The influence of soil characteristics on cadmium toxicity for Folsomia candida (Collembola: Isotomidae)

Pedobiologia 47, 387–395, 2003 © Urban & Fischer Verlag http://www.urbanfischer.de/journals/pedo

The influence of soil characteristics on cadmium toxicity for Folsomia candida (Collembola: Isotomidae) Cornelis A. M. van Gestel* and Sandra Mol Institute of Ecological Science, Vrije Universiteit, De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands Submitted February 13, 2003 · Accepted May 30, 2003

Summary Effects of cadmium on survival, growth and reproduction of the collembolan Folsomia candida was determined in four soils differing in organic matter (3.0–10.9 %) and clay content (1.4–5.2 %) but all having a pH of approx. 6.0. Total, 0.01 M CaCl2-exchangeable, water-soluble and porewater cadmium concentrations were measured in all soils, and so were internal cadmium concentrations in the surviving animals. Freundlich Kf values for cadmium desorption from soil, based on porewater or water-soluble concentrations, differed by a factor of 2 between the four soils tested, and increased only slightly during the 6-week test period. Kf values based on porewater concentrations were a factor of 10 lower than the ones for water-soluble concentrations, which can mainly, but not completely, be explained from the dilution achieved when preparing water-soluble fractions. Kf values based on CaCl2-exchangeable concentrations did not differ between the test soils. Toxicity of cadmium for the Collembola, expressed on a total soil concentration basis, differed by a factor of 1.8–6.4, with largest difference being observed after 4 weeks of exposure; at other time intervals difference was less than a factor of 3.2. EC50 values for the effect on reproduction ranged between 53.7 and 193 µg Cd/g dry soil after 4 weeks, and slightly decreased after 6 weeks to 43.3–103 µg Cd/g dry soil. Differences in toxicity between the four soils were not reduced by using CaCl2-exchangeable, water-soluble, porewater or internal concentrations instead of total cadmium concentrations as a basis for calculating EC50s. The absence of a consistent relationship between cadmium toxicity and soil properties suggests that differences of less that a factor of 3–4 in organic matter and clay content, in soils with the same pH, do not lead to significant differences in cadmium toxicity to Collembola. Key words: Folsomia candida, cadmium, bioavailability, sorption, critical body residues

Introduction Bioavailability is one of the main factors determining toxicity of metals in soil (Chapman & Wang 2000). Bioavailability may be affected by many factors, including the type of metal, properties of the soil and properties of the organism (Chapman & Wang 2000). It is therefore essential to increase our knowledge of the impact of soil characteristics on

the toxicity of metals to soil organisms (Fairbrother et al. 2002). Different methods are available to assess available fractions in soils (Lebourg et al. 1996). The meaning of these chemical available fractions for metal uptake by soil organisms remains, however, unclear. Chemical availability alone, however, is not sufficient for a metal

*E-mail corresponding author: [email protected] 0031-4056/03/47/04–387 $15.00/0

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to be taken up by organisms, also other ions may be available competing for the same uptake sites (Van Gestel 1997; Plette et al. 1999). In the end, internal metal concentrations in test organisms can provide the best insight in the real available fraction (Van Straalen 1996; Van Wensem et al. 1994). Collembola are insects occurring abundantly in the surface layers of forest soils. They feed on fungi or algae and thus indirectly contribute to the decomposition of leaf litter. Collembola may be exposed to metals accumulating in the top soil layer. The species Folsomia candida Willem is most abundant in organic soils and has been used in several laboratory studies (Usher & Stoneman 1977; ISO 1999). Among the heavy metals polluting the soil, cadmium is one of the most toxic. Cadmium is a natural contaminant of zinc ores (Aylett 1979), and has been widely distributed in the environment due to human industrial activities. Only few studies have focused on the impact of soil properties on cadmium uptake and effects in Collembola (Wohlgemuth et al. 1990; Crommentuijn et al. 1997; Sandifer & Hopkin 1996). This study aimed at gaining further insight into the impact of soil characteristics on the toxicity of cadmium to the soil-dwelling collembolan Folsomia candida, and to relate cadmium toxicity to chemical availability and internal concentrations in the test organisms. For that purpose, cadmium toxicity was determined in four soils differing in organic matter and clay content but having a similar pH. Effects of cadmium on F. candida were related to total and extractable (water-soluble, CaCl2-exchangeable) and porewater concentrations as well as to internal concentrations in the surviving animals. In addition, development of toxicity in time was assessed by performing toxicity tests over periods of two, four and six weeks.

Materials and Methods Folsomia candida (Willem 1902) were cultured on moist plaster of Paris mixed with charcoal (10 % w/w) at a temperature of 18–20 °C, and a light:dark regime of 12:12 h, with dried baker’s yeast (Dr. Oetker) as a food source. Relative humidity in the climate room was 75 %. The breeding culture originated from arable land at the experimental farm “The Lovinckhoeve” at Marknesse, The Netherlands, and has been kept in the laboratory for about 9 years before the start of the tests. To obtain synchronised cultures, adult animals were incubated in new containers to lay eggs and removed after two to three days. The eggs hatched after 14 days, and 10–12 days old juveniles were used in all experiments. To ensure equal body sizes at the start of the exPedobiologia (2003) 47, 387–395

periments, body weight of a randomly selected number of animals used for the tests was determined by weighing on a Sartorius S4 supermicrobalance. Tests were performed with an artificial soil (OECD) according to OECD (1984) and ISO (1999) and three natural soils at a similar pH (1 M KCl) of appr. 6.0. OECD soil was prepared by homogeneously mixing 70 % quartz sand, 20 % kaolin clay and c. 10 % air dried peat. The peat was finely ground and sieved over 1.0 mm. BUDEL and Kooyenburg (KOOY) are sandy soils. KOOY was collected from the top 20 cm of an agricultural field plot near Kooyenburg, The Netherlands. BUDEL is a reference soil collected 20 km Northeast of a zinc factory in Budel, The Netherlands. After removal of the litter layer, soil was collected from the top 10 cm layer. After collection, KOOY and BUDEL soils were air-dried, 4 mm sieved, well homogenized and stored at a dry place till use. The pH of OECD, BUDEL and Kooyenburg (KOOY) soils were brought to the desired levels by adding CaCO3 (Baker analyzed). LUFA 2.2 soil was purchased from the LUFA-Institute at Speyer, Germany. The pH of LUFA 2.2 soil was not manipulated. OECD, LUFA 2.2, KOOY and BUDEL soils were tested at a moisture content of 35–55 % of the Water Holding Capacity (WHC), corresponding with moisture levels (% w/w) of 50–55 %, 18.2 %, 11.2 %, and 11.0 %, respectively. Table 1 shows the main characteristics of the test soils. Cadmium (CdCl2.21/2H2O; BDH, AnalaR, 99.5 % pure) was added to the test soils as a solution in deionized water, at nominal concentrations of 0-30-90-270810-2430 µg Cd/g dry soil. The amounts of water used were sufficient to moisten the soils to the desired moisture level. Five test containers (100 ml glass jars) were prepared for each control and treatment group and each sampling time, each containing approximately 25 g wet soil. Additional containers were prepared for pH and soil moisture measurements at the beginning and at the end of the experiment. Exposure of the Collembola was started within a few days after treatment of the soils. Ten juvenile F. candida were placed in each test container, and some grains of dried baker’s yeast were added. Test containers were incubated at 20 °C, RH 70–75 % and a light/dark cycle of 12:12 hours. After two, four and six weeks, five test containers per treatment level were sacrificed for analysis of the numbers of adults and juveniles, body weight and accumulation of cadmium in the adults. The soil content of each container was transferred to a beaker glass using 100 ml deionized water and the soil/water mixture was stirred thoroughly but carefully to let all the animals present float to the surface. The water surface was then photographed on diapositive material and juveniles were counted by projecting the slide on a desk-

Cadmium toxicity to Folsomia candida

top slide projector. Ten surviving adults per test concentration were weighed to the nearest microgram using a Sartorius S4 supermicrobalance to obtain the wet weight. Consequently, the animals were lyophilised at –40 °C for 24 hours. After determination of the dry weight, the animals were individually digested in a 300 µl HClO4/HNO3 mixture (1:7 v/v; Ultrex grade, Baker) as described by Van Straalen & Van Wensem (1986) and cadmium concentrations in their bodies were measured using a Perkin Elmer 1100B AAS equipped with a graphite furnace assembly at a wavelength of 228.9 nm. Quality of the analysis was controlled by analyzing certified reference material (Bovine liver); metal concentrations usually were within 15 % of the certified values. To determine total cadmium concentrations in soil, dry soil samples were digested in a microwave using a mixture of deionized water, HCl and HNO3 (1:1:4), and analysed for cadmium by flame AAS on a Perkin Elmer 1100B AAS at a wavelength of 228.9 nm. Water-soluble and exchangeable cadmium concentrations were determined at the beginning and end of the test by shaking air-dried soil samples for 2 hours with deionized water or 0.01 M CaCl2 at a ratio of 1:5 (w/v). The resulting soil suspension was filtered over a paper filter (Schleicher & Schuell Rundfilter 595). Pore water was sampled by centrifugation of wet soil samples for 45 minutes at 4000 rpm. The pore water was 0.45 µm filtered. All solutions were analysed for cadmium concentrations by flame AAS as described above. pH was measured in all these fractions; in addition, pH-KCl was measured after shaking soil samples with a 1 M KCl solution at a ratio of 1:5 (w/v). To determine desorption of cadmium from the test soils, the relationships between total concentrations and water-soluble, CaCl2-exchangeable or porewater cadmium concentrations were fitted applying a Freundlich isotherm: log (Cs) = log (Kf) + 1/n * log(Ce)

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in which: Cs = cadmium content adsorbed to the solid phase (in µg/g) Kf = Freundlich adsorption constant (ml/g) Ce = cadmium concentration in the solution at equilibrium (in µg/ml) 1/ = shape parameter of the Freundlich isotherm n (dimensionless) For the calculation of EC50 values for the effect of cadmium on growth (fresh and dry weight) and reproduction (the number of juveniles produced), the logistic model described by Haanstra et al. (1985) was used. EC50 values were based on total, water-soluble and CaCl2-exchangeable soil concentrations, pore water concentrations and internal cadmium concentrations in the animals. In addition, EC10 values were obtained by the modified logistic model according to Van Brummelen et al. (1996). To compare EC50 and EC10 values for the different soils, a generalized likelihood ratio test was used (Sokall & Rohlf 1969). All calculations were run on a MacIntosh® computer using SYSTAT® 5.2.1. software.

Results pH values measured in the different soil extracts are given in Table 1. In a number of cases, pH decreased at high cadmium concentrations in the soils. Due to the small volumes recovered, no pH measurements could be done on porewater samples. Measured cadmium concentrations in the control soils ranged between 0.45 and 1.18 µg/g dry soil. Measured total concentrations in the test soils generally did not differ more than 10 % from the nominal ones. Nevertheless, all values in this paper are based on measured concentrations.

Table 1. Main characteristics of the soils used in this study. pH values are ranges measured at the end of the 6-week toxicity tests in the controls and the different treatments. CEC = cation exchange capacity Soil

pH-KCl

pH-CaCl2

pH-H2O

OECD KOOY BUDEL LUFA 2.2

6.09–6.42* 5.10–5.86@ 6.16–6.24 5.50–6.23@

5.76–6.35* 5.30–5.78 5.92–6.22* 5.50–6.90

6.19–7.44* 5.55–6.60 6.13–7.36* 5.80–6.44

% clay

% org. matter

CEC (meq/kg)

5.2 2.5 1.4 3.6

10.9 3.5 3.0 4.2

140.2 59.2 61.2 110.2

* highest values in control and lowest at highest test concentration @ lowest values in control and highest values at higher treatment levels

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Table 2. Freundlich constants for the desorption of cadmium from four different soils. Kf and 1/n values are calculated from log-log plots of cadmium concentrations in soil (in µg/g) versus cadmium concentrations in water and 0.01 M CaCl2 extracts and pore water (in µg/ml). Goodness-of-fit of the sorption isotherms is indicated by r2 soil

Freundlich constants for water soluble Cd 1/ r2 Kf n

OECD KOOY BUDEL LUFA 2.2

T=0 438 283 222 242

OECD KOOY BUDEL LUFA 2.2

T = 6 weeks 539 0.63 322 0.45 322 0.54 276 0.62

0.55 0.50 0.53 0.56

CaCl2 exchangeable Cd 1/ Kf r2 n

0.818 0.960 0.956 0.973

54.5 53.5 60.9 57.8

0.82 0.72 0.69 0.73

0.994 1.000 0.999 0.998

0.896 0.902 0.976 0.961

65.6 44.9 65.4 53.1

0.78 0.88 0.73 0.80

0.997 0.984 0.992 0.988

Because not enough soil was left to collect a sufficient amount of pore water, no porewater cadmium concentrations could be measured in OECD soil. In the other soils, porewater concentrations, expressed as µg/ml, generally were a factor of 10–100 higher than water-soluble concentrations, with two outliers in KOOY soil being a factor of 242–264 higher. Mean difference (±SD, n=15) between porewater and watersoluble concentrations (in µg/ml) was a factor of 66.3±77.5.

pore water Cd 1/ Kf r2 n

n.d. 42.1 57.4 32.1

n.d. 0.46 0.47 0.55

n.d. 0.987 0.932 0.990

Table 2 show the Freundlich constants based on total cadmium concentrations measured in aqueous extracts, 0.01 M CaCl2 extracts and pore water of the soils used in this study. Control values were omitted for this calculation, because they tended to deviate from the treatments leading to lower Kf and r2 values but higher 1/n values. This effect was most pronounced in the water-soluble and porewater fractions, and is probably due to the different form of cadmium in these control soils (natural versus chloride salt). In case of

Fig. 1. Effect of cadmium on the fresh weight of Folsomia candida after 4 (left) and 6 (right) weeks of exposure in four different soils. Each data point represents one animal. Lines are dose-response relationships fitted using the logistic model according to Haanstra et al. (1985) Pedobiologia (2003) 47, 387–395

OECD KOOY BUDEL LUFA 2.2

OECD KOOY BUDEL LUFA 2.2

OECD KOOY BUDEL LUFA 2.2

2

4

6

273 (214–348) 401 (329–489) 336 (186–607) 203 (170–242)

696 (515–939) 392 (336–456) 447 (211–948) 145 (123–172)

244 (167–358) 197 (140–278) 262 (154–445) 120 (95.0–152)

0.81 (0.58–1.15) 2.24 (1.14–4.42) 0.80 (0.02–2.59) 2.08 (1.63–2.65)

2.58 (1.52–4.37) 2.32 (1.40–3.84) 0.27 (–) 1.57 (1.27–1.95)

0.63 (0.27–1.42) 0.0066 (–) 0.091 (–) 1.26 (0.99–1.61)

– 119 (66.7–212) 41.6 (–) 24.1 (17.2–33.7)

– 113 (74.3–172) 4.27 (0.01–2100) 14.4 (10.6–19.5)

4.09 (0.26–65.4) 2.64 (0.01–6500) 10.6 (7.09–16.0)

–

EC50 in µg Cd/g dry weight based on concentrations total soil water soluble pore water

30.0 (22.4–40.2) 61.9 (44.4–86.2) 39.3 (15.2–102) 23.1 (18.4–29.0)

96.7 (64.0–146) 62.2 (49.0–78.9) 52.5 (12.2–226) 17.7 (14.8–21.1)

25.5 (15.3–42.6) 24.9 (15.9–38.9) 24.4 (9.61–61.8) 14.2 (11.3–17.8)

CaCl2-exchangeable

80.9 (62.9–103) 209 (160–273) 106 (69.7–163) 120 (98.9–146)

263 (207–335) 169 (148–192) 168 (78.7–357) 121 (89.4–163)

63.9 (48.4–84.7) 85.8 (65.8–112) 100 (68.5–147) 90.2 (74.8–109)

internal

soil

OECD KOOY BUDEL LUFA 2.2

OECD KOOY BUDEL LUFA 2.2

time (weeks)

4

6

70.6 (33.1–151) 103 (69.4–152) 54.4 (21.4–138) 43.3 (31.6–59.4)

193 (101–369) 141 (80.8–246) 53.7 (19.0–152) 57.9 (38.2–87.6) 0.26 (0.16–0.41) 0.29 (0.14–0.60) 0.17 (0.06–0.46) 0.47 (0.27–0.80)

0.50 (0.29–0.85) 0.41 (0.25–0.66) 0.17 (0.055–0.50) 0.85 (0.26–2.84)

– 3.05 (2.74–3.40) 0.77 (0.04–17.0) 2.62 (1.50–4.59)

– 8.24 (1.64–41.5) 0.79 (0.04–1.60) 4.39 (2.15–8.96)

EC50 in µg Cd/g dry weight based on concentrations total soil water soluble pore water

5.92 (1.87–18.7) 10.6 (7.07–16.0) 3.16 (0.89–11.3) 5.53 (3.58–8.57)

20.9 (10.8–40.3) 15.1 (8.08–28.1) 3.11 (0.76–12.0) 8.49 (4.40–16.4)

CaCl2-exchangeable

37.1 (27.0–50.6) 76.4 (57.3–103) 24.7 (20.2–31.5) 49.5 (38.2–64.1)

142 (94.4–212) 96.7 (84.4–111) 49.3 (31.6–76.6) 92.3 (65.0–131)

internal

Table 4. EC50 values with corresponding 95% confidence intervals for the effect of cadmium (tested as chloride) on the reproduction of Folsomia candida after 4 and 6 weeks of exposure in four different soils. EC50 values for pore water are given in µg/ml, and for internal in µg/g dry body weight; all other EC50 values are expressed on a µg/g dry soil basis

soil

time (weeks)

Table 3. EC50 values with corresponding 95 % confidence intervals for the effect of cadmium (tested as chloride) on the growth (fresh weight) of Folsomia candida after 2, 4 and 6 weeks of exposure in four different soils. EC50 values for pore water are given in µg/ml, and for internal in µg/g dry body weight; all other EC50 values are expressed on a µg/g dry soil basis

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Fig. 2. Effect of cadmium on the reproduction of Folsomia candida after 4 (left) and 6 (right) weeks of exposure in four different soils. Each data point represents the total number of juveniles produced in one replicate test container. Lines are dose-response relationships fitted using the logistic model according to Haanstra et al. (1985)

the 0.01 M CaCl2 exchangeable fractions omission of control values had little effect on Kf and r2 values, but also led to higher 1/n values. Differences in Kf values for the four test soils were never larger than a factor of two, except for the water-soluble fractions after 6 weeks in KOOY and BUDEL soils which showed some erratic values. Sorption constants were slightly but not significantly higher after 6 weeks. Survival of the Collembola was fairly low and ranged between 56 and 90 % after 2 weeks of exposure and between 17 and 74 % in the controls after 4 weeks. Highest control survival was recorded in LUFA 2.2. and OECD soils, lowest in BUDEL and KOOY soils. Since survival sometimes was (much) higher at low cadmium concentrations and did not show a consistent dose-related pattern, no LC50 values were calculated. Reasons for the low control survival remain unclear, especially since the animals showed normal growth and good reproduction (see below). After 4 weeks, the number of juveniles produced showed a significant linear increase (r2=0.432; p<0.01) with increasing adult survival, but after 6 weeks no such relationship was found. Because of the low and variable survival after 4 weeks, no attempts were made to quantify survival after 6 weeks of exposure. Fig. 1 shows the effects of cadmium on the fresh weight of the surviving Collembola after 4 and 6 weeks of exposure. EC50 values for the effect of cadmium on fresh weight and dry weight were lowest after 2 weeks, but did not differ much after 4 and 6 weeks. In addition, EC50 values for the effects on dry weight generally were similar to the ones for the effect Pedobiologia (2003) 47, 387–395

on fresh weight. In the Table 3 therefore only EC50 values are given for the effect of cadmium on the fresh weight of Folsomia candida after 2, 4 and 6 weeks of exposure in the four test soils. Corresponding EC10 values for the effect of cadmium on fresh weight of Folsomia candida ranged between 12.1 and 161 µg Cd/g dry soil. OECD soil had the highest EC10 value after 4 weeks, but the lowest one after 6 weeks. After 4 and 6 weeks of exposure, EC50 and EC10 values for the effect on fresh weight differed significantly between the different soils (χ23 > 11.3; p<0.01). This was the case for all EC50s, expressed on the basis of total, water-soluble or 0.01 M CaCl2-exchangeable soil concentrations, porewater concentrations or internal concentrations in the surviving animals. Only in case of the 4week EC50s expressed on a water-soluble concentration basis and the 6-week EC10s on the basis of porewater concentrations, difference between the soils was not significant (χ23 < 7.81; n.s.). Similar observations were recorded for ECx values for the effect on dry weight. In many cases, EC50 values on the basis of total soil concentrations tended to be highest in the OECD soil and lowest in LUFA2.2 soil. When expressed on the basis of extractable fractions or internal concentrations, the opposite was seen in some cases. Results were therefore not very consistent. Internal EC50 values for the effect on fresh and dry weight ranged between 50 and 291 µg Cd/g dry body weight, while corresponding EC10 values ranged between 7 and 183 µg Cd/g dry body weight. Fig. 2 shows the effect of cadmium on the reproduction of Folsomia candida after 4 and 6 weeks exposure

Cadmium toxicity to Folsomia candida

in the four test soils. On average, reproduction was high, with mean numbers of juveniles ranging between 400 and 1200 per test container after 4 weeks. EC50 values in OECD soil tended to be lower after 6 weeks of exposure, but in the other soils no time-related change in toxicity was observed (Table 4). In most cases, EC50 and EC10 values for the effect of cadmium on reproduction did not significantly (χ23 < 7.81; n.s.) differ between the test soils. Only exception were EC50s after 4 and 6 weeks of exposure based on internal concentrations and 4-week EC50s expressed on the basis of total and CaCl2-exchangeable concentrations in soil. EC50 and EC10 values for the effect on reproduction were lower than the ones for the effect on growth. Internal EC50s ranged between 25 and 142 µg Cd/g dry body weight, and corresponding EC10 values between 18 and 85 µg Cd/g dry body weight.

Discussion The decrease of soil pH with increasing cadmium concentrations (Table 1) may be explained by the excess Cd2+ ions, added to the soils at high concentrations, causing a release of H+ ions from the sorption sites on the soils. In general, difference in soil pH between the four soils was small and no larger than 0.5 pH units. Lowest pH was measured in KOOY and LUFA 2.2 soils, highest in OECD and BUDEL soils. The difference between porewater and water-soluble cadmium concentrations observed for the KOOY, BUDEL and LUFA 2.2 soils, when expressed as µg/ml, may be due to dilution of the pore water by extraction of the soil with excess water. The ratio water-to-soil used was 5:1, which at soil moisture contents (w/w) of 11.0–18.2 % would lead to a dilution of the pore water by a factor of 27–45 for the three soils. This means that concentrations in porewater are still a factor of 1.5–2.0 higher than in the water-soluble fractions. This may also be concluded when expressing water-soluble and porewater concentrations as µg/g dry soil, which reduced difference to a factor of 1.71±1.73 (range between 0.28 and 3.51, with outliers in KOOY of 5.40 and 5.90). Since pore water was 0.45 µm filtered while water-soluble fractions were only filtered over a paper filter, higher instead of lower concentrations in watersoluble fractions would have been expected because 0.45 µm filtration is expected to remove cadmium complexed to dissolved organic matter (DOM) fractions. Maybe the presence of chloride, resulting in the formation of water-soluble chloride complexes (Wolt 1994), has played a role. When chloride concentration in pore water is 40 times higher than in the water-soluble fractions, more cadmium may be complexed with

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chloride and less bound to organic matter. Cadmiumchloride complexes may pass the 0.45 µm filter more easily than cadmium-DOM complexes, which may have resulted in higher cadmium concentrations than expected in the pore water. Kf values hardly increased with time (Table 2), suggesting there was little effect of ageing on cadmium sorption. This is in agreement with Lock (2002), who, on the basis of a literature review, concluded that for a large atom like cadmium ageing has little effect on the binding strength to soil particles. When related to the water-soluble fractions, Kf values are highest in the OECD soil which corresponds with the high cation-exchange capacity (CEC). The lower Kf values measured for the other soils may either be attributed to the lower CEC (KOOY, BUDEL) and/or the lower pH (LUFA 2.2). Kf values values found in this study are in agreement with values for similar soils reported in the literature (Elliott et al. 1986; Anderson & Christensen 1988; Buchter et al. 1989; Lo et al. 1992). The low survival in our tests was unexpected and cannot be explained, especially because the animals showed normal growth (Fig. 1) and a high reproduction (Fig. 2). Reproduction was highest in LUFA 2.2 soil, but did also in the other soils meet the requirement of > 100 juveniles per test container set by ISO (1999). In all soils reproduction showed a large variation between replicate test containers. This large variation may partly be explained from the correlation between reproduction and adult survival found after 4 weeks of incubation. It does, however not explain the large variation in reproduction after 6 weeks. Such a large variation was also seen in earlier studies in our laboratory (Van Gestel & Hensbergen 1997; Van Gestel & Van Diepen 1997), also using well-synchronised populations of test organisms. It is therefore recommended to use at least five but preferably more test containers per treatment and control when testing Folsomia candida in natural soils. Cadmium toxicity in this study was comparable with that observed in earlier studies in our laboratory (Van Gestel & Hensbergen 1997; Van Gestel & Van Diepen 1997), but higher than that recorded by Crommentuijn et al. (1993, 1997), Sandifer & Hopkin (1996, 1997), Crouau et al. (1999) and Lock & Janssen (2001), using the same OECD artificial soil or natural soils. This was apparent for the effect on growth as well as reproduction. Similar conclusions could be drawn when comparing EC50 values based on watersoluble concentrations in soil or internal concentrations in surviving animals with literature data. Only little difference in toxicity between soils was observed and patterns were not consistent in time. Where the OECD artificial soil had the highest organic matter and clay contents and the highest CEC, lowest Pedobiologia (2003) 47, 387–395

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toxicity would have been expected in this soil. After 4 weeks of exposure this seemed indeed to be the case (Tables 3 and 4), but at other time intervals toxicity was lower in other soils. Soil pH may have played a role; although difference in soil pH was small and usually less than 0.5 pH units. Such a difference may still lead to a factor of 3.2 difference in the H+ concentration in the soil solution. Such a difference in H+ concentration is expected to have an impact on cadmium toxicity. Toxicity based on water-soluble concentrations indeed was highest at high soil pH (Tables 3 and 4), confirming that such a pH effect indeed may have played a role in this study. From the results of this study, it may be concluded that differences in cadmium sorption and toxicity in the four soils tested were small, and corresponded with the small differences in soil properties. The absence of a consistent relationship between cadmium toxicity and soil properties suggests that differences of less than a factor of 3-4 in organic matter and clay content, in soils with the same pH, do not lead to significant differences in cadmium toxicity to Collembola. When considering reproduction as the endpoint, toxicity only slightly increased with increasing test duration, whereas for growth no such time effect on toxicity could be seen.

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