unsaturated zone using two novel in situ field techniques: comparisons with laboratory-generated data and implications for groundwater risk assessments.

Degradation of the herbicide acetochlor in the unsaturated zone was quantified using two unique in situ field techniques. The DT50 values generated at two different sites on surface soil and two subsoil depths using these techniques were compared with values generated under aerobic laboratory-incubation conditions (typically 20 degrees C, 40% maximum water holding capacity). Additionally, laboratory-degradation data were generated on surface and subsoils from four other sites. All subsoils were treated with acetochlor at 5% of the surface soil application rate. Acetochlor degradation in both field- and laboratory-incubated subsoils was rapid and often exceeded surface soil rates. Field and laboratory DT50 values from all sites ranged from 2 to 88 days in subsoil, compared with a range of 1 to 18 days in surface soils. The DT50 results from in situ field techniques were comparable with those generated from laboratory incubations in the same soils, confirming the validity of performing laboratory-based degradation studies to determine pesticide DT50 values in subsoils. Microbiological characterisation of selected soils revealed that subsoils had a viable and active population, although direct counts of bacteria were consistently lower in subsoil (10(8)-10(9) g-1 dry soil) compared with surface soils (10(10) g-1 dry soil). The leaching models used to perform groundwater risk assessments (e.g. PELMO, PESTLA, MACRO-DB, PRZM and the FOCUS EU leaching scenarios) have provision for inclusion of subsoil degradation rates. However, conservative default estimates are typically used, as no other alternative is available. Results presented here show that these default values may significantly underestimate true subsoil degradation contributions, and therefore not accurately predict pesticide concentrations in groundwater. The degradation data generated for acetochlor were applied to the mathematical model PELMO to demonstrate the importance of the inclusion of subsoil degradation data in groundwater risk assessment models and thereby in the registration of pesticides in Europe.

Chloride-thiocyanate interactions in frog muscle anion-conducting channels at pH 5.

Bi-ionic membrane potential measurements and three-microelectrode voltage clamp experiments have been performed on surface fibres of Xenopus laevis sartorius muscle at various mole fractions of SCN- in Cl- in the perfusate, at pH 5. Potassium was replaced in the test solutions by rubidium and/or tetraethylammonium and when the mole fraction of anions was changed the measured membrane potential changed to a new constant (i.e. time-independent) value. Over a mole fraction range of 0.05-0.95 the permeability ratio of thiocyanate to chloride was independent of [SCN-]. When the bathing solution was completely changed from control to one containing thiocyanate the change in membrane potential indicated that the permeability ratio, PSCN/PCl is close to 5.9. Inward voltage clamp currents (chloride efflux) were suppressed in the presence of thiocyanate, the degree of suppression increasing with [SCN-]. Outward currents (anion influx) were not substantially altered, suggesting that it is only the voltage-dependent anion current that is sensitive to SCN-. The results are interpreted as indicating that there is a binding site in the anion-conducting channel, accessible to the extracellular space, that must be occupied by an anion in order for the channel to be "open". But that for ion traverse to be complete, the ion at the binding site must be exchanged. If the site is occupied by thiocyanate, the more strongly bound ion, the thiocyanate blocks the channel. The bi-ionic permeability ratio is thought to reflect the ratio of the binding constants for the anions at that site.

Hypertonicity and force development in frog skeletal muscle fibres.

Mechanical experiments were performed on isolated fibre bundles of frog semitendinosus muscle, using solutions made hypertonic by the addition of sucrose or NaC1. Muscle stiffness was measured with 1-ms step changes of length, from the plateau of an isometric tetanus. Within 20 min in hypertonic Ringer, isometric tetanus tension fell by 50% or more. The rate of tension redevelopment following stepwise shortening was reduced in hypertonic solution. Point voltage clamp studies showed that even when isometric tension had fallen to this extent, the stimulus strength--duration relation for mechanical activation was identical to that for controls. Point voltage clamp was used to intermittently repolarize fibres depolarized in solutions of high ionic strength, reactivating the excitation contraction coupling mechanism. Subsequent depolarization of the fibres allowed a visual test to be made of the fibres' ability to contract. The period during which contraction could be seen grew shorter as the ionic strength of the bathing medium was raised. The results of these different types of mechanical experiments suggested that the increased intracellular ionic strength caused by osmotic shrinkage in hypertonic Ringer's solution slows the rate at which individual cross bridges can form and develop tension, as well as reducing the maximum amount of tension they can generate. Muscle stiffness did not remain at control level in hypertonic solution; nor did it fall as much as did developed tension. The most obvious, although not the only, interpretation of this observation was that cross bridges are less stretched when isometric force is reduced and the length-tension relation of the elastic element is nonlinear.

Some observations on the behaviour of chloride current--voltage relations in Xenopus muscle membrane in acid solutions.

Chloride current--voltage relations in Xenopus laevis muscle membrane have been investigated in phosphate-buffered solution (pH 5.2--5.4) using a three-microelectrode voltage clamp. Resting chloride conductance in these conditions is about 10(-4) S/cm2, approximately 1/10th that at pH 8.8. When the membrane potential is stepped from the holding (resting) potential to a more negative voltage the current rises from the initial to the steady state. The instantaneous current--voltage relation is linear and the steady-state relation shows inward-going rectification. As hyperpolarization appears to "activate" the chloride conductance, the "availability" of chloride current has been measured at the beginning of a voltage step to a standard test potential following conditioning at a variety of potentials. The relationship between the test current and the conditioning voltage is sigmoid. The normalized sigmoid curve has the same slope (absolute value) but opposite sign to that obtained in the same experiment at pH 8.8. In mildly acidic solutions (pH 6.4) the current wave form is diphasic: current initially falls then rises to the steady state. This combination of transients militates against the idea that transients are due solely to accumulation--depletion effects in restricted spaces ("unstirred layers") and a hypothesis is qualitatively outlined in which pH-and voltage-dependent effects are ascribed to a single type of channel whose orientation in the membrane is unconstrained.

Mechanical activation in dystrophic C57BL mouse muscle.

Stimulus strength-duration curves for contraction threshold have been obtained from normal and dystrophic C57BL mouse muscle fibres using two microelectrode voltage clamp techniques. The threshold membrane potential for activation in dystrophic soleus fibres was further from the resting membrane potential than in normal fibres and was close to the threshold for extensor digitorum longus fibres. When a redistribution if fibre types in dystrophic soleus muscles is taken into account the dystrophic data fell within the normal range of values. It is apparent that the inability of dystrophic C57BL mice to use their hind limbs in the normal way cannot be attributed to a failure of excitation-contraction coupling.

Muscle fiber capacity in low conductivity solution.

A method is described for computing the effective capacity of muscle fibers, C = Q/V where Q is the charge stored, and V is the membrane potential, using a standard two-microelectrode, constant current injection technique. The method is used to compare physical (or effective) capacity of frog muscle fibers bathed in a low conductivity, 2.5 mMK+ solution, with circuittheory derived quantities in the same cells and in control fibers. No differences can be discerned and it is concluded that low conductivity of physiological solutions, per se, does not significantly reduce the length constant of frog muscle transverse tubules.

The capacitance of skeletal muscle fibers in solutions of low ionic strength.

The capacitance of skeletal muscle fibers was measured by recording with one microelectrode the voltage produced by a rectangular pulse of current applied with another microelectrode. The ionic strength of the bathing solution was varied by isosmotic replacement of NaCl with sucrose, the [K] [Cl] product being held constant. The capacitance decreased with decreasing ionic strength, reaching a value of some 2 microF/cm(2) in solutions of 30 mM ionic strength, and not decreasing further in solutions of 15 mM ionic strength. The capacitance of glycerol-treated fibers did not change with ionic strength and was also some 2 microF/cm(2). It seems likely that lowering the ionic strength reduces the capacitance of the tubular system (defined as the charge stored in the tubular system), and that the 2 microF/cm(2) which is insensitive to ionic strength is associated with the surface membrane. The tubular system is open to the external solution in low ionic strength solutions since peroxidase is able to diffuse into the lumen of the tubules. Twitches and action potentials were also recorded from fibers in low ionic strength solutions, even though the capacitance of the tubular system was very small in these solutions. This finding can be explained if there is an action potential-like mechanism in the tubular membrane.

A theoretical analysis of the capacitance of muscle fibers using a distributed model of the tubular system.

A model is developed to predict the changes in total capacitance (i.e. total charge stored divided by surface membrane potential) of the tubular system of muscle fibers. The tubular system is represented as a punctated disc and the area of membrane across which current flows is represented as a punctated annulus, the capacitance of the muscle fiber being proportional to this area. The area can be determined from a distributed model of the tubular system, in which the only resistance to radial current flow is presumed to be in the lumen of the tubules. Calculations are made of the variation of capacitance expected as the conductivity of the bathing solution is varied. These calculations include the effects of fixed charge in the tubular lumen and the effects of changes in the shape and volume of the tubular system in solutions of low conductivity. The calculated results fail to fit comparable experimental data, although they do qualitatively account for the known variation of the radial spread of contraction with conductivity of the bathing medium. It is pointed out that the existence of a significant "access resistance" at the mouth of the tubules might explain the discrepancy between theory and experiment.

Ultrastructure and contractures of the pigeon iris striated muscle.

1. The ultrastructure of adult pigeon iris muscle fibres has been described with emphasis on the distribution of the sarcoplasmic reticulum (SR). Contractures due to superfusion with solutions of different [K(+)] (3-150 mM) and acetylcholine (ACh) and their modification by alteration of external [Ca(2+)] and [Mg(2+)] were studied in isolated pigeon iris.2. The arrangement of the contractile myofilaments was like that of vertebrate skeletal fibres. The SR is well developed in the I-band and sparse at the A-band level. Tubular elements (T-system) which form triads with the SR were seen at all levels of the sarcomere though usually adjacent to the A-I junction.3. K(+) contractures developed monotonically to a steady level which was maintained for the duration of the high [K(+)] superfusion. The response to a standard [K(+)] stepwise change was not altered by conditioning the preparation with various [K(+)].4. Decreasing external [Ca(2+)] from 20 mM to Ca(2+)-free (i.e. no Ca(2+) added), enhanced iris contractures at all [K(+)] and in ACh enriched solutions. The K(+) response was abolished when the iris was superfused with Ca(2+) free solution plus EDTA (2 mM) for 45 min. Increasing [Mg(2+)] had little or no effect on iris contracture.5. Reducing external [Ca(2+)] from 3 to 0.3 mM caused a reduction of 3-7 mV in resting membrane potential and an increase from 3 to 10 mM-Ca(2+) caused 3 to 7 mV membrane hyperpolarization. Muscle fibre input resistance was not affected.6. It is concluded that in the pigeon iris, Ca(2+) required for contractile activation is obtained from internal stores, that membrane potential determines the degree of contractile activation and that the maintenance of the contracture is dependent on the failure of the Ca(2+) releasing mechanism to inactive. In addition, it is speculated that because the iris muscle has only sparse SR at the A-band level of the sarcomere, there may be slow Ca(2+) reaccumulation.

The maintenance of resting potentials in glycerol-treated muscle fibres.

1. A modification of a previously published method for the disruption of the T-tubules of frog skeletal muscle is described. The modification permits the disruption of the T-tubules without the decline in resting potentials which was reported previously.2. The method for the disruption of the T-tubules involves the washout of glycerol following loading in a 400 mM glycerol Ringer solution. The modification consists of elevating the concentration of divalent cations in the Ringer used for glycerol washout.3. The optimum concentrations are 5 mM-Ca(2+) and 5 mM-Mg(2+) added as their chloride salts. Neither 10 mM-Ca(2+) nor 10 mM-Mg(2+) are as effective as the combination of each at 5 mM. Other concentrations gave less satisfactory results.4. The use of the modified technique provides a preparation which maintains 85-90 mV resting potentials for up to 6 or 8 hr but which will not contract in response to membrane depolarization.