Pergamon PII: s0045-6535(%)00348-7
Chemosphere, Vol. 33, No. 12, pp. 2475-2486, 1996 Copyright 0 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 00456535/96 $15.00+0.00
EFFECTS OF pH ON THE TOXICITY OF CADMIUM, COPPER, LEAD AND ZINC TO FOLSOML4 CXNDZDA WILLEM, 1902 (COLLEMBOLA)
IN A STANDARD LABORATORY
TEST
SYSTEM.
Richard D. Sandifer and Stephen P. Hopkin. ??
Ecotoxicology Group School of Animal and Microbial Sciences University of Reading PO Box 228, Reading RG6 6AJ TelNo. 0118 931 6049 FaxNo. 0118 931 0180 E-mail:
[email protected] (Received in Gemnny
12 June 1996: accepted 29 August 1996)
ABSTRACT E&s for cadmium, copper, lead and zinc were determined for juvenile production of Folsomia candidu at pH6.0, 5.0 and 4.5 in a standard laboratory test system In contrast to most previous studies where metal toxicity was increased at low pHs, in our experiments there was no clear relationship between soil acidity and values (pg g-l) for cadmium and zinc were similar at all three ECSO-nproduet,on in this species. The ECSO_reproductlon pHs (pH6.0: Cd 590, Zn 900; pH5.0: Cd 780, Zn 600; pH4.5: Cd 480, Zn 590). In contaminated field sites adjacent to primary zinc smelters, zinc is invariably present in soils at concentrations of at least 50 times that of cadmium. Thus deleterious effects of mixtures of these metals on populations of Collembola in such sites can be attributed to zinc rather than cadmium.
Copyright 0 1996 Elsevier Science Ltd
INTRODUCTION The bioavailability and toxicity of chemicals to soil animals are influenced by physical factors that determine soil pore water concentrations (Van Gestel, 1992). For metals, toxicity and bio-concentration f&ctors (BCFs) are affected by soil characteristics that influence availability, for example the pH, cation exchange capacity
2475
2416 (CEC), clay content and the percentage organic matter content (Ma et al, 1983; Van Gestel & Van Dis, 1988). Metals are found in eight hactions within the soil: i) t&e metal cations (e.g. Pb”); ii) inorganic complexes (e.g. CdCl’), iii) organo-metal complexes (e.g. (CH3) 4Pb); iv) organic complexes, chelates; v) bound on high molecular weight organic material, vi) bound as diverse colloids; vii) adsorbed on colloids; and viii) within the soil particles (Martin & Bullock, 1994). The metals in the first four of these Imctions are in the soil solution and are more available than those bound to the soil matrix. Therefore, any soil factor that changes the relative distributions of metals between Imctions will alter their bioavailability and hence toxicity and BCFs. In general, lowering the pH of a metal-contaminated soil increases the toxicity to soil organisms (Crommentuijn, 1994; Hopkin, 1989; Spurgeon & Hopkin, 1996; Walker et al, 1996). In this paper, the effects of diierent
soil pH on the toxicity of cadmium, copper, lead and zinc to the
springtail Folsomia candida has been examined in a standard laboratory test system. The aims of the study were a) to establish whether the metals were more toxic in more acid soils, and b) to determine the relative toxicities of cadmium, copper, lead and zinc and to compare these values to ratios of the elements in contaminated field soils in the vicinity of a primary smelting works at Avonmouth, Southwest England where all four metals occur together in a “cocktail” (Hopkii & Hames, 1994).
MATERIALS AND METHODS Artificial soil was used as described in the OECD standard earthworm test Guideline 207 (OECD, 1984) and the dra8 recommendation for the Folsomiu candidu standard test (Riepert, 1993). The medium consisted (by dry weight) of 70% sand, 20% clay (kaolin clay) and 10% organic matter as Sphagnum peat. For further details, see Spurgeon et al (1994). The pH of the medium was adjusted to either 6.0, 5.0 or 4.5 * 0.05 at the start of the experiment with powdered calcium carbonate. The constituents for the artiticial soil were air- dried, mixed thoroughly, (Cd(N0&.4HrO),
and weighed into plastic boxes (275xl55x95mm). copper nitrate (Cu(N0&.3HrO),
Solutions of cadmium nitrate
lead nitrate (Pb(NO,)r) and zinc nitrate (Zn(N03)2.6H20)
(BDH chemicals, Poole, Dorset, UK) were mixed with the dry constituents to give the required percentage water content (c.30%) and metal concentrations in the soils. The same volume of distihed water was added to the controls. The concentrations of metals used in the pH6.0 test (in pg metal g-’ dry weight of soil) were; 5, 20, 80, 300 and 1200 (cadmium); 10,40, 200, 1000 and 3000 (copper); 100, 400, 2000, 5000, 8000, 10000 and 50000 (lead); and 100, 190, 350, 620 and 1200 (zinc). At both pH5.0 and pH4.5, the same concentrations were used for cadmium, copper and lead, but for zinc, the concentrations 100, 300, 1000, 2000, 3000, 6500 and 10000 were employed since some reproduction
was still observed at 12OOpgZn g-’ at pH6.0.
Consequently, an additional experiment was carried out at pH6.0 with zinc to con&m the lack of reproduction
2477
at and above 2OOOugZn g-t. Niic
acid digests of samples of contaminated soils were analysed by atomic
absorption spectrometry (for further details see Hopkin, 1989). Concentrations of metals were within 10% of the nominal values in every case. After equilibration for 48 hours, soils were added to 2OOmlplastic vending machine cups (30g into each, four replicates for each concentration) and 5mg of dried yeast was placed in each as a food source for the Collembola. Cultures of Folsomiu cundidu (Wflem) were maintained in the laboratory at 20 f 1°C under constant light, in plastic containers with a base of plaster of Paris mixed with graphite powder. A small amount of dried yeast was added weekly as a food source. All the F. cundidu used in the test were members of a “Reading strain”, derived thorn a single female isolated from a culture donated by Dr. J. Wiles of Southampton University. Ten adult springtails of equal size were added to each container using a pooter. The lid of a petri dish was lightly sprayed with distilled water and placed over the top of each cup to maintain high humidity in the containers. The tests were all carried out at 20 f 1°C under constant light conditions. The petri dish lids were sprayed every 48’hours with distilled water and another Smg of yeast placed in each experimental container after two weeks. After four weeks the containers were flooded with distilled water and photographed individually from above on Fujichrome Provia colour transparency slide film. The transparencies were projected onto a desktop viewer and the number of juveniles produced, and adults surviving in each container, were counted. ECso values for reproduction (the concentration of metal at which juvenile production was reduced to 50% of the controls) were determined for each metal treatment at each pH from the graphs (Figs. 1,2,3). Student t-tests were performed to determine the significance of differences in the responses of F. cundidu between control and metal-treated soils at the three pHs (Tables 1,2,3). The pHs of the soils were measured at this stage.
RESULTS Cadmium.
At all three pH levels, a clear reduction in reproduction was observed at 1200 ug g”, whereas at
300 pg g-’ there was little effect. Adult survival was less sensitive to cadmium (Tables 1, 2, 3, Figs lA, 2A, 3A). ECSO-~~~~~~,~ values were similar in the three treatments (Table 4).
Copper.
There was no reproduction at soil concentrations of 3OOOpgg-’ at all three pHs (Figs IB, 2B, 3B),
though at both pH6.0 and pH5.0 there was evidence of decreased juvenile numbers at 1OOOpgg-’(Tables 1,2). A slight reduction in adult survival also occurred at the highest concentrations. EC~~_~~rod~~j~ values (Table 4) were almost identical at pH6.0 and pH5.0, but copper was less toxic at pH4.5.
2478 Table 1. Adult survival and juvenile production (mean k standard errors; 4 replicates at each concentration) for each metal at pH6.0 (ns = not significantly different Corn the control; * = significantly different fkom the control at the 5% level;** =
80
:; (kO.8)
gY1 (f52)
200
300
816 (M.4)
7?6 (f42)
1200
7?“s (M.6)
;: (f8.8)
ns
??
*
61;
7n7s4
2000
71; (kO.7)
6;sO (f134
350
71; (fo.8)
504s8 (f57) 5ngsq (SO)
(fo.6)
W7)
1000
5: (fo.8)
2;” (f46) ?? *
5000
4?s8 (fo.5)
2”; (&IS) ?? *
620
61; (fo.3)
3000
3: (f1.5)
(zl)
8000
31; (fl.1)
(&
1200
G
2:7
ns
**
(fo.4) ;.“s
(f35)
(fo.3) *
(G **
4.8 (fl.1)
(i)
3
**
50000
*t
2000
10000
(f1.3)
(& **
4l”s (fo.7)
0 W)
ns
3000
*
**
??
6500
;“o (fl.1) *
(& **
10000 W.5) 0.8 *
(G **
Lead. No reproduction occurred at pH6.0, 5.0 and 4.5 at soil concentrations of 8OOOpgg” (Tables 1, 2, 3; Figs. lC, 2C, 3C). Lead was more toxic to reproduction at pH5.0 than at pH4.5 or 6.0. E&reproductimvalues (Table 4) showed no clear relationship with pH.
Zinc. At all three pHs, there was little or no reproduction at or above 2OOOugg-’and evidence of reduction at 12OOug g-’ at pH6.0 (Table 1; Fig.lD) and 1OOOugg-’ at pH5.0 and 4.5 (Tables 2,3; Figs. 2D,3D). E&.
2479 nmwim values (Table 4) showed a slight tendency for zinc to be more toxic at lower pH values but this was not significant. Table 2. Adult survival and juvenile production (mean f standard errors; 4 replicates at each concentration) fur each metal at PI-IS.0(ns = nut significantly diflkrent tican the control; ?? = significantly diflizrent from the control at the 5% level;** =
ns
IlS
80
8 (jzO.7)
26q5.3 (rt14)
200
??
300
81; (kO.6)
370 (*26)
1000
:“8 (f1.2)
3lz5 (rt29)
6.5 (k0.3)
15ngs.5 5000 (k14)
*
llS
1200
6:ss (N.8) **
3000 (JL)
3.8 (k1.3)
**
I(
DS
2000
71ss (N.8)
1 *rY.5 (*25)
1000
??
71; (*1.2)
*
38.5 (*Is)
2000
3: (rtO.9) **
(*:6) ?? *
5.3 (il.3)
(:;I,)
**
8000 (A)
81; (hO.6)
?? *
**
3000 (:)
51; (hl.0)
**
10000
50000
71; (hO.9) 31; @l.O) ?? *
**
6500 (G ** 10000 (Z) **
(Z5)
7:“o @1.3) 3: (*0.5) **
(i) ** (& ?? *
!
Effect of pH. There were no clear relationships between adult survival or juvenile production and soil pH. However, there was an overall decrease in reproduction in the control samples at pH5.0 and 4.5 in comparison to those at pH6.0 (Tables 1,2,3). The mean pHs of the soils measured at the end of the experiment were 5.84 (pH6.0), 5.08 (pH5.0) and 4.54 (pH4.5).
Relative Toxicities of Metals.(Table 5) At pH6.0, the effects of cadmium, copper and zinc on reproduction were similar, whereas lead was about five times less toxic than cadmium. At pH5.0, the toxicities of all four metals were similar. At pH4.5, although cadmium and zinc were of similar toxicity, copper and lead were less toxic than cadmium.
2480 Table 3. Adult survival and juvenile production (mean f standard errors; 4 replicates at each concentration) for each metal at pH4.5 (ns = not significantly different from the control; * = significantly different from the control at the 5% level;** =
ns 80
300
1200
6”.“5
3n2s3
(kO.6)
(*17)
6: (+0.4)
2”56 @49) ns
3:“s (M.6) *
200
1000
3000 (!c%) ?? *
E (*0.6) *
2n4s6 (*34)
2000
7.8 (+2.5)
2;“o (hi4) ns
5000
5Yl (d.6) ns
8000 (i) ** 10000
50000
E (hO.6) 71”o (&0.7) 7”.; (MI.9) 61; (a.1) i; (hO.8) ns
2Y* (AS) ns
1000
2000 $2) **
3: (LtO.3) ?? * 3.8 (*0.8) ??
3000 (ii) **
??
6500
7.8 (d.3) ??
10000 (i) **
$6) .*
5.8 W.9)
(iI) **
(+Yl) .*
3.3 (hO.3) **
(G ** (i) ** (i) **
Table 4. E&O__&
values in pg g-’ for each metal at all three pH levels, derived t?om the graphs shown in Figs. 1,2 & 3.
Table 5. ECSwU6
values of copper, lead and zinc relative to cadmium
EC50 for metal (pg g-‘) ( EC50 for cadmium (pg g-‘)
Cadmium
Copper
Lead
ZiiC
pH6.0
1
1.20
5.03
1.53
pH5.0
1
0.91
1.75
0.77
pH4.5
1
3.06
6.56
1.22
2481
,
4 ,’
a’..
?? .._
I’
.*
2482
I
H
f G
t
I
10
10 (pg g-l).
100 1000 Lead concentration (pg g-l).
C) Lead.
100 Cadmium cmcmtratioa
\ loo00
1000
looooo
10000
L
40 --
60 -50 --
70 --
80 --
1
20 --
Figure 3. Adult survival and juvenile production at pH4.5 expressed as a percentage of the control
ON
20t
120 T
I
IO --
20 --
40 --
60 --
80 --
10
10
100
(pg 8’).
Zinccmcentraticm (pg g-l).
100
D) Zinc.
Copper cmcentratim
‘.
B) Coppa.
i
1000
\
1000
I
10000
10000
2484 DISCUSSION Previous work has suggested that reducing soil pH increases metal toxicity. Spurgeon & Hopkin (1996) exposed the earthworm Eisenia fetidu to increasing concentrations of zinc in artificial soils. They calculated EC50 values for cocoon production of 462, 343 and 189pgZn g-’ at pH6.0, 5.0 and 4.0 respectively. Crommentuijn (1994) used pH levels from 7.29 to 3.12 and found a range of EC50 values for total survival of F. cundidu of 306 to 102ngCd g-’ respectively. Crommentmjn et al (1993) found an ECS+,~~~,~ value of 227 pgCd g“ at pH6.0. Whilst the figures presented in the present study are higher than the above values, Crommentuijn et al (1993) also found an LCso value of 893pgCd g” which is not exceeded here. However, other work has shown that in eqrimentally
manipulated field soils, the abundance of some Collembola
(notably Folsomiu quudrioculutu) increases with a decrease in pH (Hagvar & Abrahamsen, 1980). The results described in this paper show no clear relationships between the pH of soils and the toxicity of cadmium, copper, lead and zinc to F. cundidu. This unexpected fmding demonstrates that caution should be exercised when formulating conclusions based on the results of standard tests before they have been conducted in several laboratories. Factors including source of OECD soil components (Riepert, 1993), and clonal difference may influence the outcome. For example the cladoceran Duphniu magna has been found to have a range of L&J values for cadmium of 0.06 pg g*’to more than 1OOpgg-’depending on which of eight different clones is tested (Baird et al, 1990). Crommentuijn et al (1995) found an LCSOrange of 802 to more than 2024pgCd g” for four different clones of Folsomiu cundidu. However, the standard test is appropriate for determining relative toxicities of metals to F. cundidu, and possibly other species of Collembola (Hopkin, in press). This information can then be used in attempts to identify the most toxic metal within a “cocktail” of elements. For example, in the vicinity of the Avonmouth smelting works, the mean ratio of Cd:Cu:Pb:Zn in soils is 1:7:50:93 (Spurgeon & Hopkin, 1995). At pH5.0 (typical of Avonmouth soils near the smelter), the toxicity of cadmium, copper and zinc to F. candidu is very similar (in the Laboratory, lead is less toxic than these three metals, Tables 4 and 5). Since zinc occurs in
Avonmouth soils at more than ten times the concentration of copper, and nearly 100 times the level of cadmium, it is clear that deleterious effects of the metal pollution on soil Collembola are most likely to be due to zinc poisoning. This is clearly the case for snails (Laskowski & Hopkin, 19964 1996b), woodlice (Hopkin & Hames, 1994) and earthworms (Spurgeon & Hopkin, 1995; Spurgeon et al, 1994) which are absent close to the Avonmouth smelter due to the heavy zinc contamination.
2485 ACKNOWLEDGEMENTS This work was funded by a NERC Industry Targeted Studentship with additional support Tom National Power Plc, and the Institute of Terrestrial Ecology at Monks Wood. Aspects of the work were funded by a grant from the Leverhuhne Trust.
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