The effects of metal contamination on earthworm populations around a smelting works: quantifying species effects

Applied Soil Ecology ELSEVIER

Applied Soil Ecology 4 (1996) 147-160

I

The effects of metal contamination on earthworm populations around a smelting works: quantifying species effects David J. Spurgeon *, Stephen P. Hopkin Ecotoxicology Research Group, School of Animal and Microbial Sciences, University of Reading, PO Box 228, Reading RG6 6AJ, UK Accepted 8 March 1996

Abstract The effects of metal contaminants on the population density and species composition of earthworms were studied at 22 sites around a primary smelting works situated at Avonmouth, southwest England. All worms were absent from six sites within 1 km of the factory and numbers were also reduced significantly at an additional four sites 2 km from the plant. Total earthworm density was found to be inversely related to concentrations of metals in soils. Some species of earthworms were found to be more 'sensitive' to metals and were absent from sites where more 'tolerant' species persisted. For example, Lumbricus rubellus, Lurnbricus castaneus and Lumbricus terrestris were all present at sites close to the smelter where Aporrectodea rosea, Aporrectodea caliginosa and Allolobophora chlorotica were absent. The first three 'tolerant' species have more active calcium secretion glands in their gut than the three 'sensitive' worms. Calcium is known to be involved in the sequestration and elimination of various metals through the chlorogogenous tissue. Toxicity tests were carried out with a 'sensitive' and a 'non-sensitive' earthworm species (Lumbricus rubeUus and Aporrectodea rosea respectively) to examine if earthworm species distribution could be related to their susceptibility to the metals. In addition, the OECD recommended species Eisenia fetida was tested, to compare the sensitivity of this worm to the two commonly occurring soil species. Zinc was the metal used for these tests, since earlier comparisons of metal toxicity between the laboratory and field have shown that of the four metals released from the smelting works (Cd, Cu, Pb, Zn), it is zinc that is limiting the distribution of worms. Test results indicated that Aporrectodea rosea is more sensitive to the effects of zinc than Lumbricus rubellus. Thus, it appears that differences in the distribution of species of earthworms around the smelter can be related to variation in sensitivity to zinc. Both species were affected by zinc at lower concentrations than Eisenia fetida. Keywords: Aporrectodea rosea; Lumbricus rubellus; Populationdensity; Species sensitivity;Zinc

1. Introduction The effects of pollutants on populations have frequently been ignored in favour of laboratory toxicity studies using individuals (Bengtsson et al., 1985).

* Corresponding author: Tel. 0118 931 6409; Fax. 0118 931 0180; Email. [email protected].

For metals, a n u m b e r of contamination gradients (often centred on point-sources) already exist in the field. Thus it is possible to compare the results of laboratory tests with individuals to population effects at contaminated sites. Such studies should be an important part of any investigation that attempts to validate the ability of a standard test (for example the OECD artificial soil toxicity test for earthworms)

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D.J. Spurgeon, S.P. Hopkin / Applied Soil Ecology 4 (1996) 147-160

(OECD, 1984; EEC, 1985) to predict pollutant impact at the population level. In addition, any assessment of the effects of metals on earthworm populations will also permit variations in species sensitivity resulting from ecological, physiological and behavioural differences to be studied. Earthworm populations can be modified by a range of factors, these include soil conditions (Lee, 1985), food availability (Fraser et al., 1994), altered predator abundance (Churchfield et al., 1991), and climate (Lofs, 1992). However, if a population is exposed to an additional stress through elevated metal concentrations, this may be sufficient to amplify natural variation and reduce numbers so that recovery is no longer possible. Earthworm populations are known to be reduced by high concentrations of metals in soil. Bengtsson et al. (1983) found a lower total number and biomass of earthworms at sites close to a brass mill. All worms were absent in soils containing 600 ixg Cu g - l and 2200 jxg Zn g - l of NHaAc extractable copper and zinc. Hunter et al. (1987a) found a decrease in earthworm abundance in soils near a copper refinery containing 52 000 ixg Cu g - I , while Bisessar (1982) noted the absence of worms in soils around a lead smelter containing 230 p,g As g- l, 33 p,g Cd g - 1, 287 p,g Cu g - 1 and 4800 Ixg Pb g - i . The use of copper based fungicides, which have increased copper concentrations in soils (Morgan and Johnson, 1991; Morgan and Bowden, 1993) have led to reductions in abundance, diversity and biomass of earthworms (Van Rhee, 1977; Streit, 1984; Paoletti et al., 1988). A preliminary study of earthworm distribution in the Avonmouth area was conducted by Spurgeon and Hopkin (1995). From this work, it was apparent that all earthworms were absent at sites within 1 km of the factory and that certain species were absent from sites up to 2 km from the smelter. Read (1987) also conducted a survey of earthworms at Avonmouth. The results of this work indicated that earthworm abundance and biomass were not reduced at the two most contaminated sites visited (Haw and Pegwell Woods, 2.5 km from the smelter). However, results did indicate a higher proportion of surface active litter dwelling species were present (e.g. Lumbricus rubellus and Lumbricus castaneus) at these sites than in less contaminated woods. A number of smaller studies of earthworm density have also been

conducted at Avonmouth. Martin and Coughtrey (1976) and Hopkin et al. (1985) found a decrease in the abundance of earthworms in woodland sites 2 km from the smelter. This decline is particularly pronounced if density is calculated to determine the number of worms per unit mass of litter rather than individuals per metre square area (Martin and Bullock, 1994). In the study of Wright and Stringer (1980), earthworm populations were not reduced in pastureland 3.5 km from the smelter. To determine if differences in earthworm distribution could be related to their sensitivity to metals, toxicity tests were conducted with three species of earthworms using the adaptation of the OECD (1984) earthworm artificial soil test developed by Van Gestel et al. (1989). The species used for these tests were Aporrectodea rosea, a species that was found to be relatively sensitive during the preliminary study of Spurgeon and Hopkin (1995), and the less susceptible Lumbricus rubellus. The OECD (1984) recommended test species Eiseniafetida was also tested to determine if differences in sensitivity exist between this species and the two soil inhabiting worms. Previous work in which the toxicity of cadmium, copper, lead and zinc to Eisenia fetida was compared in artificial and field soils has established that the primary toxic effect on earthworms in the Avonmouth area is due to the effects of zinc (Spurgeon and Hopkin, 1995, Spurgeon et al., 1994). Studies (not reported here) have shown that this is also the case for other earthworm species including Lumbricus rubellus and Aporrectodea rosea. As a consequence zinc was used in toxicity assays with these two species. The objectives of these tests was to determine if differences found in the inter-species susceptibility for zinc could be used to explain any variations found in the distribution of earthworms during a large scale population survey conducted at 22 sites in the Avonmouth region. 2. Materials and methods

2.1. Assessing density and species diversity of earthworms

Twenty two sites were surveyed for earthworms. Of these, 21 were located in the Avonmouth area (Fig. 1) and a 'control' site was situated on the

149

D,I. Spurgeon, S.P. Hopkin / Applied Soil Ecology 4 (1996) 147-160

University of Reading campus, 100 km from the factory. Sites at Avonmouth were situated northeast of the factory downwind of the prevailing southwesterly winds (Jones, 1991). All sites were visited during late October 1993 to avoid seasonal bias due to the annual variation common for earthworm populations in temperate regions (RSzen, 1982, 1988). The samples taken at Avonmouth were from permanent grassland verges adjacent to minor roads at least 1 m (further if possible) from the kerb. The 'control' sample was collected from unmanaged grassland 20 m from the nearest road. At each site, four separate quadrats (0.25 m × 0.25 m) were marked on the soil surface. Soil was dug out to a depth of 40 cm from within each quadrat, hand sorted and all earthworms found returned to the laboratory for identification. Adult worms were identified using the key of Sims and Gerrard (1985),

whilst juveniles were identified using characteristics such as behaviour, coloration, prostomium form, setae pattern and size. In addition to sampling earthworm populations, soil was sampled at each site by collecting from the top 2 cm layer below the litter horizon, using a clean trowel. Soil was sampled at this depth since, due to the depth stratification of metals in soils, the top layer would contain the highest metal concentrations (Martin and Bullock, 1994). Soil samples were returned to the laboratory where they were prepared for cadmium, copper, lead and zinc analysis of total nitric acid digests. For the AAS analysis, the technique described by Hopkin (1989) was used. The percentage loss on ignition, and soil pH were also determined for all sampled soils. Loss on ignition was determined by heating of the dried soils at 500°C in a Gallenkamp 400PY furnace, while pH

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150

D,I. Spurgeon, S.P. Hopkin / Applied Soil Ecology 4 (1996) 147-160

was assessed using the method described by Haynes (1982).

2.2. Toxicity testing of earthworm species Toxicity tests were conducted with three earthworm species, Lumbricus rubellus, Aporrectodea rosea, and Eisenia fetida. All toxicity tests were conducted using the adaptation of the OECD (1984) protocol proposed by Van Gestel et al. (1989). For each test, artificial soil (70% sand, 20% kaolin clay, 10% Sphagnum peat, pH adjusted to 5.5-6.5 using Ca CO3; see Spurgeon et al. (1994) for further details) was prepared as described by the OECD (1984). Zinc was added to the test soils as an aqueous solution of Zn (NO3)E.6H20. Prepared solutions were mixed with 500 g of the dry medium to give the required water content (60% WHC) and metal concentrations in the test soil. The same volume of distilled water was added to controls. The zinc concentrations used were 0, 190, 350, 620, 1200 and 2000 Ixg Zn g - l . After the addition of the relevant solution, each soil was left to stabilise for 72 h before the introduction of the earthworms. Four replicate containers were used for each test concentration. The highest zinc concentration used in the artificial soil tests was an order of magnitude lower than the zinc level present at the most contaminated field site (2000 vs 22400 Ixg Zn g - l ) , however, it is known that these lower concentrations are sufficient to cause substantial toxic effects in Eisenia fetida. Earlier work comparing the toxicity of zinc in artificial and field soil tests has indicated that effects on earthworms occur at much lower zinc concentrations in artificial soil (Spurgeon and Hopkin, 1995). The differences found in zinc toxicity between the two soils can be related to their physical properties. Artificial soil has a lower binding capacity than the Avonmouth soils, which results in an increase in metal availability and consequently more severe effects on survival, reproduction and growth (Spurgeon and Hopkin, 1996). For the tests with Lumbricus rubellus and Aporrectodea rosea, adult, fully clittelate worms were obtained from an unpolluted beech-wood near Arhus, Denmark by digging and hand sorting. Worms were not collected from the Avonmouth area, since it was o

not clear if previous exposure and possible adaptation or resistance would influence the results of the tests. After collection, the animals were returned on native litter (Lumbricus rubellus) or soil (Aporrectodea rosea) and stored at 3°C before pre-exposure and use in tests. Eisenia fetida for testing were obtained from our laboratory culture. Before use in tests, all worms were pre-exposed to uncontaminated artificial soil. Eisenia fetida were pre-exposed for 7 days, while for Lumbricus rubellus and Aporrectodea rosea, a 5 day incubation period was used due to time restrictions. After preexposure, ten Eisenia fetida were added to each test container (plastic boxes, 175 mm × 120 mm × 60 mm), while for Aporrectodea rosea and Lumbricus rubellus eight and six worms respectively were used in each test replicate, due to the larger mean size of these species. The experimental containers were then covered to prevent water loss and kept for 21 days. Eisenia fetida were maintained at 20 _+ 2°C as described by the OECD (1984), while Lumbricus rubellus and Aporrectodea rosea were kept at 15 _+ 2°C due to the lower optimal temperature of these species (Lee, 1985). A constant light regime was used for all three tests. For all earthworm species, food was added to the soils to increase cocoon production over the duration of the experiments (Reinecke and Viljoen, 1990; Van Gestel et al., 1992; Spurgeon et al., 1994). In the test with Eisenia fetida, finely ground fresh horse manure, dried and rewetted to 75% water content, was used as food. However, for Aporrectodea rosea and Lumbricus rubellus, the rewetted manure was mixed with an equal volume of uncontaminated sandy loam soil. The addition of the soil was considered necessary for these species, since both incorporate a substantial quantity of inorganic matter in their diet (Piearce, 1978). In each of the three tests, mortality was measured every 7 days. Weight change was assessed by comparing average final weight of worms to mean initial weight, while cocoon production was assessed by wet sieving of the soil as described by Van Gestel et al. (1989) at the end of each test. The cocoons collected from each test container were incubated on moist filter paper to assess the percentage of fertile cocoons and the mean number of juveniles hatching from each fertile cocoon.

D.J. Spurgeon, S.P. Hopkin / Applied Soil Ecology 4 (1996) 147-160

2.3. Statistics Sites at which the total number of earthworms collected were reduced compared to the control site were determined using one tailed t-tests. Populations of individual species were not compared, since insufficient numbers were collected at many of the sites visited. To study the relationship between earthworm number and soil metal concentration, linear regressions of the cube root total number of earthworms plotted against log soil cadmium, copper, lead and zinc concentrations were calculated. Boag et al. (1994) found cube root to be most the appropriate transformation for use prior to statistical analysis of earthworm population data. LCs0 values were determined using the trimmed Spearman-Karber method (Hamilton et al., 1977; Hoekstra, 1991). ECs0s were determined by logistic analysis, while significant mean differences between test concentrations were determined using Student's t-test. All the toxicity values for reproduction and survival calculated in each toxicity test were superimposed onto zinc concentrations measured at 86 sites in the Avonmouth area by Jones (1991) using

the technique described by Spurgeon et al. (1994). This permitted maps of the area over which soil zinc levels exceed the calculated toxicity values for each species to be drawn. Thus, by determining the areas over which the toxicity values for each earthworm species were exceeded, the predicted effects of zinc on different earthworm species around the Avonmouth factory could be compared with the actual effects measured during the population survey.

3. Results

3.1. Chemical analysis of soil samples The pH of surface soils in the Avonmouth area (i.e. excluding the control site) ranged from 7.32 at Site 21 to 5.56 at Site 2, although this was the only site in the region with a pH below 6.5. Analysis of the relationship between pH and distance from the smelter by linear regression, indicated a slight positive correlation, however, this relationship is eliminated if the value from Site 2 is excluded from the calculation. The lower pH at Site 2 (the closest site to the smelter) is probably the result of acid deposi-

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DJ. Spurgeon, S.P. Hopkin / Applied Soil Ecology 4 (1996) 147-160

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D.I. Spurgeon, S.P. Hopkin /Applied Soil Ecology 4 (1996) 147-160

tion from the sulphuric acid plant that adjoins the factory (Martin and Bullock, 1994). No relationship was found between the percentage organic matter of soil and distance from the smelter. Highest metal concentrations (218 I~g Cd g - t , 1950 ixg Cu g - t , 13300 ~zg Pb g - l , 22400 Ixg Zn g - t ) were found at the sites close to the smelter (Sites 1 and 4), while lowest concentration values were found in the control soil (Site 22) (0.05 Ixg Cd g - l , 6.2 ixg Cu g - l , 60 p,g Pb g - l , 34 Izg Zn g- t). For each metal, the relationship between log soil concentration and distance from the Avonmouth smelter approximates to a straight line (Fig. 2a, Fig. 2(b), Fig. 2(c) and Fig. 2(d)), indicating an exponential decline with distance from the factory. This profile is in agreement with the results of previous studies on the spatial distribution of metals around a point source (e.g. Hunter et al., 1987b; Bengtsson and Rundgren, 1988). See also Hopkin et al. (1986), Hopkin (1989), Jones (1991) and Martin and Bullock (1994) for a full discussion of the distribution of metals in the Avonmouth area).

153

3.2. The distribution of earthworms in the Avonmouth area

All species were completely absent from six sites (Sites 1, 2, 3, 4, 5 and 8) and the number collected was also significantly reduced (P < 0.05) at four sites near the smelter (Sites 6, 7, 10 and 11) (Fig. 3). Analysis of the relationship between the cube root of total earthworm number and log metal concentration by linear regression indicated a strong negative correlation for all four metals studied (P < 0.001) (Fig. 4a, Fig. 4(b), Fig. 4(c) and Fig. 4(d)), though earlier artificial and field soil tests have indicated that of the four metals present, zinc is probably responsible for the reductions in earthworm populations at sites close to the smelter (Spurgeon and Hopkin, 1995). At all sites where earthworms were collected, both adult and juvenile animals were found. The presence of juvenile worms at these sites, suggests that the populations are maintained primarily by recruitment, rather than immigration (Grant et al., 1989). The effects of elevated metal concentrations were

Table 1 E f f e c t s o f z i n c o n the s u r v i v a l , g r o w t h a n d r e p r o d u c t i o n o f

Eisenia fetida, Lumbricus rubellus and Aporrectodea rosea in t o x i c i t y tests

c o n d u c t e d in O E C D a r t i f i c i a l soil Zinc

Survival

Initial

Cocoons worm- t

Fertile

Juveniles

Juvenile

( t z g g - 1)

14 d a y s ( % )

growth (%)

week- t

cocoons (%)

fertile c o c o o n - l

worm- i week- i

Eisenia fetida 0

100

17.2

0.39

72

1,97

0.55

190

100

5.3

0.43

78

1,85

0.62

350

100

2.9

0.53

77

2.04

0.83

620

100

- 0.3

0.22.

65

1,88

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1200

42.5 * * *

2000

0 ***

-13.4

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0 ***

0 ***

Lumbricus rubellus 0

92

7.0

1.10

62

1

0.68

190

88

6.3

0.75 *

68

1

0.51 *

350

88

26.8

0.55 * * *

66

1

0.36 * * *

620

67 * * *

4.2

0.47 * * *

44

1

0.21 * * *

1200

0 ***

2000

0 ***

Aporrectodea rosea 0

94

2.1

0

190

91

1.1

0

350

91

620

31 * * *

1200

0 ***

2000

0"**

-2.1

0

- 14.9

0

Values marked with asterisks show significant differences from control values. *

P < 0.05; * *

P < 0.01; * * *

P < 0.001.

154

D.I. Spurgeon, S.P. Hopkin / Applied Soil Ecology 4 (1996) 147-160

more severe for some earthworm species than for others. For example, Aporrectodea rosea, Aporrectodea caliginosa and Allolobophora chlorotica were absent from Sites 6, 7, and 10, whilst Lumbricus rubellus, Lumbricus castaneus and Lumbricus terrestris were present at some or all of these sites (Fig. 3). For Aporrectodea caliginosa the effects of metals were particularly severe, since this species was absent from seven sites close to the smelter (Sites 6, 7, 9, 10, 11, 12 and 13) where other species persisted (Fig. 3). For Lumbricus castaneus the situation was somewhat different, as highest total numbers of this species were collected close to the smelter (Sites 7, 9 and 10) (Fig. 3). Although this increase cannot be supported statistically, due to the high intra-sample variability and the low total numbers at some sites, the results do indicate that species-specific factors can be important in determining the responses of earthworm populations to pollutants.

3.3. Relative sensitivity of earthworm species assessed by toxicity tests Results of the three toxicity tests conducted are summarised in Table 1. For Eisenia fetida mortality was significantly higher at 1200 IxgZng-~ than in the controls (Table 1) and all worms exposed to soil containing 2000 p~g Zn g - i died. The 14 day LCs0 (with 95% confidence interval) for this species was 1106 (1010-1212) Ixg Zn g-1. Highest growth (17.2%) was recorded in worms in the control soils, while growth was significantly reduced at 1200 Ixg Zn g-~. The ECs0 for zinc on the growth of Eisenia fetida was 693 i~g Zn g-~. Cocoon production was the most sensitive parameter tested. This agrees with the results of previous toxicity tests for this species (see Van Gestel et al., 1993; Spurgeon et al., 1994; Spurgeon and Hopkin, 1995). The highest rate of cocoon production (0.53 cocoons worm- l week- l) was recorded from the worms exposed to 350 I*g Zn g-~. Cocoon production was reduced significantly at 620 p,g Zn g - l and no cocoons were produced in the 1200 p,g Zn g - i soil (Table 1). The 21 day ECs0 for the effects of zinc on cocoon production was 623 Ixg Zn g-~. The percentage of fertile cocoons and mean number of juveniles per hatched cocoon was not affected at any of the zinc concentrations tested (Table 1). The number of juveniles per worm per week therefore reflected cocoon production rates and

was significantly reduced at 620 ixg Zn g-~ and 1200 txg Zn g- 1 (Table 1). The mortality of Lumbricus rubellus was increased significantly at 620 Ixg Zn g - l . All worms died at 1200 and 2000 Ixg Zn g - i (Table 1). The 14 day LCs0 was 728 (627 - -845) Ixg Zn g-1. Growth relative to mean initial weight, was not reduced significantly at any of the test concentrations in which worms survived for the full duration of the test (Table 1). An ECs0 value could therefore not be calculated. Highest cocoon production rate of 1.1 cocoons worm-~ week-~ was recorded for worms in the control soil (Table 1). This rate of cocoon production is comparable with values recorded for Lumbricus rubellus in culture, for example, Satchell (1969) collected 42-106 cocoons year-l (0.92-2.04 cocoons worm- ~ week- 1) from worms in laboratory cultures and Evans and Guild (1948) collected a mean of 1.52 cocoons worm-1 week-1 over a 2 year period. Cocoon production was reduced significantly at all the zinc concentrations tested (Table 1). From these results, a 14 day ECs0 of 348 izg Zn g-l can be calculated. Cocoon fertility for Lumbricus rubellus was lower than for Eisenia fetida. Between 62% and 68% of the cocoons laid in the control, 190 and 350 ~g Zn g - i soils hatched (Table 1). At 620 I*g Zn g-~ only 44% of the cocoons were fertile, although this reduction in fertility was not significant, it may indicate that high zinc levels may influence the hatching of Lumbricus rubellus cocoons. Only one juvenile was produced from each cocoon that hatched. This agrees with the results of earlier studies (see Lee, 1985 for references). Aporrectodea rosea mortality was significantly higher at 620 Ixg Zn g-I than in the control soils, and all worms exposed to 1200 Ixg Zn g- ~ and 2000 Ixg Zn g-~ died (Table 1). The 14 day LCs0 for this species was 561 (499-633). As for Lumbricus rubellus, relative growth was not significantly reduced at any of the test concentrations used (Table 1), thus, growth ECs0 for this species could not be determined. No cocoons were produced by Aporrectodea rosea in any of the test soils used (Table 1). In previous studies with this species, cocoon production rates of between 3.13 and 8 cocoons worm- 1 year- 1 (0.06 and 0.15 worms week - I ) have been reported (Evans and Guild, 1948; Bolton and Phillipson, 1976). Even at these low rates of reproduction it

D.I. Spurgeon, S.P. Hopkin/Applied Soil Ecology4 (1996) 147-160 would be expected that between seven and 15 cocoons should have been produced in total from the control worms. The fact that no cocoons were produced during the test suggests that the OECD artificial soil may be unsuitable as a test medium for Aporrectodea rosea. Throughout the test, Aporrectodea rosea were frequently noted at the bottom of the test container, surrounded by large quantifies of mucus. Additionally during the test, worms appeared to be sluggish and semi-dormant, a condition somewhat similar to the aestivation state used by species such as Aporrectodea caliginosa and Aporrectodea longa (Ude) to survive unfavourable environmentally conditions (for details of aestivation in earthworms see Edwards and Lofty, 1977 and Lee, 1985). For each earthworm species tested, the LC50 values have been mapped onto the concentrations of zinc in soils around the Avonmouth factory measured by Jones (1991) (see Spurgeon et al., 1994 for a description of the method used). The maps generated in this manner indicate that the toxicity values for Lumbricus rubellus and Aporrectodea rosea were exceeded over a greater area than similar values for Eisenia fetida (Fig. 5a, Fig. 5(b) and Fig. 5(c)). For example, the LCsos for Lumbricus rubellus and Aporrectodea rosea were exceeded over 126 km 2 and 188 km 2 (Fig. 5b and Fig. 5(c)), while the LCs0 for Eisenia fetida were only exceeded over an area of 68 km 2 (Fig. 5a). The areas over which soil zinc levels exceed the LCs0s calculated in the tests with Lumbricus rubellus and Aporrectodea rosea can be compared to the areas over which these two species were found to be absent during the population survey. These comparisons indicate that populations of Aporrectodea rosea and in particular Lumbricus rubellus survive at sites where soil zinc concentrations exceed the zinc LC.~o calculated for these species (Fig. 5b and Fig. 5(c)).

4. Discussion

4.1. The relative sensitivities of earthworm species in the field Six species of earthworm (Lumbricus rubellus, Lumbricus castaneus, Lumbricus terrestris, Allolobophora chlorotica, Aporrectodea rosea and

155

Aporrectodea caliginosa) were regularly captured from the Avonmouth sites (Fig. 3). These worms are all typical of mull soils (Bolton and Phillipson, 1976; Edwards and Lofty, 1977; Lee, 1985). Since a similar species assemblage was found at all the less contaminated sites, it is unlikely that local differences in soil type account for the effects on earthworm distribution met during this study. The significant negative relationships between log cadmium, copper, lead and zinc concentration and eallhworm abundance (Fig. 4a, Fig. 4(b), Fig. 4(c) and Fig. 4(d)), indicate that elevated soil metal concentrations and in particular zinc are almost certainly responsible for the decrease in earthworm populations close to the smelter (Spurgeon and Hopkin, 1995). Aporrectodea rosea, Aporrectodea caliginosa and Allolobophora chlorotica were not present at sites close to the smelter at which Lumbricus rubellus, Lumbricus castaneus and Lumbricus terrestris persisted (Fig. 3). Thus, it is clear that the effects of metals on some earthworm species are more severe than for others. This finding agrees with the results of an earlier population survey in the Avonmouth area conducted by Spurgeon and Hopkin (1995). In that study, a comparison of the relative toxicity of cadmium, copper, lead and zinc and their concentrations in field soils indicated that, of the four metals, zinc is the most toxic to earthworms close to the smelter. Thus, comparisons of the highest zinc concentrations in which each species survives could be used to indicate the range of inter-species sensitivities. Populations of the most 'tolerant' worm Lumbricus castaneus survived in soil containing 3627 I~g Zn g-~, while the most 'sensitive', Aporrectodea caliginosa, was found in soils containing up to 903 Ixg Zn g-~. Thus, inter-species sensitivity of earthworm populations for zinc varied by up to a factor of four. Variations in sensitivity to a given pollutant may be the result of behavioural and ecological differences between species that alter exposure and response (Tomlin, 1992; Van Gestel, 1992), or physiological differences that determine detoxification and elimination strategies (Dallinger, 1993). For earthworms, the species that are relatively 'sensitive' to metals are represented by all three behavioural earthworm types: epigeics (Allolobophora chlorotica), endog~ics (Aporrectodea rosea) and an6queic

D.I. Spurgeon, S.P. Hopkin / Applied Soil Ecology 4 (1996) 147-160

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( Aporrectodea caliginosa) (Bouch6, 1992), and both r (Allolobophora chlorotica) and K (Aporrectodea rosea) life history strategy selected worms (Lee, 1985). Thus, it seems unlikely that behavioural differences or variations in life history strategies can explain the differences in sensitivities for metals found for populations of different earthworm species. Physiological similarities common to all three sensitive species do however exist. The 'sensitive' AlIolobophora chlorotica, Aporrectodea caliginosa and Aporrectodea rosea all have a lower calcium gland secretion activity than the relatively 'tolerant' Lumbricus terrestris, Lumbricus rubellus and Lumbricus castaneus (Piearce, 1972; Morgan and Morris, 1982; Morgan, 1982; Morgan and Morgan, 1988a; Morgan and Morgan, 1991). Calcium is known to be involved in the sequestration and elimination of various metals through the chlorogogenous tissue (PrentO, 1979; Andersen and Laursen, 1982; Morgan, 1982; Morgan, A.J. et ai., 1989; Morgan, J.E. et al., 1989; Fischer and Moln~r, 1993). In earthworm chlorogogenous tissue, lead, and to a lesser extent zinc, are detoxified by binding to phosphate ligands in chloragocyte granules in direct exchange with calcium (Morgan and Morgan, 1989; Morgan, A.J. et al., 1989). Thus, in species with low Ca metabolism, the rate of metal elimination may be lower and higher tissue metal concentrations may result (Ash and Lee, 1980; Morgan and Morris, 1982; Morgan and Morgan, 1988b; Morgan and Morgan, 1991). It is probable, therefore, that differences in calcium metabolism account for variations in sensitivity between earthworm species.

50

55

60

65

Fig. 5. Maps showing the areas over which the soil zinc concentrations at Avonmouthexceed the zinc LCs0 values calculated for (a) Eiseniafetida (1106 p.g Zn g- l ), (b) Lumbricus rubellus (728 p,g Zn g-i) and (c) Aporrectodea rosea (561 Ixg Zn g-l) in toxicity tests conducted in OECD artificial soil (dashed line) compared to the areas over which (b) Lumbricus rubellus and (c) Aporrectodea rosea are anticipated to be absent based on the results of the population survey. The areas where earthworms are absent have been mapped by extrapolating the highest zinc concentration at which each earthworm species was collected to soil zinc concentrations in the Avonmouth region. X and Y values are Ordnance Survey Grid Reference numbers (km).

D.J. Spurgeon, S.P. Hopkin /Applied Soil Ecology 4 (1996) 147-160

4.2. Relative susceptibility of earthworms to zinc related to sensitivity in the field It is postulated above that the differences found in the distribution of Lumbricus rubellus and Aporrectodea rosea at Avonmouth can be attributed to variations in the calcium metabolism which influence sensitivities of different species to zinc. However, comparisons of the LCs0s for the two species, indicate that Aporrectodea rosea is only slightly more sensitive to zinc than Lumbricus rubellus (561 Ixg Zn g-t compared to 726 I~g Zn g - i ) . Thus, the relative similarity in the sensitivity of these species, compared for example to Eisenia fetida (LCs0 1106 Ixg Zn g-~) may appear to undermine the initial hypothesis. However, if the LCs0 values for these two species are compared with soil zinc concentrations at Avonmouth (Fig. 5b and Fig. 5(c)), the area over which these values are exceeded differ considerably. For Aporrectodea rosea, the LCs0 of 561 ixg Zn g - t is exceeded in soils over 188 km 2 (Fig. 5c), while for Lumbricus rubellus the LCs0 of 728 p,g Zn g-t is only exceeded over 126 km z (Fig. 5b), a difference of 62 km 2. From these comparisons, it is clear that even relatively small differences in sensitivities may relate to larger differences in distribution in field soils, an effect resulting from the exponential decline in soil metal concentrations with distance from the factory (Fig. 2a, Fig. 2(b), Fig. 2(c) and Fig. 2(d)). Results from toxicity tests therefore support the hypothesis that differences in earthworm distribution are related to variations in their sensitivity to zinc. Earthworm populations at Avonmouth persisted in soils containing zinc at concentrations far in excess of those found to affect cocoon production and survival during the laboratory toxicity tests. For instance, Lumbricus rubellus were collected from soil containing up to 3490 Ixg Zn g - t , while the LCs0 and cocoon production EC50 determined for this species in the artificial soil test were 728 ~g Zn g - t and 348 I~g Zn g - i respectively (Fig. 5b). Although the ability of some species to persist at concentrations above those found to cause toxic effects in laboratory tests may be related in part to the increased resilience of the population compared to effects on individuals measured in toxicity tests (Van Straalen et al., 1989; Crommentuijn et al., 1993) and

157

population variability in resistance traits, the principal reason is almost certainly the increased availability of the zinc in the artificial soil (Spurgeon and Hopkin, 1995).

4.3. The suitability of the species used for toxicity testing The OECD (1984) and EEC (1985) have recommended the use of Eisenia fetida for laboratory toxicity testing despite the fact that this species may not be the most sensitive for a given chemical. Comparative toxicity results (e.g. Heimbach, 1985; Ma, 1988; Ma and Bodt, 1993) indicate that the relative sensitivities of earthworm species to a pollutant can vary considerably. These differences in sensitivities result from differences in physiology, ecology or behaviour. For example, the greater toxicity of the cholinesterase-inhibiting pesticide paroxone to Eisenia veneta (Rosa) compared to Eisenia fetida/Eisenia andrei, can be attributed to the presence of sensitive cholinesterases in Eisenia veneta (Stenersen et al., 1993), while the higher sensitivity of Lurnbricus terrestris to granular pesticides is due probably to its surface mating and feeding habits (Tomlin, 1992). If correct soil quality criteria for earthworms are to be established for a given pollutant, toxicity resuits from a broad range of physiological and ecologically distinct species should be considered. Using only Eisenia fetida for testing will limit the use of the collected data, as the range and variance of sensitivities will not be known. In such circumstances, the use of large safety factors is often required and as a consequence, only imprecise or guarded recommendations can be made. The development of test protocols for other earthworm species has therefore been recommended to support work conducted with Eisenia fetida (Greig-Smith, 1992a; Greig-Smith, 1992b). Of the three tested species used in this survey, Aporrectodea rosea is least suited to the test conditions because no cocoons were produced in any of the test containers and growth was less than for either Lumbricus rubellus or Eisenia fetida (Table 1). The reasons underlying the unsuitability of artificial soil for Aporrectodea rosea are not clear. However, the abrasive nature of the sand used in the mix

158

D.I. Spurgeon, S.P. Hopkin / A p p l i e d Soil Ecology 4 (1996) 147-160

may be, in part, responsible. Coarse sandy soils are known to irritate the skin of some earthworm species (Lee, 1985). Indeed, the distribution of Aporrectodea rosea has been found to be limited in soils with a high percentage sand content (NordstrSm and Rundgren, 1974). This skin irritation effect appears to result in Aporrectodea rosea producing large quantities of mucus and ultimately entering an aestivation-like state. The food supplied during the tests may also be unsuitable for optimal growth and cocoon production in Aporrectodea rosea. This species normally consumes well-degraded amorphous plant material (Piearce, 1978), thus, the large, partly-digested fragments present in the horse manure may not be appropriate. Of the other two species tested, both are suited to artificial soil. In the control containers, positive growth and cocoon production rates comparable with those reported from laboratory culture experiments were found (Table 1. The only problem encountered with the use of Lumbricus rubellus during toxicity tests, was control mortality which was higher than in any previous test with Eisenia fetida (Table 1), see also Van Gestel et al., 1993; Spurgeon et al., 1994; Spurgeon and Hopkin, 1995). Control mortality is considered a problem if rates of over 10% are reported in short term tests. For Lumbricus rubellus 8% mortality was recorded, thus, rates were close to the level that would cause serious concern. Clearly, more work must be undertaken to develop strategies that may optimise survival during tests. One such adaptation would be to use laboratory cultured worms, since worms collected from the field are of uncertain age and health. Butt (1992, 1993) recommended a culture system for breeding Lumbricus terrestris, Aporrectodea longa, Octolasion cyaneum (Savigny) and Lumbricus rubellus. The technique appears ideally suited for supplying the need of an active ecotoxicology research group and its further development could increase the number of earthworm species available for routine toxicity tests.

Acknowledgements Thanks go to Dr Hans Lokke (NERI, Silkeborg, Denmark) for providing laboratory facilities for some of this work and to Dr Martin Holmstrup for his

technical assistance and hospitality. This work was supported by a research grant from the Leverhulme Trust, a travel grant from The European Science Foundation and a research studentship from SERC (BBSRC) for DJS

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