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Magnesium and the deposition of lead in the shell of three populations of the garden snail Cantareus aspersus
Environmental Pollution 159 (2011) 1667e1672
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Magnesium and the deposition of lead in the shell of three populations of the garden snail Cantareus aspersus Alan Beeby*, Larry Richmond Department of Applied Science, London South Bank University, Borough Road, London SE1 0AA, UK
Snails losing shell mass on an experimental diet have higher concentrations of shell Pb and Mg, but lose Ca.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 November 2010 Received in revised form 18 February 2011 Accepted 26 February 2011
The loss of Pb from snail soft tissues may depend on the excretion of Ca, and involve the mobilization of shell Ca. Most sub-adults in three populations of Cantareus aspersus (syn. Cornu aspersum Müller) either failed to add, or lost, shell mass on a diet with 250 mg g1 Pb. Their failure to mineralize shell extensions occurred irrespective of food consumed, time or dietary Mg. Budgets of metals for 36 individuals in each of two populations showed that Ca loss would account for the reduction in shell mass. Lead concentrations were higher in the reduced shells but this may be a consequence of their smaller mass, rather than its cause. In both populations shell reduction correlated with the total mass of Pb assimilated. Any shell growth may have been dependent on the initial Ca reserve in each snail. Differential movement of Mg, Pb and Ca occurred between the shell and soft tissues. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Lead regulation Magnesium Calcium Shell Cantareus aspersus
1. Introduction The retention of lead by the soft tissues of Helicid snails is highly consistent, both within species (Cantareus aspersus e Beeby, 1985; Laskowski and Hopkin, 1996a,b) and between related species (Helix engaddensis e Swaileh and Ezzughayyar, 2001). Soft tissue concentrations rarely exceed 40% of those in the food consumed, though unlike several other toxic metals, Pb does not appear to be regulated by a process involving inducible proteins. There is some indication that Pb assimilation may be linked to growth of the soft tissues (Beeby and Richmond, 2010) or, as with other toxic metals, cell turnover in the hepatopancreas (Hodl et al., 2010; Soto et al., 1997; Zaldibar et al., 2008). Lead may be incorporated into the soft tissues because the snail is unable to distinguish the toxic metal from essential metals, most probably Ca (Behra, 1993; Schanne et al., 1989), but also possibly Mg (Dowd et al., 1990; Srivastava et al., 1995). Dietary Mg does not affect Pb assimilation by the soft tissues of C. aspersus (Beeby and Richmond, 2010), but they are able to exclude Pb from certain Carich tissues (Beeby and Richmond, 1998). Lead is found in the shell, albeit at low concentrations (Laskowski and Hopkin, 1996a), where it has a retention time much greater than that of the soft tissues
* Corresponding author. E-mail addresses: [email protected] (A. Beeby), [email protected] (L. Richmond). 0269-7491/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2011.02.040
(Beeby and Richmond, 1989). It is also known to inhibit the mineralization of shells in juvenile snails (Beeby et al., 2002). Calcium, or more particularly its availability, is one of the most important factors governing the distribution of snails in a range of habitats and their capacity to build shells (Gardenfors, 1992; Ireland, 1991; Kalisz and Powell, 2003; Mulvey et al., 1996; Ondina et al., 2004). Most of the mass of the shell is calcium carbonate, but Mg is also part of the crystalline structure of the shell (Fournie and Chetail, 1984). These mineral components are deposited beneath a proteinaceous outer layer, the periostracum, in a process of reinforcement following the growth of this horn-like shell extension, normally at the beginning of the growing season (Fournie and Chetail, 1984). Snails actively mineralizing their shells might incorporate more Pb into the crystalline matrix, and this may be affected by the availability of Mg and Ca in the diet. Snails are known to soil-feed (and drink) to supplement their supply of Ca (Crowell, 1973; Gomot et al., 1989; Heller and Magaritz, 1983), in the upper soil layers where Pb accumulates in contaminated habitats. In a previous paper we described an experiment in which three populations were fed a Pb-dosed diet with three different levels of Mg (Beeby and Richmond, 2010). The populations differed in their responses but soft tissue assimilation of Pb slowed in all as soft tissue growth slowed, and perhaps as shell mineralization commenced. This paper examines the deposition of Pb in the shell of these sub-adult snails over the 64-day experimental period, as they begin to reinforce their shell extensions.
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2. Methods The details of the collection and culturing of Cantareus aspersus from the three locations are given in Beeby and Richmond (2010). Sub-adults were collected in March and April, 2008, towards the end of their hibernation and prior to their new season shell growth. Two populations were from Ca-rich habitats (LE in southern England, RF in southern France) and the third from a Ca-poor soil in southern England (SB). Concentrations of soil Mg were almost twice as high at LE as the other two sites; RF was uncontaminated and SB the site most contaminated with Pb.
Table 1 The mean and standard error (n ¼ 12) for the estimated initial and final shell weights for three populations of Cantareus aspersus at each sampling interval (combining data from the three diets). Whilst the growth in the soft tissues continued throughout the experiment, the average shell mass declined in LE and SB. Population
Time days
Estimated initial shell weight mg
Final shell weight mg
Growth in soft tissues mg
LE
16 32 64
432 (39) 390 (41) 387 (30)
408 (35) 375 (45) 358 (33)
244 (31) 271 (36) 298 (44)
SB
16 32 64
389 (34) 384 (42) 421 (29)
400 (50) 392 (52) 370 (27)
310 (34) 340 (58) 466 (69)
RF
16 32 64
269 (25) 292 (29) 293 (17)
283 (34) 280 (30) 299 (32)
306 (35) 311 (36) 313 (28)
2.1. Experimental design After 3e4 weeks depuration feeding on commercially-grown lettuce, four individuals from each population were sacrificed and analysed for their metal content (day 0). Thereafter 36 snails of each population were randomly allocated to an artificial diet with one of three levels of Mg but with similar Pb and Ca levels. Four snails on each dose for each population were sampled at 16, 32 and 64 days. The details of the feeding regime and the analysis of the soft tissues are given in Beeby and Richmond (2010). For each snail the amount of food consumed, and therefore the consumption of Mg, Ca and Pb, was measured and compared to the mass recovered from the soft tissues to give percentage assimilation rates, or for the shell and soft tissues combined, the total assimilation rates. 2.2. Sample treatment and analysis After 24 h depuration by starvation each snail was sacrificed by freezing. The shell was separated from the thawed soft tissues, dried for 12 h at 80 C, weighed and then digested by slowly introducing 10 ml of 50% HNO3. After complete dissolution of the mineral component the vessel was sealed with the solution unfiltered and stored in a refrigerator prior to analysis. Solutions were analysed using a Varian 820 ICP-MS, using the isotopes Mg24, Ca43, Pb108 and Sr88. The linearity of the calibration curve for each metal was confirmed over four orders of magnitude against standard solutions (Inorganic Ventures IV-ICPMS-71A). The shells required a 40-fold dilution using ultra-pure water (up to 18.2 MU cm) prior to final analysis for Ca. Four glassware controls were used to measure and correct for any background contamination in each run. Average recovery rates for Pb from four replicates of a reference material (Lobster hepatopancreas TORT-2 e Environment Canada) were within the certified range. Certified values for Ca and Mg are not provided for TORT-2 but the uniformity of their results between digestion runs was taken to indicate consistent recovery rates. 2.3. Statistical analysis Growth or loss of shell mass was determined for each snail with reference to its estimated initial shell weight. This was calculated as the proportion of the initial total fresh weight attributable to the shell, based on the four snails sacrificed at day 0 for each population and from which a median was calculated. Medians were used because of the small sample size and the occasional outlier amongst some results. The inter-quartile range of this proportion was used to derive the limits within which the initial shell weight of each dosed snail most probably fell. By comparing its final shell weight to this range, a snail was classified as having added to its shell mass (above the upper quartile limit), lost mass (below the lower quartile limit) or indistinguishable from its initial starting weight. This conservative approach produced a wide range of values within which no change in shell weight would be attributed. Only those snails in the first two classes were regarded as showing positive or negative shell growth and used in statistical comparisons between the groups. One population did not produce groups with sufficient numbers to enable comparison e LE had 21 snails that were indistinguishable from their starting shell weight, and only three with positive shell growth. SB and RF each had 12 snails showing shell growth, 15 with losses, and 9 that could not be distinguished from their starting shell weights. Comparisons between the groups showing positive or negative shell growth were thus confined to the SB and RF populations, using t-tests assuming unequal variances. Growth in the shell and soft tissues, and the assimilation of both food and metals were examined using regression and correlation analysis. c2 analysis was used to test for any association of shell growth with diet or feeding duration in these two populations.
not, and in LE and SB average shell mass declined. These diets thus supported soft tissue growth, though this was lower in snails losing shell mass in both SB and RF (Fig. 1). This may be indicative of some more general cost or response to Pb in those snails unable to add to their shell mass. Of the three populations LE had the largest initial shell ratios (median 2.1) but the smallest change in shell mass during the experiment. In contrast, a third of SB and RF snails increased their shell mass (by 5e39% and 9e44% respectively) whilst almost half of each population lost shell (by 8e50% and 5e33% respectively). These changes were irrespective of a snail’s diet or duration of feeding: a c2 test of the combined data for these two populations found no significant association of either group with feeding duration (c2 ¼ 0.693, 2df, P > 0.9) or Mg diet (c2 ¼ 1.672, 2df, P > 0.5). There was no significant difference in the soft tissue assimilation of Ca in the two groups of either population (SB t ¼ 1.05 NS; RF t ¼ 1.37 NS) and there were few other indications of why the groups differed (Table 2). They did differ in their shell Mg and Pb concentrations (Table 3). However, the SB group losing shell had smaller fresh weights at the start of the experiment than those snails adding shell (2.44 vs. 3.05 g; t ¼ 1.82, P ¼ 0.04), whereas RF just failed to show a significant difference (2.03 vs. 2.34 g; t ¼ 1.43, P ¼ 0.08). The
3. Results 3.1. Shell growth With a single exception, each snail added a shell extension soon after the start of the experiment, though none had completed their mineralization by its end. Median shell ratios (shell dry weight/soft tissue dry weight) were above 1.5 in day 0 snails but these declined to 0.8 or less in each population by day 64: whilst the soft tissues increased in weight in all snails over time (Table 1), most shells did
Fig. 1. Mean growth rate in the soft tissues of Cantareus aspersus showing a reduction in shell mass (light fill) or adding to their shells (dark fill), for each of two populations (irrespective of feeding duration or diet). In each population snails losing shell mass have significantly lower rates of soft tissue growth (SB t ¼ 2.10, P ¼ 0.05; RF t ¼ 2.15, P ¼ 0.02).
A. Beeby, L. Richmond / Environmental Pollution 159 (2011) 1667e1672
Strontium, a known analogue for Ca, followed the same pattern. This was supplied at a uniform level in all diets e around 6 mg g1 e and again the groups were distinguished by the amount of Sr lost or gained in each population (Table 3). This is further indication of a demineralization of the shell and mobilization of Ca and Sr. There were, however, differences between the populations: a large proportion of the SB snails growing their shells also appeared to have lost Sr, at least in comparison to their estimated starting mass. Most shells had Mg concentrations between 150 and 200 mg g1, but these rose with dietary level in both RF (F ¼ 6.176, P ¼ 0.006) and LE (F ¼ 7.498, P ¼ 0.002) though not SB (F ¼ 0.99, P ¼ 0.384). The highest levels were achieved by RF snails at the highest dose on day 64 (average 490 mg g1). Lead concentrations rose in the shells of all populations from around 2e3 mg g1 at day 16 to 6 mg g1 by day 64: duration of feeding was a significant factor for both LE (F ¼ 14.131, P ¼ 0.000) and SB (F ¼ 7.69, P ¼ 0.002), and probably for RF as well (F ¼ 3.165, P ¼ 0.058). Dietary Mg was also a significant factor for shell Pb in RF (F ¼ 3.763, P ¼ 0.036) but this was again largely attributable to the high concentrations in one group e the snails on the highest Mg diet at day 64. Magnesium dose did not appear to affect shell assimilation of Pb in LE and SB, but the mass of Pb and Mg were correlated in both SB and RF across all shells (Table 5). Lead and Mg concentrations were higher in shells that had lost mass in both SB and RF (Table 3). However the weight of these metals in the shell did not differ between the groups in either population (Table 2) and so concentrations rose as shell mass fell. Comparing the groups on a standardised weight basis (by calculating the equivalent concentration using their measured final Pb mass and estimated initial shell mass), Pb concentrations did not differ between them in either population (SB t ¼ 0.28, NS; RF t ¼ 1.53, NS). The increased Pb concentrations could result solely from the reduction in shell mass, rather than any increase in the assimilation or retention of Pb. This loss of mass does not, however, account for the higher Mg concentrations in these shells (Fig. 2) e RF had significantly higher Mg concentrations in this group, even after standardisation to the initial shell weight (263 vs. 185 mg g1; t ¼ 2.19, P ¼ 0.04 (two-tailed test)). The same pattern was not significant in SB. Average concentrations of Mg in the shell prior to exposure ranged between 110e130 mg g1 across the three populations and were also highly consistent in the soft tissues (1840e1940 mg g1). Soft tissue Mg concentrations fell in all populations on the experimental diets (Beeby and Richmond, 2010), but mean shell concentrations rose (Fig. 2). Again, to allow for the differential growth of shells, a standardised concentration shows that snails increased Mg in their shell, with most adding 10e20 mg during the experiment. The mass of Mg in the entire snail increased by a small amount (total assimilation rates were typically less than 2%), with no effect of Mg dose, so Mg was probably being added to the shell by depleting the soft tissues. The release of Ca and Sr from the shells thus appears to be a highly specific mechanism which does not entrain either Mg or Pb.
Table 2 Non-significant differences in several key parameters between snails growing or losing shell mass in two populations of C. aspersus, compared using a two-tailed t- test. The results are shown with their associated probability value (n ¼ 12 and 15 respectively, in each population). These factors are therefore are unlikely to explain the different shell growth responses in either population on this experimental diet. Parameter
SB t (probability)
RF t (probability)
Food consumed day1 Pb consumed day1 Total Pb Assimilation % Mg consumed day1 Soft tissue Pb mg g1 Soft tissue Mg mg g1 Soft tissue Ca mg g1 Shell Ca mg g1 Shell Pb mg Shell Mg mg
0.33 0.45 1.69 0.77 0.03 1.71 1.09 1.67 0.6 0.08
0.65 0.68 0.82 0.47 0.32 1.68 0.84 0.035 0.88 0.96
(0.74) (0.65) (0.11) (0.45) (0.97) (0.11) (0.28) (0.11) (0.55) (0.94)
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(0.52) (0.50) (0.42) (0.64) (0.75) (0.11) (0.41) (0.97) (0.39) (0.34)
initial shell mass was also significantly smaller in SB snails losing shell (0.35 vs. 0.44 g; t ¼ 1.96, P ¼ 0.03) and again, RF show the same pattern but it is not significant (0.26 vs. 0.30 g; t ¼ 1.43, P ¼ 0.08). Shell size relative to the soft tissues, the shell ratio, is a measure of the Ca reserve available to meet demand during the experiment: LE snails started with the largest shell ratios and varied the least from their initial shell weights over the experiment. Notably, amongst those snails losing shell mass, both SB and RF show a correlation of total Pb mass (shell þ soft tissues) with percentage reduction in shell mass (Table 4), some indication that the capacity to restrict soft tissue levels of Pb may be linked to the Ca losses from their shell.
3.2. Food consumption and metal assimilation The amount of food consumed per day was not significantly different between groups in either SB or RF, nor the daily consumption of Mg or Pb (Table 2). Neither did their total Pb assimilation efficiency differ, so roughly equal proportions of Pb were retained by those growing or losing shell mass in both populations. Since soft tissue Pb concentrations did not differ between the groups (Table 2), there was no indication that loss of shell mass caused any change in the amount of Pb retained.
3.3. Metal content of shells Shell Ca concentrations did not differ between the two groups in either population (Table 2), but their differential shell growth meant the groups differed in their total Ca (Table 3). Since average shell Ca concentrations approached 400 mg g1, these changes in mass can be explained by the loss or addition of calcium carbonate alone. A reduction in shell mass (based on their estimated initial shell weight) represented average losses of 8e10 mg Ca, whilst shell growth was equivalent, on average, to 20 mg (SB) or 34 mg Ca (RF).
Table 3 Mean (and SE) concentrations of Pb and Mg in the shells of two populations of C. aspersus grouped by their increase or loss in shell mass, and compared between groups for each population using two-tailed t-tests. The change in shell Ca and Sr mass, relative to their estimated starting mass, is also shown to differ significantly between groups in both populations. Population group parameter
SB Shell loss (n ¼ 15)
Shell growth (n ¼ 12)
t
RF
Shell Pb mg g1 Shell Mg mg g1 Change in shell Ca mg Change in shell Sr mg
6.3 223.0 10.1 83.9
3.8 121.1 19.7 2.8
2.31 2.66 3.16 4.44
(0.9) (37.5) (5.4) (10.1)
(0.6) (8.5) (7.6) (17.9)
(P (P (P (P
¼ ¼ ¼ ¼
0.030) 0.009) 0.005) 0.000)
Shell loss (n ¼ 15)
Shell growth (n ¼ 12)
t
7.3 344.0 8.5 5.31
3.4 153.4 33.9 16.7
3.42 3.79 4.38 4.32
(0.9) (48.1) (2.7) (1.4)
(0.7) (14.9) (9.3) (4.9)
(P (P (P (P
¼ ¼ ¼ ¼
0.002) 0.001) 0.000) 0.000)
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Table 4 Regression analysis of the total Pb mass (soft tissues þ shell) with the percentage reduction in the shell mass in those snails losing shell in two populations of C. aspersus (n ¼ 15 in both cases). The significance of each regression coefficient is indicated by its t-statistic and the proportion of variation in shell loss explained by the model by R2. Population
Intercept
Regression coefficient (standard error)
t-value for regression coefficient (probability)
R2
SB RF
0.04 0.02
0.0005 (0.0001) 0.0007 (0.002)
3.41 (0.005) 2.99 (0.01)
43% 36%
Combining the data from all sampling intervals and doses, the mass of Pb in the shell of each population was correlated with that consumed (Table 5). With the exception of RF, it was also correlated with the mass of Pb in the soft tissues. Partial regression analysis demonstrates these factors account for around 70% of the variation in shell Pb in SB and LE, but only 52% in RF. The association of Mg and Pb mass in SB and RF may be a consequence of their relative immobility as shell mass changes. 3.4. The partitioning of Mg and Pb between the soft tissues and the shell There appears to be little correspondence between the proportion of Pb and Mg added to the shell or soft tissues in any population (Fig. 3). Whilst RF is somewhat higher, all populations add roughly equivalent proportions of their daily consumption of Pb to the shell and soft tissues throughout the experiment, with each declining as consumption declined. Despite the consistency in Mg consumption rates between populations and sampling dates, differences emerge between the populations (Fig. 3b). Unlike RF and LE, SB showed a net daily loss of Mg from its soft tissues, yet a consistent addition to the shell. RF added more Mg to its shell than its soft tissues, whilst LE, from the site with the highest ambient Mg, always retained the largest fraction in its soft tissues. 4. Discussion 4.1. Calcium loss from the shell Calcium is readily mobilised from the shell during both reproduction and shell repair (Abolins-Krogis, 1968; Tompa and Wilbur, Table 5 Regression analysis of the mass of Pb (mg) in the shell with (a) Pb in the soft tissues (mg), (b) Pb consumed (mg), and (c) shell Mg (mg), in three populations of C. aspersus. Each regression combines the data from all sampling intervals and all diets (omitting four snails with no detectable Pb in the soft tissues). The significance of a regression coefficient is given by its t-statistic and the proportion of variation in shell Pb explained in each model by R2. Population
Intercept
Regression coefficient (standard error)
t-value for regression coefficient (probability)
R2
Fig. 2. Magnesium concentrations in the shells of C. aspersus. In SB and RF the average (SE) is shown for shells losing mass (light fill; n ¼ 15 in both) and those growing (dark fill; n ¼ 12 in both) and were significantly different in each population (Table 3). The unfilled bars show the average shell Mg concentration for all snails in each population (SE; n ¼ 36 for each population) standardised to their initial weight (measured Mg mass in each shell/estimated initial shell weight). Compared to their measured starting concentrations (SB ¼ 109, RF ¼ 129, LE ¼ 123 mg g1) these concentrations increased and Mg had been added to the shell.
1977; Wagge, 1952) increasing the labile reserve in the soft tissues. The loss of Ca-rich intracellular granules from the hepatopancreas is the main route by which protein-bound toxic metals are excreted (Dallinger and Wieser, 1984) and this may increase demand for Ca. However, the extent to which the soft tissue reserves are depleted by a toxic metal, or induce any demineralization of the shell, remains largely unquantified. In juvenile and sub-adults, demineralization of the shell in the presence of Pb has been reported for marine bivalves (Almeida et al., 1998) and terrestrial gastropods (Beeby et al., 2002). During embryonic development, Pt may cause the shell to develop internally in freshwater gastropods, though Ca supplements do not limit Pt uptake (Osterauer et al., 2010). Such effects were probably due to changes in the Ca signalling pathways rather than the availability of Ca available for shell building: small shells resulted from the limited size of the shell field during embryonic development, rather than any impact of Pt directly on its mineralization (Osterauer et al., 2010). The experiment described here was designed to assess the effects of Mg on the assimilation of Pb in sub-adults reinforcing their shell extension, and included no control for the effect of Pb itself (Beeby and Richmond, 2010). Consequently, it cannot demonstrate that dietary Pb has caused the loss of shell Ca. The association of total Pb with the percentage loss of shell mass may be an indication of Ca being released from the shell in response to the toxic metal, but further experimentation would be needed to confirm that Pb was inducing the loss. These results demonstrate that Mg levels in the diet do not affect the deposition of Pb in the shell, and also that the movement of Mg into the shell continues even as it is losing Ca (and Sr). 4.2. Soft tissue and shell growth
(a) LE (n ¼ 35) SB (n ¼ 34) RF (n ¼ 35)
0.45 0.94 1.00
0.023 (0.006) 0.015 (0.003) 0.003 (0.005)
4.14 (0.000) 4.81 (0.000) 0.71 (0.48)
31% 40% >1%
(b) LE n ¼ 35) SB (n ¼ 34) RF (n ¼ 35)
0.09 0.572 0.224
0.008 (0.001) 0.005 (0.001) 0.005 (0.001)
5.49 (0.000) 5.71 (0.000) 3.82 (0.005)
45% 49% 28%
(c) LE (n ¼ 35) SB (n ¼ 34) RF (n ¼ 35)
1.11 0.91 0.42
0.005 (0.005) 0.014 (0.004) 0.013 (0.003)
0.95 (0.349) 3.49 (0.001) 3.52 (0.001)
>1% 25% 25%
There can be considerable variation in growth rates within a population. Juveniles from the same brood, raised under identical conditions, will grow at markedly different rates, even when food is supplied in excess (Beeby and Richmond, 2007; Wagge, 1952). Here some snails grew their shell and soft tissues on the experimental diet, but most were unable to add to their shell mass. The difference between the two groups was not obvious from this experiment, but there were indications that it reflected the size of their Ca reserve: smaller snails with lighter shells in the SB population were less likely to grow new shell and the same was possibly true of the RF snails. In contrast, most of the heavily-shelled
A. Beeby, L. Richmond / Environmental Pollution 159 (2011) 1667e1672
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Fig. 3. The amount of Pb (a) and Mg (b) consumed per day (dark fill), added (or lost) from the soft tissues (light fill) and shell (no fill) in each population of C. aspersus. In (a) the amount of Pb added to the shell is multiplied by 10 to allow comparison between populations and sampling intervals; for the same reason, the amount of Mg consumed is divided by 10 in (b).
LE population maintained their shell mass over the course of the experiment, despite consuming equivalent amounts of Pb. Soft tissue concentrations of Ca were reduced and then restored in both SB and LE, perhaps as Ca was released from their shells. This may explain the observed change in all populations on day 64, when Pb assimilation became uncoupled from the soft tissue growth rate in each of the three populations (Beeby and Richmond, 2010). If this is the case, the difference between those growing shells and those losing shell mass was the size of the Ca reserve, indicated by the shell ratio, perhaps mobilised to facilitate the excretion of Pb. 4.3. Metal partitioning between the soft tissues and the shell Magnesium and Ca assimilation and metabolism in snails have long been thought to be associated (Wagge, 1952). Soil feeding in C. aspersus allows them to supplement their Ca supply: Gomot et al. (1989) found that Ca concentrations were reduced in soils in which snails had been allowed to feed. There was no equivalent decline in soil Mg concentrations, though the details of this differential
uptake are not described. The snails in the present experiment appear to show the same discrimination in the movements of these essential metals in and out of their shells. Despite the loss of Ca, all populations added Mg to the shell throughout the experiment. After day 32, most snails ingested about 25 mg/day and added 0.4 mg/day to the shell. SB was consistent in losing Mg from the soft tissues throughout the experiment and RF added little Mg to this compartment from day 32. LE continued to add around 0.5 mg/day to its soft tissues but since these snails showed little shell growth (or loss), this may reflect their small additions to the shell towards the end of the experiment (0.2 mg/day). Notably LE is a site with over twice as much Mg in its soil than the two other habitats (Beeby and Richmond, 2010) and these differences may represent a locally adapted Mg metabolism. Lead consumption and assimilation in the shell and soft tissues became relatively consistent after day 16 and there were few differences between populations or diets e all ingested around 5 mg of Pb each day, with a net addition of 1 mg to the soft tissues and 0.03 mg to the shell. There was no significant difference in these
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A. Beeby, L. Richmond / Environmental Pollution 159 (2011) 1667e1672
figures between the snails growing or losing shell mass in SB and RF. On average, around 3% of the total Pb burden was held in the shell, compared to 12% of total Mg. The mass of Pb in the soft tissues was correlated with that of Ca in all populations (Beeby and Richmond, 2010) and here the lowest shell ratios were found in most treatments at the end of the experiment, as soft tissue Ca concentrations began to be restored. This suggests that Ca had been mobilised from the shell, perhaps in response to the Pb in the diet. Magnesium did not appear to play a significant role either in soft tissue or shell assimilation of Pb. However Pb and Mg additions to the shell were shown to be independent of the major process in shell mineralization, its Ca dynamics. 5. Conclusions The capacity of different sub-adult Cantareus aspersus from three populations to reinforce their shells was not determined by the length of time feeding, amount of food or Ca consumed. Most snails lost shell weight on this diet, despite significant growth of their soft tissues and of a shell extension that needed to be mineralized. In two of these populations at least, the loss of Ca from the shell was not matched by a loss of Mg, suggesting that the movement of these two essential metals in the shell, under these demands, was not closely coupled. The loss of shell mass was primarily due to the loss of Ca, released from the existing shell and subsequently lost from the soft tissues. Whilst this may be associated with the excretion of Pb, this experiment could not demonstrate this to be the causative agent. However, Mg and Pb were retained in the shell, and thus had different dynamics to shell Ca. The mass of Pb and Mg in the shell appeared to be closely matched in shells undergoing changes in mass. There were indications that the size of the Ca reserves, indicated by the shell ratio, may be a key determinant of the different responses to the dosed diet. Acknowledgement We are grateful for the helpful comments of two referees. References Abolins-Krogis, A., 1968. Shell regeneration in Helix pomatia with special reference to the elementary calcifying particles. Symposium of the Zoological Society of London 22, 75e92. Almeida, M.J., Moura, G., Pinheiro, T., Machado, J., Coimbra, J., 1998. Modifications in Crassostrea gigas shell composition exposed to high concentrations of lead. Aquatic Toxicology 40, 323e334. Beeby, A., 1985. The role of Helix aspersa as a major herbivore in the transfer of lead through a polluted ecosystem. Journal of Applied Ecology 22, 267e275. Beeby, A., Richmond, L., 1989. The shell as a site of lead deposition in Helix aspersa. Archives of Environmental Contamination and Toxicology 18, 623e628. Beeby, A., Richmond, L., 1998. Variation in the mineral composition of eggs of the snail, Helix aspersa between populations exposed to different levels of metal contamination. Environmental Pollution 101, 25e31. Beeby, A., Richmond, L., Herpé, F., 2002. Lead reduces shell mass in juvenile garden snails (Helix aspersa). Environmental Pollution 120, 283e288. Beeby, A., Richmond, L., 2007. Differential growth rates and calcium-allocation strategies in the garden snail Cantareus aspersus. Journal of Molluscan Studies 73, 105e112.
Beeby, A., Richmond, L., 2010. Magnesium and the regulation of lead in three populations of the garden snail Cantareus aspersus. Environmental Pollution 158, 2288e2293. Behra, R., 1993. In vitro effects of cadmium, zinc and lead on calmodulin-dependent actions in Oncoryhncus mykiss, Mytilus sp. and Chlamydomonas reinhardii. Archives of Environmental Contamination and Toxicology 24, 21e27. Crowell, H., 1973. Laboratory study of calcium requirements of the brown garden snail, Helix aspersa Müller. Proceedings of the Malacological Society of London 40, 491e503. Dallinger, R., Wieser, W., 1984. Patterns of accumulation, distribution and liberation of Zn, Cu, Cd and Pb in different organs of the land snail Helix pomatia L. Comparative Biochemistry and Physiology 79C, 117e122. Dowd, T.L., Rosen, J.F., Gupta, RK., 1990. 31P NMR and saturation transfer studies of the effect of Pb2þ on cultured osteoblastic bone cells. Journal of Biological Chemistry 265, 20833e20838. Fournie, J., Chetail, M., 1984. Calcium dynamics in land gastropoda. American Zoologist 24, 857e870. Gardenfors, U., 1992. Effects of artificial liming on land snail populations. Journal of Applied Ecology 29, 50e54. Gomot, A., Gomot, L., Boukraa, S., Bruckert, S., 1989. Influence of soil on the growth of the land snail Helix aspersa. An experimental study of the route for the stimulating factors. 55, 1e7. Heller, J., Magaritz, M., 1983. From where do land snails obtain the chemicals to build their shells? Journal of Molluscan Studies 49, 116e121. Hodl, E., Felder, E., Chabicovsky, M., Dallinger, R., 2010. Cadmium stress stimulates tissue turnover in Helix pomatia: increasing cell proliferation from metal tolerance to exhaustion in molluscan midgut gland. Cell and Tissue Research 341, 159e171. Ireland, M.P., 1991. The effect of dietary calcium on growth, shell thickness and tissue calcium distribution in the snail Achatina fulica. Comparative Biochemistry and Physiology 98A, 111e116. Kalisz, P.J., Powell, J.E., 2003. Effect of calcareous road dust on land snails (Gastropoda, Pulmonata) and millipedes (Diplopoda) in acid forest soils of the Daniel Boone National Forest of Kentucky, USA. Forest Ecology & Management 186, 177e183. Laskowski, R., Hopkin, S.P., 1996a. Accumulation of Zn, Cu, Pb and Cd in the garden snail (Helix aspersa): implications for predators. Environmental Pollution 91, 289e297. Laskowski, R., Hopkin, S.P., 1996b. Effect of Zn, Cu, Pb and Cd in snails (Helix aspersa). Ecotoxicology and Environmental Safety 34, 59e69. Mulvey, M., Newman, M.C., Beeby, A.N., 1996. Genetic and conchological comparison of snails (Helix aspersa) differing in shell deposition of lead. Journal of Molluscan Studies 62, 213e223. Ondina, P., Hermida, J., Outeiro, A., Mato, J., 2004. Relationships between terrestrial gastropod distribution and soil properties in Galicia (N.W. Spain). Applied Soil Ecology 26, 1e9. Osterauer, R., Marschner, L., Betz, O., Gerberding, M., Sawasdee, B., Cloetens, P., Haus, N., Sures, B., Triebskorn, R., Köhler, H.-R., 2010. Turning snails into slugs: induced body plan changes and formation of an internal shell. Evolution & Development 12, 474e483. Schanne, F.A.X., Dowd, T.L., Gupta, R.K., Rosen, J.F., 1989. Lead increases free Ca2þ concentration in cultured osteoblastic bone cells: simultaneous detection of intracellular free Pb2þ by 19F NMR. Proceedings of the National Academy of Sciences USA 86, 5133e5135. Soto, M., Ireland, M.P., Marigómez, I., 1997. The contribution of metal/shell-weight index in target-tissues to metal body burden in sentinel marine molluscs. 1. Littorina littorea. The Science of the Total Environment 198, 135e147. Srivastava, D., Hurwitz, R.L., Fox, D.A., 1995. Lead- and calcium-mediated inhibition of bovine rod cGMP phosphodiesterase: interactions with magnesium. Toxicology & Applied Pharmacology 134, 43e52. Swaileh, K.M., Ezzughayyar, A., 2001. Dose-dependent effects of dietary Pb and Zn on feeding and growth rates of the landsnail Helix engaddensis. Ecotoxicology and Environmental Safety 50, 9e14. Tompa, A.S., Wilbur, K.M., 1977. Calcium mobilisation during reproduction in snail Helix aspersa. Nature 270, 53e54. Wagge, L.E., 1952. Quantitative studies of calcium metabolism in Helix aspersa. Journal of Experimental Zoology 120, 311e342. Zaldibar, B., Cancio, I., Soto, I., Marigómez, I., 2008. Changes in cell-type composition in digestive gland of slugs and its influence on biomarkers following transplantation between a relatively unpolluted and a chronically metal polluted site. Environmental Pollution 156, 367e379.
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Magnesium and the deposition of lead in the shell of three populations of the garden snail Cantareus aspersus Alan Beeby*, Larry Richmond Department of Applied Science, London South Bank University, Borough Road, London SE1 0AA, UK
Snails losing shell mass on an experimental diet have higher concentrations of shell Pb and Mg, but lose Ca.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 November 2010 Received in revised form 18 February 2011 Accepted 26 February 2011
The loss of Pb from snail soft tissues may depend on the excretion of Ca, and involve the mobilization of shell Ca. Most sub-adults in three populations of Cantareus aspersus (syn. Cornu aspersum Müller) either failed to add, or lost, shell mass on a diet with 250 mg g1 Pb. Their failure to mineralize shell extensions occurred irrespective of food consumed, time or dietary Mg. Budgets of metals for 36 individuals in each of two populations showed that Ca loss would account for the reduction in shell mass. Lead concentrations were higher in the reduced shells but this may be a consequence of their smaller mass, rather than its cause. In both populations shell reduction correlated with the total mass of Pb assimilated. Any shell growth may have been dependent on the initial Ca reserve in each snail. Differential movement of Mg, Pb and Ca occurred between the shell and soft tissues. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Lead regulation Magnesium Calcium Shell Cantareus aspersus
1. Introduction The retention of lead by the soft tissues of Helicid snails is highly consistent, both within species (Cantareus aspersus e Beeby, 1985; Laskowski and Hopkin, 1996a,b) and between related species (Helix engaddensis e Swaileh and Ezzughayyar, 2001). Soft tissue concentrations rarely exceed 40% of those in the food consumed, though unlike several other toxic metals, Pb does not appear to be regulated by a process involving inducible proteins. There is some indication that Pb assimilation may be linked to growth of the soft tissues (Beeby and Richmond, 2010) or, as with other toxic metals, cell turnover in the hepatopancreas (Hodl et al., 2010; Soto et al., 1997; Zaldibar et al., 2008). Lead may be incorporated into the soft tissues because the snail is unable to distinguish the toxic metal from essential metals, most probably Ca (Behra, 1993; Schanne et al., 1989), but also possibly Mg (Dowd et al., 1990; Srivastava et al., 1995). Dietary Mg does not affect Pb assimilation by the soft tissues of C. aspersus (Beeby and Richmond, 2010), but they are able to exclude Pb from certain Carich tissues (Beeby and Richmond, 1998). Lead is found in the shell, albeit at low concentrations (Laskowski and Hopkin, 1996a), where it has a retention time much greater than that of the soft tissues
* Corresponding author. E-mail addresses: [email protected] (A. Beeby), [email protected] (L. Richmond). 0269-7491/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2011.02.040
(Beeby and Richmond, 1989). It is also known to inhibit the mineralization of shells in juvenile snails (Beeby et al., 2002). Calcium, or more particularly its availability, is one of the most important factors governing the distribution of snails in a range of habitats and their capacity to build shells (Gardenfors, 1992; Ireland, 1991; Kalisz and Powell, 2003; Mulvey et al., 1996; Ondina et al., 2004). Most of the mass of the shell is calcium carbonate, but Mg is also part of the crystalline structure of the shell (Fournie and Chetail, 1984). These mineral components are deposited beneath a proteinaceous outer layer, the periostracum, in a process of reinforcement following the growth of this horn-like shell extension, normally at the beginning of the growing season (Fournie and Chetail, 1984). Snails actively mineralizing their shells might incorporate more Pb into the crystalline matrix, and this may be affected by the availability of Mg and Ca in the diet. Snails are known to soil-feed (and drink) to supplement their supply of Ca (Crowell, 1973; Gomot et al., 1989; Heller and Magaritz, 1983), in the upper soil layers where Pb accumulates in contaminated habitats. In a previous paper we described an experiment in which three populations were fed a Pb-dosed diet with three different levels of Mg (Beeby and Richmond, 2010). The populations differed in their responses but soft tissue assimilation of Pb slowed in all as soft tissue growth slowed, and perhaps as shell mineralization commenced. This paper examines the deposition of Pb in the shell of these sub-adult snails over the 64-day experimental period, as they begin to reinforce their shell extensions.
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2. Methods The details of the collection and culturing of Cantareus aspersus from the three locations are given in Beeby and Richmond (2010). Sub-adults were collected in March and April, 2008, towards the end of their hibernation and prior to their new season shell growth. Two populations were from Ca-rich habitats (LE in southern England, RF in southern France) and the third from a Ca-poor soil in southern England (SB). Concentrations of soil Mg were almost twice as high at LE as the other two sites; RF was uncontaminated and SB the site most contaminated with Pb.
Table 1 The mean and standard error (n ¼ 12) for the estimated initial and final shell weights for three populations of Cantareus aspersus at each sampling interval (combining data from the three diets). Whilst the growth in the soft tissues continued throughout the experiment, the average shell mass declined in LE and SB. Population
Time days
Estimated initial shell weight mg
Final shell weight mg
Growth in soft tissues mg
LE
16 32 64
432 (39) 390 (41) 387 (30)
408 (35) 375 (45) 358 (33)
244 (31) 271 (36) 298 (44)
SB
16 32 64
389 (34) 384 (42) 421 (29)
400 (50) 392 (52) 370 (27)
310 (34) 340 (58) 466 (69)
RF
16 32 64
269 (25) 292 (29) 293 (17)
283 (34) 280 (30) 299 (32)
306 (35) 311 (36) 313 (28)
2.1. Experimental design After 3e4 weeks depuration feeding on commercially-grown lettuce, four individuals from each population were sacrificed and analysed for their metal content (day 0). Thereafter 36 snails of each population were randomly allocated to an artificial diet with one of three levels of Mg but with similar Pb and Ca levels. Four snails on each dose for each population were sampled at 16, 32 and 64 days. The details of the feeding regime and the analysis of the soft tissues are given in Beeby and Richmond (2010). For each snail the amount of food consumed, and therefore the consumption of Mg, Ca and Pb, was measured and compared to the mass recovered from the soft tissues to give percentage assimilation rates, or for the shell and soft tissues combined, the total assimilation rates. 2.2. Sample treatment and analysis After 24 h depuration by starvation each snail was sacrificed by freezing. The shell was separated from the thawed soft tissues, dried for 12 h at 80 C, weighed and then digested by slowly introducing 10 ml of 50% HNO3. After complete dissolution of the mineral component the vessel was sealed with the solution unfiltered and stored in a refrigerator prior to analysis. Solutions were analysed using a Varian 820 ICP-MS, using the isotopes Mg24, Ca43, Pb108 and Sr88. The linearity of the calibration curve for each metal was confirmed over four orders of magnitude against standard solutions (Inorganic Ventures IV-ICPMS-71A). The shells required a 40-fold dilution using ultra-pure water (up to 18.2 MU cm) prior to final analysis for Ca. Four glassware controls were used to measure and correct for any background contamination in each run. Average recovery rates for Pb from four replicates of a reference material (Lobster hepatopancreas TORT-2 e Environment Canada) were within the certified range. Certified values for Ca and Mg are not provided for TORT-2 but the uniformity of their results between digestion runs was taken to indicate consistent recovery rates. 2.3. Statistical analysis Growth or loss of shell mass was determined for each snail with reference to its estimated initial shell weight. This was calculated as the proportion of the initial total fresh weight attributable to the shell, based on the four snails sacrificed at day 0 for each population and from which a median was calculated. Medians were used because of the small sample size and the occasional outlier amongst some results. The inter-quartile range of this proportion was used to derive the limits within which the initial shell weight of each dosed snail most probably fell. By comparing its final shell weight to this range, a snail was classified as having added to its shell mass (above the upper quartile limit), lost mass (below the lower quartile limit) or indistinguishable from its initial starting weight. This conservative approach produced a wide range of values within which no change in shell weight would be attributed. Only those snails in the first two classes were regarded as showing positive or negative shell growth and used in statistical comparisons between the groups. One population did not produce groups with sufficient numbers to enable comparison e LE had 21 snails that were indistinguishable from their starting shell weight, and only three with positive shell growth. SB and RF each had 12 snails showing shell growth, 15 with losses, and 9 that could not be distinguished from their starting shell weights. Comparisons between the groups showing positive or negative shell growth were thus confined to the SB and RF populations, using t-tests assuming unequal variances. Growth in the shell and soft tissues, and the assimilation of both food and metals were examined using regression and correlation analysis. c2 analysis was used to test for any association of shell growth with diet or feeding duration in these two populations.
not, and in LE and SB average shell mass declined. These diets thus supported soft tissue growth, though this was lower in snails losing shell mass in both SB and RF (Fig. 1). This may be indicative of some more general cost or response to Pb in those snails unable to add to their shell mass. Of the three populations LE had the largest initial shell ratios (median 2.1) but the smallest change in shell mass during the experiment. In contrast, a third of SB and RF snails increased their shell mass (by 5e39% and 9e44% respectively) whilst almost half of each population lost shell (by 8e50% and 5e33% respectively). These changes were irrespective of a snail’s diet or duration of feeding: a c2 test of the combined data for these two populations found no significant association of either group with feeding duration (c2 ¼ 0.693, 2df, P > 0.9) or Mg diet (c2 ¼ 1.672, 2df, P > 0.5). There was no significant difference in the soft tissue assimilation of Ca in the two groups of either population (SB t ¼ 1.05 NS; RF t ¼ 1.37 NS) and there were few other indications of why the groups differed (Table 2). They did differ in their shell Mg and Pb concentrations (Table 3). However, the SB group losing shell had smaller fresh weights at the start of the experiment than those snails adding shell (2.44 vs. 3.05 g; t ¼ 1.82, P ¼ 0.04), whereas RF just failed to show a significant difference (2.03 vs. 2.34 g; t ¼ 1.43, P ¼ 0.08). The
3. Results 3.1. Shell growth With a single exception, each snail added a shell extension soon after the start of the experiment, though none had completed their mineralization by its end. Median shell ratios (shell dry weight/soft tissue dry weight) were above 1.5 in day 0 snails but these declined to 0.8 or less in each population by day 64: whilst the soft tissues increased in weight in all snails over time (Table 1), most shells did
Fig. 1. Mean growth rate in the soft tissues of Cantareus aspersus showing a reduction in shell mass (light fill) or adding to their shells (dark fill), for each of two populations (irrespective of feeding duration or diet). In each population snails losing shell mass have significantly lower rates of soft tissue growth (SB t ¼ 2.10, P ¼ 0.05; RF t ¼ 2.15, P ¼ 0.02).
A. Beeby, L. Richmond / Environmental Pollution 159 (2011) 1667e1672
Strontium, a known analogue for Ca, followed the same pattern. This was supplied at a uniform level in all diets e around 6 mg g1 e and again the groups were distinguished by the amount of Sr lost or gained in each population (Table 3). This is further indication of a demineralization of the shell and mobilization of Ca and Sr. There were, however, differences between the populations: a large proportion of the SB snails growing their shells also appeared to have lost Sr, at least in comparison to their estimated starting mass. Most shells had Mg concentrations between 150 and 200 mg g1, but these rose with dietary level in both RF (F ¼ 6.176, P ¼ 0.006) and LE (F ¼ 7.498, P ¼ 0.002) though not SB (F ¼ 0.99, P ¼ 0.384). The highest levels were achieved by RF snails at the highest dose on day 64 (average 490 mg g1). Lead concentrations rose in the shells of all populations from around 2e3 mg g1 at day 16 to 6 mg g1 by day 64: duration of feeding was a significant factor for both LE (F ¼ 14.131, P ¼ 0.000) and SB (F ¼ 7.69, P ¼ 0.002), and probably for RF as well (F ¼ 3.165, P ¼ 0.058). Dietary Mg was also a significant factor for shell Pb in RF (F ¼ 3.763, P ¼ 0.036) but this was again largely attributable to the high concentrations in one group e the snails on the highest Mg diet at day 64. Magnesium dose did not appear to affect shell assimilation of Pb in LE and SB, but the mass of Pb and Mg were correlated in both SB and RF across all shells (Table 5). Lead and Mg concentrations were higher in shells that had lost mass in both SB and RF (Table 3). However the weight of these metals in the shell did not differ between the groups in either population (Table 2) and so concentrations rose as shell mass fell. Comparing the groups on a standardised weight basis (by calculating the equivalent concentration using their measured final Pb mass and estimated initial shell mass), Pb concentrations did not differ between them in either population (SB t ¼ 0.28, NS; RF t ¼ 1.53, NS). The increased Pb concentrations could result solely from the reduction in shell mass, rather than any increase in the assimilation or retention of Pb. This loss of mass does not, however, account for the higher Mg concentrations in these shells (Fig. 2) e RF had significantly higher Mg concentrations in this group, even after standardisation to the initial shell weight (263 vs. 185 mg g1; t ¼ 2.19, P ¼ 0.04 (two-tailed test)). The same pattern was not significant in SB. Average concentrations of Mg in the shell prior to exposure ranged between 110e130 mg g1 across the three populations and were also highly consistent in the soft tissues (1840e1940 mg g1). Soft tissue Mg concentrations fell in all populations on the experimental diets (Beeby and Richmond, 2010), but mean shell concentrations rose (Fig. 2). Again, to allow for the differential growth of shells, a standardised concentration shows that snails increased Mg in their shell, with most adding 10e20 mg during the experiment. The mass of Mg in the entire snail increased by a small amount (total assimilation rates were typically less than 2%), with no effect of Mg dose, so Mg was probably being added to the shell by depleting the soft tissues. The release of Ca and Sr from the shells thus appears to be a highly specific mechanism which does not entrain either Mg or Pb.
Table 2 Non-significant differences in several key parameters between snails growing or losing shell mass in two populations of C. aspersus, compared using a two-tailed t- test. The results are shown with their associated probability value (n ¼ 12 and 15 respectively, in each population). These factors are therefore are unlikely to explain the different shell growth responses in either population on this experimental diet. Parameter
SB t (probability)
RF t (probability)
Food consumed day1 Pb consumed day1 Total Pb Assimilation % Mg consumed day1 Soft tissue Pb mg g1 Soft tissue Mg mg g1 Soft tissue Ca mg g1 Shell Ca mg g1 Shell Pb mg Shell Mg mg
0.33 0.45 1.69 0.77 0.03 1.71 1.09 1.67 0.6 0.08
0.65 0.68 0.82 0.47 0.32 1.68 0.84 0.035 0.88 0.96
(0.74) (0.65) (0.11) (0.45) (0.97) (0.11) (0.28) (0.11) (0.55) (0.94)
1669
(0.52) (0.50) (0.42) (0.64) (0.75) (0.11) (0.41) (0.97) (0.39) (0.34)
initial shell mass was also significantly smaller in SB snails losing shell (0.35 vs. 0.44 g; t ¼ 1.96, P ¼ 0.03) and again, RF show the same pattern but it is not significant (0.26 vs. 0.30 g; t ¼ 1.43, P ¼ 0.08). Shell size relative to the soft tissues, the shell ratio, is a measure of the Ca reserve available to meet demand during the experiment: LE snails started with the largest shell ratios and varied the least from their initial shell weights over the experiment. Notably, amongst those snails losing shell mass, both SB and RF show a correlation of total Pb mass (shell þ soft tissues) with percentage reduction in shell mass (Table 4), some indication that the capacity to restrict soft tissue levels of Pb may be linked to the Ca losses from their shell.
3.2. Food consumption and metal assimilation The amount of food consumed per day was not significantly different between groups in either SB or RF, nor the daily consumption of Mg or Pb (Table 2). Neither did their total Pb assimilation efficiency differ, so roughly equal proportions of Pb were retained by those growing or losing shell mass in both populations. Since soft tissue Pb concentrations did not differ between the groups (Table 2), there was no indication that loss of shell mass caused any change in the amount of Pb retained.
3.3. Metal content of shells Shell Ca concentrations did not differ between the two groups in either population (Table 2), but their differential shell growth meant the groups differed in their total Ca (Table 3). Since average shell Ca concentrations approached 400 mg g1, these changes in mass can be explained by the loss or addition of calcium carbonate alone. A reduction in shell mass (based on their estimated initial shell weight) represented average losses of 8e10 mg Ca, whilst shell growth was equivalent, on average, to 20 mg (SB) or 34 mg Ca (RF).
Table 3 Mean (and SE) concentrations of Pb and Mg in the shells of two populations of C. aspersus grouped by their increase or loss in shell mass, and compared between groups for each population using two-tailed t-tests. The change in shell Ca and Sr mass, relative to their estimated starting mass, is also shown to differ significantly between groups in both populations. Population group parameter
SB Shell loss (n ¼ 15)
Shell growth (n ¼ 12)
t
RF
Shell Pb mg g1 Shell Mg mg g1 Change in shell Ca mg Change in shell Sr mg
6.3 223.0 10.1 83.9
3.8 121.1 19.7 2.8
2.31 2.66 3.16 4.44
(0.9) (37.5) (5.4) (10.1)
(0.6) (8.5) (7.6) (17.9)
(P (P (P (P
¼ ¼ ¼ ¼
0.030) 0.009) 0.005) 0.000)
Shell loss (n ¼ 15)
Shell growth (n ¼ 12)
t
7.3 344.0 8.5 5.31
3.4 153.4 33.9 16.7
3.42 3.79 4.38 4.32
(0.9) (48.1) (2.7) (1.4)
(0.7) (14.9) (9.3) (4.9)
(P (P (P (P
¼ ¼ ¼ ¼
0.002) 0.001) 0.000) 0.000)
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Table 4 Regression analysis of the total Pb mass (soft tissues þ shell) with the percentage reduction in the shell mass in those snails losing shell in two populations of C. aspersus (n ¼ 15 in both cases). The significance of each regression coefficient is indicated by its t-statistic and the proportion of variation in shell loss explained by the model by R2. Population
Intercept
Regression coefficient (standard error)
t-value for regression coefficient (probability)
R2
SB RF
0.04 0.02
0.0005 (0.0001) 0.0007 (0.002)
3.41 (0.005) 2.99 (0.01)
43% 36%
Combining the data from all sampling intervals and doses, the mass of Pb in the shell of each population was correlated with that consumed (Table 5). With the exception of RF, it was also correlated with the mass of Pb in the soft tissues. Partial regression analysis demonstrates these factors account for around 70% of the variation in shell Pb in SB and LE, but only 52% in RF. The association of Mg and Pb mass in SB and RF may be a consequence of their relative immobility as shell mass changes. 3.4. The partitioning of Mg and Pb between the soft tissues and the shell There appears to be little correspondence between the proportion of Pb and Mg added to the shell or soft tissues in any population (Fig. 3). Whilst RF is somewhat higher, all populations add roughly equivalent proportions of their daily consumption of Pb to the shell and soft tissues throughout the experiment, with each declining as consumption declined. Despite the consistency in Mg consumption rates between populations and sampling dates, differences emerge between the populations (Fig. 3b). Unlike RF and LE, SB showed a net daily loss of Mg from its soft tissues, yet a consistent addition to the shell. RF added more Mg to its shell than its soft tissues, whilst LE, from the site with the highest ambient Mg, always retained the largest fraction in its soft tissues. 4. Discussion 4.1. Calcium loss from the shell Calcium is readily mobilised from the shell during both reproduction and shell repair (Abolins-Krogis, 1968; Tompa and Wilbur, Table 5 Regression analysis of the mass of Pb (mg) in the shell with (a) Pb in the soft tissues (mg), (b) Pb consumed (mg), and (c) shell Mg (mg), in three populations of C. aspersus. Each regression combines the data from all sampling intervals and all diets (omitting four snails with no detectable Pb in the soft tissues). The significance of a regression coefficient is given by its t-statistic and the proportion of variation in shell Pb explained in each model by R2. Population
Intercept
Regression coefficient (standard error)
t-value for regression coefficient (probability)
R2
Fig. 2. Magnesium concentrations in the shells of C. aspersus. In SB and RF the average (SE) is shown for shells losing mass (light fill; n ¼ 15 in both) and those growing (dark fill; n ¼ 12 in both) and were significantly different in each population (Table 3). The unfilled bars show the average shell Mg concentration for all snails in each population (SE; n ¼ 36 for each population) standardised to their initial weight (measured Mg mass in each shell/estimated initial shell weight). Compared to their measured starting concentrations (SB ¼ 109, RF ¼ 129, LE ¼ 123 mg g1) these concentrations increased and Mg had been added to the shell.
1977; Wagge, 1952) increasing the labile reserve in the soft tissues. The loss of Ca-rich intracellular granules from the hepatopancreas is the main route by which protein-bound toxic metals are excreted (Dallinger and Wieser, 1984) and this may increase demand for Ca. However, the extent to which the soft tissue reserves are depleted by a toxic metal, or induce any demineralization of the shell, remains largely unquantified. In juvenile and sub-adults, demineralization of the shell in the presence of Pb has been reported for marine bivalves (Almeida et al., 1998) and terrestrial gastropods (Beeby et al., 2002). During embryonic development, Pt may cause the shell to develop internally in freshwater gastropods, though Ca supplements do not limit Pt uptake (Osterauer et al., 2010). Such effects were probably due to changes in the Ca signalling pathways rather than the availability of Ca available for shell building: small shells resulted from the limited size of the shell field during embryonic development, rather than any impact of Pt directly on its mineralization (Osterauer et al., 2010). The experiment described here was designed to assess the effects of Mg on the assimilation of Pb in sub-adults reinforcing their shell extension, and included no control for the effect of Pb itself (Beeby and Richmond, 2010). Consequently, it cannot demonstrate that dietary Pb has caused the loss of shell Ca. The association of total Pb with the percentage loss of shell mass may be an indication of Ca being released from the shell in response to the toxic metal, but further experimentation would be needed to confirm that Pb was inducing the loss. These results demonstrate that Mg levels in the diet do not affect the deposition of Pb in the shell, and also that the movement of Mg into the shell continues even as it is losing Ca (and Sr). 4.2. Soft tissue and shell growth
(a) LE (n ¼ 35) SB (n ¼ 34) RF (n ¼ 35)
0.45 0.94 1.00
0.023 (0.006) 0.015 (0.003) 0.003 (0.005)
4.14 (0.000) 4.81 (0.000) 0.71 (0.48)
31% 40% >1%
(b) LE n ¼ 35) SB (n ¼ 34) RF (n ¼ 35)
0.09 0.572 0.224
0.008 (0.001) 0.005 (0.001) 0.005 (0.001)
5.49 (0.000) 5.71 (0.000) 3.82 (0.005)
45% 49% 28%
(c) LE (n ¼ 35) SB (n ¼ 34) RF (n ¼ 35)
1.11 0.91 0.42
0.005 (0.005) 0.014 (0.004) 0.013 (0.003)
0.95 (0.349) 3.49 (0.001) 3.52 (0.001)
>1% 25% 25%
There can be considerable variation in growth rates within a population. Juveniles from the same brood, raised under identical conditions, will grow at markedly different rates, even when food is supplied in excess (Beeby and Richmond, 2007; Wagge, 1952). Here some snails grew their shell and soft tissues on the experimental diet, but most were unable to add to their shell mass. The difference between the two groups was not obvious from this experiment, but there were indications that it reflected the size of their Ca reserve: smaller snails with lighter shells in the SB population were less likely to grow new shell and the same was possibly true of the RF snails. In contrast, most of the heavily-shelled
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Fig. 3. The amount of Pb (a) and Mg (b) consumed per day (dark fill), added (or lost) from the soft tissues (light fill) and shell (no fill) in each population of C. aspersus. In (a) the amount of Pb added to the shell is multiplied by 10 to allow comparison between populations and sampling intervals; for the same reason, the amount of Mg consumed is divided by 10 in (b).
LE population maintained their shell mass over the course of the experiment, despite consuming equivalent amounts of Pb. Soft tissue concentrations of Ca were reduced and then restored in both SB and LE, perhaps as Ca was released from their shells. This may explain the observed change in all populations on day 64, when Pb assimilation became uncoupled from the soft tissue growth rate in each of the three populations (Beeby and Richmond, 2010). If this is the case, the difference between those growing shells and those losing shell mass was the size of the Ca reserve, indicated by the shell ratio, perhaps mobilised to facilitate the excretion of Pb. 4.3. Metal partitioning between the soft tissues and the shell Magnesium and Ca assimilation and metabolism in snails have long been thought to be associated (Wagge, 1952). Soil feeding in C. aspersus allows them to supplement their Ca supply: Gomot et al. (1989) found that Ca concentrations were reduced in soils in which snails had been allowed to feed. There was no equivalent decline in soil Mg concentrations, though the details of this differential
uptake are not described. The snails in the present experiment appear to show the same discrimination in the movements of these essential metals in and out of their shells. Despite the loss of Ca, all populations added Mg to the shell throughout the experiment. After day 32, most snails ingested about 25 mg/day and added 0.4 mg/day to the shell. SB was consistent in losing Mg from the soft tissues throughout the experiment and RF added little Mg to this compartment from day 32. LE continued to add around 0.5 mg/day to its soft tissues but since these snails showed little shell growth (or loss), this may reflect their small additions to the shell towards the end of the experiment (0.2 mg/day). Notably LE is a site with over twice as much Mg in its soil than the two other habitats (Beeby and Richmond, 2010) and these differences may represent a locally adapted Mg metabolism. Lead consumption and assimilation in the shell and soft tissues became relatively consistent after day 16 and there were few differences between populations or diets e all ingested around 5 mg of Pb each day, with a net addition of 1 mg to the soft tissues and 0.03 mg to the shell. There was no significant difference in these
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figures between the snails growing or losing shell mass in SB and RF. On average, around 3% of the total Pb burden was held in the shell, compared to 12% of total Mg. The mass of Pb in the soft tissues was correlated with that of Ca in all populations (Beeby and Richmond, 2010) and here the lowest shell ratios were found in most treatments at the end of the experiment, as soft tissue Ca concentrations began to be restored. This suggests that Ca had been mobilised from the shell, perhaps in response to the Pb in the diet. Magnesium did not appear to play a significant role either in soft tissue or shell assimilation of Pb. However Pb and Mg additions to the shell were shown to be independent of the major process in shell mineralization, its Ca dynamics. 5. Conclusions The capacity of different sub-adult Cantareus aspersus from three populations to reinforce their shells was not determined by the length of time feeding, amount of food or Ca consumed. Most snails lost shell weight on this diet, despite significant growth of their soft tissues and of a shell extension that needed to be mineralized. In two of these populations at least, the loss of Ca from the shell was not matched by a loss of Mg, suggesting that the movement of these two essential metals in the shell, under these demands, was not closely coupled. The loss of shell mass was primarily due to the loss of Ca, released from the existing shell and subsequently lost from the soft tissues. Whilst this may be associated with the excretion of Pb, this experiment could not demonstrate this to be the causative agent. However, Mg and Pb were retained in the shell, and thus had different dynamics to shell Ca. The mass of Pb and Mg in the shell appeared to be closely matched in shells undergoing changes in mass. There were indications that the size of the Ca reserves, indicated by the shell ratio, may be a key determinant of the different responses to the dosed diet. Acknowledgement We are grateful for the helpful comments of two referees. References Abolins-Krogis, A., 1968. Shell regeneration in Helix pomatia with special reference to the elementary calcifying particles. Symposium of the Zoological Society of London 22, 75e92. Almeida, M.J., Moura, G., Pinheiro, T., Machado, J., Coimbra, J., 1998. Modifications in Crassostrea gigas shell composition exposed to high concentrations of lead. Aquatic Toxicology 40, 323e334. Beeby, A., 1985. The role of Helix aspersa as a major herbivore in the transfer of lead through a polluted ecosystem. Journal of Applied Ecology 22, 267e275. Beeby, A., Richmond, L., 1989. The shell as a site of lead deposition in Helix aspersa. Archives of Environmental Contamination and Toxicology 18, 623e628. Beeby, A., Richmond, L., 1998. Variation in the mineral composition of eggs of the snail, Helix aspersa between populations exposed to different levels of metal contamination. Environmental Pollution 101, 25e31. Beeby, A., Richmond, L., Herpé, F., 2002. Lead reduces shell mass in juvenile garden snails (Helix aspersa). Environmental Pollution 120, 283e288. Beeby, A., Richmond, L., 2007. Differential growth rates and calcium-allocation strategies in the garden snail Cantareus aspersus. Journal of Molluscan Studies 73, 105e112.
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