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Seasonal changes in metal levels (Cu, Pb, Cd, Zn and Ca) within the grey field slug, Deroceras reticulatum, living in a highly polluted habitat
Environmental Pollution 59 (1989) 287-303
Seasonal Changes in Metal Levels (Cu, Pb, Cd, Zn and Ca) within the Grey Field Slug, Deroceras reticulatum, Living in a Highly Polluted Habitat R. W. Greville & A. J. M o r g a n School of Pure and Applied Biology,Universityof Wales College of Cardiff, PO Box 915, CardiffCFI 3TL, UK (Received 2 February 1988; revised version received 3 February 1989; accepted 17 February 1989)
ABSTRACT Slugs (Deroceras reticulatum (Muller)) were collected from a disused Pb/Zn mine site for a period encompassing 3 years. The analysis of whole body replicates, collected monthly for a period of one year, demonstrated a large variability in metal concentrations for each of the five metals ( Cu, Pb, Cd, Zn and Ca) analysed. Nevertheless, a number of significant monthly differences in metal levels were found, the most regular being for Ca, and the least regular for Cu. Significant differences in metal body burdens were also found between slugs collected during the same month, but in consecutive years. The presence of such large and irregular variability greatly restricts the potential value of terrestrial slugs as biomonitors of heavy metal contamination.
INTRODUCTION The assimilation and accumulation of bioavailable environmental heavy metals has been well studied in a wide range of aquatic and terrestrial organisms (Phillips, 1980; Lepp, 1981; Martin & Coughtrey, 1982). Several models of heavy metal accumulation and detoxification have been proposed (Simkiss, 1977; Simkiss et al., 1982), as well as the importance of intermetallic relationships, especially interactions between non-essential toxic metals and metabolically active metals (Schutte, 1964). 287 Environ. Pollut. 0269-7491/89/$03"50 © 1989 Elsevier Science Publishers Ltd, England. Printed in Great Britain
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R. IV. Greville, A. J. Morgan
Although the analysis of living organisms is conceptually attractive integrating the physical and chemical parameters of the environment (Krivolutzky, 1986), examples in which biological monitoring of terrestrial pollution has been used by regulatory bodies are few and far between (Martin & Coughtrey, 1982). Variability is an inherent biological problem, and to optimise biomonitoring, this must be kept to a minimum (Herricks & Schaeffer, 1985). The use of biological material to monitor heavy metal pollution introduces a complexity, not encountered by direct measurement of the abiotic environment. The following sources of variability have been suggested as being of particular importance (Martin & Coughtrey, 1982): (1) intraindividual variation, e.g. between tissues, organs and fluids; (2) intra-species variation, e.g. between ecotypes, races and populations; (3) variation due to vigour, age or exposure of the selected organism; (4) variation due to microhabitat, or foraging areas of particular individual populations or individuals; (5) variation due to locality, and related climatic factors; (6) seasonal variation; (7) analytical variability between subsamples of individual samples. Terrestrial slugs accumulate heavy metals (Coughtrey & Martin, 1976, 1977) and have been suggested as possibly useful biomonitoring organisms (Martin & Coughtrey, 1982) and, in principle, they fulfil the majority of the biomonitoring prerequisites (Greville & Morgan, 1987). Several sources of natural variability which complicate and detract from the use of slugs as biomonitors of terrestrial pollution have already been considered (Ireland, 1984; Greville & Morgan, 1989). It is the purpose of this study to examine the seasonal variations in both essential and non-essential heavy metals within D. reticulatum living in a highly contaminated Pb/Zn habitat. The variability that exists is discussed in the context of the possible utility of this gastropod for biomonitoring heavy metal pollution. MATERIALS AND METHODS
(a) Monthly metal concentrations D. reticulatum were collected indiscriminately by hand from a small mixed deciduous woodland at Llantrisant, South Wales (British Ordnance Survey ref. no. STO48823). This is a revegetated calcareous site that has been contaminated by the activities of a disused Pb/Zn mine (Greville & Morgan, 1989). Sampling was performed during the third week of each month in 1986. Monthly variations in sample size reflect the seasonal fluctuations in population size and distribution. Slugs were transferred with minimal delay to the laboratory. Adherent
Seasonal changes in metal levels within the grey field slug
289
soil and litter were carefully removed and the gut contents were cleared by maintenance on damp filter paper (deionised water: Whatman No. 1) in a dark constant temperature room (5°C) for 7 days. The filter paper was changed daily to minimise coprophagy. Slugs were killed by immersion in liquid nitrogen, and stored prior to analysis in a deep freeze.
(b) Tissue distribution Animals collected during the month of December 1986, were dissected into four tissue components (digestive gland, reproductive organ, intestine and foot/head), and processed for atomic absorption spectrophotometry (AAS) analysis, as below.
(c) Effects of starvation Slugs collected during the month of December 1986, were kept on damp filter paper (deionised water: Whatman No. 1) for varying periods up to 16 days, in a dark constant temperature room (5°C). The filter paper was renewed daily to minimise coprophagy. Slugs were killed for analysis at intervals of 3, 7, 11 and 16 days.
(d) Yearly variations in metal concentration D. reticulatum were collected during the third week of May 1985, 1986 and 1987 for metal analyses.
(e) Atomic absorption spectrophotometry Individual slugs and tissue samples were dried to constant mass at 90°C in pre-weighed glass tubes, or on pieces of filter paper (Millipore S.A.). All weighings were performed in a silica gel dried atmosphere on a Mettler AE163 microbalance (range 0-30 g, stability 1, integration 3). Metals were extracted from the dried slug tissue in 2 ml concentrated 'Analar' HNO 3 and made up to 10 ml with deionised water (final HNO 3 concentration = 10%). Five metals (Cu, Pb, Cd, Zn and Ca) were analysed in a Varian Techtron AA6 atomic absorption spectrophotometer, with standards also made up in a 10% HNO 3 matrix. Samples and standards used to measure Ca contained 1% La.
(f) Weather data Weather data were supplied by the Cardiff Meteorological Office.
R. W. Greville, A. J. Morgan
290
(g) Statistical analyses Data were expressed as mean + SE and differences between the means were assessed by non-parametric analysis (Mann-Whitney U-test). The P>0.005 level of significance was chosen because it introduces a level of rigour which circumvents problems which might arise due to high variability.
RESULTS
(a) Distribution of metals in the tissues The digestive gland appeared to contain the highest concentration of three metals (Pb, Cd and Zn) (Table 1). When expressed as a percentage of the total body burden of metals, the digestive gland contained 48% Pb, 63.5% Cd, 78.5% Zn, 31.2% Cu and 7.1% Ca.
(b) Starvation effects The time course of metal elimination during a 16-day starvation period is shown in Fig. 1. Cu concentration decreased steadily and significantly during the starvation period. Both Pb and Zn concentrations remained relatively constant during this period of metal deprivation; Cd concentrations were variable and displayed no obvious pattern. There was no significant drop in slug dry weight during this period.
TABLE I Organ Metal Distribution within the Slug Deroceras reticulatum (December Sample)
Metal concentration (#g g - 1 dry weight)
Organ Cu
Pb
Cd
Zn
Ca
Foot/head (n = 10)
~ + SE
145 31
23 8
22 7
308 16
65 409 1 307
Digestive gland (n = 10)
.~ + SE
90 16
362 86
203 60
3 968 829
13 935 2 299
Reproductive organs ( n = 10) Intestine (n = 10)
~ _+SE .~ _+SE
43 4 89 8
9 1 332 86
10 3 66 20
1! 8 4 380 53
4 667 ! 687 4 765 1088
1@.
0.
(a)
6.
8
g
4
@ Do~js oF S t a r v a t i o n 10.
(b)
0_ 2
4
6 8 10 Days oF S t o r v o t l o n
4
~
12
14
16
10_
(c)
o
0 0
6
ie
12
~,
~6
Doys oF S t o r v a t l o n
Fig. l(a-c).
Time course o f recta| (Cu, Pb, Cd) elimination during a 16-day starvation period (mean 4- SE).
-I-÷
JAN FEB MAR APR HAY JUN JUL AUG SEP IIET NOV 0EE Months of the Year
Months of the Year
SO
JAN FIB MAR APR HAY JUN JUL AUG SEP O[T NOV DEC
,rf' I00~
d-
Fig. 2(a--e). M e a n ( + SE) metal contents and c o n c e n t r a t i o n s in the m o n t h l y samples o f D. reticulatum. (N.B. Jan sample: dry b o d y weight = 43 ___2, n = 40; Feb; dry weight = 28 _ 4, n = 11; Mar: dry w e i g h t = 31 _ 2, n = 35; Apr: dry weight = 53 _+ 3, n = 22; May: dry weight = 54 + 4, n = 16; June: dry weight = 58 ___4, n = 16; July: dry w e i g h t = 60 _ 1, n = 8; Aug: dry w e i g h t = 91 + 2, n = 15; Sept: dry weight = 43 +__4, n = 12; N o v : dry weight = 27 + 2, n = 32; Dec: dry w e i g h t = 70 + 6, n = 13).
2O
~0
150"
2ool ÷
÷ Jr
'°°1
Months of the Year
JAN FEB MAR APR MAY JUN /UL AUG SEP ()CT NOV DEC
Lead Concentration {jJg/g)
2
6
8 ¸
÷--I--
%
Lead Content (~g}
I0
tO tO
Fig. 2b
Copper Concentration (JJg/g}
Months of the Year
JAN FEB MAR APR HAY JUN JUL AUG SEP OCT NOV DEC
Fi, • 2a Cop t e r Content (IJg)
tO
2O
30
+
,1'
qt
Months of the Year
JAN FEB MAR APR I~IAY JUN JUL AUG SEP OCT NOV DEC
I
Cadmium Concentration I ~ )
~0
]'
Fig. 2.--contd.
200
z.00
600
800
1000
Months of the Year
JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC
Zinc Concentration (.ug/g)
Months of the Year
F10
20
30
z~O
JAN FEB MAR APR MAY JUN JUL AUG SEP DLI NOV DEC
Jr
Mnnthr. of the Year
-I-
JAN FEB MAR APP M-~Y JUN JUL AUG SEP ,'LT NOV DEC
!
SO
60
Zinc Content (J.Ig)
+ -I-+
Fig. 2d
Fig. 2c
Cadmium Content (.uo)
O~
.T..
~b
B,
294
R. HI. Greville, A. J. Morgan Fig. 2e Calcium Content (~g)
20OO
1500
1000
5OO
JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV OEC HonPh$ of the Year
Catcium Concentration(JJ(J/9)
30000
20000
-I 10000
JAN FEB H~,R APR PlAY JUN JUL AUG SEP OCT NOV DEC Honths of the Year
Fig. 2.--contd.
(c) Seasonal variation in metal content and concentration
Whole body metal burdens change significantly between consecutive months during three periods of the year (Fig. 2). First, in the Spring: the contents of Cu, Cd and Zn increased significantly between March and April; Ca content increased steadily throughout the Spring, with a sharp, significant increase between April and May; Pb content also increased steadily during the period February to April, but the differences between adjacent months were not statistically significant. Secondly, in the Summer: the content of all five metals (Cu, Pb, Cd, Zn and Ca) decreased significantly between June and July, and increased significantly between July and August; the July sample contained the lowest absolute amounts of each metal (expressed as #g) and the lowest concentrations (/~gg-z) of any of the monthly samples. Thirdly, in the Winter: the contents of all five metals increased significantly between November and December.
Seasonal changes in metal levels within the grey field slug
295
Fig. 3b Lied Content IJig)
Fig. 3c cidmium Content(A)
Fg3a Copper Content t~g)
II ll
? MAY85 MAY85
t~AY 86
MAY 87 05
MAY07
HA
flAY 66
MAY 07
Copper Concentration(~g~)
60@~ ÷
cadmium Con{.enfralion{yg/g) Lead Conctmirafion (Jig/g) 300' 120 - - [
~
250'
lOO.
200
80
1,0
150
60
100
I
2O
5O
20
I
MAY 05
MAY87
MAY86
MAY 85
Fig 3d Zinc Coelent (.Pg)
MAY86
MAY85
MAY 07
MAY86
Fig. 3e
Ca~ium Contenf(.~) t
1200
¢0
1000 8O0 6OO
20
~00 @ 10 2OO
PLAY8$
MAY06
Zinc Concentration (jig/g}
SO0'
200
100
20000.
10000.
' '
MAY05
Fig. 3(a-e).
MAY 07
15000.
kO0 300
HAyH6
Calriu Concentration (jJg/g)
-t-
700' 600.
PP~Y05
MAY 8"/
5000,
i MAY66
MAY07
MAY 65
MAY 06
MAY $?
Mean (-I- SE) metal contents and concentrations in D. reticulatum collected in the month of May in three successive years.
296
R. IV. Greville, A. J. Morgan
TABLE 2 The Regression Analysis of Body Metal Content (pg): Monthly Dry Body Weight Showing Correlation Coefficients and Levels of Significance Cu
Pb
Cd
Zn
January
1.77 24.33 0.23
1'80 90'73 0-0
1.08 23.56 0.22
16"80 115"99 0" 11
n/s
n/s
n/s
n/s
February
-0"34 95"40 0"89 ** 0"19 4-57 0.71
- 1'90 239"34 0"88 ** 0-19 215-40 0-71
-- 1"63 2"94 0"02 n/s -0.10 49.14 0.74
5"75 538"9 0'49 n/s 2'88 449.94 0"69
144"95 8 095"46 0"82 ** 57-90 13756-12 0.69
0.81 57.18 0.84 ** 8.33 94.27 0.70
1.47 151-34 0"50 ** --6-58 249-42 0-78
-0-42 58"6 0.65 ** -0.37 61.25 0.85
40.21 8-87 0.004 n/s -22.67 1 026.61 0-73
163"80 8068.12 0-62 ** 798.50 7617.23 0-26
5.09 7.45 0-06
-4"53 237"56 0.61
0"66 41.51 0-39
22-1 759-39 0.23
728.60 2664.46 0" 10
March
April
May
June
n/s July
August
2"88 60-14 0"55 n/s 0-80 28-40 0.31
** - 17"92 331"58 0"50 n/s - 4.60 113-45 0"45
n/s -5'66 100-96 0'58 n/s 0"35 19.94 0.19
n/s -23"64 509"37 0-34 n/s - 23.10 623-87 0.31
Ca 509.42 5 192.33 0" 17
n/s
n/s -614"03 13777"73 0"63 n/s - 590.49 14591.12 0.42
n/s
n/s
n/s
n/s
0-24 65" 11 0'38 n/s
3"50 37"70 0"35 n/s
0'54 50"49 0"49 n/s
3"74 773"77 0"52 n/s
547"26 17 747-22 0"33 n/s
(lost)
(lost)
(lost)
(lost)
(lost)
November
0-28 57.53 0.74
-0.69 113.47 0.78
-0.58 61.37 0.82
11.85 413.26 0"56
91.78 22 606.97 0-68
December
0'77 53"25 0"35
--3"41 211"36 0"67
- 1-41 74-61 0"77
-5.61 849"50 0'56
I 629"24 7 990"09 0"65
n/s
**
**
**
September
n/s
October
I = intercept, S = slope, r = correlation coefficient. ** = significant at P < if05. n/s = non-significant at P < 0.05 (see legend o f Fig. 2 for n values).
**
Seasonal changes in metal levels within the grey field slug
TABLE
3
Statistical Analysis (First Order Partial Correlations, r ~) of the Relationships Between C d : Z n , and Pb:Ca, respectively
Month
Metal concentration (llgg- l dry weight) Cd:Zn
Pb:Ca 0'127 n/s 0.162
January
r
February
r
0.680 *** 0'596
March
r
0.414
*
n/s April
r
0"311 n/s 0.400 n/s 0.709
May
r
June
r
July
r
August
r
September
r
0.963 ** 0.661 ** 0.629
October November
r r
(Lost) 0'199
December
r
**
*
n/s 0-127
n/s
n/s
0-00
n/s 0'124 n/s O-181 n/s -0.309 n/s -0-512 n/s - 2-459 n/s 0'211 n/s (Lost) -0'246
n/s - 0" 132
n/s
(Of the metals analysed, these two metal pairs are those that are most often considered to be biologically significant; the interrelationships are based on shared chemical affinities.) t = in the partial correlation analysis, the possible influence of dry body weight on the metal-metal relationships was removed. *=P<0'05; **=P<0-01, ***=P<0"001; n/s=non significant (see legend of Fig. 2 for n values).
297
298
R. W. Greville, A. J. Morgan
(d) Yearly differences in metal content and concentration Slug samples were collected and analysed during the month of May in three consecutive years (1985, 1986, 1987) in an attempt to assess the year to year variability of metal levels. Metal content did not differ significantly except for Ca, which was significantly higher in May 1986 than either of the other two samples. Metal concentration was found to be more variable than metal content; Pb concentration in May 1987, and Ca concentrations in May 1986, were found to be higher than both other samples, respectively (Fig. 3). (e) Body weight: body burden correlations Metal content tends to be positively correlated with dry body weight for each of the five metals analysed. These relationships do not, in each case, 26 24.
22. 20_ 18_ tJ v
o
113. 14
.,a a~
12 10 8.
o~
G. 4_ 2_ O. -2. X10 ~ 15
10
0
I JAN
FEB
MAR
APR
HAY
JUN
JUL
AUG
SEP
OCT
NOV
OEC
Months of the Year
Fig. 4.
Monthly mean temperature (°C) and rainfall (mm) during the main sampling year, 1986.
Seasonal changes in metal levels within the grey field slug
299
produce a significant coefficient of correlation (P > 0.05). Of the 11 monthly slug samples, Pb versus dry weight relationships were significant on seven occasions, Cu, Cd and Ca on five occasions, and Zn on four occasions. During the months of March and November, each of the five metals yielded significant relationships between metal content and dry weight (Table 2). Logarithmic transformation of data did not substantially increase the significance of the metal burden versus dry weight relationship.
(f) Inter-metallic relationships Due to the presence of a strong tendency for metal content to be positively correlated with slug dry weight, the examination of possible inter-metallic relationships was performed by partial correlation. A significant correlation between Cd:Zn was found in seven of the 11 months analysed. No other regular metal:metal correlation was observed, except that the Pb:Ca relationship was not significant for any of the 11 months (Table 3).
(g) Weather data Mean weather conditions at the sampling area for each month during the year of 1986 are shown in Fig. 4. Maximum temperature was recorded during June (20-4°C), minimum temperature during February (-1.6°C). Total rainfall was least in February (l'7mm) and highest in November (142.2 mm). DISCI3SSION Seasonal variations in heavy metal concentrations have been extensively recorded for aquatic organisms (Phillips, 1980). A large proportion of this variability has often been attributed to episodic exposure of the organism depending on the physical variations in pollutant delivery. In fact, it is the smoothing out of such variations in pollutant delivery which makes the use of aquatic biomonitors most appealing. Terrestrial organisms, on the other hand, do not normally suffer from such wide seasonal fluxes in pollutant delivery, although terrestrial ecosystems in temperate regions are subject to wide fluctuations in local climatic conditions. In this study, large seasonal differences in both the content and concentration of the essential and non-essential metals were observed in the grey field slug D. reticulatum, and these are probably caused by interactions of various seasonal changes in food availability/choice, local climate and physiological responses/requirements. Weather conditions have been shown to affect the activity and
300
R. W. Greville, A. J. Morgan
distribution of slugs (Barnes & Weil, 1945; Crawford-Sidebotham, 1972) with maximum activity, and thereby maximum effective exposure to environmental contaminants, tending to occur on warm still nights with plenty of surface moisture. Therefore, the very low temperature and exceptionally low rainfall of February may have led to a lower net metal assimilation, resulting in lowered metal contents during February and March. Behaviour-related seasonal fluxes, if taken in isolation, do not however, explain the seasonal fluctuations in metal levels, because the results of the depuration experiment indicate that (with the exception of Cu) short periods of starvation do not significantly affect tissue metal burdens. Whilst predominantly herbivorous, D. reticulatum consumes a very catholic range of different living plant material (Pallant, 1969). Metal uptake and distribution in plants has been shown to display interspecies and (seasonal) intraspecies differences. For example, several deciduous plants tend to accumulate a large part of their total toxic metal burdens in old leaves just prior to senescence (Martin & Coughtrey, 1982), and dicotyledons tend to accumulate higher metal concentrations in their aerial parts than do species of monocotyledon. A temporal surge in the metal content of the leaves of their preferred food plant could explain increases in the mean metal concentrations of the grazing slugs during the months of August and September. The fact that this slug species tends to be a relatively nondiscriminating grazer may also explain some of the variation in metal content and concentration between samples. The steady increase in metal content and concentration from July to September is also noteworthy for another reason. During this period none of the metals displayed any significant correlation with dry body weight (Table 2). This relationship has been recommended by Boyden (1974) for comparing the degree of pollution at different marine sites. The present results suggest that this relationship is less satisfactory for comparing pollution in the less stable and uniform terrestrial ecosystems. The high metabolic rate accompanying rapid growth and high environmental temperatures could explain the very low levels of metal found in D. reticulatum during July. During this month, one would expect the slug to be most active, and it has been shown that the efficiency with which zinc and cadmium are assimilated by a variety of organisms decreases as the rate of food consumption increases (Russell et al., 1981; Joose et al., 1981). Wieser et al. (1977) offered a similar explanation for the observed maximal Cu concentration in the terrestrial isopod, Porcellio scaber, during Winter; they suggested that in cold weather the passage of food along the gut is slow, thereby increasing the contact between the metal and the absorbtive epithelial surfaces.
Seasonal changes in metal levels within the grey.fieM slug
301
Calcium reserve mobilisation has been linked to reproductive requirements in D. reticulaturn (Fournie & Chetail, 1982). Certain essential and nonessential metals may also be prone to similar cyclical mobilisation during phases in the reproductive cycle (Burton, 1972; Phillips, 1980; Williamson, 1980; Ireland, 1984), although the evidence for a direct physiological link to Ca fluxes is not compelling (see Table 3). Unfortunately, the simple scenario described by Fournie & Chetail (1982) of a steady increase in body calcium prior to and after each egg batch is laid cannot easily be studied in free living slugs. Up to three generations of the slug may be present within any population (Runham, 1970). Each generation would therefore be at differing stages of reproductive development, with even their rate of development depending on their time of hatch (Bett, 1960). Problems associated with asynchrony can be alleviated in some species by choosing individuals of a fairly narrow size cohort (Williamson, 1980; Coughtrey & Martin, 1977). This option is not effective in D. reticulatum because body weight does not reliably reflect the stage of sexual development (Runham & Laryea, 1968). Therefore, a large natural variation in metal content and concentration, as was found by Williamson (1980) in the snail, Cepaea hortensis, has to be endured. In the present study all slugs encountered during a field sampling session were collected for chemical analysis, irrespective of their sizes, so that the relationship between metal content and dry body weight could be examined, as recommended by Boyden (1974). Our observations showed that the metal content: dry weight relationships, although tending to be positive throughout the year, suffered from subtle deviations. For example, the regression coefficients (metal content :body weight) were not consistently positive for all metals during a given month of the year; neither were they consistently positive for a given metal at all monthly intervals throughout the year. A complex pattern also emerged when considering inter-metal relationships. Positive Cd: Zn correlations have been observed in several organisms living on contaminated sites (Martin & Coughtrey, 1982) due to the chemical affinities of the metals and their association with cysteine-rich, metallothionein proteins (Coughtrey & Martin, 1977). In our study, even such a well documented metal-metal relationship was not statistically significant for each monthly sample, although this metal pairing yielded more significant correlations than any other. Also noteworthy was the lack of correlation between Ca and Pb, although positive correlations between these two metals have been observed in the tissues of woodlice (Beeby, 1978) and earthworms (Ireland, 1974). These seasonal inconsistencies in inter-metal relationships confirmed the findings of Coughtrey & Martin (1977) for Helix aspersa. Furthermore, our analyses of slugs collected in the same calendar month in
302
R. W. Greville, A. J. Morgan
three successive years showed that, whilst the pattern of seasonal flux may be reproducible from year to year, the precise timing of metal fluxes is not. These findings cast considerable doubt on the value of using terrestrial slugs as monitors of environmental pollution. In conclusion, our study demonstrates that D. reticulatum is not an ideal biomonitor for comparing heavy metal contamination between geographically separated sites. Certainly, generalisations concerning metal accumulation and assimilation in this terrestrial slug cannot be stated with the same precision and confidence as those put forward to describe the metal relationships of certain aquatic molluscs (Boyden, 1974, 1977; Phillips, 1977). Finally, any biomonitoring programme that seeks to use slugs to assess or compare intra-site and/or inter-site differences in pollution levels must take into account the seasonal and year-to-year fluxes that the present study highlights.
ACKNOWLEDGEMENTS We would like to thank the following: Mr V. Williams for photographic assistance, Mrs A. Robson for typing the text, and Dr J. E. Morgan for his constructive comments on an early draft of the manuscript.
REFERENCES Barnes, H. F. & Weil, J. W. (1945). Slugs in gardens: Their numbers, activities and distribution. Part 2. J. Anim. Ecol., 14, 71-96 Beeby, A. N. (1978). Interaction of lead and calcium uptake by the woodlouse Porcellio scaber (Isopoda, Porcellinoidae). Oecologia (Berl.), 32, 255 62. Bett, J. A. (1960). The breeding season of slugs in gardens. Proc. Zool. Soc. London, 135, 311-14. Boyden, C. R. (1974). Trace element content and body size in molluscs. Nature, Lond., 251, 311-14. Boyden, C. R. (1977). Effects of size upon metal content of shellfish. J. Mar. Biol. Assoc. (U/Q, 57, 675 714. Burton, R. F. (1972). The storage of calcium and magnesium phosphate and of calcite in the digestive glands of the pulmonata (Gastropoda). Comp. Biochem. Physiol. 43A, 655 63. Coughtrey, P. J. & Martin, M. H. (1976). The distribution of Pb, Zn, Cd and Cu within the pulmonate mollusc, Helix aspersa Muller. Oecologia (Berl.), 23, 315-22. Coughtrey, P. J. & Martin, M. H. (1977). The uptake of lead, zinc, cadmium and copper by the pulmonate mollusc Helix aspersa Muller, and its relevance to the monitoring of heavy metal contamination of the environment. Oecologia (Berl.), 27, 65-74. Crawford-Sidebotham, T. J. (1972). The influence of weather upon the activity of slugs. Oecologia (Berl.), 9, 141-54.
Seasonal changes in metal levels within the grey field slug
303
Fournie, J. & Chetail, M. (1982). Evidence for a mobilization of calcium reserves for reproductive requirements in Deroceras reticulatum. Malacologia, 22, 285-91. Greville, R. W. & Morgan, A. J. (1987). The accumulation and effects of heavy metal assimilation on selected terrestrial slug species. In Heavy Metals in the Environment, New Orleans, Voi. II. CEP Consultants, Edinburgh, UK, pp. 48-51. Greville, R. W. & Morgan, A. J. (1989). Concentrations of metals (Cu, Pb, Cd, Zn and Ca) in 6 species of British terrestrial gastropods living near a disused Pb/Zn mine. J. Mollusc. Stud., 51, 31-6. Herricks, E. E. & Schaeffer, D. J. (1985). Can we optimise biomonitoring? Environ. Manage., 19, 487-92. Ireland, M. P. (1974). Metal content of Dendrobaena rubida (Oligochaeta) in a base metal mining area. Oikos, 26, 1-8. Ireland, M. P. (1984). Seasonal changes in zinc, manganese, magnesium, copper and calcium content in the digestive gland of the slug Arion ater L. Comp. Biochem. Physiol., 78A, 855-8. Joose, E. N. G., Wulfraat, K. J. & Glas, H. P. (1981). Tolerance and acclimation to zinc of the isopod Porcellio scaber Latr. In Heavy Metals in the Environment, Amsterdam, Sept. 1981, CEP Consultants, Edinburgh, UK, pp. 425-8. Krivolutzky, D. A. (1986). Animals as bioindicators. In Biological Monitoring of the State of the Environment: Bioindicators, IUBS Monograph Series No. 1. IRL Press, Oxford, New York, Tokyo. Lepp, N. W. (1981). Effects of Heavy Metal Pollution on Plants. Vol. 2. Applied Science Publishers, London. Martin, M. H. & Coughtrey, P. J. (1982). Biological Monitoring of Heavy Metal Pollution, Applied Science Publishers, London. Pallant, D. (1969). The food of the grey field slug (Agriolimax reticulatus (Muller)) in woodland. J. Anim. EcoL, 38, 391-7. Phillips, D. J. H. (1977). The use of biological indicator organisms to monitor trace metal pollution in marine and estuarine environments--A review. Environ. Pollut., 13, 281-317. Phillips, D. J. H. (1980). Quantitative Aquatic Biological Indicators. Applied Science Publishers, London. Runham, N. W. (1970). Terrestrial Slugs. Hutchinson and Co. Ltd, London. Runham, N. W. & Laryea, A. A. (1968). Studies on the maturation of the reproductive system of Agriolimax reticulatus (Pulmonata: Limacidae) Malacologia, 7(1), 93-108. Russell, L. K., Dehaven, J. I. & Botts, R. P. (1981). Toxic effects of cadmium on the garden snail Helix aspersa. Bull. Environ. Contain. Toxicol., 26, 634~,0. Schutte, K. H. (1964). The Biology of the Trace Metals: Their Role in Nutrition. Crosby Lockwood and Son, Ltd, London. Simkiss, K. (1977). Biomineralization and detoxification. Calcif. Tissue Res., 24, 199-200. Simkiss, K., Taylor, M. & Mason, A. Z. (1982). Metal detoxification and accumulation--A review. Mar. Biol. Lett., 3, 187-201. Weiser, W., Dallinger, R. & Busch, G. (1977). The flow of copper through a terrestrial food chain, 2: Factors influencing the copper content of isopods. Oecologia (Berl.), 30, 265-72. Williamson, P. (1980). Variables affecting body burdens of lead, zinc and cadmium in a roadside population of the snail Cepaea hortensis Muller. Oecologia (Berl.), 44, 213-20.
Seasonal Changes in Metal Levels (Cu, Pb, Cd, Zn and Ca) within the Grey Field Slug, Deroceras reticulatum, Living in a Highly Polluted Habitat R. W. Greville & A. J. M o r g a n School of Pure and Applied Biology,Universityof Wales College of Cardiff, PO Box 915, CardiffCFI 3TL, UK (Received 2 February 1988; revised version received 3 February 1989; accepted 17 February 1989)
ABSTRACT Slugs (Deroceras reticulatum (Muller)) were collected from a disused Pb/Zn mine site for a period encompassing 3 years. The analysis of whole body replicates, collected monthly for a period of one year, demonstrated a large variability in metal concentrations for each of the five metals ( Cu, Pb, Cd, Zn and Ca) analysed. Nevertheless, a number of significant monthly differences in metal levels were found, the most regular being for Ca, and the least regular for Cu. Significant differences in metal body burdens were also found between slugs collected during the same month, but in consecutive years. The presence of such large and irregular variability greatly restricts the potential value of terrestrial slugs as biomonitors of heavy metal contamination.
INTRODUCTION The assimilation and accumulation of bioavailable environmental heavy metals has been well studied in a wide range of aquatic and terrestrial organisms (Phillips, 1980; Lepp, 1981; Martin & Coughtrey, 1982). Several models of heavy metal accumulation and detoxification have been proposed (Simkiss, 1977; Simkiss et al., 1982), as well as the importance of intermetallic relationships, especially interactions between non-essential toxic metals and metabolically active metals (Schutte, 1964). 287 Environ. Pollut. 0269-7491/89/$03"50 © 1989 Elsevier Science Publishers Ltd, England. Printed in Great Britain
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R. IV. Greville, A. J. Morgan
Although the analysis of living organisms is conceptually attractive integrating the physical and chemical parameters of the environment (Krivolutzky, 1986), examples in which biological monitoring of terrestrial pollution has been used by regulatory bodies are few and far between (Martin & Coughtrey, 1982). Variability is an inherent biological problem, and to optimise biomonitoring, this must be kept to a minimum (Herricks & Schaeffer, 1985). The use of biological material to monitor heavy metal pollution introduces a complexity, not encountered by direct measurement of the abiotic environment. The following sources of variability have been suggested as being of particular importance (Martin & Coughtrey, 1982): (1) intraindividual variation, e.g. between tissues, organs and fluids; (2) intra-species variation, e.g. between ecotypes, races and populations; (3) variation due to vigour, age or exposure of the selected organism; (4) variation due to microhabitat, or foraging areas of particular individual populations or individuals; (5) variation due to locality, and related climatic factors; (6) seasonal variation; (7) analytical variability between subsamples of individual samples. Terrestrial slugs accumulate heavy metals (Coughtrey & Martin, 1976, 1977) and have been suggested as possibly useful biomonitoring organisms (Martin & Coughtrey, 1982) and, in principle, they fulfil the majority of the biomonitoring prerequisites (Greville & Morgan, 1987). Several sources of natural variability which complicate and detract from the use of slugs as biomonitors of terrestrial pollution have already been considered (Ireland, 1984; Greville & Morgan, 1989). It is the purpose of this study to examine the seasonal variations in both essential and non-essential heavy metals within D. reticulatum living in a highly contaminated Pb/Zn habitat. The variability that exists is discussed in the context of the possible utility of this gastropod for biomonitoring heavy metal pollution. MATERIALS AND METHODS
(a) Monthly metal concentrations D. reticulatum were collected indiscriminately by hand from a small mixed deciduous woodland at Llantrisant, South Wales (British Ordnance Survey ref. no. STO48823). This is a revegetated calcareous site that has been contaminated by the activities of a disused Pb/Zn mine (Greville & Morgan, 1989). Sampling was performed during the third week of each month in 1986. Monthly variations in sample size reflect the seasonal fluctuations in population size and distribution. Slugs were transferred with minimal delay to the laboratory. Adherent
Seasonal changes in metal levels within the grey field slug
289
soil and litter were carefully removed and the gut contents were cleared by maintenance on damp filter paper (deionised water: Whatman No. 1) in a dark constant temperature room (5°C) for 7 days. The filter paper was changed daily to minimise coprophagy. Slugs were killed by immersion in liquid nitrogen, and stored prior to analysis in a deep freeze.
(b) Tissue distribution Animals collected during the month of December 1986, were dissected into four tissue components (digestive gland, reproductive organ, intestine and foot/head), and processed for atomic absorption spectrophotometry (AAS) analysis, as below.
(c) Effects of starvation Slugs collected during the month of December 1986, were kept on damp filter paper (deionised water: Whatman No. 1) for varying periods up to 16 days, in a dark constant temperature room (5°C). The filter paper was renewed daily to minimise coprophagy. Slugs were killed for analysis at intervals of 3, 7, 11 and 16 days.
(d) Yearly variations in metal concentration D. reticulatum were collected during the third week of May 1985, 1986 and 1987 for metal analyses.
(e) Atomic absorption spectrophotometry Individual slugs and tissue samples were dried to constant mass at 90°C in pre-weighed glass tubes, or on pieces of filter paper (Millipore S.A.). All weighings were performed in a silica gel dried atmosphere on a Mettler AE163 microbalance (range 0-30 g, stability 1, integration 3). Metals were extracted from the dried slug tissue in 2 ml concentrated 'Analar' HNO 3 and made up to 10 ml with deionised water (final HNO 3 concentration = 10%). Five metals (Cu, Pb, Cd, Zn and Ca) were analysed in a Varian Techtron AA6 atomic absorption spectrophotometer, with standards also made up in a 10% HNO 3 matrix. Samples and standards used to measure Ca contained 1% La.
(f) Weather data Weather data were supplied by the Cardiff Meteorological Office.
R. W. Greville, A. J. Morgan
290
(g) Statistical analyses Data were expressed as mean + SE and differences between the means were assessed by non-parametric analysis (Mann-Whitney U-test). The P>0.005 level of significance was chosen because it introduces a level of rigour which circumvents problems which might arise due to high variability.
RESULTS
(a) Distribution of metals in the tissues The digestive gland appeared to contain the highest concentration of three metals (Pb, Cd and Zn) (Table 1). When expressed as a percentage of the total body burden of metals, the digestive gland contained 48% Pb, 63.5% Cd, 78.5% Zn, 31.2% Cu and 7.1% Ca.
(b) Starvation effects The time course of metal elimination during a 16-day starvation period is shown in Fig. 1. Cu concentration decreased steadily and significantly during the starvation period. Both Pb and Zn concentrations remained relatively constant during this period of metal deprivation; Cd concentrations were variable and displayed no obvious pattern. There was no significant drop in slug dry weight during this period.
TABLE I Organ Metal Distribution within the Slug Deroceras reticulatum (December Sample)
Metal concentration (#g g - 1 dry weight)
Organ Cu
Pb
Cd
Zn
Ca
Foot/head (n = 10)
~ + SE
145 31
23 8
22 7
308 16
65 409 1 307
Digestive gland (n = 10)
.~ + SE
90 16
362 86
203 60
3 968 829
13 935 2 299
Reproductive organs ( n = 10) Intestine (n = 10)
~ _+SE .~ _+SE
43 4 89 8
9 1 332 86
10 3 66 20
1! 8 4 380 53
4 667 ! 687 4 765 1088
1@.
0.
(a)
6.
8
g
4
@ Do~js oF S t a r v a t i o n 10.
(b)
0_ 2
4
6 8 10 Days oF S t o r v o t l o n
4
~
12
14
16
10_
(c)
o
0 0
6
ie
12
~,
~6
Doys oF S t o r v a t l o n
Fig. l(a-c).
Time course o f recta| (Cu, Pb, Cd) elimination during a 16-day starvation period (mean 4- SE).
-I-÷
JAN FEB MAR APR HAY JUN JUL AUG SEP IIET NOV 0EE Months of the Year
Months of the Year
SO
JAN FIB MAR APR HAY JUN JUL AUG SEP O[T NOV DEC
,rf' I00~
d-
Fig. 2(a--e). M e a n ( + SE) metal contents and c o n c e n t r a t i o n s in the m o n t h l y samples o f D. reticulatum. (N.B. Jan sample: dry b o d y weight = 43 ___2, n = 40; Feb; dry weight = 28 _ 4, n = 11; Mar: dry w e i g h t = 31 _ 2, n = 35; Apr: dry weight = 53 _+ 3, n = 22; May: dry weight = 54 + 4, n = 16; June: dry weight = 58 ___4, n = 16; July: dry w e i g h t = 60 _ 1, n = 8; Aug: dry w e i g h t = 91 + 2, n = 15; Sept: dry weight = 43 +__4, n = 12; N o v : dry weight = 27 + 2, n = 32; Dec: dry w e i g h t = 70 + 6, n = 13).
2O
~0
150"
2ool ÷
÷ Jr
'°°1
Months of the Year
JAN FEB MAR APR MAY JUN /UL AUG SEP ()CT NOV DEC
Lead Concentration {jJg/g)
2
6
8 ¸
÷--I--
%
Lead Content (~g}
I0
tO tO
Fig. 2b
Copper Concentration (JJg/g}
Months of the Year
JAN FEB MAR APR HAY JUN JUL AUG SEP OCT NOV DEC
Fi, • 2a Cop t e r Content (IJg)
tO
2O
30
+
,1'
qt
Months of the Year
JAN FEB MAR APR I~IAY JUN JUL AUG SEP OCT NOV DEC
I
Cadmium Concentration I ~ )
~0
]'
Fig. 2.--contd.
200
z.00
600
800
1000
Months of the Year
JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC
Zinc Concentration (.ug/g)
Months of the Year
F10
20
30
z~O
JAN FEB MAR APR MAY JUN JUL AUG SEP DLI NOV DEC
Jr
Mnnthr. of the Year
-I-
JAN FEB MAR APP M-~Y JUN JUL AUG SEP ,'LT NOV DEC
!
SO
60
Zinc Content (J.Ig)
+ -I-+
Fig. 2d
Fig. 2c
Cadmium Content (.uo)
O~
.T..
~b
B,
294
R. HI. Greville, A. J. Morgan Fig. 2e Calcium Content (~g)
20OO
1500
1000
5OO
JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV OEC HonPh$ of the Year
Catcium Concentration(JJ(J/9)
30000
20000
-I 10000
JAN FEB H~,R APR PlAY JUN JUL AUG SEP OCT NOV DEC Honths of the Year
Fig. 2.--contd.
(c) Seasonal variation in metal content and concentration
Whole body metal burdens change significantly between consecutive months during three periods of the year (Fig. 2). First, in the Spring: the contents of Cu, Cd and Zn increased significantly between March and April; Ca content increased steadily throughout the Spring, with a sharp, significant increase between April and May; Pb content also increased steadily during the period February to April, but the differences between adjacent months were not statistically significant. Secondly, in the Summer: the content of all five metals (Cu, Pb, Cd, Zn and Ca) decreased significantly between June and July, and increased significantly between July and August; the July sample contained the lowest absolute amounts of each metal (expressed as #g) and the lowest concentrations (/~gg-z) of any of the monthly samples. Thirdly, in the Winter: the contents of all five metals increased significantly between November and December.
Seasonal changes in metal levels within the grey field slug
295
Fig. 3b Lied Content IJig)
Fig. 3c cidmium Content(A)
Fg3a Copper Content t~g)
II ll
? MAY85 MAY85
t~AY 86
MAY 87 05
MAY07
HA
flAY 66
MAY 07
Copper Concentration(~g~)
60@~ ÷
cadmium Con{.enfralion{yg/g) Lead Conctmirafion (Jig/g) 300' 120 - - [
~
250'
lOO.
200
80
1,0
150
60
100
I
2O
5O
20
I
MAY 05
MAY87
MAY86
MAY 85
Fig 3d Zinc Coelent (.Pg)
MAY86
MAY85
MAY 07
MAY86
Fig. 3e
Ca~ium Contenf(.~) t
1200
¢0
1000 8O0 6OO
20
~00 @ 10 2OO
PLAY8$
MAY06
Zinc Concentration (jig/g}
SO0'
200
100
20000.
10000.
' '
MAY05
Fig. 3(a-e).
MAY 07
15000.
kO0 300
HAyH6
Calriu Concentration (jJg/g)
-t-
700' 600.
PP~Y05
MAY 8"/
5000,
i MAY66
MAY07
MAY 65
MAY 06
MAY $?
Mean (-I- SE) metal contents and concentrations in D. reticulatum collected in the month of May in three successive years.
296
R. IV. Greville, A. J. Morgan
TABLE 2 The Regression Analysis of Body Metal Content (pg): Monthly Dry Body Weight Showing Correlation Coefficients and Levels of Significance Cu
Pb
Cd
Zn
January
1.77 24.33 0.23
1'80 90'73 0-0
1.08 23.56 0.22
16"80 115"99 0" 11
n/s
n/s
n/s
n/s
February
-0"34 95"40 0"89 ** 0"19 4-57 0.71
- 1'90 239"34 0"88 ** 0-19 215-40 0-71
-- 1"63 2"94 0"02 n/s -0.10 49.14 0.74
5"75 538"9 0'49 n/s 2'88 449.94 0"69
144"95 8 095"46 0"82 ** 57-90 13756-12 0.69
0.81 57.18 0.84 ** 8.33 94.27 0.70
1.47 151-34 0"50 ** --6-58 249-42 0-78
-0-42 58"6 0.65 ** -0.37 61.25 0.85
40.21 8-87 0.004 n/s -22.67 1 026.61 0-73
163"80 8068.12 0-62 ** 798.50 7617.23 0-26
5.09 7.45 0-06
-4"53 237"56 0.61
0"66 41.51 0-39
22-1 759-39 0.23
728.60 2664.46 0" 10
March
April
May
June
n/s July
August
2"88 60-14 0"55 n/s 0-80 28-40 0.31
** - 17"92 331"58 0"50 n/s - 4.60 113-45 0"45
n/s -5'66 100-96 0'58 n/s 0"35 19.94 0.19
n/s -23"64 509"37 0-34 n/s - 23.10 623-87 0.31
Ca 509.42 5 192.33 0" 17
n/s
n/s -614"03 13777"73 0"63 n/s - 590.49 14591.12 0.42
n/s
n/s
n/s
n/s
0-24 65" 11 0'38 n/s
3"50 37"70 0"35 n/s
0'54 50"49 0"49 n/s
3"74 773"77 0"52 n/s
547"26 17 747-22 0"33 n/s
(lost)
(lost)
(lost)
(lost)
(lost)
November
0-28 57.53 0.74
-0.69 113.47 0.78
-0.58 61.37 0.82
11.85 413.26 0"56
91.78 22 606.97 0-68
December
0'77 53"25 0"35
--3"41 211"36 0"67
- 1-41 74-61 0"77
-5.61 849"50 0'56
I 629"24 7 990"09 0"65
n/s
**
**
**
September
n/s
October
I = intercept, S = slope, r = correlation coefficient. ** = significant at P < if05. n/s = non-significant at P < 0.05 (see legend o f Fig. 2 for n values).
**
Seasonal changes in metal levels within the grey field slug
TABLE
3
Statistical Analysis (First Order Partial Correlations, r ~) of the Relationships Between C d : Z n , and Pb:Ca, respectively
Month
Metal concentration (llgg- l dry weight) Cd:Zn
Pb:Ca 0'127 n/s 0.162
January
r
February
r
0.680 *** 0'596
March
r
0.414
*
n/s April
r
0"311 n/s 0.400 n/s 0.709
May
r
June
r
July
r
August
r
September
r
0.963 ** 0.661 ** 0.629
October November
r r
(Lost) 0'199
December
r
**
*
n/s 0-127
n/s
n/s
0-00
n/s 0'124 n/s O-181 n/s -0.309 n/s -0-512 n/s - 2-459 n/s 0'211 n/s (Lost) -0'246
n/s - 0" 132
n/s
(Of the metals analysed, these two metal pairs are those that are most often considered to be biologically significant; the interrelationships are based on shared chemical affinities.) t = in the partial correlation analysis, the possible influence of dry body weight on the metal-metal relationships was removed. *=P<0'05; **=P<0-01, ***=P<0"001; n/s=non significant (see legend of Fig. 2 for n values).
297
298
R. W. Greville, A. J. Morgan
(d) Yearly differences in metal content and concentration Slug samples were collected and analysed during the month of May in three consecutive years (1985, 1986, 1987) in an attempt to assess the year to year variability of metal levels. Metal content did not differ significantly except for Ca, which was significantly higher in May 1986 than either of the other two samples. Metal concentration was found to be more variable than metal content; Pb concentration in May 1987, and Ca concentrations in May 1986, were found to be higher than both other samples, respectively (Fig. 3). (e) Body weight: body burden correlations Metal content tends to be positively correlated with dry body weight for each of the five metals analysed. These relationships do not, in each case, 26 24.
22. 20_ 18_ tJ v
o
113. 14
.,a a~
12 10 8.
o~
G. 4_ 2_ O. -2. X10 ~ 15
10
0
I JAN
FEB
MAR
APR
HAY
JUN
JUL
AUG
SEP
OCT
NOV
OEC
Months of the Year
Fig. 4.
Monthly mean temperature (°C) and rainfall (mm) during the main sampling year, 1986.
Seasonal changes in metal levels within the grey field slug
299
produce a significant coefficient of correlation (P > 0.05). Of the 11 monthly slug samples, Pb versus dry weight relationships were significant on seven occasions, Cu, Cd and Ca on five occasions, and Zn on four occasions. During the months of March and November, each of the five metals yielded significant relationships between metal content and dry weight (Table 2). Logarithmic transformation of data did not substantially increase the significance of the metal burden versus dry weight relationship.
(f) Inter-metallic relationships Due to the presence of a strong tendency for metal content to be positively correlated with slug dry weight, the examination of possible inter-metallic relationships was performed by partial correlation. A significant correlation between Cd:Zn was found in seven of the 11 months analysed. No other regular metal:metal correlation was observed, except that the Pb:Ca relationship was not significant for any of the 11 months (Table 3).
(g) Weather data Mean weather conditions at the sampling area for each month during the year of 1986 are shown in Fig. 4. Maximum temperature was recorded during June (20-4°C), minimum temperature during February (-1.6°C). Total rainfall was least in February (l'7mm) and highest in November (142.2 mm). DISCI3SSION Seasonal variations in heavy metal concentrations have been extensively recorded for aquatic organisms (Phillips, 1980). A large proportion of this variability has often been attributed to episodic exposure of the organism depending on the physical variations in pollutant delivery. In fact, it is the smoothing out of such variations in pollutant delivery which makes the use of aquatic biomonitors most appealing. Terrestrial organisms, on the other hand, do not normally suffer from such wide seasonal fluxes in pollutant delivery, although terrestrial ecosystems in temperate regions are subject to wide fluctuations in local climatic conditions. In this study, large seasonal differences in both the content and concentration of the essential and non-essential metals were observed in the grey field slug D. reticulatum, and these are probably caused by interactions of various seasonal changes in food availability/choice, local climate and physiological responses/requirements. Weather conditions have been shown to affect the activity and
300
R. W. Greville, A. J. Morgan
distribution of slugs (Barnes & Weil, 1945; Crawford-Sidebotham, 1972) with maximum activity, and thereby maximum effective exposure to environmental contaminants, tending to occur on warm still nights with plenty of surface moisture. Therefore, the very low temperature and exceptionally low rainfall of February may have led to a lower net metal assimilation, resulting in lowered metal contents during February and March. Behaviour-related seasonal fluxes, if taken in isolation, do not however, explain the seasonal fluctuations in metal levels, because the results of the depuration experiment indicate that (with the exception of Cu) short periods of starvation do not significantly affect tissue metal burdens. Whilst predominantly herbivorous, D. reticulatum consumes a very catholic range of different living plant material (Pallant, 1969). Metal uptake and distribution in plants has been shown to display interspecies and (seasonal) intraspecies differences. For example, several deciduous plants tend to accumulate a large part of their total toxic metal burdens in old leaves just prior to senescence (Martin & Coughtrey, 1982), and dicotyledons tend to accumulate higher metal concentrations in their aerial parts than do species of monocotyledon. A temporal surge in the metal content of the leaves of their preferred food plant could explain increases in the mean metal concentrations of the grazing slugs during the months of August and September. The fact that this slug species tends to be a relatively nondiscriminating grazer may also explain some of the variation in metal content and concentration between samples. The steady increase in metal content and concentration from July to September is also noteworthy for another reason. During this period none of the metals displayed any significant correlation with dry body weight (Table 2). This relationship has been recommended by Boyden (1974) for comparing the degree of pollution at different marine sites. The present results suggest that this relationship is less satisfactory for comparing pollution in the less stable and uniform terrestrial ecosystems. The high metabolic rate accompanying rapid growth and high environmental temperatures could explain the very low levels of metal found in D. reticulatum during July. During this month, one would expect the slug to be most active, and it has been shown that the efficiency with which zinc and cadmium are assimilated by a variety of organisms decreases as the rate of food consumption increases (Russell et al., 1981; Joose et al., 1981). Wieser et al. (1977) offered a similar explanation for the observed maximal Cu concentration in the terrestrial isopod, Porcellio scaber, during Winter; they suggested that in cold weather the passage of food along the gut is slow, thereby increasing the contact between the metal and the absorbtive epithelial surfaces.
Seasonal changes in metal levels within the grey.fieM slug
301
Calcium reserve mobilisation has been linked to reproductive requirements in D. reticulaturn (Fournie & Chetail, 1982). Certain essential and nonessential metals may also be prone to similar cyclical mobilisation during phases in the reproductive cycle (Burton, 1972; Phillips, 1980; Williamson, 1980; Ireland, 1984), although the evidence for a direct physiological link to Ca fluxes is not compelling (see Table 3). Unfortunately, the simple scenario described by Fournie & Chetail (1982) of a steady increase in body calcium prior to and after each egg batch is laid cannot easily be studied in free living slugs. Up to three generations of the slug may be present within any population (Runham, 1970). Each generation would therefore be at differing stages of reproductive development, with even their rate of development depending on their time of hatch (Bett, 1960). Problems associated with asynchrony can be alleviated in some species by choosing individuals of a fairly narrow size cohort (Williamson, 1980; Coughtrey & Martin, 1977). This option is not effective in D. reticulatum because body weight does not reliably reflect the stage of sexual development (Runham & Laryea, 1968). Therefore, a large natural variation in metal content and concentration, as was found by Williamson (1980) in the snail, Cepaea hortensis, has to be endured. In the present study all slugs encountered during a field sampling session were collected for chemical analysis, irrespective of their sizes, so that the relationship between metal content and dry body weight could be examined, as recommended by Boyden (1974). Our observations showed that the metal content: dry weight relationships, although tending to be positive throughout the year, suffered from subtle deviations. For example, the regression coefficients (metal content :body weight) were not consistently positive for all metals during a given month of the year; neither were they consistently positive for a given metal at all monthly intervals throughout the year. A complex pattern also emerged when considering inter-metal relationships. Positive Cd: Zn correlations have been observed in several organisms living on contaminated sites (Martin & Coughtrey, 1982) due to the chemical affinities of the metals and their association with cysteine-rich, metallothionein proteins (Coughtrey & Martin, 1977). In our study, even such a well documented metal-metal relationship was not statistically significant for each monthly sample, although this metal pairing yielded more significant correlations than any other. Also noteworthy was the lack of correlation between Ca and Pb, although positive correlations between these two metals have been observed in the tissues of woodlice (Beeby, 1978) and earthworms (Ireland, 1974). These seasonal inconsistencies in inter-metal relationships confirmed the findings of Coughtrey & Martin (1977) for Helix aspersa. Furthermore, our analyses of slugs collected in the same calendar month in
302
R. W. Greville, A. J. Morgan
three successive years showed that, whilst the pattern of seasonal flux may be reproducible from year to year, the precise timing of metal fluxes is not. These findings cast considerable doubt on the value of using terrestrial slugs as monitors of environmental pollution. In conclusion, our study demonstrates that D. reticulatum is not an ideal biomonitor for comparing heavy metal contamination between geographically separated sites. Certainly, generalisations concerning metal accumulation and assimilation in this terrestrial slug cannot be stated with the same precision and confidence as those put forward to describe the metal relationships of certain aquatic molluscs (Boyden, 1974, 1977; Phillips, 1977). Finally, any biomonitoring programme that seeks to use slugs to assess or compare intra-site and/or inter-site differences in pollution levels must take into account the seasonal and year-to-year fluxes that the present study highlights.
ACKNOWLEDGEMENTS We would like to thank the following: Mr V. Williams for photographic assistance, Mrs A. Robson for typing the text, and Dr J. E. Morgan for his constructive comments on an early draft of the manuscript.
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