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Interaction and effects of molybdenum compounds on growth and mineral content of Achatina fulica and Arion ater (Gastropoda:Pulmonata)
Comp. Biochem. Physiol. Vol. 107C, No. 3, pp. 441-446, 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0305-0491/94 $6.00 + 0.00
Pergamon
Interaction and effects of molybdenum compounds on growth and mineral content of Achatina fulica and Arion ater (Gastropoda:Pulmonata) M. P. Ireland Institute of Biological Sciences, University of Wales, Aberystwyth SY23 3DA, U.K. The reduction of Cu in the tissues occurred within the first week of experimentation. The reduction in Cu concentration was most pronounced in the foot tissues after treatment with TTM (ammonium tetrathiomolybdate). Molybdate compounds had little effect on tissue metal levels except for Cu. Molybdate compounds affected Cu metabolism in the two tissues investigated in different ways. Molybdate was higher in the foot tissue after treatment with sodium molybdate but higher in the digestive gland after treatment with the thiomolybdate compounds. The final concentration of Cu after treatment with TTNI for 6 weeks was similar in the same tissue of both species. Key words: Molybdenum; Achatina fulica; Cu; Ammonium tetrathiomolybdate.
Comp. Biochern. Physiol. 107C, 441-446, 1994.
Introduction
Inorganic salts of molybdenum are usually regarded as relatively non-toxic to animals and they are currently being used in paints and hydraulic fluids, etc. as corrosion inhibition elements (Banke, 1980; Vukasovich, 1980). However, animals grazing in pastures where the vegetation is high in molybdenum can develop hypocuprosis (Thornton, 1976). Ruminants such as cows and sheep are particularly sensitive to molybdenum. Horses and pigs are less sensitive and can tolerate levels as high as 1000ppm in the diet, while molybdenum toxicity in humans has rarely been reported (Rajagopalan, 1988). The reason for the sensitivity of ruminants is apparently due to the molybdenum combining with the hydrogen sulphide in the rumen to form di- and trithiomolybdates which are absorbed into the blood stream (Hynes et al., 1985). Experimental studies using sheep have established that tetrathiomolybdate compounds are not absorbed from the intestinal
tract while di- and trithiomolybdates can be absorbed into the body and effect copper metabolism (Kincaid and White, 1988). Molybdenum can accumulate in numerous organs of animals but mostly in the bone and liver after elevated dietary intake (Underwood, 1976). High levels of molybdenum have been found in the Harderian gland of male hamsters (Hoffman and Jones, 1981). Molybdenum can reduce the net calcium transport into bone in rats (Solomons and Bekemans, 1976), while liver molybdenum increases significantly with increasing concentrations of dietary calcium (Gunshin et al., 1988). Molybdenum has also been reported to influence phosphorus and copper absorption (O'Moore, 1953). The effects of sulphate upon molybdenum retention and excretion and the interrelationship with copper have been the subject of extensive study (Chappell and Peterson, 1976). Sodium molybdate can inhibit weight gain in rats (Johnson et al., 1969). The purpose of the present study was to investigate the effects of various dietary doses Correspondence to: M. P. Ireland, Institute of Biological Sciences, Universityof Wales,AberystwythSY23 3DA, of molybdenum compounds on the elemental content of the tissues and growth of the snail U.K. Received 10 September 1993; accepted 15 October 1993. Achatina fulica and the slug Arion ater. 441
442
M.P. Ireland
Materials and Methods All snails were taken from the same batch of eggs laid in the laboratory. Because of the high mortality during early post-hatching stages, Achatina fulica were used after the snails were approximately 5 g in wet weight. Throughout the experimental period, snails were fed proprietary 350 Coney Brand Rabbit Pellets (BOCM Silcock Ltd) referred to in the text as RP. Snails were fed RP supplemented with 20% Ca as the carbonate salt and moistened with 5" 1 v/w of a salt solution or distilled water for controls. The diets were fed ad libitum and changed every 3 days. All groups were maintained at 25-30°C in a saturated environment at a 12/12 photoperiod. For the experiments using 0.2 mg/ml of molybdate compounds, snails and slugs were initially weight matched (5.2 +_ 1.1 and 5.1 + 0.2 respectively) and divided into four groups which were not significantly different from each other. Arion ater were collected locally and maintained under the same experimental conditions as Achatina. The RP Casupplemented diet was given for 1 week before experimentation. Tissues were removed from gastropods, after overnight starvation and decapitation, rinsed in distilled water, dried at 105°C for 48 hr and extracted with concentrated nitric acid. Sodium trithiomolybdate was prepared according to Mason et al. (1982) by passing hydrogen sulphide gas through a solution of buffered sodium molybdate for 30 min. It was subsequently purified by eluting from a Sephadex G-25 column where the thiomolybdate was initially retained (Zumft, 1978). The spectra for the purified compound corresponded to those reported by Aymonino et al. (1969). Ammonium tetrathiomolybdate (TTM) was purchased as black-brown crystals (Aldrich & Co) and the sodium molybdate as the hydrated
salt (BDH, Analar). Metals were estimated by atomic absorption spectrophotometry with background correction when necessary. Samples used to estimate Ca and Mg contained 0.1% lanthanum chloride. Molybdenum (Mo) was estimated using an air/acetylene flame with 0.1% sodium sulphate added to reduce interference from Ca and Fe. Inorganic phosphate was determined by the method of Rockstein and Herron (1951). Data were analysed by ANOVA with Duncan's multiple-range test or by Student's t-test. The 5% level of significance was used in all tests.
Results Growth
Preliminary experiments with Achatina using 2mg/ml trithiomolybdate and T T M compounds revealed inhibition of feeding and no significant increase in growth over 6 weeks. Sodium molybdate at the same concentration did not significantly effect growth rate. These concentrations of molybdate salts contain approximately 760 ppm Mo. After treatment with 1 mg/ml of molybdate and the T T M salts, there was less inhibition of the feeding rate as judged by faecal deposition and growth rate increase. Dietary treatment with 0.2mg/ml of all molybdate salts had no effect on the feeding rate and growth of both pulmonate species. Growth rate is illustrated for Achatina in Fig. 1. Copper
Possibly as the result of reduced feeding with 2 mg/ml molybdate compounds, there was no significant drop in the concentration of copper in the whole soft body of Achatina. After treatment with 1 mg/ml of TTM, there was a highly significant reduction in whole soft body copper (Table 1). Except for treatment with TTM, A. fulica
g 20.
10-
0
4
2
6
WEEKS
Fig. 1. Whole body wet weights of snails fed diets containing different Mo compounds (0.2mg/ml) for 6 weeks. (A) Control; (B) Na molybdate; (C) trithiomolybdate;(D) TTM (n = 6).
Effect of molybdate on snail and slug growth and Cu content Table 1. Copper concentrations (/~g/g dry wt) in whole soft body tissues o f Achatina after treatment with molybdenum c o m p o u n d s for 6 weeks (n = 6) Treatment Control Sodium molybdate Sodium trithiomolybdate A m m o n i u m tetrathiomolybdate
2 mg/ml
1 mg/ml
66.85 + 6.7 84.02 __+11.2 54.56 __+3.1
75.59 __+4.4 ---
56.63 -+ 11.4
48.22 _ 3.6
contained less Cu in the digestive gland than in foot tissue while A. ater contained more Cu in the digestive gland compared with the foot tissue. In snails treated with 0.2 mg/ml as the molybdate and TTM salts, there was significantly more copper in the digestive gland than control snails but not after treatment with trithiomolybdate. After treatment of Arion with 0.2 mg/ml of TTM, the concentration of Cu in the digestive gland was significantly lower than the control value but there was no significant drop in digestive gland Cu after treatment with molybdate or trithiomolybdate compounds. It is interesting to point out that the Cu concentration in both species after treatment with the same dose of TTM was not significantly different (Tables 2 and 3). The lowest significant concentration of copper was located in the foot tissue of Achatina and Arion fed a diet containing TTM for 6 weeks. None of the other treatments was significantly different (Tables 2 and 3). As pointed out for digestive gland, and despite the initial differences in tissue Cu, the concentration of Cu in the foot tissue after treatment with the same dose of TTM was not significantly different for both species of gastropod. As shown in Fig. 2, there was a rapid drop in Cu concentration in the tissues of Arion within a week of TTM treatment and it did not significantly change for the following 5 weeks. After 6 weeks, the percentage drop in Cu level in the digestive gland to 37% and 42% in foot tissue of the original values was close to the drop of 40% and 37% respectively presented in Table 3.
443
Molybdate No molybdate could be detected in control tissues. Much higher concentrations of molybdenum were found in the digestive gland of both gastropod species compared with foot tissue after the same treatment. In Achatina there was significantly more Mo in the digestive gland after ingestion of trithiomolybdate and TTM compounds compared with the molybdate salt but the thiomolybdate treated Mo values were not significantly different from each other (Fig. 3). Arion showed a similar high concentration of Mo after treatment with TTM, but after treatment with trithiomolybdate, the Mo concentration was not significantly different from molybdate treatment (Table 4). Molybdenum values in the digestive gland of Arion increased rapidly within the first week but did not increase in concentration over the next 5 weeks. In the foot tissue, there was significantly less Mo after treatment with trithiomolybdate and TTM compared with sodium molybdate but the thiomolybdate treatment Mo concentrations were not significantly different from each other in both species (Table 4, Fig. 3). Zinc, magnesium, calcium and phosphate Despite an apparent gain in the concentrations of Zn, Mg, Ca and P after treatment with TTM in the digestive gland of Achatina, the concentrations of these metals in both digestive gland and foot tissue were not significantly different from control values in both species investigated over 6 weeks of experimentation (Tables 2 and 3).
Discussion No animals died as the result of molybdate treatment over the 6 week experimental period, although growth was inhibited and feeding stopped at 2mg/ml. At a concentration of 1.0mg/ml of TTM, feeding and growth occurred but at a slow rate. The response was
Table 2. Copper, Zn ( # g / g dry wt) Mg, Ca and P (mg/g dry wt) in digestive gland and foot tissue 6 weeks of Achatina after treatment with molybdenum compounds (0.2 mg/ml) for 6 weeks (n = 6) Treatment
Cu
Control Sodium molybdate Sodium trithiomolybdate A m m o n i u m tetrathiomolybdate
20.35 37.02 25.51 29.89
+ 2.6 -+ 3.5 __+1.8 _ 2.0
Control Sodium molybdate Sodium trithiomolybdate A m m o n i u m tetrathiomolybdate
81.00+2.2 78.45 _+ 16.6 84.11 + 8 . 3 19.99 __+3.4
Zn 237.1 291.3 308.6 382.8
+ 60 +__49 +__39 + 40
Mg
Ca
Digestive gland 3.63 __+0.4 3.06 + 0.7 3.26 + 0.5 4.14 __+0.4
4.86 _+ 0.7 7.88 _+ 1.3 8.58 _+ 1.8 5.16 + 0.4
Foot tissue 3.30_+0.1 3.45 ___0.2 3.28+0.2 3.09 + 0.3
11.56+0.2 6.21 + 1.4 9 . 4 2 + 1.8 12.39 + 0.9
52.7+3.0 45.3 + 3.5 55.7+5.3 69.4 _ 23.4
P 9.90 8.53 8.45 12.79
__+0.15 + 1.5 + 1.0 + 1.4
5.91 ___ 1.1 4.68 + 0.7 4.25+0.6 4.83 _+ 0.9
M. P. Ireland
444
Table 3. Copper, Zn (/tg/g dry wt), Mg, Ca and P (mg/g dry wt) in digestive gland and foot tissue of Arion ater after treatment with molybdenum compounds (0.2 mg/ml) for 6 weeks (n = 5) Treatment
Cu
Zn
Mg
Ca
Control Sodium molybdate Sodium trithiomolybdate Ammonium tetrathiomolybdate
Digestive gland 76.85_+11.5 1232.6_+244 9.29_+0.4 71.69_+22.7 918.5_+ 198 7.71 _+0.8 76.54_+ 17.3 1022.5 _+ 172 7.67 _+0.8 30.59_+6.4 924.3_+163 7.36_+0.6
Control Sodium molybdate Sodium trithiomolybdate Ammonium tetrathiomolybdate
41.17+7.9 33.05+8.6 33.65+6.5 15.13_+3.3
90.72+2.9 81.84+13.7 79.52+10.8 72.71_+4.7
associated with a significant drop in the concentration of Cu in the whole soft body of Achatina. This decrease in body Cu may be the effect of reduced absorption. According to Kincaid and White (1988), TTM is not absorbed from the intestinal tract of sheep but it binds to Cu and reduces the absorption of this metal and may subsequently produce Moinduced hypocupraemia (Mason et al., 1982). Using a molybdate compound concentration of 0.2 mg/ml resulted in a feeding and growth rate comparable to control gastropods, Tissue Mo increases significantly with increasing concentration of dietary Ca (Gunshin et al., 1988) and accumulates mostly in bone and liver (Underwood, 1976). The present high dietary Ca in the standard RP diet could be responsible for the elevated concentration in the digestive gland compared with the foot tissue. Unlike most micronutrients, availability of Mo to plants is enhanced under alkaline conditions (Adriano, 1986). Molybdenum supplements decrease the concentration of Cu in the liver of sheep (White et al., 1989) and Mo is readily and rapidly absorbed from most diets. In Arion, the response of increased Mo uptake occurred within the first week of treatment with TTM.
Foot tissue 3.17+0.2 3.65_+0.3 4.18___0.5 4.82_+0.3
P
31.18__+4.8 29.97_+ 10.6 36.37 _ 4.2 21.05_+1.4 15.06+2.6 23.72_+4.1 15.96+2.1 15.55_+1.4
37.44+3.8 34.07_+4.3 34.50 _+4.7 39.66_+1.6 4.15+0.3 4.77+0.3 3.47+0.2 4.05_+0.2
An analysis of Cu distribution in the tissues revealed a species difference for A. fulica and A. ater. There was more Cu in the control Achatina foot tissue than in the slug foot tissue. The respiratory pigment of both species of gastropod investigated is haemocyanin (Redfield, 1934) which contains about 0.25% Cu (Burton, 1965) and the foot tissue contains large blood filled sinuses (Martin et al., 1958). The bulk of foot tissue is unlikely to be the sole answer to the differential Cu distribution since the blood volume of both gastropods is not significantly different (Martin et al., 1958) although the blood volume of the foot has not been determined. A similar discussion pertaining to the blood volume in the digestive gland can be used to explain the high concentration of Cu in this organ of control Arion compared with the snail species. Again, the blood volume of the digestive gland was not determined. Like the foot tissue, this organ is richly vascularized in Arion (Duval and Runham, 1981) and there is no obvious reason to suspect any significant diversity in Achatina. The difference in the Cu distribution in organs and between species has been noted elsewhere (Ireland, 1979, 1993). The Cu concentration in the digestive gland
50
o~
25
=L
i
0
2
3
4
5
6
WEEKS
Fig. 2. Copper concentration (/~g/g dry wt) in digestive gland (A) and foot tissue (B) of Arion ater after treatment with TTM for 6 weeks (n = 5).
Effect of molybdate on snail and slug growth and Cu content
30
iitl Dlgelltlve glend
20
Foot tissue
Fig. 3. Molybdenum concentration (#g/g dry wt) in the digestive gland and foot tissue of Achatina fulica after treatment with molybdate compounds (0.2 mg/ml) for 6 weeks. (A) Sodium molybdate. (B) trithiomolybdate. (C) TTM (n = 5).
appears to show a species dissimilarity in response to Mo compounds. There is a general increase in Cu values in Achatina and a decrease in Arion except after trithiomolybdate treatment which did not significantly affect Cu levels in either species. Trithiomolybdate is absorbed into the blood stream of sheep (Haynes et al., 1985) where it can compete with liver Cu-proteins for Cu in cattle (Price et al., 1987) or bind by ionic interactions to albumin protein (Clarke et al., 1987). It can also produce molybdenosis (Mason et al., 1982). From the present Mo results, trithiomolybdate clearly enters the digestive gland of the gastropod species investigated. It has been suggested that an irreversible chemical reaction between Cu and trithiomolybdate similar to the reaction with copper such as copper sulphide does not occur (Haynes et al., 1985) In terrestrial gastropods, not only soluble but also fine particulate material passes from the stomach to the digestive gland where it is absorbed (Runham and Hunter, 1970). This may account for the high concentration of Mo in the Table 4. Molybdenum concentrations (~ug/g dry wt) in digestive gland and foot tissue of Arion ater after treatment with molybdenum compounds (0.2mg/ml) for 6 weeks (n = 6) Treatment
Digestive gland
Foot tissue
Control Sodium molybdate Sodium trithiomolybdate Ammonium tetrathiomolybdate
ND 254.7 _ 48 198.6 -I- 18 517.8 + 23
ND 18.6 _ 11 4.6 _ 2 3.4 _ 2
ND none detected
445
digestive gland compared with the foot tissue and indicates that Mo from both thiomolybdate sources enters the digestive gland. If TTM is injected into sheep, it will dramatically reduce the Cu level in the liver (Kumaratilake and Howell, 1987). A decrease in Cu concentration after TTM treatment and not after trithiomolybdate is difficult to explain. It may reside in the fact that thiomolybdates are inorganic compounds containing Mo and S and TTM has the higher value of S. High intake of Mo and sulphate produce Cu deficiency in sheep (Chappel and Peterson, 1976). The dose of TTM administered appears to produce the same reduction in copper level in both species and types of tissue. Irrespective o f the initial Cu concentration in control digestive gland, the Cu concentration of both species was the same after treatment with TTM for 6 weeks. Despite the 2-fold difference in the initial Cu concentration in control foot tissue, Cu was significantly affected by TTM to the extent that the drop and final concentration were not significantly different in either species. Copper (Ryder and Bowen, 1977) as well as Ca (Simkiss and Wilbur, 1977) and Zn (Ireland, 1982) are taken up by the foot epithelium of terrestrial gastropods. If TTM is acting specifically by inhibiting Cu uptake (Kincaid and White, 1988), it would account for the response of Cu to TTM and the lack of response of Zn, Mg, Ca and P in foot tissue after treatment in both species. Sodium and ammonium molybdates are particularly well absorbed by cattle (Underwood, 1976) and could account for the high concentration of Mo in foot tissue after molybdate treatment, if thiomolybdates form less absorbable salts by this route, as indicated by the Mo values in both species. Molybdenum disulphide is not absorbed by rabbits and guinea pigs but not because it is insoluble (Underwood, 1976).
References Adriano D. C. (1986) Trace Elements in the Terrestrial Environment. Springer, New York. Aymonino P. J., Ranade A. C., Diemann E. and Muller A. (1969) Study of formation and relative reaction rates of different thioanions of molybdenum and tungsten. Z. anorg, allg. Chem. 371, 300-305. Banke W. J. (1980) Non-toxic molybdate pigments provide corrosion inhibition. Mon. Paint Coat. 70, 45-47. Burton R. F. (1965) Possible factors limiting the concentration of haemocyanin in the blood of the snail, Helix pomatia L. Can J. Zool. 43, 433-438. Chappell W. R. and Petersen K. K. (1976) Molybdenum in the Environment. Vol. 1. Marcel Dekker, Basle. Clarke N. J., Laurie S. H. and Pratt D. E. (1987) The copper-molybdenum antagonism in ruminants. IV. Reac-
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tion of thiomolybdate ions with proteins and metalloproteins. Inorg. Chim. Acta 138, 103-104. Duval A. and Runham N. W. (1981) The arterial system of six species of terrestrial slug. J. molec. Stud. 47, 43 52. Gunshin H., Ohchi H. and Kato N. (1988) Effects of dietary lactose and calcium level on tissue levels of trace elements, molybdenum, nickel, cobalt and chromium in rats. Nutr. Rept. Internl. 37, 1021 1026. Hynes M., Woods M., Poole D., Rogers P. and Mason J. (1985) Some studies on the metabolism of labelled molybdenum compounds in cattle. J. Inorg. Biochem. 24, 279 288. Hoffman R. A. and Jones J. W. (1981) Concentrations of metals in the Harderian glands of male and remale hamsters. J. Comp. Biochem. Physiol. 69A, 153-156. Ireland M. P. (1979) Distribution of essential and toxic metals in the terrestrial gastropod Arion ater. Envir. Pollut. 20, 271-278. Ireland M. P. (1982) Sites of water, zinc and calcium uptake and distribution of these metals after cadmium administration in Arion ater (Gastropoda:Pulmonata). Comp. Biochem. Physiol. 73A, 217-22 I. Ireland M. P. (1993) The effect of diamox at two dietary calcium levels on growth, shell thickness and distribution of Ca, Mg, Zn, Cu, P in the tissues of the snail Achatina fulica. Comp. Biochem. Physiol. 104C, 21-28. Johnson R. H., Little J. W. and Bickley H. C. (1969) Some effects of molybdenum on connective tissue. J. Dent. Res. 48, 1290-1295. Kincaid R. L. and White C. L. (1988) The effects of ammonium tetrathiomolybdate intake of tissue copper and molybdenum in pregnant ewes and lambs. J. Anita. Sci. 66, 3252 3258. Kumaratilake J. S. and Howell J. McC. (1987) Effects of intravenously administered tetra-thiomolybdate on the distribution of copper in the liver and kidney of copper loaded sheep: a histochemical study. Res. Vet. Sci. 42, 154-161. Martin A. W., Harrison F. M., Huston M. J. and Stewart D. M. (1958) The blood volumes of some representative molluscs. J. exp. Biol. 35, 260 279. Mason J., Lamand M. and Kelleher C. A. (1982) The effect of duodenal infusion of tri- and dithiomolybdate on plasma copper and on the diamine oxidase activity of caeruloplasmin (E.C1.16.3.1) in sheep. J. comp. Path. 92, 509-518.
O'Moore L. B. (1953) Excess of molybdenum in herbage as a possible contributary factor in equine osteodystrophia. Nature 171, 1166. Price J., Will A. M., Paschaleris G. and Chesters J. K. (1987) Identification of thiomolybdate in digesta and plasma from sheep after administration of 99Mo-labelled compounds in the rumen. Brit. J. Nutr. 58, 127-132. Rajagopalan K. V. (1988) Molybdenum: an essential trace element in human nutrition. A. Rev. Nutr. g, 401-427. Redfield A. C. (1934) The haemocyanins. Biol. Rev. 9, 175 212. Rockstein M. and Herron P. W. (1951) Colorimetric determination of inorganic phosphate in microgram quantities. Analyt. Chem. 23, 1500-1501. Runham N. W. and Hunter P. J. (1970) Terrestrial Slugs. Hutchinson, London. Ryder T. A. and Bowen I. D. (1977) The slug foot as a site of uptake of copper molluscicide. J. Invert. Path. 30, 381-386. Simkiss K. and Wilbur K. M. (1977) The molluscan epidermis and its secretions. In Comparative Biology of Skin (Edited by Spearman R.I.C.), pp. 35-76. Academic Press, London. Solomons C. C. and Bekemans K. (1976) Biological effects of molybdenum. In Molybdenum in the Environment (Edited by Chappell W. R. and Petersen K. K.), Vol. l, pp. 229-241. Marcel Dekker, Basel. Thornton I. (1976) Biogeochemical studies on molybdenum in the United Kingdom. In Molybdenum in the Environment (Edited by Chappell W. R. and Peterson K. K.), Vol. 1, pp. 341 369. Marcel Dekker, Basel. Underwood E. J. (1976) Molybdenum in animal nutrition. In Molybdenum in the Environment (Edited by Chappell W. R. and Petersen K. K.), Vol. 1, pp. 9-13. Marcel Dekker, Basel. Vukasovich M. S. (1980) Sodium molybdate corrosion inhibition of synthetic metalworking fluids. Lubric. Enginr. 36, 708-712. White C. L., Caldwalader T. K., Hoekstra W. G. and Pope A. L. (1989) Effects of copper and molybdenum supplements on the copper and selenium status of pregnant ewes and lambs. J. Anita. Sei. 67, 803-809. Zumft W. G. (1978) Isolation of thiomolybdate compounds from the molybdenum iron protein of clostridial nitrogenase. Eur. J. Biochem. 91, 345-350.
Pergamon
Interaction and effects of molybdenum compounds on growth and mineral content of Achatina fulica and Arion ater (Gastropoda:Pulmonata) M. P. Ireland Institute of Biological Sciences, University of Wales, Aberystwyth SY23 3DA, U.K. The reduction of Cu in the tissues occurred within the first week of experimentation. The reduction in Cu concentration was most pronounced in the foot tissues after treatment with TTM (ammonium tetrathiomolybdate). Molybdate compounds had little effect on tissue metal levels except for Cu. Molybdate compounds affected Cu metabolism in the two tissues investigated in different ways. Molybdate was higher in the foot tissue after treatment with sodium molybdate but higher in the digestive gland after treatment with the thiomolybdate compounds. The final concentration of Cu after treatment with TTNI for 6 weeks was similar in the same tissue of both species. Key words: Molybdenum; Achatina fulica; Cu; Ammonium tetrathiomolybdate.
Comp. Biochern. Physiol. 107C, 441-446, 1994.
Introduction
Inorganic salts of molybdenum are usually regarded as relatively non-toxic to animals and they are currently being used in paints and hydraulic fluids, etc. as corrosion inhibition elements (Banke, 1980; Vukasovich, 1980). However, animals grazing in pastures where the vegetation is high in molybdenum can develop hypocuprosis (Thornton, 1976). Ruminants such as cows and sheep are particularly sensitive to molybdenum. Horses and pigs are less sensitive and can tolerate levels as high as 1000ppm in the diet, while molybdenum toxicity in humans has rarely been reported (Rajagopalan, 1988). The reason for the sensitivity of ruminants is apparently due to the molybdenum combining with the hydrogen sulphide in the rumen to form di- and trithiomolybdates which are absorbed into the blood stream (Hynes et al., 1985). Experimental studies using sheep have established that tetrathiomolybdate compounds are not absorbed from the intestinal
tract while di- and trithiomolybdates can be absorbed into the body and effect copper metabolism (Kincaid and White, 1988). Molybdenum can accumulate in numerous organs of animals but mostly in the bone and liver after elevated dietary intake (Underwood, 1976). High levels of molybdenum have been found in the Harderian gland of male hamsters (Hoffman and Jones, 1981). Molybdenum can reduce the net calcium transport into bone in rats (Solomons and Bekemans, 1976), while liver molybdenum increases significantly with increasing concentrations of dietary calcium (Gunshin et al., 1988). Molybdenum has also been reported to influence phosphorus and copper absorption (O'Moore, 1953). The effects of sulphate upon molybdenum retention and excretion and the interrelationship with copper have been the subject of extensive study (Chappell and Peterson, 1976). Sodium molybdate can inhibit weight gain in rats (Johnson et al., 1969). The purpose of the present study was to investigate the effects of various dietary doses Correspondence to: M. P. Ireland, Institute of Biological Sciences, Universityof Wales,AberystwythSY23 3DA, of molybdenum compounds on the elemental content of the tissues and growth of the snail U.K. Received 10 September 1993; accepted 15 October 1993. Achatina fulica and the slug Arion ater. 441
442
M.P. Ireland
Materials and Methods All snails were taken from the same batch of eggs laid in the laboratory. Because of the high mortality during early post-hatching stages, Achatina fulica were used after the snails were approximately 5 g in wet weight. Throughout the experimental period, snails were fed proprietary 350 Coney Brand Rabbit Pellets (BOCM Silcock Ltd) referred to in the text as RP. Snails were fed RP supplemented with 20% Ca as the carbonate salt and moistened with 5" 1 v/w of a salt solution or distilled water for controls. The diets were fed ad libitum and changed every 3 days. All groups were maintained at 25-30°C in a saturated environment at a 12/12 photoperiod. For the experiments using 0.2 mg/ml of molybdate compounds, snails and slugs were initially weight matched (5.2 +_ 1.1 and 5.1 + 0.2 respectively) and divided into four groups which were not significantly different from each other. Arion ater were collected locally and maintained under the same experimental conditions as Achatina. The RP Casupplemented diet was given for 1 week before experimentation. Tissues were removed from gastropods, after overnight starvation and decapitation, rinsed in distilled water, dried at 105°C for 48 hr and extracted with concentrated nitric acid. Sodium trithiomolybdate was prepared according to Mason et al. (1982) by passing hydrogen sulphide gas through a solution of buffered sodium molybdate for 30 min. It was subsequently purified by eluting from a Sephadex G-25 column where the thiomolybdate was initially retained (Zumft, 1978). The spectra for the purified compound corresponded to those reported by Aymonino et al. (1969). Ammonium tetrathiomolybdate (TTM) was purchased as black-brown crystals (Aldrich & Co) and the sodium molybdate as the hydrated
salt (BDH, Analar). Metals were estimated by atomic absorption spectrophotometry with background correction when necessary. Samples used to estimate Ca and Mg contained 0.1% lanthanum chloride. Molybdenum (Mo) was estimated using an air/acetylene flame with 0.1% sodium sulphate added to reduce interference from Ca and Fe. Inorganic phosphate was determined by the method of Rockstein and Herron (1951). Data were analysed by ANOVA with Duncan's multiple-range test or by Student's t-test. The 5% level of significance was used in all tests.
Results Growth
Preliminary experiments with Achatina using 2mg/ml trithiomolybdate and T T M compounds revealed inhibition of feeding and no significant increase in growth over 6 weeks. Sodium molybdate at the same concentration did not significantly effect growth rate. These concentrations of molybdate salts contain approximately 760 ppm Mo. After treatment with 1 mg/ml of molybdate and the T T M salts, there was less inhibition of the feeding rate as judged by faecal deposition and growth rate increase. Dietary treatment with 0.2mg/ml of all molybdate salts had no effect on the feeding rate and growth of both pulmonate species. Growth rate is illustrated for Achatina in Fig. 1. Copper
Possibly as the result of reduced feeding with 2 mg/ml molybdate compounds, there was no significant drop in the concentration of copper in the whole soft body of Achatina. After treatment with 1 mg/ml of TTM, there was a highly significant reduction in whole soft body copper (Table 1). Except for treatment with TTM, A. fulica
g 20.
10-
0
4
2
6
WEEKS
Fig. 1. Whole body wet weights of snails fed diets containing different Mo compounds (0.2mg/ml) for 6 weeks. (A) Control; (B) Na molybdate; (C) trithiomolybdate;(D) TTM (n = 6).
Effect of molybdate on snail and slug growth and Cu content Table 1. Copper concentrations (/~g/g dry wt) in whole soft body tissues o f Achatina after treatment with molybdenum c o m p o u n d s for 6 weeks (n = 6) Treatment Control Sodium molybdate Sodium trithiomolybdate A m m o n i u m tetrathiomolybdate
2 mg/ml
1 mg/ml
66.85 + 6.7 84.02 __+11.2 54.56 __+3.1
75.59 __+4.4 ---
56.63 -+ 11.4
48.22 _ 3.6
contained less Cu in the digestive gland than in foot tissue while A. ater contained more Cu in the digestive gland compared with the foot tissue. In snails treated with 0.2 mg/ml as the molybdate and TTM salts, there was significantly more copper in the digestive gland than control snails but not after treatment with trithiomolybdate. After treatment of Arion with 0.2 mg/ml of TTM, the concentration of Cu in the digestive gland was significantly lower than the control value but there was no significant drop in digestive gland Cu after treatment with molybdate or trithiomolybdate compounds. It is interesting to point out that the Cu concentration in both species after treatment with the same dose of TTM was not significantly different (Tables 2 and 3). The lowest significant concentration of copper was located in the foot tissue of Achatina and Arion fed a diet containing TTM for 6 weeks. None of the other treatments was significantly different (Tables 2 and 3). As pointed out for digestive gland, and despite the initial differences in tissue Cu, the concentration of Cu in the foot tissue after treatment with the same dose of TTM was not significantly different for both species of gastropod. As shown in Fig. 2, there was a rapid drop in Cu concentration in the tissues of Arion within a week of TTM treatment and it did not significantly change for the following 5 weeks. After 6 weeks, the percentage drop in Cu level in the digestive gland to 37% and 42% in foot tissue of the original values was close to the drop of 40% and 37% respectively presented in Table 3.
443
Molybdate No molybdate could be detected in control tissues. Much higher concentrations of molybdenum were found in the digestive gland of both gastropod species compared with foot tissue after the same treatment. In Achatina there was significantly more Mo in the digestive gland after ingestion of trithiomolybdate and TTM compounds compared with the molybdate salt but the thiomolybdate treated Mo values were not significantly different from each other (Fig. 3). Arion showed a similar high concentration of Mo after treatment with TTM, but after treatment with trithiomolybdate, the Mo concentration was not significantly different from molybdate treatment (Table 4). Molybdenum values in the digestive gland of Arion increased rapidly within the first week but did not increase in concentration over the next 5 weeks. In the foot tissue, there was significantly less Mo after treatment with trithiomolybdate and TTM compared with sodium molybdate but the thiomolybdate treatment Mo concentrations were not significantly different from each other in both species (Table 4, Fig. 3). Zinc, magnesium, calcium and phosphate Despite an apparent gain in the concentrations of Zn, Mg, Ca and P after treatment with TTM in the digestive gland of Achatina, the concentrations of these metals in both digestive gland and foot tissue were not significantly different from control values in both species investigated over 6 weeks of experimentation (Tables 2 and 3).
Discussion No animals died as the result of molybdate treatment over the 6 week experimental period, although growth was inhibited and feeding stopped at 2mg/ml. At a concentration of 1.0mg/ml of TTM, feeding and growth occurred but at a slow rate. The response was
Table 2. Copper, Zn ( # g / g dry wt) Mg, Ca and P (mg/g dry wt) in digestive gland and foot tissue 6 weeks of Achatina after treatment with molybdenum compounds (0.2 mg/ml) for 6 weeks (n = 6) Treatment
Cu
Control Sodium molybdate Sodium trithiomolybdate A m m o n i u m tetrathiomolybdate
20.35 37.02 25.51 29.89
+ 2.6 -+ 3.5 __+1.8 _ 2.0
Control Sodium molybdate Sodium trithiomolybdate A m m o n i u m tetrathiomolybdate
81.00+2.2 78.45 _+ 16.6 84.11 + 8 . 3 19.99 __+3.4
Zn 237.1 291.3 308.6 382.8
+ 60 +__49 +__39 + 40
Mg
Ca
Digestive gland 3.63 __+0.4 3.06 + 0.7 3.26 + 0.5 4.14 __+0.4
4.86 _+ 0.7 7.88 _+ 1.3 8.58 _+ 1.8 5.16 + 0.4
Foot tissue 3.30_+0.1 3.45 ___0.2 3.28+0.2 3.09 + 0.3
11.56+0.2 6.21 + 1.4 9 . 4 2 + 1.8 12.39 + 0.9
52.7+3.0 45.3 + 3.5 55.7+5.3 69.4 _ 23.4
P 9.90 8.53 8.45 12.79
__+0.15 + 1.5 + 1.0 + 1.4
5.91 ___ 1.1 4.68 + 0.7 4.25+0.6 4.83 _+ 0.9
M. P. Ireland
444
Table 3. Copper, Zn (/tg/g dry wt), Mg, Ca and P (mg/g dry wt) in digestive gland and foot tissue of Arion ater after treatment with molybdenum compounds (0.2 mg/ml) for 6 weeks (n = 5) Treatment
Cu
Zn
Mg
Ca
Control Sodium molybdate Sodium trithiomolybdate Ammonium tetrathiomolybdate
Digestive gland 76.85_+11.5 1232.6_+244 9.29_+0.4 71.69_+22.7 918.5_+ 198 7.71 _+0.8 76.54_+ 17.3 1022.5 _+ 172 7.67 _+0.8 30.59_+6.4 924.3_+163 7.36_+0.6
Control Sodium molybdate Sodium trithiomolybdate Ammonium tetrathiomolybdate
41.17+7.9 33.05+8.6 33.65+6.5 15.13_+3.3
90.72+2.9 81.84+13.7 79.52+10.8 72.71_+4.7
associated with a significant drop in the concentration of Cu in the whole soft body of Achatina. This decrease in body Cu may be the effect of reduced absorption. According to Kincaid and White (1988), TTM is not absorbed from the intestinal tract of sheep but it binds to Cu and reduces the absorption of this metal and may subsequently produce Moinduced hypocupraemia (Mason et al., 1982). Using a molybdate compound concentration of 0.2 mg/ml resulted in a feeding and growth rate comparable to control gastropods, Tissue Mo increases significantly with increasing concentration of dietary Ca (Gunshin et al., 1988) and accumulates mostly in bone and liver (Underwood, 1976). The present high dietary Ca in the standard RP diet could be responsible for the elevated concentration in the digestive gland compared with the foot tissue. Unlike most micronutrients, availability of Mo to plants is enhanced under alkaline conditions (Adriano, 1986). Molybdenum supplements decrease the concentration of Cu in the liver of sheep (White et al., 1989) and Mo is readily and rapidly absorbed from most diets. In Arion, the response of increased Mo uptake occurred within the first week of treatment with TTM.
Foot tissue 3.17+0.2 3.65_+0.3 4.18___0.5 4.82_+0.3
P
31.18__+4.8 29.97_+ 10.6 36.37 _ 4.2 21.05_+1.4 15.06+2.6 23.72_+4.1 15.96+2.1 15.55_+1.4
37.44+3.8 34.07_+4.3 34.50 _+4.7 39.66_+1.6 4.15+0.3 4.77+0.3 3.47+0.2 4.05_+0.2
An analysis of Cu distribution in the tissues revealed a species difference for A. fulica and A. ater. There was more Cu in the control Achatina foot tissue than in the slug foot tissue. The respiratory pigment of both species of gastropod investigated is haemocyanin (Redfield, 1934) which contains about 0.25% Cu (Burton, 1965) and the foot tissue contains large blood filled sinuses (Martin et al., 1958). The bulk of foot tissue is unlikely to be the sole answer to the differential Cu distribution since the blood volume of both gastropods is not significantly different (Martin et al., 1958) although the blood volume of the foot has not been determined. A similar discussion pertaining to the blood volume in the digestive gland can be used to explain the high concentration of Cu in this organ of control Arion compared with the snail species. Again, the blood volume of the digestive gland was not determined. Like the foot tissue, this organ is richly vascularized in Arion (Duval and Runham, 1981) and there is no obvious reason to suspect any significant diversity in Achatina. The difference in the Cu distribution in organs and between species has been noted elsewhere (Ireland, 1979, 1993). The Cu concentration in the digestive gland
50
o~
25
=L
i
0
2
3
4
5
6
WEEKS
Fig. 2. Copper concentration (/~g/g dry wt) in digestive gland (A) and foot tissue (B) of Arion ater after treatment with TTM for 6 weeks (n = 5).
Effect of molybdate on snail and slug growth and Cu content
30
iitl Dlgelltlve glend
20
Foot tissue
Fig. 3. Molybdenum concentration (#g/g dry wt) in the digestive gland and foot tissue of Achatina fulica after treatment with molybdate compounds (0.2 mg/ml) for 6 weeks. (A) Sodium molybdate. (B) trithiomolybdate. (C) TTM (n = 5).
appears to show a species dissimilarity in response to Mo compounds. There is a general increase in Cu values in Achatina and a decrease in Arion except after trithiomolybdate treatment which did not significantly affect Cu levels in either species. Trithiomolybdate is absorbed into the blood stream of sheep (Haynes et al., 1985) where it can compete with liver Cu-proteins for Cu in cattle (Price et al., 1987) or bind by ionic interactions to albumin protein (Clarke et al., 1987). It can also produce molybdenosis (Mason et al., 1982). From the present Mo results, trithiomolybdate clearly enters the digestive gland of the gastropod species investigated. It has been suggested that an irreversible chemical reaction between Cu and trithiomolybdate similar to the reaction with copper such as copper sulphide does not occur (Haynes et al., 1985) In terrestrial gastropods, not only soluble but also fine particulate material passes from the stomach to the digestive gland where it is absorbed (Runham and Hunter, 1970). This may account for the high concentration of Mo in the Table 4. Molybdenum concentrations (~ug/g dry wt) in digestive gland and foot tissue of Arion ater after treatment with molybdenum compounds (0.2mg/ml) for 6 weeks (n = 6) Treatment
Digestive gland
Foot tissue
Control Sodium molybdate Sodium trithiomolybdate Ammonium tetrathiomolybdate
ND 254.7 _ 48 198.6 -I- 18 517.8 + 23
ND 18.6 _ 11 4.6 _ 2 3.4 _ 2
ND none detected
445
digestive gland compared with the foot tissue and indicates that Mo from both thiomolybdate sources enters the digestive gland. If TTM is injected into sheep, it will dramatically reduce the Cu level in the liver (Kumaratilake and Howell, 1987). A decrease in Cu concentration after TTM treatment and not after trithiomolybdate is difficult to explain. It may reside in the fact that thiomolybdates are inorganic compounds containing Mo and S and TTM has the higher value of S. High intake of Mo and sulphate produce Cu deficiency in sheep (Chappel and Peterson, 1976). The dose of TTM administered appears to produce the same reduction in copper level in both species and types of tissue. Irrespective o f the initial Cu concentration in control digestive gland, the Cu concentration of both species was the same after treatment with TTM for 6 weeks. Despite the 2-fold difference in the initial Cu concentration in control foot tissue, Cu was significantly affected by TTM to the extent that the drop and final concentration were not significantly different in either species. Copper (Ryder and Bowen, 1977) as well as Ca (Simkiss and Wilbur, 1977) and Zn (Ireland, 1982) are taken up by the foot epithelium of terrestrial gastropods. If TTM is acting specifically by inhibiting Cu uptake (Kincaid and White, 1988), it would account for the response of Cu to TTM and the lack of response of Zn, Mg, Ca and P in foot tissue after treatment in both species. Sodium and ammonium molybdates are particularly well absorbed by cattle (Underwood, 1976) and could account for the high concentration of Mo in foot tissue after molybdate treatment, if thiomolybdates form less absorbable salts by this route, as indicated by the Mo values in both species. Molybdenum disulphide is not absorbed by rabbits and guinea pigs but not because it is insoluble (Underwood, 1976).
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