Bioaccumulation of Metals in Earthworms

Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By Shuo Yu, M.S. Environmental Science Graduate Program

The Ohio State University

2009

Dissertation Committee: Roman P. Lanno, Advisor Nicholas T. Basta Susan W. Fisher Timothy J. Buckley

Copyright by Shuo Yu 2009

Abstract

Contamination of soil with metals poses a risk to ecosystem health, particularly to organisms that are in direct contact with the soil (i.e., plants, soil invertebrates). Earthworms are often used as test organisms in laboratory and field studies in order to estimate metal risk in contaminated soils. Bioaccumulation is a direct biological measure of metal bioavailability since it measures the actual amount of metal taken up by the earthworm and integrates all of the effects of biotic (e.g., earthworm behavior) and abiotic (e.g., soil pH) modifying factors over the duration of the exposure period. This dissertation used earthworms as a model to examine the effects of various factors on bioaccumulation of cadmium (Cd) in soil. Chemical bioaccumulation factors (BAF) are considered a simple tool to estimate the bioaccumulation of contaminants in a particular organism in a specific medium. However, there can be considerable uncertainty associated with the application of BAF to metals in soil systems. In this study, data from a comprehensive review of studies reporting bioaccumulation of Cd by earthworms was used to perform statistical analyses on factors that affected variation in Cd BAF. Seventy-five papers from 1973 to 2006 with 727 observations from both lab and field studies were reviewed. Data on earthworm

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species, deputation time, earthworm tissue Cd, and soil properties including pH, organic matter content, clay content, cation exchange capacity (CEC), soil texture, and total soil Ca and Cd, were included in our data set. Due to inconsistent reporting of chemical/physical soil properties, statistical analyses of the more commonly reported variables (i.e., species, depuration time, and experiment type [field vs. laboratory]) were first performed using a generalized linear model (GLM). Because earthworm species was the only significant factor among the commonly reported variables, effects of soil properties on Cd BAF were subsequently evaluated on a subset of the data set using multiple linear regressions of residual values from the GLM. The two most significant parameters affecting mean Cd BAF in earthworms were earthworm species and soil matrix properties. Soil clay content, soil pH, and organic matter content independently comprised 61.3%, 6.0%, and 0.4% of the soil property effect on Cd BAF, respectively. Covariance among the three soil property parameters accounted for the remaining 32.3% of the soil matrix effect. Acclimation of earthworms to cadmium (Cd) levels exceeding background concentrations may result in the development of Cd-tolerance mechanisms and allow the accumulation of Cd with minimum adverse effects. In this study, earthworms (Eisenia andrei) were acclimated by exposure to 20 mg/kg Cd in Webster soil for 28 days. A 224day bioaccumulation test was subsequently conducted with both acclimated and unacclimated worms exposed in Webster soils spiked with 20 mg/kg and 100 mg/kg Cd. Uptake kinetics and subcellular compartmentalization of Cd were examined. Results

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suggest that acclimated earthworms accumulated more Cd and required longer time to reach steady state than unacclimated worms. Most of the Cd was present in the metallothionein (MT) fraction. Cadmium in the MT fraction increased approximately linearly with time, and required a relatively longer time to reach steady state, while Cd in the cell debris and granule fractions quickly reached steady state. Cadmium in the cell debris fraction is considered potentially toxic, but low steady state concentrations observed in this study suggested no adverse effects occurred. Future bioaccumulation tests should report earthworm species and use standardized methods to measure critical soil properties (i.e., clay content, pH, organic matter) in order to produce reliable results and make comparison with other studies. Also, use of earthworms in ecological risk assessment should take into consideration pre-exposure histories of the test organisms, and a prolonged test period may be required for a comprehensive understanding of uptake kinetics and compartmentalization. Laboratory toxicity and bioaccumulation models along with measurements of key soil properties could be used as a screening tool to evaluate metal bioavailability in the baseline ecological risk assessment.

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Dedication

Dedicated to my parents Lingbo Yu Zhitao Zhang

Acknowledgments

First, I would like to express my sincere appreciation to Dr. Roman P. Lanno, my academic advisor, for his encouragement, advice, and support in every aspect of my three-year experience in his lab. I would like to thank Dr. Nicholas T. Basta, Dr. Susan W. Fisher, and Dr. Timothy J. Buckley for serving as my committee members. I also would like to thank Richard H. Anderson from Office of Research and Development of U.S. Environmental Protection Agency (EPA) for helping me conduct statistical analysis of Cd BAF database. I am sincerely thankful to Anthony D. Lutton, Research Associate from Microscopic and chemical Analysis Research Center for his guidance of metal analysis. I would like to express a special thank to Chris Hurdzan for his work in maintenance of earthworm culture. I a, also thankful to my lab mates and my fellow graduate students and staff members in Department of Entomology and in Environmental Science Graduate Progam for their suggestions and friendship all throughout. Finally, I am deeply thankful to my parents and my grandparents in China who always give me support, encouragement and best wishes.

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Vita

January 1980 .............................Changchun, Jilin, Peoples Republic of China 2003............................................B.S. Environmental Science, Beijing University 2006.............................................M.S. Environmental Science, The Ohio State University 2006 to present .......................Ph. D. student, Environmental Science Graduate Program, The Ohio State University

Fields of Study

Major Field: Environmental Science

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Table of Contents

Abstract ......................................................................................................................... ii Dedication ......................................................................................................... v Acknowledgments ............................................................................................................ vi Vita .............................................................................................................................. vii List of Tables ................................................................................................................. x List of Figures ............................................................................................................... xi

Chapters 1. Introduction ................................................................................ 1 2. Factors affecting the bioaccumulation factor (BAF) for cadmium by earthworms: A literature review .......................................... 6 3. Uptake kinetics and subcellular compartmentalization of cadmium in acclimated and unacclimated earthworms (Eisenia andrei) ... 34 4. Conclusion ... 55

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References ................................................................................................................... 58 Appendix A. Earthworm cadmium bioaccumulation database ....... 68 Appendix B. References of studies included in earthworm cadmium bioaccumulation database . 153 Appendix C. Equations used for normalizing pH values measured using different soil: solution ratios .... 161

ix

List of Tables

Table 2.1. Summary of soil properties in Cd BAF data set 19 Table 2.2 Parameter distribution . 19 Table 2.3. Soil property correlation matrix with Pearson correlation coefficients ......... 24 Table 3.1 Total Cd and CaCl2 extractable-Cd concentrations in three test soils ..... 42 Table 3.2. Estimated half-lives for Cd uptake and steady-state Cd concentrations of the whole organism and three subcellular fractions. The values in brackets are 95% confidence intervals ....... 46 Table 3.3. BAF values of unacclimated and acclimated earthworms exposed in 20 and 100 mg/kg of Cd . 52

List of Figures

Figure 1.1. Schematic model of bioavailability ............................................................... 2 Figure 2.1. Effect of depuration time on BAF ........ 13 Figure 2.2. Coefficient of variation in BAF at three depuration time intervals .............. 13 Figure 2.3. Effect of experiment type (laboratory vs field) on BAF ........................... 14 Figure 2.4. Effect of earthworm species on BAF ........................................................ 15 Figure 2.5. Effect of earthworm species on BAF, excluding An. icterica and P. lauta ... 16 Figure 2.6. Effect of species and experiment type interaction on BAF ...... 17 Figure 2.7. Effect of species and experiment type interaction on BAF, excluding Ap. tuberculata ..... 18 Figure 2.8. Effect of soil pH on BAF ...... 20 Figure 2.9. Effect of soil organic matter content on BAF ... 21 Figure 2.10. Effect of soil clay content on BAF ...... 21 Figure 2.11. Effect of soil pH on BAF, excluding outliers .. 22 Figure 2.12. Effect of soil organic matter content on BAF, excluding outliers ... 23 Figure 2.13. Effect of soil clay content on BAF, excluding outliers ....... 23

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Figure 2.14. Effect of GM on BAF, excluding outliers....... 25 Figure 2.15. Relative soil property importance in BAF model ... 26 Figure 2.16. Raw residuals from the regression of BAF on GM 27 Figure 2.17. Raw residuals from the regression of BAF on GM after adjusting BAF values for the Species effect using a generalized linear model .... 28 Figure 3.1. Time course distribution profiles for the depuration of Cd in subcellular fractions and whole organism in acclimated earthworms in control soil .......... 44 Figure 3.2. Time course distribution profiles for Cd accumulation in subcellular fractions and whole organism in unacclimated earthworms in soil spiked with 20 mg/kg of Cd .,.. 44 Figure 3.3. Time course distribution profiles for Cd accumulation in subcellular fractions and whole organism in acclimated earthworms in soil spiked with 20 mg/kg of Cd .... 45 Figure 3.4. Time course distribution profiles for Cd accumulation in subcellular fractions and whole organism in unacclimated earthworms in soil spiked with 100 mg/kg of Cd .......... 45 Figure 3.5. Time course distribution profiles for Cd accumulation in subcellular fractions and whole organism in acclimated earthworms in soil spiked with 100 mg/kg of Cd .. 46

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Chapter 1: Introduction

Contamination of soil with metals poses a risk to ecosystem health, particularly to organisms that are in direct contact with the soil (i.e., plants, soil invertebrates). Earthworms are often used as test organisms in laboratory and field studies in order to estimate ecological risk in metal-contaminated soils. One of the biggest problems in earthworm toxicity and bioaccumulation bioassays is determining the actual exposure concentration of metal to earthworms. Most risk assessments of metals in soil use total metal levels as exposure concentrations, but actually during the whole process of metal uptake, metals interact with soil constituents, reducing bioavailability, and are distributed and detoxified among different fractions within the earthworm. Therefore, only a small portion of absorbed metal is available for causing biological effects. Metal bioaccumulation and toxicity are often poorly related to total metal content, and the calculated risk based upon total metal content may overestimate the true risk of exposure to metal contaminated soils. The concept of bioavailability is very important in metal risk assessment. Its broad definition is a measure of the potential of a chemical for entry into biological receptors. It is specific to the receptor, the route of entry, time of exposure, and the soil matrix

containing the contaminant (Anderson et al., 1999). Within this broad definition, bioavailability can be defined and measured more specifically according to Figure 1.1 (adapted from Lanno, 2003), a concept diagram of bioavailability within the whole process of chemical uptake by earthworm.

Figure 1.1. Schematic model of bioavailability.

First, chemicals present in soil interact with specific soil constituents in a dynamic manner over time, resulting in the sequestration of a portion of the chemicals by soil

constituents, making them unavailable for interaction with biological receptors (Adriano 2001; Basta et al. 2005; Peijnenburg and Jager 2003). For example, soil pH has the potential to modify metal solubility by controlling metal dissolution/precipitation, and it also regulates the ionization of pH-dependent ion-exchange sites on organic matter (OM) and metal oxide clay minerals. Soil chemical reactions between clay mineral or OM surfaces and metal are attributed primarily to adsorption of metal to cation-exchange sites or strong specific adsorption of metals to Fe and Al oxides or OM. The portion of total chemical in the soil that is not sequestered by soil constituents may be referred to as the environmentally available fraction or bioaccessible fraction. This fraction of chemical remains available for all fate and transport processes, including interaction with earthworms. During movements through the soil, earthworms encounter and interact with only a specific portion of the environmentally available chemical. Interaction may be through either direct dermal contact with chemicals in soil solution or ingestion of soil, and the fraction of the environmentally available chemical that the earthworm interacts with is termed the bioavailable fraction. It is dependent on the physiology and behavior of the earthworm as well as the route of exposure (Lanno et al., 2004). As an example, only the chemical dissolved in the soil solution is thought to be bioavailable to the earthworm for dermal uptake (Belfroid et al., 1996). However, for metals taken up by soil ingestion, the bioavailable fraction is subjected to the chemical conditions present in the gastrointestinal tract of earthworm (e.g., gut pH at 7.0). The bioavailable fraction of the chemical may be

absorbed across the external membranes of the earthworm (e.g., epidermis, gastrointestinal tract). After uptake, some portion of metals may be excreted, while other portions may be distributed and accumulated in different subcellular compartments within the body of earthworm. Detoxification systems, including metallothionein (MT) and granules, could detoxify and sequester a certain amount of metals, and isolate them from interacting with site(s) of toxic action. The portion of metals that is not sequestered into MT and granule fractions, but reaches and interacts with sites of toxic action is termed the toxicologically bioavailable fraction (Lanno et al., 1998, 1999), the fraction of absorbed metals that is actually related to toxicity. There are several methods to measure metal exposure. Actually, different methods provide different measures of bioavailability (Figure 1.1). Chemical extraction using weak salt solutions is a measurement of the environmental availability or bioaccessibility of metals, integrating soil matrix factors, but not biological factors. Bioaccumulation is a direct biological measure of metal bioavailability since it measures the actual amount of metal taken up by the earthworm and integrates all of the effects of biotic (e.g., earthworm behavior) and abiotic (e.g., soil pH) modifying factors over the duration of the exposure period. Furthermore, if toxicity is an endpoint, toxicological bioavailability may be measured using a fractionation procedure that isolates the amount of metal at sites of toxic action. A modification of the fractionation procedure used by Wallace et al. (2003) could be

used to separate earthworm metal body burdens into operationally defined subcellular compartments through tissue homogenization followed by differential centrifugation, heat treatment, and chemical digestion (Jones et al. 2009). This procedure is very inexpensive and rapid, and produces a pellet fraction containing granules, a cell debris fraction containing tissue fragments, cuticle, organelles, and gut content, and a supernatant fraction containing cytosol with intact cell organelles (nuclei, mitochondria, etc.) plus dissolved heat-stable proteins (e.g., MT) and heat- labile proteins (e.g., enzymes). Different subcellular compartments have differing biological significance for different metals. This dissertation used earthworms as a model to examine bioaccumulation of cadmium (Cd) in soil. Our objectives were: 1) to conduct meta-analysis of factors that affect variation in Cd bioaccumulation factors (BAF) in earthworms; 2) to examine the differences in Cd uptake kinetics and Cd subcellular compartmentalization between acclimated and unacclimated worms.

Chapter 2: Factors affecting the bioaccumulation factor (BAF) for cadmium by earthworms: A literature review (Submitted to Environmental Toxicology and Chemistry)

2.1 Introduction Metal bioaccumulation can be defined as the net accumulation of a metal in a whole organism or a tissue of interest that results from exposure from all relevant metal sources (e.g. water, food, and sediment). The simplest tools for estimating bioaccumulation include direct measures of tissue levels and the derivation of bioaccumulation factors (BAF). The BAF is the ratio of the metal concentration in an organism to that in a specified medium at steady state. BAFs for metals are not absolute values, but vary widely with the specific metal, the specific organism, the specific exposure circumstance, and the status/age/condition of the specific organism measured. Different metals have different chemical and toxicological properties, and different organisms have different toxicological and physiological behaviors under different exposure circumstances and life stages. All the preceding factors contribute to variation of metal bioaccumulation, as measured by BAFs. Various databases (e.g., see USEPA 2007) summarize BAF values reported in the

literature. It is well recognized that there can be considerable uncertainty associated with the application of BAF to metals risk assessment. Due to the fundamental differences in the physical, chemical, and toxicological properties between inorganic metals and organic chemicals, the principal assumptions and features of the BAF model that make it applicable to neutral organic chemicals also make it inapplicable to inorganic metals (McGeer et al. 2004). For example, for the vast majority of the metal/taxonomic group combinations assessed, the assumptions regarding the independence of BAF with exposure concentration and proportionality of hazard with increasing BAF do not hold true (McGeer et al. 2003). Also, the latest scientific data on bioaccumulation do not currently support the use of BAF data when applied as generic threshold criteria for the hazard potential of inorganic metals (USEPA 2007). Inorganic metals and metal compounds have unique characteristics that should be considered when assessing their risks. Metals are naturally occurring, and are neither created nor destroyed by biological or chemical processes. Some metals, such as copper (Cu) and zinc (Zn) are nutritionally essential elements at low levels but toxic at higher levels, while others metals, such as lead (Pb), cadmium (Cd), and mercury (Hg) have no known biological functions. Many organisms have evolved mechanisms to regulate metal accumulation, especially accumulation of essential metals. The environmental chemistry of metals strongly influences their exposure and effects on human and ecological receptors. It is well known that in soil, physical and chemical interactions between metals and soil constituents could alter metal

bioaccessibility. Several studies (Pierzynski and Schwab 1993; Dayton et al. 2006; Bradham et al. 2006; McLaughlin, 2002; Basta et al., 2005) indicate that metal uptake by organisms living in soil is directly related to the bioaccessible fraction of metal in soil, not the total metal concentration. Therefore, soil properties that affect metal bioaccessibility also affect metal bioaccumulation. Metal bioaccumulation as measured by BAFs is actually a way of measuring metal bioavailability, which is the fraction of metal that can be absorbed across external membranes and enter the body of an organism. It excludes the fraction of metal that is sorbed strongly by soil components and cannot be taken up by organisms. Soil parameters that generally contribute the most to explaining the variability of BAFs are pH, cation exchange capacity (CEC), organic matter content, and clay content. Multiple regression models are available (Janssen et al. 1997; Peijnenburg et al. 1999a, b) that help explain BAF as a function of soil characteristics. In this study, we focused on Cd bioaccumulation because: 1) Cd is a non-essential metal, and there is little uptake regulation by organisms, so physiology-based variability should be minimal, and 2) there was a significant number of papers on Cd bioaccumulation published, providing substantial data with various reported parameters. To our knowledge, no comprehensive literature review on Cd bioaccumulation by earthworms has been published since Sample et al. (1999) who reported a data set of Cd bioaccumulation by earthworms for field studies conducted from 1973 to 1996. Because much additional information has accrued in the literature since 1996, an additional

review is warranted. This chapter was a descriptive literature review with no hypothesis, and the objectives were: 1) To perform a comprehensive review of literature reporting bioaccumulation of Cd by earthworms, and 2) to statistically analyze and explore various physical, chemical, biological, and experimental parameters that affect variation in Cd BAF in earthworms.

2.2 Methods Seventy-five published studies from 1973 to 2006 with 727 observations from both lab and field studies were examined. These studies used either naturally polluted soils or soils spiked with Cd salts, and total soil Cd concentrations ranged from 0.02 mg/kg to 1570 mg/kg. Data concerning earthworm species, soil pH, organic matter content, clay content, CEC, soil texture, depuration time, exposure time, Cd source term, aging time, total soil Ca and Cd concentrations, and earthworm tissue Cd concentrations were recorded whenever possible. BAFs were calculated based upon worm Cd and total soil Cd concentrations. All data were tabulated in an Excel spreadsheet (Microsoft 2000, 9.0.2812) for statistical analyses. The earthworm Cd bioaccumulation database and references of studies included are presented in Appendix A and B. All parameters affecting BAF were divided into two groups: primary and secondary parameters. Primary parameters, such as earthworm species and soil pH were reported by most studies, and were considered applicable to the whole data set (i.e., applicable to both field studies and lab studies). Secondary parameters were reported by

relatively fewer studies, and were only applicable to a specific subset of the entire data set. They were dependent upon a certain primary parameter; for example, exposure time was under the primary parameter called experiment type (i.e., field vs. laboratory) and could only be evaluated for lab studies, but not for the whole data set because there was no exposure time in field studies. Statistical analyses of primary parameters were performed first, and only when the primary parameter resulted in a significant effect on BAF, statistical analyses of the corresponding secondary parameters could be performed (e.g., exposure time effects on lab studies). In this paper, only results of analyses of primary parameters are reported. Due to inconsistent reporting of chemical/physical soil properties, the resulting data set contained little information on soil characterization relative to the other parameters. Evaluating all parameters simultaneously would have resulted in a dramatic reduction of sample size. Therefore, statistical analyses of the more commonly reported experimental factors (i.e., species, depuration time, and experiment type [field vs. laboratory]) and soil property parameters were performed separately. First, main and interactive effects of species, depuration time, and experiment type on BAF were examined using a generalized linear model (GLM) in SAS Version 9.1.3 for windows. Because the data set was so unbalanced, the least-squares mean of BAF values are reported for qualitative parameters. Least-squares mean values are estimated weighted to the respective sample size of that parameter and reflect the perceived mean adjusted for the other parameters in the model (SAS 1999).

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Secondly, the effects of soil properties on BAF were evaluated. Three soil property parameters were included: pH, organic matter content, and clay content. Soil CEC and soil Ca concentration were not included because of insufficient data. Also, CEC is pH dependent and is positively correlated with both clay and organic matter content, so it is accounted for by pH, clay, and organic matter (Haghiri 1974; Martel et al. 1978). Soil Ca may affect metal bioavailability and bioaccumulation through the competition of Ca2+ with other metal cations on CEC sites. Free Ca2+ concentrations are mechanistically regulated by soil chemical reactions, which predominantly involve pH, free CaCO3 content, organic matter, and clay. To utilize as much reported information as possible, soil parameters were normalized to remove study-specific reporting and/or methodological differences. First, the qualitative parameter soil texture was converted to quantitative clay content values using the midpoint value from the USDA Soil Texture Triangle (USDA 1993) for each textural class. Second, all soil pH values were normalized to those reported using 0.01 M CaCl2 as the extractant solution and under a soil: solution ratio of 1:5 by various transformations among different soil pH measurement methods. Studies (e.g., Lathwell and Peech 1964; Okusami et al. 1987) have shown that the pH value is about 0.5 units higher when measured in H2O, and 1 unit lower when measured in 1 M KCl, compared to that measured in CaCl2. The higher the concentration of the extractant, the lower the pH measured. Equations from EPD Analytical Information Sheet MC2 (EPD 2005) were used for normalizing pH values measured using different soil: solution ratios (Appendix

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C). After normalizing all the soil property parameters, their effects on BAF were examined using the REG procedure in SAS Version 9.1.3 for windows by fitting residuals from the previously described GLM model, where only the significant factor(s) were included, to linear regression models with soil properties as model parameters. In all the statistical analyses, we chose the 95% level of significance ( = 0.05).

2.3 Results Depuration time had a significant effect (P=0.0013) on BAF with slight positive slope (Figure 2.1). However, the perceived depuration time effect is an artifact because there is no biological explanation for BAF values increasing with increasing depuration time. They should only decrease, since longer depuration period may remove soilassociated Cd in the gut content of earthworms more completely (Stafford and McGrath, 1986). Only one study used a depuration time of 13 days (Pizl and Josens 1995), while other studies used depuration times of less than 7 days. The artifact is most likely due to the leverage associated with the observations at 13 days on the fitted regression line (Kutner et al. 2004). Another reason was the effect of unequal variances across depuration times. Figure 2.2 shows the coefficient of variation (CV) in BAF values at three depuration time intervals. Notably apparent is that BAF values were more variable at low depuration times (0-2 days), which may be due to the effects of other factors. Beyond a depuration time of 2 days, variability in BAF values reached a minimum suggesting constant Cd BAFs.

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BAF
400 P=0.0013 300 200 100 0 0 2 4 6 8 10 12 14

Depuration Time (day)

Figure 2.1. Effect of depuration time on BAF.

250

Coefficient of Variation in BAF

200 150 100 50 0

0~2

2~4 Depuration Time (day)

>4

Figure 2.2. Coefficient of variation in BAF at three depuration time intervals.

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We found that experiment type did not have a significant (P=0.8072) effect on Cd BAFs. Laboratory studies reported similar BAFs to that from field studies (Figure 2.3).

BAF LSMEAN 50 40 30 20 10 0 field lab

P=0.8072

Figure 2.3. Effect of experiment type (laboratory vs field) on BAF.

A significant (P<0.0001) effect of earthworm species on BAF was found, indicating that of the 21 species in the data set, Pheretima lauta and Anacanthiptera icterica produced significantly higher BAFs than other species (Figure 2.4). However, they are not commonly used species in earthworm bioaccumulation tests, and in our data set, only one observation was found for both An. icterica (Pizl and Josens 1995) and P. lauta (Hsu et al. 2005).

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BAF LSMEAN

250 200 150 100 50 0

Ds. rubidus L. terrestris Es. carolinensis L. rubellus N. caliginosus

P<0.0001

Ap. trapezoides

An. icterica

Ap. tuberculata

Ap. caliginosa

Da. octaedra

Al. chlorotica

Ela. tetraedra

Figure 2.4. Effect of earthworm species on BAF.

After removing the first two species from the data set, a re-analysis of the data showed a significant (P<0.0001) effect of earthworm species on BAF (Figure 2.5). Differences in earthworm species are partly due to their habits in the soil and their food selection (Morgan and Morgan 1992). Endogeic species, such as Aporrectodea rosea, Ap. tuberculata, Ap. turgida, and Ap. caliginosa, rarely come to the soil surface, and spend their lives in the burrow systems they build under the soil. They ingest small particles of mineral soil and actually consume significant volumes of soil. Conversely, epigeic species, such as Dendrobaena veneta, Eisenia veneta, Eia. fetida, Eiseniella tetraedra, and Lumbricus rubellus, live in the top soil, and feed on organic surface debris (Bouche

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Eia. veneta

Da. veneta

P. lauta

Ap. turgida

Eia. andrei

Eia. fetida

Ap. rosea

Ap. longa

1972). Figure 2.5 showed that endogeic earthworms feeding on soil often accumulated more Cd in their tissues than epigeic worms feeding on surface litter, and several studies (Morgan and Morgan 1992, 1993, 1999; Dai et al. 2004; Beyer et al. 1987) have also indicated similar results.

BAF LSMEAN

120 90 60 30 0
N. caliginosus Es. carolinensis Ap. trapezoides Ds. rubidus L. terrestris Ap. turgida L. rubellus Ap. caliginosa Ela. tetraedra Eia. fetida Ap. rosea Ap. longa Eia. veneta Ap. tuberculata Da. octaedra Al. chlorotica Da. veneta Eia. andrei

P<0.0001

Figure 2.5. Effect of earthworm species on BAF, excluding An. icterica and P. lauta.

Of the 21 species in the data set, only eight species were common to both field and laboratory studies. We used these eight species to test the interaction between species and experiment type on BAF. Significant (P<0.0001) interaction was observed where Ap.

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tuberculata lab studies produced relatively higher BAFs (Figure 2.6). Four laboratory observations of Ap. tuberculata occured and were from the same paper (Neuhauser et al. 1995). In this study, earthworms were pre-exposed to Cd in the field before the laboratory study was performed. Removing Ap. tuberculata from the data set resulted in no significant (P=0.8685) interaction (Figure 2.7).

BAF LSMEAN
180

P<0.0001
120

60

0 Ap. caliginosa Al. chlorotica Ap. tuberculata Da. octaedra Da. veneta Eia. fetida Ap. caliginosa Ap. chlorotica Ap. tuberculata Da. octaedra Da. veneta L. rubellus L. terrestris Eia. fetida L. rubellus L. terrestris

field

lab

Figure 2.6. Effect of species and experiment type interaction on BAF.

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BAF LSMEAN
35 30 25 20 15 10 5 0
Ap. Al. Da. Da. veneta Eia. fetida L. rubellus L. Ap. Ap. Da. Da. veneta Eia. fetida L. rubellus L. terrestris lab caliginosa chlorotica octaedra field terrestris caliginosa chlorotica octaedra

P=0.8685

Figure 2.7 Effect of species and experiment type interaction on BAF, excluding Ap. tuberculata.

The effects of soil properties on BAF were examined on a subset of the original data set. Table 2.1 indicates the wide ranges of soil properties in the data set. We also tested the normality of BAF as well as three soil properties, and they were all not normally distributed (Table 2.2). However, Bivariate regressions are robust to variable distributions except when extreme observations influence Least-Square lines (Kutner et al. 2004). Even if distributions of BAF were far from normal and soil property

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parameters had the property of asymptotic normality, their distributions approach normality under very general conditions as the sample size increases. Therefore, with our sufficiently large sample size, linear regression model could still be applied. We recovered the residuals from the previous analysis of species effect, and fitted residuals to linear regression models with soil properties as model parameters. Residuals are simply the magnitude of deviation of observed BAFs from predicted BAFs by the model with species as the only factor. This approach allowed us to examine the effect of soil properties on BAF values in the absence of the confounding effect of species.

Soil property Clay content Normalized pH Organic matter content

Minimum Maximum Total N. of observations 2.00% 2.89 0.30% 70.0% 9.71 40.0% 235 581 432

N. of observations in low region 71 (30.2%) 155 (26.7%) 25 (5.79%)

N. of observations in moderate region 115 (48.9%) 388 (66.8%) 151 (35.0%)

N. of observations in high region 49 (20.9) 38 (6.54%) 256 (59.3%)

Table 2.1. Summary of soil properties in Cd BAF data set.

Three pH regions: <5.5 (low), 5.5-7.5 (moderate), >7.5(high). Three clay content regions: <10% (low), 10%-30% (moderate), >30% (high). Three organic matter content regions: <2% (low), 2%-6% (moderate), >6% (high).

Parameter BAF Clay content Normalized pH Organic matter content

Minimum Maximum Mean 0.02 2.00% 2.89 0.30% 190 70.0% 9.71 40.0% 14.0 19.4% 6.13 10.4%

25% 2.79 10.0% 5.46 4.20

50% 6.66 17.0% 6.18 7.70

75% 17.0 28.1% 7.00 12.4%

P <0.0001 <0.0001 <0.0001 <0.0001

Table 2.2. Parameter distribution.

Student t test was used to examine whether the parameter fits normal distribution. When P<, normal distribution was rejected.

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Soil pH was the only soil property parameter that had a significant effect (P<0.0001) on BAF (Figure 2.8). Organic matter content (P=0.0824) and clay content (P=0.0507) were not significant parameters on BAF (Figure 2.9, 2.10).

BAF Residual 400 300 200 100 0 -100 2 3 4 5 6 7 8 9 10 Normalized Soil pH

Y=-6.81x+43 (P<0.0001)

Figure 2.8. Effect of soil pH on BAF.

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BAF Residual 400 300 200 100 0 -100 Y=-0.15x+2.11 (P=0.0824)

10

20

30

40

50

60

70

80

Soil Organic Matter Content (%)

Figure 2.9. Effect of soil organic matter content on BAF.

BAF Residual 400 300 200 100 0 -100

0 10 20

Y=-0.38x+7.12 (P=0.0507)

Soil Clay Content (%)

30

40

50

60

70

Figure 2.10. Effect of soil clay content on BAF.

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When 16 outliers (detected by Walsh Test, about 2% of the total data) were removed from the analysis, all the three soil property parameters had a significant effect on BAF (Figure 2.11, 2.12, 2.13).

BAF Residual 100 80 60 40 20 0 -20 -40 2 3 4 5 6

Y=-4.38x+28 (P<0.0001)

10

Normalized Soil pH

Figure 2.11. Effect of soil pH on BAF, excluding outliers.

22

BAF Residual 100 80 60 40 20 0 -20 -40

0 10 20

Y=-0.18x+1.47 (P=0.0207)

Soil Organic Matter Content (%)

30

40

Figure 2.12. Effect of soil organic matter content on BAF, excluding outliers.

BAF Residual 100 80 60 40 20 0 -20 -40 0 10 20 30 40 Soil Clay Content (%) 50 60 70

Y=-0.37x+6.99 (P<0.0001)

Figure 2.13. Effect of soil clay content on BAF, excluding outliers.

23

There was significant correlation among the three soil properties (Table 2.3). However, such correlation was just statistical, which may be due to the large size of the data set, since the correlation coefficients were all very small. There was just too much methodological variability, which prevented strong intercorrelation as commonly reported (Mallarino et al. 1996; Mulla and McBratney 2000). Ridge regression (see Anderson and Basta 2009 a, b) also indicated that multicollinearity was not an issue (Variance Inflation Factors (VIF) were all less than 2). Nonetheless, soil pH, organic matter content, and clay content values were collectively indexed into a variable called Growing Medium (GM) to more clearly illustrate the effect of soil matrix composition on Cd BAF. GM was quantified as the first component scores from a principal component analysis (PCA) of the three selected soil properties. The parameter GM was subsequently evaluated against BAF. We found that GM had a significant effect (P=0.0007) on BAF (Figure 2.14).

Soil property Clay content

Normalized pH Organic matter content 0.36 0.28 (P<0.0001) (P<0.0001) Normalized pH 0.36 1.00 0.23 (P<0.0001) (P=0.0013) Organic matter content 0.28 0.23 1.00 (P<0.0001) (P=0.0013) Table 2.3. Soil property correlation matrix with Pearson correlation coefficients.

Clay content 1.00

24

BAF Residual 100 80 60 40 20 0 -20 -40 -3 -2 -1 0 GM

Y=-2.88x-1.38 (P=0.0007)

Figure 2.14. Effect of GM on BAF, excluding outliers.

In order to assess the relative importance of each soil property, soil parameters were standardized (mean=0, standard deviation=1) and reevaluated. The resulting regression model obtained was of the form: Species adjusted BAF = -0.22*StdOM 1.05*StdpH-3.68*StdClay+12.52 (2.1) All the three parameters had negative slopes, indicating that BAF decreased with increases in soil pH, organic matter content, and clay content. The overall model was significant (p=0.0035), but the R2 value was low (0.0544). Consequently, the partial R2 values for each property were even lower (0.0044 for pH, 0.0436 for clay, and 0.00009 for organic matter). Although the above equation was not good for prediction, the relative importance

25

of each soil property on BAF can be assessed. Scale normalized parameter estimates can be evaluated for their relative effects on a response variable in multiple regression models (Kutner et al. 2004). Clay content was the most important soil property affecting BAF since it had the largest parameter estimate (Equation 2.1). In order to present the results more clearly, we plotted the relative percentage of the total model sum of squares each soil property independently accounted for (Figure 2.15). We found that clay content accounted for the largest percentage (61.3%), followed by pH (6.0%), and organic matter (0.4%). Despite the fact that the influence of multicollinearity on parameter estimates was minimal (Anderson and Basta 2009 a, b) there was still some residual covariance that accounted for a substantial portion of the total model sum of squares (32.3%).

70 Model Percentage (%) 60 50 40 30 20 10 0 Clay pH Organic Matter Soil Property Covariance

Figure 2.15. Relative soil property importance in BAF model.

26

We also conducted the residual analysis to validate our results. The residual plots are shown in Figure 2.16 and 2.17. The results validated our overall conclusion that soil properties and earthworm species are the primary contributors to variation in BAF values. Also, ANOVA analysis for homogeneity of variances for residuals among species groups indicated that using residual values illustrating the significant Species effect were valid.

Figure 2.16. Raw residuals from the regression of BAF on GM.

27

Figure 2.17. Raw residuals from the regression of BAF on GM after adjusting BAF values for the Species effect using a generalized linear model.

2.4 Discussion There are several papers that developed Cd bioaccumulation models for earthworms (Sample et al. 1999; Neuhauser et al. 1995; Abdul Rada and Bouche 1995; Janssen et al. 1997), but most studies used data from a limited number of locations with limited parameters within a limited range reported and measured by limited analytical methods. Such limitations would introduce bias into their models and make the models

28

only applicable to specific situations. Sample et al. (1999) provided a relatively broader literature review, which summarized 21 studies with 229 observations worldwide from 1973 to 1996, assembled a larger database of Cd bioaccumulation by earthworms, and developed bioaccumulation models from these data. The maximum total concentration of Cd in soil included in the database was 467 mg/kg, and only field studies in which resident earthworms were collected, were considered. In order to expand this database, our literature review of Cd bioaccumulation by earthworms included lab data and more recent studies (1997-2006). Our database contained a total number of 75 studies with 727 observations worldwide from 1973 to 2006 with a maximum total Cd concentration in soil up to 1570 mg/kg. Sample et al. (1999) examined the effects of soil total Cd, soil pH, and soil Ca on the variation of worm tissue Cd, and did not consider parameters of earthworm species, clay content, and organic matter content. Soil total Cd and soil Ca were found to be significant parameters in Cd bioaccumulation. As to our data set, the preliminary analysis suggested nonlinearity between worm tissue Cd and soil total Cd as determined by a significant (P< 0.0001) quadratic term in a regression model without an intercept (tissue Cd = soil Cd+ (soil Cd)2 ). Total soil Cd effect accounted for 60.6% of the variation in tissue Cd concentration. The remaining 39.4% of the variation was either random of a result of numerous biotic and abiotic factors. Because of nonlinearity between tissue Cd levels and total soil Cd levels, we opted to evaluate BAF values as opposed to tissue Cd values using the total soil Cd concentration as a covariate. Using a linear covariate for a

29

nonlinear effect would have resulted in biased results. Our results indicate that total soil Cd, earthworm species, soil clay content, soil pH, and soil organic matter content are all significant parameters affecting Cd bioaccumulation. The results of our analysis could provide some new insights into Cd bioaccumulation by earthworms and some guidance for the design of laboratory bioassays. First, earthworm species is a factor which must be taken into consideration. The choice between endogeic species and epigeic species could produce significant differences in Cd bioaccumulation which is not related to the Cd contamination in soil. Also, when making comparisons among studies using different species, its effect must be accounted for to evaluate the relative effect of other factors. Second, soil properties are important parameters affecting Cd bioaccumulation, but just a few studies reported comprehensive results of soil analysis. For example, clay content was found to be the soil property that contributed the most to the variation in Cd BAFs, but in our database, only about 235 out of 727 observations had it reported. Soil pH was the property reported most frequently (581 out of 727 observations), but there was too much variability in the way it was measured, using different extractants in different concentrations and different soil:solution ratios. Situations may be the same for other soil properties. For example, soil organic matter content could be measured by either K2Cr2O7 digestion, dry combustion, or loss on ignition (Nelson and Sommers 1996), while the pipette method (Claydon 1989) was commonly used to determine clay content although a FRITSCH Laser Particle Sizer could also be used (Hobbelen et al.

30

2006). We used various transformations to normalize the values of soil properties before testing their effects on BAF, but there was still a substantial portion of covariance (32.3%) in our model. Standardizing the methods of soil property measurement and having them reported in all bioaccumulation studies would be a better way to produce more reliable results, and make the comparison among studies much easier. There was no significant difference in Cd bioaccumulation between lab and field studies, contrary to what we expected. Field worms could develop some mechanisms to accumulate metals to elevated degree without impacts. Also, their tissue burdens were thought to be at steady state with soil concentrations, while for laboratory studies, exposure time was often shorter than that required to reach steady state. That begs the question: Can bioaccumulation test results from laboratory bioassays be used to predict bioaccumulation in field situations? Our data suggest that this was the case for Cd, but due to the variability in the data set, we cannot use our results to make any prediction to a specific situation. Also, Cd has its own unique characteristics. First, Cd uptake kinetics in earthworms tends to be linear over a prolonged period of time and no studies have shown when the steady state could be achieved. Second, Cd is a nonessential metal and there is little regulation of bioaccumulation by earthworms. Therefore, for other metals, especially essential metals whose uptake is regulated by earthworms, we cannot simply extrapolate our results on Cd to other metals. Our study incorporated data from multiple studies in multiple sites with multiple earthworm species and multiple analytical methods used, which produced variability in

31

Cd BAFs. Moreover, there were still some parameters related to Cd bioaccumulation that we did not include into our analysis, such as soil Zn, which has been shown to be negatively correlated with Cd concentration in earthworm tissue (Beyer et al. 1982). Including additional parameters would make the model more complicated and less operational. Actually, it seems impossible to develop a model that could be applied to every situation. Therefore, our goal was not to produce a model for prediction, but to provide a conceptual tool in ecological risk assessment which could be used to guide the design of earthworm bioassays with important biotic and abiotic parameters measured and reported. Thus, our study provides a qualitative and semi-quantitative interpretation of the tendency of Cd bioaccumulation by earthworms, and should serve to facilitate more accurate estimation of Cd bioaccumulation when combined with site-specific data. Our approach could also be extrapolated to other metals when their unique characteristics are taken into consideration.

2.5 Conclusion Results from our study clearly show that earthworm species and soil matrix composition are the two most significant parameters affecting Cd BAF in earthworms. Endogeic species accumulate more Cd than epigeic species due to behavioral differences, and clay content could account for 61.3% of the soil property effect on Cd BAF. Bioaccumulation tests should report earthworm species and use standardized methods to measure critical soil properties (i.e., clay content, pH, organic matter) in order to produce

32

reliable results and make comparison with other studies. Our approach may be extrapolated to other metals, and could be used as a conceptual tool to improve decision making in ecological risk assessment of metal-contaminated sites.

33

Chapter 3: Uptake kinetics and subcellular compartmentalization of cadmium in acclimated and unacclimated earthworms (Eisenia andrei) (Submitted to Environmental Toxicology and Chemistry)

3.1 Introduction Earthworms inhabit various metal contaminated sites throughout the world, and are commonly used to assess biological impacts of metal contaminants in soil. Resident earthworms exposed to elevated background levels of metals may develop metaltolerance mechanisms to accumulate metals with minimum adverse effects, and this phenomenon is called acclimation. A prime example of a metal to which earthworms may acclimate is the non-essential element cadmium (Cd). The world average soil Cd concentration is 0.62 mg/kg, but geological features and a variety of anthropogenic activities can give rise to local Cd concentrations that are elevated by several orders of magnitude (Davies et al. 2003). It has been documented that viable populations of earthworms inhabit contaminated soils with elevated Cd levels (Morgan and Morris 1982; Morgan and Morgan 1988; Bengtsson et al. 1992; Lock and Janssen 2001), some even exceeding the Cd Ecological Soil Screening Level (Eco-SSL) for soil invertebrates (140 mg/kg, USEPA 2005). Thus, some earthworm populations appear to be relatively

34

insensitive to Cd toxicity (Spurgeon et al., 2004). Ma (1982) found differences in internal Cd levels between introduced earthworms from laboratory experiments and resident earthworms from contaminated sites when exposed to the same level of Cd in soil. Evidence of increased Cd tolerance has been found in populations of a number of earthworm species, e.g., Eisenia fetida (Suzuki et al. 1980, Yamamura et al. 1981), Lumbricus rubellus, and Dendrodrilus rubidus (Morgan et al. 1989). Differences in Cd accumulation between acclimated and unacclimated earthworms may be related to uptake kinetics and subcellular compartmentalization. Earthworms show a variety of uptake patterns for trace metals, ranging from uptake curves reaching steady-state for essential metals to linear uptake curves for non-essential metals (Marinussen et al. 1997, Peijnenburg et al. 1999, Spurgeon and Hopkin 1999). Several studies have indicated that earthworms tend to accumulate Cd over long time periods (Van Gestel et al. 1993, Honeycutt et al. 1995, Neuhauser et al. 1995, Peramaki et al. 1992, Sheppard et al. 1997, Spurgeon and Hopkin 1999), and steady state conditions were not reached during the exposure period (usually less than 100 days) in these laboratory bioaccumulation tests. However, in field studies, steady-state assumptions are usually made. Therefore, when conducting ecological risk assessment of Cdcontaminated soils, two questions need to be answered: 1) Is there a steady state for Cd uptake over a prolonged exposure period? 2) How long should the duration of a Cd bioaccumulation test be? Metals are detoxified and sequestered into subcellular compartments after uptake

35

by earthworms (Morgan et al. 1999; Rainbow 2002). Three major detoxification mechanisms have been described: 1) excretion from the earthworms; 2) storage in the earthworms within the inorganic matrix of granules and 3) binding to special proteins (e.g., metallothioneins (MT)) and other ligands (Wallace and Lopez 1997; Vijver et al. 2004; Tessier et al. 1994). When a significant portion of the total body burden has been acted upon by any of these three mechanisms, the amount of metal that is distributed to tissues at sites of toxic action and can render a toxic response is diminished. Different metals elicit different mechanisms of detoxification and subcellular compartmentalization in earthworms (Morgan and Morgan 1990), resulting in different patterns of accumulation (Morgan et al. 2002). For Cd, a non-essential metal, excretion is slow or absent as revealed by depuration experiments (Sheppard et al. 1997, Spurgeon 1997). Morgan et al. (1995) also indicated that Cd is detoxified by storage rather than excretion. Cadmium exposure can induce the production of cysteine-rich metalloproteins called metallothioneins (MT) (Morgan et al. 1989, Bengtsson et al. 1992, Melancon et al. 1992, Dallinger 1993, Reinecke et al. 1999, Spurgeon and Hopkin 1999). Cadmium can also be stored in a distinct subtype of sulfur-rich granule termed cadmosomes (Morgan et al. 1989, 1995, 2002). This suggests that Cd accumulation may be a continuous process over the lifetime of an organism. Thus, an understanding of Cd uptake kinetics over a long time course is necessary to predict Cd bioaccumulation. In this study, earthworms (Eisenia andrei) were acclimated by exposure to 20 mg/kg Cd in Webster soil for 28 days. A 224-day bioaccumulation test was subsequently

36

conducted with both acclimated and unacclimated worms under three different Cd exposure concentrations. There were two hypotheses in this study: 1) Even though literature data suggest that Cd uptake by earthworms is linear with time, if earthworms are exposure to soil Cd for a prolonged period of time (e.g. more than 200 days), a steady state concentration can be reached or modeled, and 2) Since a greater induction of MT occurs in Cd-acclimated earthworms, more binding sites for Cd are present. Therefore, Cd uptake in acclimated earthworms should take a longer time to reach a steady state and the steady state concentration should be higher than in unacclimated earthworms. The objectives of this study were: 1) To examine Cd uptake kinetics over a prolonged exposure period; 2) To evaluate subcellular compartmentalization of Cd within the earthworm over this time period, and 3) To compare differences in Cd uptake kinetics and subcellular compartmentalization between acclimated and unacclimated earthworms under different exposure concentrations.

3.2 Methods 3.2.1 Earthworm acclimation Six kilograms of Webster soil (Ames, IA; 2.4% OC, 35.6% clay, pH 5.5) was spiked with Cd(NO3)2 (Fisher Scientific, Pittsburgh, PA) solution to achieve a soil Cd concentration of 20 mg/kg and left overnight for equilibration at 202 C. The acclimation substrate was further amended with 3 kg of separated dairy solids (SDS; Ohio Agricultural Research and Development Center, Wooster, OH). Approximately 650

37

adult E. andrei obtained from a lab culture maintained in SDS, were exposed to the acclimation substrate for 28 days. During the acclimation period, moisture content was monitored and SDS was provided weekly.

3.2.2 Test soils Test soils were prepared by spiking aliquots of Webster soil with Cd(NO3)2 to achieve total soil Cd concentrations of 20 and 100 mg/kg. Unamended Webster soil was used as a control. All the test soils were hydrated with deionized water to achieve a moisture content of 50% of the Webster water-holding capacity (48%) and equilibrated over night at 202 C.

3.2.3 Bioassays A 224-day bioaccumulation test was conducted in test soils with both acclimated and unacclimated worms according to procedures for bioaccumulation tests provided by Environment Canada (2004). Ten worms were placed on the surface of the soil (200 g dry weight) in each test chamber (glass mason jars; 500 mL; Ball, Muncie, IN), and test chambers were sealed with a perforated metal lid (one hole, ~2.0 mm, to allow gas exchange) and screw collar. During the tests, all the test chambers were maintained under continuous fluorescent lighting at 202 C. Worms were fed with 25 g of SDS once a week. Both acclimated and unacclimated worms were sampled from four replicates of each exposure concentration on Day 0, 14, 28, 56, 112, and 224. Harvested worms were

38

placed on moistened filter paper for 24 h to void their gut contents and frozen at -70 C in Nalgene bottles (Fisher Scientific, Pittsburgh, PA).

3.2.4 Tissue fractionation and separation into subcellular compartments Subcellular fractionation of whole earthworms was conducted according to Jones et al. (2009) and Vijver et al. (2006). Five earthworms from each replicate were thawed, weighed, and homogenized (Gruber et al. 2000) in 3 mL of cold Tris/HCl buffer (Fisher Scientific, Pittsburgh, PA). Then, the homogenate was centrifuged at 1450 g for 15 min at 4 C, and the supernatant was decanted and termed as the protein fraction. Giguere et al. (2006) found that MT is the major ligand in this faction. The resulting pellet was digested in 1 mL of 1 N NaOH (Fisher Scientific, Pittsburgh, PA) in a hot water bath at 70 C for 1 h (Silverman et al. 1983), and then further centrifuged at 5000 g for 10 min at 4 C, yielding a final pellet fraction containing granules and a resulting supernatant defined as a cell debris fraction containing tissue fragments (Wallace et al. 2003). All fractions were brought to 5 mL in volumetric flasks with deionized water for metal analysis.

3.2.5 Soil extraction Test soils on Day 0 and Day 224 were extracted by mechanically shaking a mixture of 20 mL of 0.01 M CaCl2 (Fisher Scientific, Pittsburgh, PA) solution and 10 g of airdried test soil for 2 h. Mixtures were centrifuged for 10 min at 1300 g, and filtered (0.22 m) according to Lebourg et al. (1998). To avoid precipitation, the soil extract were

39

added with 2.5 mL of trace metal-grade concentrated HNO3 (Fisher Scientific, Pittsburgh, PA) and diluted to 50 mL in volumetric flasks with deionized water. Extracts were analyzed directly for Cd content by inductively coupled plasma mass spectroscopy (ICPMS; Elan 6000; Perkin Elmer Sciex., Woodbridge, ON Canada), and reported based upon the dry weight of soil.

3.2.6 Tissue and soil Cd analysis Each earthworm fraction sample was mixed with 5 mL of 50% (v/v) concentrated trace metal-grade HNO3 (Fisher Scientific, Pittsburgh, PA), and digested in a closed Teflon bottle in a microwave oven (Ethos 320; Milestone Inc., Monroe, CT) at 180 C for 10 minutes. After cooling at ambient temperature, the solution was diluted to 50 mL with deionized water in a volumetric flask. Cadmium concentrations were determined by ICPMS (Elan 6000; Perkin Elmer Sciex., Woodbridge, ON Canada). Cadmium concentrations in the different fractions were calculated based upon the dry weight of earthworms assuming 84% water content (USEPA 1993). The whole organism Cd burden was estimated by summing Cd concentrations in the three subcellular fractions. Total soil Cd concentrations were measured to confirm spike concentrations. First, 0.5 g of test soil was oven-dried at 105 C for 2 h, weighed, mixed with 10 mL of concentrated trace metal-grade HNO3 (Fisher Scientific, Pittsburgh, PA), and digested in a closed Teflon bottle in a microwave oven (Ethos 320; Milestone Inc., Monroe, CT) at 180 C for 10 minutes. After cooling at ambient temperature, the solution was diluted to

40

50 mL with deionized water, and then any residual soil was removed by filtration (0.22 m). Cadmium concentrations in the digests were determined with ICP-MS (Elan 6000; Perkin Elmer Sciex., Woodbridge, ON Canada), and reported based upon the dry weight of soil.

3.2.7 Data analysis A one-compartment model was used to analyze the dynamic change of Cd in worms over time, following the generalized formula: dCw/dt=k1*Cs-k2*Cw (3.1)

where t is exposure time (days), CW is Cd content in the whole organism or in a subcellular fraction (mg/kg dry weight), Cs is Cd concentration in soil (mg/kg), k1 is the uptake rate constant (day-1), and k2 is the elimination rate constant (day-1). The general solution of Eq. (3.1) is: Cw(t)= Cw(0)*e-k t+k1*Cs/ k2(1- e-k t)
2 2

(3.2)

where CW(0) is the initial Cd content in the whole body or subcellular fraction of worms (mg/kg dry weight). When the elimination rate constant (k2) equals zero, Eq. (3.2) becomes a linear regression: Cw(t)= Cw(0)+k1*Cs*t (3.3)

While in an elimination test where the uptake rate constant equals zero, Eq. (3.2) is converted into the following regression: Cw(t)= Cw(0)*e-k t
2

(3.4)

41

3.3 Results Acclimated and unacclimated earthworms were exposed to three different Cd concentrations: control (unamended), 20 mg/kg, and 100 mg/kg. Total Cd and CaCl2 extractable-Cd concentrations are shown in Table 3.1. CaCl2 extractable-Cd concentrations did not change significantly over the 224-day exposure period with earthworms present in the soils. Worms were sampled at six exposure times, and the Cd content of the three different subcellular fractions, as well as the whole organism, were reported. No significant Cd accumulation (P=0.0830) occurred over the 224 days in unacclimated earthworms exposed in control soil. Average total tissue Cd concentration was 3.78 (0.89) mg/kg, about 69% of which was distributed to the MT fraction.

Soil

CaCl2 extractable-Cd (mg/kg) Day 0 Day 224 Control soil 0.500.08 0.080.01 0.070.02 Soil spiked with 20 mg/kg of Cd 22.12.36 1.920.16 1.780.22 Soil spiked with 100 mg/kg of Cd 10410.7 6.660.40 6.100.45 Table 3.1. Total Cd and CaCl2 extractable-Cd concentrations in three test soils (meanSD).

Total Cd (mg/kg)

Acclimated worms harvested from control soil can be regarded as an elimination test (Figure 3.1). The Cd content in the three subcellular fractions, as well as in the whole organism, were significantly (P=0.0385) higher than in unacclimated earthworms on Day 0 due to the 28-day acclimation and decreased with exposure time. After 224 days of

42

elimination, the Cd level in the whole organism (23.4 mg/kg) still had not returned to the initial level (3.78 mg/kg) before acclimation, and the residual amount of unexcreted Cd was about 51% of the Cd content on Day 0 (46.0 mg/kg). Eq. (3.4) was used to describe the elimination of Cd, and all the elimination rate constants (k2) were significant but very small with the lowest in the MT fraction, indicating that elimination is a very slow process. According to the regression equations shown in Figure 1, the half-life (t1/2) for whole organism Cd elimination was 239 days. The estimated time required for whole organism Cd content to decrease to initial level before acclimation (3.78 mg/kg) was 833 days (about 2.28 years), which which is less than the documented life span for E. andrei (4.25 years). Time course distribution profiles for Cd in subcellular fractions and whole organism in unacclimated and acclimated earthworms in soil spiked with 20 mg/kg or 100 mg/kg of Cd are shown in Figures 3.2-3.5. Cadmium content of the whole organism as well as the different subcellular fractions increased significantly (P<0.0001) with exposure time, and could be described by one-compartment model (Eq. (3.2)). Half-lives (t1/2) for Cd uptake and steady-state Cd concentrations (Css) of the whole organism as well as three subcellular fractions were calculated (Table 3.2) according to regression equations shown in Figures 3.2-3.5.

43

Worm Cd concentration (mg/kg)

60 50 40 30 20 10 0 0

MT cell debris granule total y = 42.315e-0.0029x (R2 = 0.9017, P=0.0037)

y = 31.526e-0.0026x (R2 = 0.9359, P=0.0016) y = 6.3322e-0.0031x (R2 = 0.7413, P=0.0277) y = 4.4021e-0.005x (R2 = 0.8167, P=0.0135)

56

Exposure time (day)

112

168

224

Figure 3.1. Time course distribution profiles for the depuration of Cd in subcellular fractions and whole organism in acclimated earthworms in control soil.

Worm Cd concentration (mg/kg)

120 MT cel debris granule 80 total y=9.32*e-0.008x +129*(1- e-0.008x ) (R2 = 0.9962, P<0.0001)

y=6.46*e-0.007x +107*(1- e-0.007x ) (R2 = 0.9963, P<0.0001) 40 y=1.71*e-0.011x +16.6*(1- e-0.011x ) (R2 = 0.995, P<0.0001) y=1.45*e-0.014x +8.84*(1- e-0.014x ) (R2 = 0.9881, P<0.0001) 0 0 56

Exposure time (day)

112

168

224

Figure 3.2. Time course distribution profiles for Cd accumulation in subcellular fractions and whole organism in unacclimated earthworms in soil spiked with 20 mg/kg of Cd.

44

Worm Cd concentration (mg/kg)

200

MT cell debris granule total

y=37.7*e-0.004x +270*(1- e-0.004x ) (R 2 = 0.9909, P<0.0001)

150

100 y = 23.9*e-0.004x +229*(1- e-0.004x ) (R2 = 0.9733, P<0.0001) 50 y=2.61*e-0.022x +27.4*(1- e-0.022x ) (R 2 = 0.9925, P<0.0001) y=2.59*e-0.029x +10.1*(1- e-0.029x )(R2 = 0.9819, P=0.0001)

0 0 56

Exposure time (day)

112

168

224

Figure 3.3. Time course distribution profiles for Cd accumulation in subcellular fractions and whole organism in acclimated earthworms in soil spiked with 20 mg/kg of Cd.

Worm Cd concentration (mg/kg)

270

MT cell debris granule total y=13.6*e-0.009x +267*(1- e-0.009x ) (R2 = 0.9989, P<0.0001)

180

y=9.90*e-0.008x +219*(1- e-0.008x ) (R2 = 0.9932, P<0.0001) 90 y=0.17*e-0.019x +44.6*(1- e-0.019x ) (R2 = 0.9983, P<0.0001) y=1.22*e-0.016x +12.5*(1- e-0.016x ) (R2 = 0.9956, P<0.0001)

0 0 56

Exposure time (day)

112

168

224

Figure 3.4. Time course distribution profiles for Cd accumulation in subcellular fractions and whole organism in unacclimated earthworms in soil spiked with 100 mg/kg of Cd.

45

Worm Cd concentration (mg/kg)

350 300 250 200 150 100 50 0 0

MT cell debris granule total y =64.1*e-0.004x+490*(1- e-0.004x) (R2 = 0.9915, P<0.0001)

y=40.4*e-0.003x+426*(1- e-0.003x) (R2 = 0.9873, P<0.0001) y=11.2*e-0.020x+48.6*(1- e-0.020x) (R2 = 0.9938, P<0.0001) y=4.76*e-0.009x+22.8*(1- e-0.009x ) (R2 = 0.996, P<0.0001)

56

Exposure time (day)

112

168

224

Figure 3.5. Time course distribution profiles for Cd accumulation in subcellular fractions and whole organism in acclimated earthworms in soil spiked with 100 mg/kg of Cd.

Test

Whole organism t1/2 Css (day) (mg/kg) 129 (122-140) 270 (251-310) 267 (254-282) 490 (400-593) 76.9 (61.1-92.4) 135 (108-174) 71.7 (61.0-77.3) 139 (120-167)

MT t1/2 (day) 90.1 (74.3-108) 146 (137-251) 80.4 (76.7-95.6) 198 (151-247) Css (mg/kg) 107 (98.1-118) 229 (218-243) 219 (202-239) 426 (381-470) t1/2

Cell debris Css (mg/kg) 16.6 (16.0-17.3) 27.4 (27.3-27.5) 44.6 (44.2-45.0) 48.6 (48.4-48.8) t1/2 (day) 36.4 (day) 52.7 (40.9-72.9) 26.8 (11.7-45.6) 36.5 (29.5-46.7) 21.6 (8.81-37.1)

Granule Css (mg/kg) 8.84 (8.69-9.00) 10.1 (10.0-10.2) 12.5 (12.3-12.7) 22.8 (21.6-24.2)

Unacclimated worms exposed to 20 mg/kg Cd Acclimated worms exposed to 20 mg/kg Cd Unacclimated worms exposed to 100 mg/kg Cd Acclimated worms exposed to 100 mg/kg Cd

(18.7-63.4) 13.7 (5.28-21.2) 37.1 (26.1-52.9) 51.4 (35.4-67.6)

Table 3.2. Estimated half-lives (t1/2) for Cd uptake and steady-state Cd concentrations (Css) of the whole organism and three subcellular fractions. The values in brackets are 95% confidence intervals.

46

3.4 Discussion Acclimated and unacclimated earthworms showed different Cd accumulation patterns in different subcellular fractions. In uptake tests for both acclimated and unacclimated earthworms, Cd content in cell debris fractions, as well as in granule fractions, were nonlinear as a function of exposure time. Half-lives (Table 3.2) were all less than 60 days, and the Cd levels in these fractions on Day 224 approximately reached steady-state concentrations, which were low relative to in the MT fractions. Cadmium content in the MT fractions and in the whole organism also increased nonlinearly with exposure time in unacclimated earthworms with half-lives ranging from 71.7 to 90.1 days. The Cd levels on Day 224 reached approximately 90% of the steady-state concentrations. In contrast, in acclimated earthworms, although Cd content in the MT fractions and in the whole organism could be described using a one-compartment, nonlinear regression model (Eq. (3.2)), within the 224 days of exposure time, Cd content could also be described well with a linear regression model (Eq. (3.3)). Half-lives for Cd uptake in the MT fraction and in the whole organism for acclimated worms ranged from 135 to 198 days, and only about 68% of the steady-state concentrations were reached on Day 224. Since the median longevity for E. andrei is 4.25 years (Mulder et al 2007), steady-state concentrations in acclimated earthworms would only be reached in even longer-term bioaccumulation studies. Moreover, the steady-state concentrations in acclimated earthworms were twice as high as those in unacclimated worms, indicating that earthworms could accumulate more Cd after acclimation. For either acclimated or

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unacclimated earthworms, soil exposure concentration had no effect on the Cd accumulation pattern. Different subcellular fractions have their own specific uptake and elimination kinetics because of their different binding affinities for Cd. Cadmium incorporated into the MT fraction is tightly bound and did not easily dissociate, resulting in slow depuration kinetics (Harrahy and Clements 1997) and a linear accumulation pattern. Cadmium in cell debris or granule fractions is loosely bound and might easily be eliminated from the organism (Vijver et al. 2006). These fractions had larger elimination rates, and showed accumulation curves quickly reaching steady state. These results are consistent with several other studies (Conder et al. 2002; Vijver et al. 2006). Due to the differences in accumulation kinetics of the different subcellular fractions, Cd levels in the MT fraction contained an increasing proportion of total Cd body burden over time. After 224 days of exposure, the proportionate distribution of Cd in the three subcellular fractions between acclimated and unacclimated earthworms under different exposure concentrations were approximately similar (78% in the MT fraction, 16% in the cell debris fraction, and 6% in the granule fraction). Information on subcellular compartmentalization provides clues to the Cd detoxification strategies adopted by earthworms. In all tests, Cd was mainly measured in the MT fraction suggesting the ability of the MT detoxification system to sequester incoming Cd over the entire exposure period (Holloway et al. 1990). Due to the 28-day acclimation to low-level of Cd, more MT was induced for acclimated earthworms. MT

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detoxifies absorbed Cd by sequestering it and preventing interactions with sites of toxic action in the organism (Conder et al. 2002). With greater MT induction, acclimated earthworms could accumulate Cd to fairly high levels with minimum adverse effects. Also, due to the high affinity of MT for Cd, acclimated worms exhibited lower elimination rates than unacclimated worms (Figure 2-5), which was another reason for higher levels of accumulation. Metal acclimation and long-term exposure may impose a cost of energy and other resources on earthworms (Bengtsson et al. 1983; Sibly and Calow 1989). The costs can be of several different types, in the specific case of Cd-resistance, including the metabolic cost of synthesizing MT and transporting Cd to the tissue and subcellular sequestration sites, the possible loss of certain essential metals incidentally sequestered by MT (Klerks and Levinton 1993; Posthuma and Van Straalen 1993), and a diminution of the ability to invest in other processes; e.g., the energy available for reproduction is reduced (Holloway et al. 1990). Direct damage to reproductive tissues, and indirect impairment of reproductive success due to depleted energy reserves, can adversely affect earthworm populations (Morgan and Morgan 1990). The exposure concentrations applied in our study were 20 mg/kg (bioaccumulation level) and 100 mg/kg (presumptive sublethal level lower than the Cd Eco-SSL for soil invertebrates (140 mg/kg)). However, no lethal or sublethal effects were observed under either exposure concentration. No significant mortality of earthworms was observed and in all jars, greater than 80% of worms were recaptured. The change in earthworm wet weight was negligible over 224 days of

49

exposure (RSD=11.7%). Cocoons were produced and hatched in all test jars, but this observation is qualitative, as the study was not designed to quantify reproduction. Overall, it did not appear that survival or growth of earthworms was affected and cocoons and hatchlings were present in all containers. Although earthworms accumulated Cd to very high levels, most was deposited in the MT fractions, which was not bioavailable and, hence, non-toxic. Only Cd deposited in cell debris fractions could be considered as potentially toxic, and the uptake of Cd in cell debris fractions quickly reached steady state. In an acute toxicity test, Conder et al. (2002) exposed E. fetida to 1574 mg/kg soil Cd for 14 days and observed 50% mortality when steady-state Cd concentrations of 132 mg/kg were reached in cell debris+granule fractions. In the current study, maximum Cd level in cell debris+granule fractions was 71.4 mg/kg which was significantly lower than that in Conder et al. (2002), and this is likely the reason why no toxic responses were observed. Toxicity cannot be predicted from total metal burden in earthworms (Rainbow 2002) since the biological significance of a metal concentration depends on the specific tissue in which the metal is deposited. Typical earthworm bioaccumulation tests do not differentiate toxic from non-toxic fractions leading to the potential for overestimation of risk to earthworms for Cd contaminated soils. According to the spillover hypothesis (Rainbow 1993; Spurgeon and Hopkin 1999), another explanation for the absence of toxic response is that under the exposure concentrations used in this study, MT may have been significantly induced, and its

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binding capacity matched net Cd uptake. Therefore, no adverse effects can be observed. Alternatively, when the external Cd bioavailability is very high and the exposure time is very long, the capacity of detoxicification mechanisms is exceeded and excess Cd would spill over into the toxic fractions where Cd could interact with biomolecules that represent potential sites of toxic action, resulting in adverse effects. In this case, only when exposure duration is long, toxic body concentrations may be reached. Potential toxic effects of Cd may be underestimated when determined by short-term studies. If earthworms are exposed to lethal concentrations, the complete induction of MT synthesis will not be achieved, and adverse effects will already occur at a very low internal concentration. In the extreme situation where earthworms are exposed to a very high concentration of Cd, animals die within a few minutes, while no elevated internal concentrations can be detected (Lock and Janssen 2001). This may form the distinction between mechanisms of acute and chronic metal toxicity. Even though acclimation did not affect survival, reproduction, and growth of earthworms in some cases, high levels of metal accumulation in earthworm tissues could be a source of toxic elements for worm predators (e.g., birds or small mammals) through trophic transfer, and may be critical to their survival and fitness (Cooke et al., 1992; Abdul Rida and Bouch, 1994). Contrary to the risk to earthworms, the whole body burden of metal needs to be taken into account when estimating the risk to worm predators. Bioaccumulation factor (BAF) is a simple way to estimate bioaccumulation and is

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usually expressed as the ratio of the total Cd tissue burden in the earthworm to total soil Cd concentration, at steady state. In Table 3.3, three types of BAF were calculated based upon total soil Cd or CaCl2 extractable-Cd values in Table 3.1 as well as steady state Cd concentrations in the whole organism or cell debris fractions shown in Table 3.2. Within the same group of earthworms (unacclimated or acclimated), BAF-total values decreased about 59% as total soil Cd increased from 20 mg/kg to 100 mg/kg. However, when Cd bioavailability in soil and Cd distribution in earthworm were taken into account, the difference in BAF values between two exposure concentrations was marginally decreased (44% for BAF-extract and 36% for BAF-cb).

Test BAF-total BAF-extract BAF-cb Unacclimated worms 5.85 67.3 8.63 exposed to 20 mg/kg Cd Acclimated worms 12.2 140 14.2 exposed to 20 mg/kg Cd Unacclimated worms 2.57 40.1 6.69 exposed to 100 mg/kg Cd Acclimated worms 4.72 73.5 7.29 exposed to 100 mg/kg Cd Table 3.3. BAF values of unacclimated and acclimated earthworms exposed in 20 and 100 mg/kg of Cd.
BAF-total=total Cd tissue burden/total soil Cd; BAF-extract= total Cd tissue burden/CaCl2 extractable-Cd; BAF-cb=Cd concentration in cell debris fraction/ CaCl2 extractable-Cd.

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Kinetic studies yield important toxicological information, and may also provide useful insight on the design of laboratory bioassays. In this study, we found that Cd showed continuous linear accumulation within the 224-day exposure period, especially in acclimated earthworms, and longer exposure times would be needed to achieve steady state. The situation is the same for other persistent chemicals. Edwards and Bohlen (1996) indicated that for long-lived invertebrates such as earthworms, accumulation of persistent chemicals may occur over the life span of the organisms which can be up to four years for some earthworm species. Several studies published in recent years also advocated the need for full life history exposure studies to allow more accurate extrapolation of toxicity and bioaccumulation data to population level effects (Calow et al., 1997; Forbes et al., 2001).

3.5 Conclusions Acclimated earthworms accumulated more Cd and required longer time to reach steady state than unacclimated worms. Most of the Cd was present in the MT fraction. Cadmium in the MT fraction increased approximately linearly with time, and required a relatively longer time to reach steady state, while Cd in the cell debris and granule fractions quickly reached steady state. Cadmium in the cell debris fraction is considered potentially toxic, but low steady state concentrations observed in this study suggested no adverse effects occurred. Future use of earthworms in ecological risk assessment should take into consideration pre-exposure histories of the test organisms since evolved

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resistance or acclimation to a toxicant could bias results. Also, a prolonged test period may be required for a comprehensive understanding of uptake kinetics and compartmentalization. Our study focused on one metal, one earthworm species, one soil type, and just a few exposure concentrations. Additional work with a wide variety of metals, species, soil types and exposure concentrations are needed for a more comprehensive understanding.

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Chapter 4: Conclusion

Cadmium bioaccumulation is related to various abiotic and biotic factors. Earthworm species and soil matrix composition are the two most significant parameters affecting Cd BAF in earthworms. Also, acclimated earthworms accumulated more Cd and required longer time to reach steady state than unacclimated worms. Most of the Cd was detoxified and present in the MT fraction, while Cd in the cell debris fraction, although considered potentially toxic, quickly reached steady state and the steady state concentration was fairly low, not resulting in any observed adverse effects on earthworms. This study yielded important toxicological information related to metal bioaccumulation, and may also provide useful insight on the design of laboratory bioassays. First, earthworm species is a significant factor on metal bioaccumulation. The choice between endogeic species and epigeic species could produce significant differences in metal bioaccumulation which is not related to the metal contamination in soil. Also, in order to utilize the models developed from a specific species to other species, interspecies transfer coefficients may need to be considered.

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Second, soil properties are also important parameters affecting metal bioaccumulation, but just a few studies reported comprehensive results of soil analysis, and there was too much variability in the way soil properties were measured. Standardizing the methods of soil property measurement and having them reported in all bioaccumulation studies would be a better way to produce more reliable results, and make the comparison among studies much easier. Third, future use of earthworms in ecological risk assessment should take into consideration pre-exposure histories of the test organisms since evolved resistance or acclimation to a toxicant could bias results. Also, a prolonged test period may be required for a comprehensive understanding of metal uptake kinetics and compartmentalization. This study focused on the Cd uptake kinetics by one earthworm species (E. andrei) in one soil type (Webster soil) with just a few exposure concentrations. Additional work with a wide variety of metals, species, soil types and exposure concentrations are needed for a more comprehensive understanding. In summary, laboratory toxicity and bioaccumulation models along with measurements of key soil properties could be used as a screening tool to evaluate metal bioavailability in the baseline risk assessment. It can be used to determine whether more definitive site-specific metal bioavailability studies are warranted, to facilitate more accurate estimation of Cd bioaccumulation when combined with site-specific data, and to help better prioritize site cleanup. The approach used in this study could also be extrapolated to various metals when their unique characteristics are taken into

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consideration. Compared to the standard procedure using total soil metal concentrations, incorporating bioavailability data into risk assessment could save huge expenses in unnecessary remedial costs.

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Soc. 37: 101-116. Silverman, H., W. L. Steffens, and T. H. Dietz. 1983. Calcium concentrations in the gills of a freshwater mussel serve as a calcium reservoir during periods of hypoxia. J. Exp. Zool. 227: 177189. Spurgeon, D. J. 1997. Comparison of cadmium, copper, lead and zinc kinetics in the earthworm Eisenia fetida. Abstracts of the Second Int. Workshop on Earthworm Ecotoxicology, Amsterdam, p. 40. Spurgeon, D. J. and S. P. Hopkin. 1999. Comparisons of metal accumulation and excretion kinetics in earthworms (Eisenia fetida) exposed to contaminated field and laboratory soils. Applied Soil Ecology 11: 227-243. Spurgeon, D. J., C. Svendsen, P. Kille, A. J. Morgan, and J. M. Weeks. 2004. Responses of earthworms (Lumbricus rubellus) to copper and cadmium as determined by measurement of juvenile traits in a specifically designed test system. Ecotoxicol. Environ. Saf. 57: 54-64. Stafford, E. A. and S. P. McGrath. 1986. The use of acid insoluble residue to correct for the presence of soil-associated metals in the gut of earthworms used as bioindicator organisms. Environ. Pollut. 42: 233-246. Suzuki, K.T., M. Yamamura, and T. Muri. 1980. Cadmium-binding proteins induced in the earthworm. Arch. Environ. Contain. Toxicol. 9: 415-424. Tessier, L., G. Vaillancourt, and L. Pazdernik. 1994. Temperature effects on cadmium and mercury kinetics in freshwater mollusks under laboratory conditions. Arch. Environ. Contam. Toxicol. 26: 179-184. USDA 1993. Soil Survey Manual. Handbook 18. United States Department of Agriculture, Washington, DC, United States. USEPA 1993. Wildlife Exposure Factors Handbook. U. S. Environmental Protection Agency.Vol. 1. EPA/600/R-93/187. Washington, D.C., United States. USEPA 2005. Eco-SSL for Cadmium. U. S. Environmental Protection Agency. Washington, D.C., United States. USEPA 2007. Framework for Metals Risk Assessment. U. S. Environmental Protection Agency Washington, D.C., United States.

66

USEPA 3051a. 2007. Microwave assisted acid digestion of sediments, sludges, soils, and oils. SW-846. Environmental Protection Agency, Washington, DC, United States. Van Gestel C. A. M, E. M. Dirven-van Breemen, and R. Baerselman. 1993. Accumulation and elimination of cadmium, chromium and zinc and effects on growth and reproduction in Eisenia andrei (Oligochaeta, Annelida). Sci. Total Environ. (Suppl.): 585597. Van Straalen, N. M., M. H. Donker, M. G. Vijver, and C. A. M. van Gestel. 2005. Bioavailability of contaminants estimated from uptake rates into soil invertebrates. Environ. Pollut. 136: 409-417. Vijver, M. G., C. A. M. Van Gestel, R. P. Lanno, N. M. Van Straalen, and W. J. G. M. Peijnenburg. 2004. Internal metal sequestration and its ecotoxicological relevance: a review. Environ. Sci. Technol. 38, 47054712. Vijver, M. G., C. A. M. Van Gestel, N. M. Van Straalen, R. P. Lanno, and W. J. G. M. Peijnenburg. 2006. Biological significnce of metals partitioned to subcellular fractions within earthworms (Aporrectodea caliginosa). Environmental Toxicology and Chemistry 25 (3): 807814. Wallace, W. G. and G. R. Lopez. 1997. Bioavailability of biologically sequestered cadmium and the implications of metal detoxification. Mar. Ecol. Prog. Ser. 147: 149157. Wallace, W. G. and S. N. Luoma. 2003. Subcellular compartmentalization of Cd and Zn in two bivalves. II. Significance of trophically available metal (TAM). Mar. Ecol. Prog. Ser. 257: 125137. Yamamura M., T. Mori, and K. T. Suzuki. 1981. Metallothionein induced in the earthworm. Experientia 37:1187-1189.

67

Appendix A. Earthworm Cadmium Bioaccumulation Database

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference 1 Netherlands Ap. rosea field 1 5.7 CaCl2 2 Netherlands L. terrestris field 1 5.9 CaCl2 3 Netherlands Ap. rosea field 1 5.8 CaCl2 4 Netherlands Al.chlorotica field 1 5.8 CaCl2 5 Netherlands L. terrestris field 1 5.8 CaCl2 6 Netherlands Ap. longa field 1 5.8 CaCl2 7 Netherlands Ap. longa field 1 6.0 CaCl2 8 Netherlands Ap. caliginosa field 1 5.8 CaCl2

2134 21.2 0.14 151.43 16.9 0.29 58.28 10.90 0.65 16.77 10.90 0.65 16.77 16.80 0.65 25.85 5.70 0.65 8.77 9.20 0.99 9.29 6.90 0.65 10.62

HNO3+HClO4 NPK-fertilized

HNO3+HClO4 K-fertilized

HNO3+HClO4 sludge-amended

HNO3+HClO4 sludge-amended

HNO3+HClO4 sludge-amended

HNO3+HClO4 sludge-amended

HNO3+HClO4 sludge-amended

HNO3+HClO4 sludge-amended

Andersen 1979

68

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

9 Netherlands Ap. caliginosa field 1 6.0 CaCl2

10 Netherlands Al. chlorotica field 1 6.0 CaCl2

11 Netherlands Ap. rosea field 1 6.0 CaCl2

12 Netherlands Ap. longa field 1 5.9 CaCl2

13 Netherlands L. terrestris field 1 6.0 CaCl2

14 Netherlands Ap. rosea field 1 5.9 CaCl2

15 Netherlands L. terrestris field 1 6.9 CaCl2

16 Netherlands L. terrestris field 1 6.4 CaCl2

25300 10.90 0.99 11.01 16.20 0.99 16.36 19.60 0.99 19.80 11.80 0.29 40.69 8.80 0.99 8.89 26.90 0.29 92.76 21.00 1.04 20.19

25300 23.80 1.04 22.88

HNO3+HClO4 sludge-amended

HNO3+HClO4 sludge-amended

HNO3+HClO4 sludge-amended

HNO3+HClO4 K-fertilized

HNO3+HClO4 sludge-amended

HNO3+HClO4 K-fertilized

HNO3+HClO4

HNO3+HClO4

Andersen 1979

69

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

17 Netherlands L. terrestris field 1 7.1 CaCl2

18 Netherlands L. terrestris field 1 6.5 CaCl2

19 Netherlands L. terrestris field 1 7.2 CaCl2

20 Netherlands L. terrestris field 1 6.8 CaCl2

21 Netherlands L. terrestris field 1 7.4 CaCl2

22 Netherlands L. terrestris field 1 7.3 CaCl2

23 Netherlands Ap. longa field 1 5.7 CaCl2

24 Denmark Ap. longa field

23000 30.80 0.76 40.53

23000 15.60 0.76 20.53

24000 32.50 0.72 45.14

24000 19.80 0.72 27.50

24000 29.10 0.29 100.34

161000 12.50 0.45 27.78 10.00 0.14 71.43

24 9.20 1.00 9.20

HNO3+HClO4

HNO3+HClO4

HNO3+HClO4

HNO3+HClO4

HNO3+HClO4

HNO3+HClO4

HNO3+HClO4 NPK-fertilized

HNO3+HClO4

Andersen 1979

Andersen and Laursen 1982

70

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

25 Denmark L. terrestris field 6.9 CaCl2

26 USA field 3 6.9 H2O 1:1 18

27 USA Ap. longa field 3 7.2 H2O 1:1 7.1

28 USA L. terrestris field 3 6.6 H2O 1:1 11

29 USA Ap. trapezoides field 3 5.8 H2O 1:1 7.5

30 USA L. terrestris field 3 6.0 H2O 1:1 8.1

31 USA Ap. longa field 3 5.6 H2O 1:1 13

32 USA Ap. trapezoides field 3 5.8 H2O 1:1 7.5

28000 21.00 1.00 21.00 11.00 8.20 1.34 12.00 2.70 4.44 18.00 11.00 1.64 6.50 0.46 14.13 16.00 1.70 9.41 10.00 0.47 21.28 4.00 0.46 8.70

HNO3+HClO4

HNO3+HCl mine

HNO3+HCl industry

HNO3+HCl mine

HNO3+HCl industry

HNO3+HCl industry

HNO3+HCl industry

HNO3+HCl industry

Andersen and Laursen 1982

Beyer and Cromartie 1987

71

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

33 USA L. terrestris field 3 7.0 H2O 1:1 8.9

34 USA Ap. tuberculata field 3 7.0 H2O 1:1 8.9

35 USA Ap. turgida field 2 4.6

36 USA L. terrestris field 2 5.5

37 USA L. terrestris field 2 5.5

38 USA Ap. longa field 2 4.9

39 USA Ap. tuberculata field 2 5.4

40 USA Ap. turgida field 2 4.9

41 USA L. terrestris field 2 5.3

2.8 loam 8

3.8 silt loam 11

2.8 loam

4.9 silt loam 13

3.0 silt loam 9

2.5 loam 8

2.6 silt loam 10

23 2.80 8.21

8.3 2.80 2.96

101 1.90 53.16

118 8.20 14.39

51 3.80 13.42

18 0.91 19.78

5.3 0.18 29.44

8.6 0.14 61.43

3.60 0.09 40.00

HNO3+HCl mine

HNO3+HCl mine

HNO3+HCl

HNO3+HCl

HNO3+HCl

HNO3+HCl

HNO3+HCl

HNO3+HCl

HNO3+HCl

Beyer and Cromartie 1987

Beyer et al. 1982

72

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

42 USA Ap. tuberculata field 2 5.9

43 USA Ds. rubidus field 5.0

44 USA Es. carolinensis field 3 5.0

45 Canada L. rubellus field 2 4.1 0.1 M CaCl2 1:2 silt clay loam

46 Canada L. rubellus field 2 4.1 0.1 M CaCl2 1:2 silt clay loam

47 Canada field 2 4.1 0.1 M CaCl2 1:2 silt clay loam

48 Canada Al. chlorotica field 2 4.1 0.1 M CaCl2 1:2 silt clay loam

49 UK L. rubellus lab 1.5 7.2 H2O 1:10 4

50 UK L. rubellus lab 1.5 6.6 H 2O 1:10 15

2.5 loam

23 loam 19

23 loam 19

209000 4.00 0.06 66.67 140.00 2.70 51.85 62.00 2.70 22.96 6.00 0.40 15.00 5.10 0.40 12.75 8.00 0.40 20.00 8.00 0.40 20.00 487.00 318.00 1.53 31 HNO3

4910 102.30 8.31 12.31 31 HNO3

HNO3+HCl

HNO3+HCl

HNO3+HCl

HNO3+HCl

HNO3+HCl

Beyer et al. 1982

Beyer et al. 1985

Carter 1983

Corp and Morgan 1991

73

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

51 UK L. rubellus lab 1.5 6.4 H2O 1:10 12

52 UK L. rubellus lab 1.5 8.1 H2O 1:10 6

53 UK L. rubellus lab 1.5 5.3 H2O 1:10 12

54 UK L. rubellus lab 1.5 6.4 H2O 1:10 35

55 UK L. rubellus lab 1.5 6.0 H2O 1:10 17

56 UK L. rubellus lab 1.5 5.2 H2O 1:10 21

57 UK L. rubellus lab 1.5 5.8 H2O 1:10 17

58 UK L. rubellus lab 1.5 3.5 H2O 1:10 19

59 UK L. rubellus lab 1.5 6.6 H2O 1:10 15

60 UK L. rubellus lab 1.5 5.2 H2O 1:10 21

61 UK L. rubellus lab 1.5 5.3 H2O 1:10 12

55500 331.30 207.00 1.60 31 HNO3

176000 214.00 124.00 1.73 31 HNO3

1860 205.10 20.80 9.86 31 HNO3

62100 70.90 94.40 0.75 31 HNO3

15400 61.00 6.75 9.04 31 HNO3

3200 29.50 3.56 8.29 31 HNO3

2761 9.30 0.69 13.48 31 HNO3

274 10.40 0.46 22.61 31 HNO3

6750 30.30 8.43 3.59 31 HNO3

3350 17.10 3.84 4.45 31 HNO3

1850 52.50 23.40 2.24 31 HNO3

Corp and Morgan 1991

74

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

62 UK L. rubellus lab 1.5 6.4 H2O 1:10 35

63 UK L. rubellus lab 1.5 3.5 H2O 1:10 19

64 UK L. rubellus lab 1.5 6.0 H2O 1:10 17

65 UK L. rubellus lab 1.5 6.4 H2O 1:10 12

66 UK L. rubellus lab 1.5 8.1 H2O 1:10 6

67 UK L. rubellus lab 1.5 7.2 H2O 1:10 4

68 Poland L. terrestris field 6.9 KCl 6.9 silty loamy sand

69 Poland L. terrestris field 6.6 KCl 2.8 silty loamy sand

70 Poland L. terrestris field 6.8 KCl 6.6 silty loamy sand

60200 21.40 91.90 0.23 31 HNO3

285 11.70 0.33 35.45 31 HNO3

18900 19.00 6.63 2.87 31 HNO3

62000 54.40 173.00 0.31 31 HNO3

183000 65.00 138.00 0.47 31 HNO3

199000 140.00 266.00 0.53 31 HNO3 16.10 1.10 14.64 10.39 0.30 34.63 14.55 0.92 15.82

Corp and Morgan 1991

Czarnowska and Jopkiewicz 1978

75

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

71 Poland L. terrestris field 6.6 KCl 2.0 silty loamy sand

72 Poland L. terrestris field 7.2 KCl 4.8 silty loamy sand

73 Poland L. terrestris field 6.1 KCl 3.8 silty loamy sand

74 Poland L. terrestris field 6.0 KCl 3.1 silty loamy sand

75 Germany field

76 Germany field

77 USA field 1 6.9

78 USA field 1 7.0

79 USA field 1 6.9

4.8 loamy sand

5.0 silt clay

3.3

9.09 0.28 32.46

12.21 0.62 19.69

7.53 0.29 25.97

3.12 0.11 28.36

1.88 0.07 27.17

7.00 0.11 66.04

7.20 0.62 11.61

7.80 0.64 12.19

3.00 0.66 4.55

HCl+HNO3

HCl+HNO3

HCl

HCl

HCl

Czarnowska and Jopkiewicz 1978

Diercxsens et al. 1985

Gish and Christensen 1973

76

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

80 USA field 1 7.0

81 USA field 1 10

82 UK L. rubellus field 4

83 UK Da. veneta field 4

84 UK L. rubellus field 4

85 UK Ela. tetraedra field 4

86 UK L. rubellus field 4

87 Netherlands L. terrestris field 3 6.5 KCl 10.2 sand

88 Netherlands L. terrestris field 3 4.0 KCl 5.7 sand

89 Netherlands L. terrestris field 3 6.0 KCl 7.3 sand

6.4 silt loam

7.3 silt clay 4921 32129 7.00 4.00 1.75 32129 4.00 4.00 1.00 32129 3.00 4.00 0.75 998 15.00 2.00 7.50

8.70 0.88 9.89

10.80 1.14 9.47

25.00 4.00 6.25

114.00 9.20 12.39

19.00 0.10 190.00

79.00 6.00 13.17

HCl

HCl

HNO3

HNO3

HNO3

HNO3

HNO3

HCl

HCl

HCl

Gish and Christensen 1973

Ireland 1979

Ma 1987

77

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

90 UK L. rubellus field 4 6.8 H2O 1:10

91 UK L. rubellus field 4 4.3 H2O 1:10

92 UK L. rubellus field 4 5.2 H2O 1:10

93 UK L. rubellus field 4

94 UK Ds. rubidus field 4 4.3 H 2O 1:10

95 UK Ds. rubidus field 4

96 UK L. rubellus field 4

97 UK Ds. rubidus field 4

98 UK Ds. rubidus field 4 5.2 H2O 1:10

99 UK L. rubellus field 4

5080 130.00 19.00 6.84

170 8.00 0.09 88.89

1790 14.00 0.90 15.56

48920 160.00 80.00 2.00

170 10.00 0.09 111.11

6780 17.00 0.84 20.24

6780 14.00 0.84 16.67

1390 12.00 1.21 9.92

1790 30.00 0.90 33.33

58120 180.00 90.00 2.00

HNO3 mine

HNO3 mine

HNO3 mine

HNO3

HNO3 mine

HNO3

HNO3

HNO3

HNO3 mine

HNO3

Morgan and Morgan 1991

78

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

100 UK L. rubellus field 4

101 UK Ds. rubidus field 4

102 UK L. rubellus field 4

103 UK Ds. rubidus field 4

104 UK Ds. rubidus field 4 6.8 H2O 1:10

105 UK Ds. rubidus field 4

106 UK Ds. rubidus field 4

107 UK Ds. rubidus field 4 6.5 H 2O 1:10

108 UK L. rubellus field 4 6.5 H 2O 1:10

109 UK L. rubellus field 4

69440 220.00 50.00 4.40

69440 320.00 50.00 6.40

1390 17.00 1.21 14.05

48920 190.00 80.00 2.38

5080 430.00 19.00 22.63

91850 270.00 280.00 0.96

58120 410.00 90.00 4.56

26420 1790.00 350.00 5.11

26420 580.00 350.00 1.66

91850 120.00 280.00 0.43

HNO3

HNO3

HNO3

HNO3

HNO3 mine

HNO3

HNO3

HNO3 mine

HNO3 mine

HNO3

Morgan and Morgan 1991

79

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

110 UK Ds. rubidus field 4 6.8 H2O 1:2 carboniferous limestone

111 UK L. rubellus field 4 6.8 H2O 1:2 carboniferous limestone

112 USA field 7 8.0 H2O 1:1 silt loam

113 USA Ap. tuberculata field 7 7.9 H2O 1:1 silt clay loam

114 USA Ap. tuberculata field 7 7.5 H 2O 1:1 silt clay loam

115 USA field 7 5.9 H2O 1:1 silt loam

116 Belgium Al. chlorotica field 13 7.0 H2O 1:10 4.0

9523 1320.00 467.00 2.83 823.00 467.00 1.76 12.00 1.00 12.00 3.80 0.60 6.33 22.00 2.00 11.00 36.00 3.50 10.29 18.48 0.83 22.27

HNO3 mine

HNO3 mine

HNO3+ H2SO4

HNO3+ H2SO4 mine

HNO3+ H2SO4 mine

HNO3+ H2SO4

HNO3

Morgan and Morris 1982

Pietz et al. 1984

Pizl and Josens 1995

80

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

117 Belgium L. rubellus field 13 5.9 H2O 1:10 3.2

118 Belgium L. castaneus field 13 6.7 H2O 1:10 4.1

119 Belgium L. rubellus field 13 7.0 H2O 1:10 4.4

120 Belgium L. terrestris field 13 7.0 H2O 1:10 4.0

121 Belgium L. rubellus field 13 7.1 H2O 1:10 3.5

122 Belgium Ap. caliginosa field 13 6.0 H2O 1:10 2.3

123 Belgium Ap. rosea field 13 6.0 H2O 1:10 2.3

124 Belgium Ap. rosea field 13 5.9 H2O 1:10 3.2

125 Belgium L. terrestris field 13 7.1 H2O 1:10 3.5

126 Belgium L. terrestris field 13 6.7 H2O 1:10 4.1

1733 14.39 0.28 51.39

5320 18.45 0.70 26.36

20397 16.68 0.70 23.83

9523 19.80 0.83 23.86

7050 28.24 0.68 41.53

2287 16.89 0.21 80.43

2287 9.78 0.21 46.57

1733 45.17 0.28 161.32

7050 18.78 0.68 27.62

5320 22.68 0.70 32.40

HNO3

HNO3

HNO3

HNO3

HNO3

HNO3

HNO3

HNO3

HNO3

HNO3

Pizl and Josens 1995

81

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

127 Belgium L. castaneus field 13 6.0 H2O 1:10 2.3

128 Belgium An. icterica field 13 6.0 H2O 1:10 2.3

129 Belgium L. rubellus field 13 6.0 H2O 1:10 2.3

130 Belgium L. castaneus field 13 7.0 H2O 1:10 4.4

131 Belgium L. terrestris field 13 5.9 H2O 1:10 3.2

132 Belgium Ap. caliginosa field 13 5.9 H2O 1:10 3.2

133 USA field 4

134 USA field 4

135 USA field 4

2287 3.44 0.21 16.38

2287 39.90 0.21 190.00

2287 8.14 0.21 38.76

20397 15.30 0.70 21.86

1733 15.82 0.28 56.50

1733 45.23 0.28 161.54 3.40 0.23 14.78 3.10 0.20 15.50 6.10 0.28 21.79

HNO3

HNO3

HNO3

HNO3

HNO3

HNO3

HCl+HNO3

HCl+HNO3

HCl+HNO3

Pizl and Josens 1995

Van Hook 1974

82

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

136 USA field 4

137 USA field 4

138 USA field 4

139 Taiwan P. lauta field 0

140 UK Ds.rubidus field 4 6.4

141 China Eia. fetida lab 2 6.4

142 China Eia. fetida lab 2 5.8

143 China Eia. fetida lab 2 5.8

24.1

24.2

25.1

92100 7.20 0.32 22.50 9.30 0.80 11.63 5.10 0.28 18.21 3.65 0.02 158.70 1250.00 510.00 2.45 3.07 1.83 1.68 60 H2SO4+HClO4 sewage sludge 8.81 6.83 1.29 60 H2SO4+HClO4 CdCl2 sewage sludge yes 14 Liu et al. 2005 13.30 16.83 0.79 60 H2SO4+HClO4 CdCl2 sewage sludge yes 14

HCl+HNO3

HCl+HNO3

HCl+HNO3

HCl+HNO3

HNO3 mine

Van Hook 1974

Hsu et al. 2005

Cotter-Howells et al. 2005

83

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

144 China Eia. fetida lab 2 5.7

145 UK L. rubellus field 1 6.7

146 UK L. rubellus field 1 5.1

147 UK L. rubellus field 1

148 UK L. rubellus field 1 6.1

149 UK L. rubellus lab 6.7

150 UK L. rubellus lab 6.7

151 UK L. rubellus lab 6.7

152 UK L. rubellus lab 5.1

153 UK L. rubellus lab 5.1

25.2

6.99 21.83 0.32 60 H2SO4+HClO4 CdCl2 sewage sludge yes 14 Liu et al. 2005

45.30 3.42 13.25

145.10 20.17 7.19

59.90 27.00 2.22

117.20 41.88 2.80

25.00 2.00 12.50 30 yes

45.00 2.00 22.50 30 yes smelter

140.00 2.00 70.00 30 yes smelter

70.00 10.90 6.42 30 yes smelter

70.00 10.90 6.42 30 yes smelter

smelter

smelter

smelter

smelter

smelter

Simonsen and Fordsmand 2004

84

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

154 UK L. rubellus lab 5.1

155 UK L. rubellus lab

156 UK L. rubellus lab

157 UK L. rubellus lab

158 UK L. rubellus lab 6.1

159 UK L. rubellus lab 6.1

160 UK L. rubellus lab 6.1

161 France Ap. caliginosa field 7 5.8 18.5 9.2

162 France Ap. caliginosa field 7 5.8 27 14.5

163 France Ap. caliginosa field 7 6.2 20 18.8

130.00 10.90 11.93 30 yes smelter

35.00 27.00 1.30 30 yes smelter

65.00 27.00 2.41 30 yes smelter

138.00 27.00 5.11 30 yes smelter

60.00 41.30 1.45 30 yes smelter

80.00 41.30 1.94 30 yes smelter

152.00 41.30 3.68 30 yes

54.36 4.50 12.08

69.94 5.20 13.45

47.55 5.00 9.51

HNO3+HCl smelter smelter

HNO3+HCl smelter

HNO3+HCl smelter

Simonsen and Fordsmand 2004

Dai et al. 2004

85

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

164 France Ap. caliginosa field 7 6.1 15.6 5.1

165 France Ap. caliginosa field 7 6.3 13.2 3.7

166 France Ap. caliginosa field 7 6.4 11.8 4.0

167 France L. rubellus field 7 5.8 18.5 9.2

168 France L. rubellus field 7 5.8 27 14.5

169 France L. rubellus field 7 6.2 20 18.8

170 France L. rubellus field 7 6.1 15.6 5.1

171 France L. rubellus field 7 6.3 13.2 3.7

172 France L. rubellus field 7 6.4 11.8 4.0

54.46 3.20 17.02

18.54 3.00 6.18

20.93 2.70 7.75

23.00 4.50 5.11

18.93 5.20 3.64

18.40 5.00 3.68

20.29 3.20 6.34

16.68 3.00 5.56

11.12 2.70 4.12

HNO3+HCl smelter

HNO3+HCl smelter

HNO3+HCl smelter

HNO3+HCl smelter

HNO3+HCl smelter

HNO3+HCl smelter

HNO3+HCl smelter

HNO3+HCl smelter

HNO3+HCl smelter

Dai et al. 2004

86

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

173 Poland Al. chlorotica field 2 7.5

174 Poland Al. chlorotica lab 2 7.6

175 Poland Da. veneta lab 4.6

176 Poland Da. veneta lab 7.5

177 Poland Da. veneta lab 7.5

178 Poland Da. veneta lab 7.5

179 Poland Da. veneta lab 7.5

180 Poland Da. veneta lab 7.6

181 Poland Da. veneta lab 7.6

182 Poland Da. veneta lab 7.6

5.0

7.7

74.2

5.0

5.0

5.0

5.0

7.7

7.7

7.7

3.20 1.40 2.29

27.70 72.20 0.38 28 yes HNO3

0.69 0.50 1.38 0 HNO3

1.57 1.43 1.10 14 HNO3

0.89 1.43 0.62 14 HNO3

3.46 1.43 2.42 28 HNO3

1.40 1.43 0.98 28 HNO3

3.62 72.07 0.05 14 HNO3

1.72 72.07 0.02 14 HNO3

6.92 72.07 0.10 28 HNO3

HNO3

Homa et al. 2003

Olchawa et al. 2003

87

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

183 Poland Da. veneta lab 7.6

184 USA Eia. fetida lab 5.9 20

185 Iran Eia. fetida lab 0 5.5

186 Australia Eia. fetida lab 0 7.2

187 Canada Eia. andrei lab 1 6.3

188 Canada Eia. andrei lab 1 6.3

189 Canada Eia. andrei lab 1 6.3

7.7

10.2 loamy sand

loamy sand

loamy sand

8.60 72.07 0.12 28 HNO3

614.00 1574.00 0.39

6.80 17.40 0.39 60 HNO3 sewage sludge

7.10 17.40 0.41 60 HNO3 sewage sludge

14.00 0.28 50.00 14 HF+HClO4+HNO3

14.001 0.36
1

12.701 0.431 29.53 14 HF+HClO4+HNO3 sludge-amended

38.89 14 HF+HClO4+HNO3 sludge-amended

HNO3 Cd(NO3)2 OECD yes

Olchawa et al. 2003

Conder et al. 2002

Shahmansouri et al. 2005

Renoux et al. 2001

Estimated by figure.

88

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

190 Canada Eia. andrei lab 1 6.3

191 Canada Eia. andrei lab 1 6.3

192 Canada Eia. andrei lab 1 6.3

193 Canada Eia. andrei lab 1 6.3

194 Canada Eia. andrei lab 1 6.3

195 Canada Eia. andrei lab 1 6.3

196 Canada Eia. andrei lab 1 6.3

loamy sand

loamy sand

loamy sand

loamy sand

loamy sand

loamy sand

loamy sand

11.721 0.29
1

9.771 0.37
1

106.451 4.77
1

136.38 5.17
1

24.411 0.90
1

33.201 0.85
1

88.871 4.631 19.19 14 HF+HClO4+HNO3 Cd(NO3)2 sludge-amended yes

40.41 14 HF+HClO4+HNO3 sludge-amended

26.41 14 HF+HClO4+HNO3 sludge-amended

22.32 14 HF+HClO4+HNO3 Cd(NO3)2 sludge-amended yes

26.38 14 HF+HClO4+HNO3 Cd(NO3)2 sludge-amended yes Renoux et al. 2001

27.12 14 HF+HClO4+HNO3 Cd(NO3)2 sludge-amended yes

39.06 14 HF+HClO4+HNO3 Cd(NO3)2 sludge-amended yes

89

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

197 Canada Eia. andrei lab 1 6.3

198 Canada Eia. andrei lab 1 6.3

199 Canada Eia. andrei lab 1 6.3

200 Slovakia field 7.4

201 UK L. terrestris field 1 6.5

202 UK L. terrestris field 1 6.8

203 UK Ap. caliginosa field 1 6.5

204 UK Ap. caliginosa field 1 6.8

0.3 loamy sand loamy sand loamy sand

10.741 5.50 1.95 14 HF+HClO4+HNO3 Cd(NO3)2 sludge-amended yes

29.301 0.81
1

27.341 1.17
1

5.30 0.39 13.59

16.00 1.00 16.00

55.00 10.00 5.50

35.00 1.00 35.00

63.00 10.00 6.30

36.17 14 HF+HClO4+HNO3 Cd(NO3)2 sludge-amended yes Renoux et al. 2001

23.37 14 HF+HClO4+HNO3 Cd(NO3)2 sludge-amended yes

HCl+HNO3 refinery

Spiegel 2002

Wright & Stringer 1980

90

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

205 UK Ap. tuberculata field 1 6.5

206 UK Al. chlorotica field 1 6.5

207 UK Al. chlorotica field 1 6.8

208 UK Ap. longa field 1 6.5

209 UK Ap. longa field 1 6.8

210 UK Ap. rosea field 1 6.5

211 UK Ap. rosea field 1 6.8

212 Eia. fetida lab 7.2 10 17 10

213 Eia. fetida lab 7.5 12.9 3.6 9.8

214 Eia. fetida lab 7.5 10 17 10

19.00 1.00 19.00

16.00 1.00 16.00

55.00 10.00 5.50

17.00 1.00 17.00

39.00 10.00 3.90

17.00 1.00 17.00

49.00 10.00 4.90

1.90 0.30 6.33 28

47.00 23.00 2.04 28

1.90 0.30 6.33 28

OECD

OECD

Wright & Stringer 1980

Ma et al. 1991

91

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

215 Italy Ap. caliginosa field 2

216 Italy Ap. caliginosa field 2

217 Italy Ap. caliginosa field 2

218 Poland Da. octaedra field 2

219 Poland Da. octaedra field 2

220 Poland Da. octaedra field 2

221 Poland Da. octaedra lab 2

24.63 2.90 8.49

19.13 3.69 5.18

59.75 3.84 15.56

89.43 18.64 4.80

16.70 3.23 5.17

8.68 1.94 4.47

162.00 3.95 41.01 270 yes HNO3 cattle manure+OECD

HNO3+H2O2

HNO3+H2O2

HNO3+H2O2

HNO3 smelter

HNO3

HNO3

Carpene et al. 2006

Rozen 2006

92

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

222 Poland Da. octaedra lab 2

223 Poland Da. octaedra lab 2

224 Poland Da. octaedra lab 2

225 Poland Da. octaedra lab 2

226 Poland Da. octaedra lab 2

227 Poland Da. octaedra lab 2

1176.50 26.23 44.85 270 yes HNO3 CdCl2 cattle manure+OECD yes

425.00 26.23 16.20 270 yes HNO3 CdCl2 cattle manure+OECD yes

44.90 3.95 11.37 270 yes HNO3 cattle manure+OECD

174.70 26.23 6.66 270 yes HNO3 CdCl2 cattle manure+OECD yes

12.90 3.95 3.27 270 HNO3 cattle manure+OECD

14.20 3.95 3.59 270 HNO3 cattle manure+OECD

Rozen 2006

93

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

228 Poland Da. octaedra lab 2

229 Netherlands L. rubellus field 2 7.8 30.3 12.2

230 Netherlands L. rubellus field 2 7.6 7.8 12.4

231 Netherlands L. rubellus field 2 7.4 24 17.5

232 Netherlands L. rubellus field 2 7.6 35.6 15.5

233 Netherlands L. rubellus field 2 7.5 26.3 14.4

234 Netherlands L. rubellus field 2 7.4 33.1 18.2

235 Netherlands L. rubellus field 2 7.5 32.7 17.8

236 Netherlands L. rubellus field 2 7.4 43.7 22.4

10.40 3.95 2.63 270 HNO3 cattle manure+OECD

23.10 0.20 115.50

60.90 5.09 11.96

115.00 10.80 10.65

63.50 11.60 5.47

86.00 15.30 5.62

111.00 15.70 7.07

201.00 16.20 12.41

50.40 18.10 2.78

HCl+HNO3

HCl+HNO3

HCl+HNO3

HCl+HNO3

HCl+HNO3

HCl+HNO3

HCl+HNO3

HCl+HNO3

Rozen 2006

Hobbelen et al. 2006

94

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

237 Netherlands L. rubellus field 2 7.5 39.9 21.1

238 Netherlands L. rubellus field 2 7.4 37.4 18.5

239 Netherlands L. rubellus field 2 7.5 38 17.2

240 Netherlands L. rubellus field 2 7.5 32.9 17.3

241 Netherlands L. rubellus field 2 7.2 39.4 20.4

242 Netherlands L. rubellus lab 2 7.6 7.8 12.4

243 Netherlands L. rubellus lab 2 7.4 24 17.5

244 Netherlands L. rubellus lab 2 7.6 35.6 15.5

245 Netherlands L. rubellus lab 2 7.5 26.3 14.4

96.60 18.40 5.25

133.00 18.80 7.07

81.90 19.50 4.20

90.60 20.30 4.46

111.00 27.80 3.99

41.80 5.09 8.21 54 yes HCl+HNO3

36.00 10.80 3.33 54 yes HCl+HNO3

45.20 11.60 3.90 54 yes HCl+HNO3

48.40 15.30 3.16 54 yes HCl+HNO3

HCl+HNO3

HCl+HNO3

HCl+HNO3

HCl+HNO3

HCl+HNO3

Hobbelen et al. 2006

95

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

246 Netherlands L. rubellus lab 2 7.4 33.1 18.2

247 Netherlands L. rubellus lab 2 7.5 32.7 17.8

248 Netherlands L. rubellus lab 2 7.4 43.7 22.4

249 Netherlands L. rubellus lab 2 7.5 39.9 21.1

250 Netherlands L. rubellus lab 2 7.4 37.4 18.5

251 Netherlands L. rubellus lab 2 7.5 38 17.2

252 Netherlands L. rubellus lab 2 7.5 32.9 17.3

253 Netherlands L. rubellus lab 2 7.2 39.4 20.4

254 Netherlands Ap. caliginosa field 2 7.5 15.1 9.8

42.80 15.70 2.73 54 yes HCl+HNO3

56.30 16.20 3.48 54 yes HCl+HNO3

39.80 18.10 2.20 54 yes HCl+HNO3

42.60 18.40 2.32 54 yes HCl+HNO3

51.40 18.80 2.73 54 yes HCl+HNO3

36.10 19.50 1.85 54 yes HCl+HNO3

51.40 20.30 2.53 54 yes HCl+HNO3

81.60 27.80 2.94 54 yes HCl+HNO3

39.341 2.811 14.00

HCl+HNO3

Hobbelen et al. 2006

van Vliet et al. 2005

96

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

255 Netherlands Ap. caliginosa field 2 7.5

256 Netherlands Ap. caliginosa field 2 7.2 27.5 13.3

257 Netherlands Ap. caliginosa field 2 7.4

258 Netherlands Al. chlorotica field 2 7.5 15.1 9.8

259 Netherlands Al. chlorotica field 2 7.5

260 Netherlands Al. chlorotica field 2 7.2 27.5 13.3

261 Netherlands Al. chlorotica field 2 7.4

262 Netherlands L. rubellus field 2 7.5 15.1 9.8

263 Netherlands L. rubellus field 2 7.2 27.5 13.3

9.8

11.0

9.8

11.0

16.861 2.81
1

40.471 4.50
1

25.291 4.50
1

34.291 2.81
1

16.861 2.81
1

37.341 4.50
1

22.481 4.50
1

35.411 2.81
1

47.211 4.501 10.49

6.00

8.99

5.62

12.20

6.00

8.30

5.00

12.60

HCl+HNO3

HCl+HNO3

HCl+HNO3

HCl+HNO3

HCl+HNO3

HCl+HNO3

HCl+HNO3

HCl+HNO3

HCl+HNO3

van Vliet et al. 2005

97

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

264 Netherlands L. rubellus field 2 7.4

265 UK Ds. rubidus field 4 6.3

266 UK Ds. rubidus field 4 4.3

267 UK Ds. rubidus field 4 6.3

268 UK L. rubellus field 4 6.3

269 UK L. rubellus field 4 4.3

270 UK L. rubellus field 4 6.3

271 UK L. rubellus lab 1 7.1 41

272 UK L. rubellus lab 1 7.1 41 clay loam

11 clay loam

23.041 4.50
1

30.35 0.90 33.72

10.12 0.10 101.20

1798.58 350.00 5.14

14.61 0.90 16.23

7.87 0.10 78.70

562.06 350.00 1.61

6.741 0.20
1

67.391 19.111 3.53 70 yes HNO3 CdCl2 yes 14

5.12

33.70 70 yes HNO3

HCl+HNO3

HNO3

HNO3 mine

HNO3 mine

HNO3

HNO3 mine

HNO3 mine

van Vliet et al. 2005

Morgan & Turner 2005

Spurgeon et al. 2005

98

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

273 UK L. rubellus lab 1 7.1 41 clay loam

274 UK L. rubellus lab 1 7.1 41 clay loam

275 UK L. rubellus lab 1 7.1 41 clay loam

276 UK L. rubellus lab

277 UK L. terrestris lab 1 6.8 0.01 M CaCl2 6 sand

278 UK L. terrestris lab 1 6.6 0.01 M CaCl2 8.1 sand

279 UK L. terrestris lab 1 6.0 0.01 M CaCl2 4.5

280 UK L. terrestris lab 1 6.0 0.01 M CaCl2 14.2

281 UK L. terrestris lab 1 6.2 0.01 M CaCl2 12.9

52.181 38.22 1.37 70 yes HNO3 CdCl2 yes 14

1

52.181 159.00 0.33 70 yes HNO3 CdCl2 yes 14 Spurgeon et al. 2005
1

187.511 632.00 0.30 70 yes HNO3 CdCl2 yes 14

1

1129.00 279.00 4.05 49 HNO3 mine

5.86 2.12 2.76 12 HNO3

5.46 0.86 6.35 12 HNO3

4.54 0.70 6.49 12 HNO3

5.44 1.24 4.39 12 HNO3

4.30 0.25 17.20 12 HNO3

Sturzenbaum et al. 2004

Hankard et al. 2004

99

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

282 Poland Ap. caliginosa field 4 7.0 H2O 1:2.5

283 Poland Ap. caliginosa field 4 7.3 H2O 1:2.5

284 Poland Ap. caliginosa field 4 7.4 H2O 1:2.5

285 Poland Ap. caliginosa field 4 7.5 H 2O 1:2.5

286 Poland L. terrestris field 4 7.0 H 2O 1:2.5

287 Poland L. terrestris field 4 7.3 H2O 1:2.5

288 Poland L. terrestris field 4 7.4 H 2O 1:2.5

289 Poland L. terrestris field 4 7.5 H2O 1:2.5

290 Poland Eia. fetida field 4 7.3 H 2O 1:2.5

217.40 51.00 4.26

133.40 18.00 7.41

115.30 3.30 34.94

36.40 0.84 43.33

281.10 51.00 5.51

94.20 18.00 5.23

59.80 3.30 18.12

30.70 0.84 36.55

96.50 18.00 5.36

smelter

smelter

smelter

smelter

smelter

smelter

smelter

smelter

smelter

Laszczyca et al. 2004

100

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

291 Poland Eia. fetida field 4 7.4 H2O 1:2.5

292 Netherlands field 2

293 Netherlands field 2

294 UK L. rubellus lab 2 7.8 H2O 1:2.5 41 10.2 clay loam

295 UK L. rubellus lab 2 7.6 H2O 1:2.5 41 10.4 clay loam

296 UK L. rubellus lab 2 7.8 H2O 1:2.5 41 10.3 clay loam

297 UK L. rubellus lab 2 7.9 H2O 1:2.5 41 9.9 clay loam

298 UK L. rubellus lab 2 7.6 H 2O 1:2.5 41 10.4 clay loam

299 Belgium Eia. fetida lab 1 6.0 1 M KCl 1:2.5 20 10 14.1

20.10 3.30 6.09

16.00 2.00 8.00

3.60 0.30 12.00

1.781 0.20 8.90 294 HNO3

112.411 18.00 6.25 294 HNO3 CdCl2 yes 14

562.001 51.30 10.96 294 HNO3 CdCl2 yes 14 Spurgeon et al. 2004

1415.001 180.00 7.86 294 HNO3 CdCl2 yes 14

5634.001 906.00 6.22 294 HNO3 CdCl2 yes 14

40.001 1.00 40.00 21 HCl+HNO3 CdCl2 OECD yes Lock & Janssen 2001

HNO3+H2SO4 smelter

HNO3+H2SO4

Laszczyca et al. 2004

Van den Brink et al. 2003

101

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

300 Belgium Eia. fetida lab 1 6.0 1 M KCl 1:2.5 20 10 14.1

301 Belgium Eia. fetida lab 1 6.0 1 M KCl 1:2.5 20 10 14.1

302 Belgium Eia. fetida lab 1 6.0 1 M KCl 1:2.5 20 10 14.1

303 Belgium Eia. fetida lab 1 6.0 1 M KCl 1:2.5 20 10 14.1

304 Belgium Eia. fetida lab 1 6.0 1 M KCl 1:2.5 20 10 14.1

305 Belgium Eia. fetida lab 1 6.0 1 M KCl 1:2.5 20 10 14.1

306 Belgium Eia. fetida lab 1 6.0 1 M KCl 1:2.5 20 10 14.1

307 Belgium Eia. fetida lab 1 6.0 1 M KCl 1:2.5 20 10 14.1

308 Belgium Eia. fetida lab 1 6.0 1 M KCl 1:2.5 20 10 14.1

53.351 1.80 29.64 21 HCl+HNO3 CdCl2 OECD yes

70.731 3.20 22.10 21 HCl+HNO3 CdCl2 OECD yes

93.041 5.60 16.61 21 HCl+HNO3 CdCl2 OECD yes

123.611 10.00 12.36 21 HCl+HNO3 CdCl2 OECD yes

164.871 18.00 9.16 21 HCl+HNO3 CdCl2 OECD yes Lock & Janssen 2001

218.571 32.00 6.83 21 HCl+HNO3 CdCl2 OECD yes

287.521 56.00 5.13 21 HCl+HNO3 CdCl2 OECD yes

382.001 100.00 3.82 21 HCl+HNO3 CdCl2 OECD yes

509.501 180.00 2.83 21 HCl+HNO3 CdCl2 OECD yes

102

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

309 Belgium Eia. fetida lab 1 6.0 1 M KCl 1:2.5 20 10 14.1

310 Belgium Eia. fetida lab 1 6.0 1 M KCl 1:2.5 20 10 14.1

311 Belgium Eia. fetida lab 1 6.0 1 M KCl 1:2.5 20 10 14.1

312 Belgium & Netherlands Eia. veneta lab 3 5.2 0.01 M CaCl2 7 4.4

313 Belgium & Netherlands Eia. veneta lab 3

314 Belgium & Netherlands L. rubellus lab 3 4.4 0.01 M CaCl2

7 4.4

6.5 3.4

675.431 320.00 2.11 21 HCl+HNO3 CdCl2 OECD yes

888.531 560.00 1.59 21 HCl+HNO3 CdCl2 OECD yes Lock & Janssen 2001

1180.481 1000.00 1.18 21 HCl+HNO3 CdCl2 OECD yes

42.001 3.40 12.35 56 yes HCl+HNO3 smelter

32.001 3.40 9.41 56 yes HCl+HNO3 smelter

13.30 1.60 8.31 28 yes HCl+HNO3 smelter

Oste et al. 2001

103

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

315 Belgium & Netherlands L. rubellus lab 3 5.4 0.01 M CaCl2 6.5 3.4

316 Belgium & Netherlands L. rubellus lab 3 6.1 0.01 M CaCl2 6.5 3.4

317 Belgium & Netherlands Eia. veneta lab 3 5.3 0.01 M CaCl2 7 4.2

318 Belgium & Netherlands Eia. veneta lab 3

319 Belgium & Netherlands Eia. veneta lab 3

7 4.2

7 4.2

11.90 1.60 7.44 28 yes HCl+HNO3 smelter

14.80 1.60 9.25 21 yes HCl+HNO3 smelter

35.001 9.60 3.65 21 yes HCl+HNO3 smelter

28.001 9.60 2.92 21 yes HCl+HNO3 smelter

30.001 9.60 3.13 42 yes HCl+HNO3 smelter

Oste et al. 2001

104

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

320 Netherlands L. rubellus lab 3 5.4 0.01 M CaCl2 2 5.8

321 Netherlands Eia. veneta lab 3 4.4 0.01 M CaCl2 3 3.2

322 UK Ap. caliginosa field 4 5.2 H 2O 1:2 9.4

323 UK Ap. caliginosa field 4 6.5 H 2O 1:2 18.0

324 UK Ap. caliginosa field 4 6.5 H 2O 1:2 11.2

325 UK Ap. caliginosa field 4 6.5 H 2O 1:2 8.2

326 UK Ap. caliginosa field 4 6.8 H 2O 1:2 10.0

327 UK Ap. caliginosa field 4 6.7 H 2O 1:2 13.0

1790 4.001 0.12 33.33 35 yes HCl+HNO3 4.001 0.20 20.00 24.00 0.90 26.67

6080 90.00 3.60 25.00

7560 233.00 14.70 15.85

5320 135.00 6.50 20.77

8300 229.00 10.50 21.81

5120 344.00 17.00 20.24

HCl+HNO3

HNO3 mine

HNO3 mine

HNO3 mine

HNO3 mine

HNO3 mine

HNO3 mine

Oste et al. 2001

Morgan & Morgan 1999

105

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

328 UK Ap. caliginosa field 4 5.0 H2O 1:2 5.8

329 UK L. rubellus field 4 5.2 H2O 1:2 9.4

330 UK L. rubellus field 4 6.5 H2O 1:2 18

331 UK L. rubellus field 4 6.5 H2O 1:2 11.2

332 UK L. rubellus field 4 6.5 H 2O 1:2 8.2

333 UK L. rubellus field 4 6.8 H 2O 1:2 10

334 UK L. rubellus field 4 6.7 H 2O 1:2 13

335 UK L. rubellus field 4 5.0 H 2O 1:2 5.8

336 UK L. rubellus field 6.1 H2O 1:10 40

337 UK L. rubellus lab 6.1 H2O 1:10 40

1050 214.00 2.70 79.26

1790 12.00 0.90 13.33

6080 42.00 3.60 11.67

7560 123.00 14.70 8.37

5320 144.00 6.50 22.15

8300 57.00 10.50 5.43

5120 147.00 17.00 8.65

1050 46.00 2.70 17.04

30725 126.00 74.00 1.70

30725 79.00 74.00 1.07 90 yes HNO3 mine

HNO3 mine

HNO3 mine

HNO3 mine

HNO3 mine

HNO3 mine

HNO3 mine

HNO3 mine

HNO3 mine

HNO3 mine

Morgan & Morgan 1999

Marino & Morgan 1999a

106

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

338 UK L. rubellus lab 6.1 H2O 1:10 40

339 UK L. rubellus field 3.9 H2O 1:10 18.8

340 UK L. rubellus lab 3.9 H2O 1:10 18.8

341 UK L. rubellus field 4.9 H2O 1:10 26.9

342 UK L. rubellus lab 4.9 H2O 1:10 26.9

343 UK L. rubellus field 5.5 H2O 1:10 29.4

344 UK L. rubellus lab 5.5 H2O 1:10 29.4

345 UK L. rubellus field 6.0 H 2O 1:10 29.3

346 UK L. rubellus lab 6.0 H2O 1:10 29.3

347 UK L. rubellus lab 6.0 H2O 1:10 29.3

30725 60.00 74.00 0.81 90 yes HNO3 mine

175 11.00 0.50 22.00

175 3.00 0.50 6.00 90 yes HNO3 mine

660 209.00 3.50 59.71

660 31.00 3.50 8.86 90 yes HNO3 mine

552 27.00 0.50 54.00

552 10.00 0.50 20.00 90 yes HNO3 mine

20175 789.00 89.00 8.87

20175 199.00 89.00 2.24 90 yes HNO3 mine

20175 238.00 89.00 2.67 90 yes HNO3 mine

HNO3 mine

HNO3 mine

HNO3 mine

HNO3 mine

Marino & Morgan 1999a

Marino & Morgan 1999b

107

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

348 UK L. rubellus field 6.1 H2O 1:10 38.2

349 UK L. rubellus lab 6.1 H2O 1:10 38.2

350 UK L. rubellus lab 6.1 H2O 1:10 38.2

351 UK L. rubellus field 6.1 H2O 1:10 32

352 UK L. rubellus lab 6.1 H 2O 1:10 32

353 UK L. rubellus field 6.4 H 2O 1:10 35.6

354 UK L. rubellus lab 6.4 H 2O 1:10 35.6

355 UK L. rubellus lab 6.4 H 2O 1:10 35.6

356 UK L. rubellus field 4.1 H2O 1:10 19.9

357 UK L. rubellus field 5.8 H2O 1:10 17.8

34975 121.00 82.00 1.48

34975 79.00 82.00 0.96 90 yes HNO3 mine

34975 53.00 82.00 0.65 90 yes HNO3 mine

126000 736.00 110.50 6.66

126000 231.00 110.50 2.09 90 yes HNO3 mine

127600 539.00 117.50 4.59

127600 208.00 117.50 1.77 90 yes HNO3 mine

127600 361.00 117.50 3.07 90 yes HNO3 mine

132 13.00 0.20 65.00

2100 23.00 0.80 28.75

HNO3 mine

HNO3 mine

HNO3 mine

HNO3

HNO3

Marino & Morgan 1999b

108

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

358 South Africa Eia. fetida lab 1

359 South Africa Eia. fetida lab 1

360 South Africa Eia. fetida lab 1

361 South Africa Eia. fetida lab 1

362 South Africa Eia. fetida lab 1

363 UK L. rubellus lab 4 6.2 H2O 1:10

20 10

40

1724.00 600.00 2.87 35 yes HClO4+HNO3 CdSO4 cattle manure amended yes 2

3094.00 1200.00 2.58 35 yes HClO4+HNO3 CdSO4 cattle manure amended yes 2

916.00 600.00 1.53 35 HClO4+HNO3 CdSO4 cattle manure amended yes 2 Reinecke et al. 1999

1383.00 1200.00 1.15 35 HClO4+HNO3 CdSO4 cattle manure amended yes 2

1598.00 336.00 4.76 960 HClO4+HNO3 CdSO4 cattle manure+OECD yes

220.00 280.00 0.79 28 yes HNO3 mine

Marino et al. 1998

109

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

364 UK L. rubellus lab 4 6.2 H2O 1:10 40

365 UK L. rubellus field 4 6.2 H2O 1:10 40

366 UK L. rubellus lab 4 6.2 H2O 1:10 40

367 UK L. rubellus lab 4 6.3 H 2O 1:10 38

368 UK L. rubellus lab 4 6.3 H2O 1:10 38

369 UK L. rubellus field 4 6.3 H2O 1:10 38

370 UK L. rubellus lab 4 6.3 H2O 1:10 38

371 UK L. rubellus field 4 6.0 H2O 1:10 16

372 UK Ap. caliginosa field 4 5.2 H2O 1:10 4.7

373 UK Ap. caliginosa field 4 6.8 H2O 1:10 6.5

1791 278.00 280.00 0.99 28 yes HNO3 mine 364.00 280.00 1.30 1149.00 280.00 4.10 28 yes HNO3 mine 168.00 350.00 0.48 28 yes HNO3 mine 196.00 350.00 0.56 28 yes HNO3 mine 575.00 350.00 1.64 1288.00 350.00 3.68 28 yes HNO3 mine 6.00 0.60 10.00 29.00 0.90 32.22

5083 339.00 19.00 17.84

HNO3 mine

HNO3 mine

HNO3

HNO3

HNO3 mine

Marino et al. 1998

Morgan & Morgan 1998

110

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

374 Netherlands Da. veneta lab 3 5.4 0.01 M CaCl2 1:10 7 3.0 sandy loam

375 Netherlands Da. veneta lab 3 5.4 0.01 M CaCl2 1:10 7 3.0 sandy loam

376 Netherlands Da. veneta lab 3 5.4 0.01 M CaCl2 1:10 7 3.0 sandy loam

377 Netherlands Da. veneta lab 3 5.4 0.01 M CaCl2 1:10 7 3.0 sandy loam

378 France Eia. fetida lab 6.8

379 France Eia. fetida lab 6.8

380 Netherlands Ap. caliginosa lab 2 6.5 CaCl2 8.6 light clay 2550

381 Netherlands Ap. caliginosa field 2 4.6 CaCl2 5.3 sand 950 56.251 2.04 27.57

5.621 0.45 12.49 21 HNO3

11.241 3.37 3.34 21 HNO3 Cd(NO3)2 yes 270

17.991 6.74 2.67 21 HNO3 Cd(NO3)2 yes 270

30.351 12.37 2.45 21 HNO3 Cd(NO3)2 yes 270

55.601 8.00 6.95 56

215.001 80.00 2.69 56

141.001 582.00 0.24 28 yes HNO3

HNO3

CdCl2 compost yes

CdCl2 compost yes

Cd acetate yes 70 vijver et al. 2006

Marinussen et al. 1997

Scaps et al.1997

111

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

382 UK Eia. fetida lab 1 6.1 H2O 20 10

383 UK Eia. fetida lab 1 6.1 H2O 20 10

384 UK Eia. fetida lab 1 6.1 H2O 20 10

385 Netherlands L. rubellus lab 2 7.3 0.01 M CaCl2 25 18

386 Canada L. terrestris lab 3 6.1 H2O 1:1 9.3 23.1

387 Canada L. terrestris lab 3 7.8 H2O 1:1 13.3

388 USA Ap. tuberculata field 1 6.5

389 USA Ap. tuberculata field 1 5.1

silt loam 7 7600 0.011 0.05 0.25 42 HNO3 OECD 0.381 4.60 0.08 42 HNO3 Cd(NO3)2 OECD yes 7 1.271 13.70 0.09 42 HNO3 Cd(NO3)2 OECD yes 7 Vijver et al. 2005 Kennette et al. 2002 13.50 12.97 1.04 14 HNO3 5.14 0.20 25.70 14 HClO4+HNO3 3.38 2.50 1.35 21 HClO4+HNO3 6.70 0.29 23.10

silt loam

129.00 8.40 15.36

HNO3

HNO3

Spurgeon & Hopkin 1999

Neuhauser et al. 1995

112

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

390 USA Ap. tuberculata field 1 4.9

391 USA Ap. tuberculata lab 1 5.1

392 USA Ap. tuberculata lab 1 4.9

393 USA Ap. tuberculata lab 1 6.5

394 USA Ap. tuberculata lab 1 6.5

395 Netherlands Eia. andrei lab 1 3.4 CaCl2 3.61

396 Netherlands Eia. andrei lab 1 5.7 CaCl2 22.41 44.71

397 Netherlands Eia. andrei lab 1 5.4 CaCl2 11.21 31.61

silt loam

silt loam

silt loam

silt loam

silt loam 4.01

179.00 23.00 7.78

54.00 8.40 6.43 112 yes HNO3

217.00 23.00 9.43 112 yes HNO3

67.00 0.29 231.03 112 yes HNO3

108.00 0.29 372.41 112 yes HNO3

5.17 0.04
1

5.96 0.37 16.11

5.06 0.36 14.06

143.61

HNO3

HNO3

HNO3

HNO3

Neuhauser et al. 1995

Peijnenburg et al. 1999

113

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

398 Netherlands Eia. andrei lab 1 7.2 CaCl2 3.21 12.61

399 Netherlands Eia. andrei lab 1 6.5 CaCl2 5.01 101

400 Netherlands Eia. andrei lab 1 6.5 CaCl2 8.91 17.81

401 Netherlands Eia. andrei lab 1 6.8 CaCl2 101 201

402 Netherlands Eia. andrei lab 1 3.5 CaCl2 2.81 2.51

403 Netherlands Eia. andrei lab 1 3.0 CaCl2 4.01 4.51

404 Netherlands Eia. andrei lab 1 5.0 CaCl2 6.31 7.91

405 Netherlands Eia. andrei lab 1 3.8 CaCl2 3.61 2.81

406 Netherlands Eia. andrei lab 1 6.8 CaCl2 8.911 201

25.851 3.59 7.20

39.201 7.31 5.36 63 HNO3

46.281 16.86 2.74 63 HNO3

134.891 10.68 12.63

14.411 1.46 9.87 63 HNO3

5.85 1.05 5.57

500.221 49.46 10.11 63 HNO3

3.93 0.12 32.75

10.45 0.92 11.36

HNO3

HNO3

HNO3

HNO3

HNO3

Peijnenburg et al. 1999

114

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

407 Netherlands Eia. andrei lab 1 3.8 CaCl2 2.51 5.01

408 Netherlands Eia. andrei lab 1 3.9 CaCl2 21 1.81

409 Netherlands Eia. andrei lab 1 5.3 CaCl2 5.01 11.21

410 Netherlands Eia. andrei lab 1 6.8 CaCl2 3.21 101

411 Netherlands Eia. andrei lab 1 6.7 CaCl2 12.61 35.51

412 Netherlands Eia. andrei lab 1 6.9 CaCl2 7.91 28.21

413 Netherlands Eia. andrei lab 1 7.0 CaCl2 5.61 17.81

414 Netherlands Eia. andrei lab 1 7.0 CaCl2 6.31 15.91

415 Netherlands Eia. andrei lab 1 4.9 CaCl2 20 101

5.17 0.04 129.25

5.40 0.04 135.00

337.231 13.49 25.00

32.601 6.07 5.37

7.421 0.92 8.07

34.851 5.06 6.89

31.481 0.73 43.12

40.471 15.74 2.57

4.83 0.02 241.50

HNO3

HNO3

HNO3

HNO3

HNO3

HNO3

HNO3

HNO3

HNO3 OECD

Peijnenburg et al. 1999

115

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

416 UK L. rubellus field 4 4.3 H2O 1:10

417 UK L. rubellus field 4 5.2 H2O 1:10

418 UK L. rubellus field 4 6.8 H2O 1:10

419 UK L. rubellus field 4 7.1 H 2O 1:10

420 UK L. rubellus field 4 6.5 H 2O 1:10

421 UK Ap. caliginosa field 4 6.8 H2O 1:10

422 UK L. rubellus field 4 6.8 H 2O 1:10

423 UK L. rubellus field 4 6.8 H 2O 1:10

424 UK Ap. caliginosa field 4 6.8 H 2O 1:10

425 UK L. rubellus field 4 6.8 H2O 1:10

168 9.30 0.10 93.00

1791 12.30 0.90 13.67

5083 169.00 19.00 8.89

31277 100.00 157.00 0.64

26421 811.50 347.00 2.34

5083 360.00 17.00 21.18

5083 120.00 17.00 7.06

5083 180.00 17.00 10.59

5083 330.00 19.00 17.37

5083 100.00 19.00 5.26

HNO3 mine

HNO3 mine

HNO3 mine

HNO3 mine

HNO3 mine

HNO3 mine

HNO3 mine

HNO3 mine

HNO3 mine

HNO3 mine

Morgan and Morgan 1990

Morgan and Morgan 1993

116

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

426 UK Ap. caliginosa field 4 6.8 H2O 1:10

427 UK L. rubellus field 4 6.8 H2O 1:10

428 UK Ap. caliginosa field 4 6.8 H2O 1:10

429 UK L. rubellus field 4 6.8 H 2O 1:10

430 UK Ap. caliginosa field 4 6.8 H 2O 1:10

431 UK L. rubellus field 4 6.8 H 2O 1:10

432 UK Ap. caliginosa field 4 6.8 H 2O 1:10

433 UK L. rubellus field 4 6.8 H 2O 1:10

434 UK Ap. caliginosa field 4 6.8 H2O 1:10

5083 180.00 19.00 9.47

5083 170.00 19.00 8.95

5083 410.00 19.00 21.58

5083 140.00 19.00 7.37

5083 330.00 19.00 17.37

5083 100.00 19.00 5.26

5083 380.00 21.00 18.10

5083 170.00 21.00 8.10

5083 330.00 22.00 15.00

HNO3 mine

HNO3 mine

HNO3 mine

HNO3 mine

HNO3 mine

HNO3 mine

HNO3 mine

HNO3 mine

HNO3 mine

Morgan and Morgan 1993

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

435 UK L. rubellus field 4 6.8 H2O 1:10

436 UK L. rubellus field 4 6.8 H2O 1:10

437 UK Ap. caliginosa field 4 6.8 H2O 1:10

438 UK L. rubellus field 4 6.8 H 2O 1:10

439 UK Ap. caliginosa field 4 6.8 H2O 1:10

440 UK Ap. caliginosa field 4 6.8 H2O 1:10

441 UK L. rubellus field 4 6.8 H 2O 1:10

442 UK Ap. caliginosa field 4 6.8 H2O 1:10

443 UK L. rubellus field 4 6.8 H2O 1:10

5083 120.00 22.00 5.45

5083 150.00 23.00 6.52

5083 340.00 23.00 14.78

5083 120.00 23.00 5.22

5083 380.00 23.00 16.52

5083 389.00 23.00 16.91

5083 190.00 23.00 8.26

5083 370.00 24.00 15.42

5083 190.00 24.00 7.92

HNO3 mine

HNO3 mine

HNO3 mine

HNO3 mine

HNO3 mine

HNO3 mine

HNO3 mine

HNO3 mine

HNO3 mine

Morgan and Morgan 1993

117

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

444 UK L. rubellus field 4 6.8 H2O 1:10

445 UK Ap. caliginosa field 4 6.8 H2O 1:10

446 Spain L. friendi

447 Spain Da. octaedra

448 Spain Ap. caliginosa

449 Spain L. friendi

450 Spain Ap. rosea

451 Spain Da. octaedra

5083 140.00 25.00 5.60

5083 410.00 25.00 16.40 0.79 0.15 5.27 1.06 0.15 7.07 0.86 0.15 5.73 0.76 0.15 5.07 1.70 0.15 11.33 0.86 0.15 5.73

HNO3 mine

HNO3 mine

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

Morgan and Morgan 1993

Marino et al. 1992

118

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

452 Spain Ap. caliginosa

453 Spain D. madeirensis

454 Spain Ap. caliginosa

455 Spain Ap. rosea

456 Spain Ap. rosea

457 Spain Ap. caliginosa

458 Spain Da. octaedra

459 Spain L. friendi

0.83 0.15 5.53

2.48 0.24 10.33

1.18 0.24 4.92

1.27 0.24 5.29

2.00 0.24 8.33

1.34 0.24 5.58

0.92 0.24 3.83

0.78 0.24 3.25

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

Marino et al. 1992

119

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

460 Spain L. friendi

461 Spain D. madeirensis

462 Spain Ap. rosea

463 Spain Ap. caliginosa

464 Spain Ap. caliginosa

465 Spain Ap. rosea

466 Spain D. madeirensis

467 Spain L. friendi

0.98 0.35 2.80

1.57 0.35 4.49

1.06 0.35 3.03

1.30 0.35 3.71

1.14 0.35 3.26

1.98 0.35 5.66

0.74 0.35 2.11

1.22 0.35 3.49

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

Marino et al. 1992

120

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

468 Spain L. friendi

469 Spain Ap. rosea

470 Spain Ap. caliginosa

471 Spain Da. octaedra

472 Spain Ap. caliginosa

473 Spain Ap. rosea

474 Spain L. friendi

475 Spain D. madeirensis

1.14 0.37 3.08

3.25 0.37 8.78

1.79 0.37 4.84

0.59 0.37 1.59

1.33 0.37 3.59

0.22 0.37 0.59

0.89 0.37 2.41

1.69 0.37 4.57

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

Marino et al. 1992

121

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

476 Spain D. madeirensis

477 Spain Ap. caliginosa

478 Spain Da. octaedra

479 Spain L. friendi

480 Spain D. madeirensis

481 Spain D. madeirensis

482 Spain Ap. rosea

483 Spain Ap. caliginosa

2.06 0.37 5.57

1.09 0.39 2.79

0.32 0.39 0.82

1.09 0.39 2.79

1.85 0.39 4.74

1.25 0.39 3.21

1.80 0.39 4.62

1.01 0.39 2.59

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

Marino et al. 1992

122

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

484 Spain L. friendi

485 Spain Ap. rosea

486 Spain Ap. caliginosa

487 Spain Ap. rosea

488 Spain L. friendi

489 Spain Ap. caliginosa

490 Spain L. friendi

491 Spain D. madeirensis

0.63 0.39 1.62

3.21 0.43 7.47

1.32 0.43 3.07

2.90 0.43 6.74

0.39 0.43 0.91

1.52 0.43 3.53

0.33 0.43 0.77

2.63 0.43 6.12

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

Marino et al. 1992

123

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

492 Spain L. friendi

493 Spain Ap. caliginosa

494 Spain Da. octaedra

495 Spain D. madeirensis

496 Spain L. friendi

497 Spain D. madeirensis

498 Spain L. friendi

499 Spain D. madeirensis

0.63 0.44 1.45

1.16 0.44 2.67

1.56 0.44 3.59

3.49 0.44 8.02

1.19 0.44 2.74

1.53 0.44 3.52

0.80 0.44 1.84

1.76 0.44 4.05

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

Marino et al. 1992

124

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

500 Spain L. friendi

501 Spain Da. octaedra

502 Spain Da. octaedra

503 Spain Da. octaedra

504 Spain Ap. caliginosa

505 Spain D. madeirensis

506 Spain Ap. caliginosa

507 Spain Ap. caliginosa

0.72 0.44 1.66

1.53 0.44 3.52

4.03 0.44 9.26

0.83 0.44 1.91

1.40 0.44 3.22

1.52 0.44 3.49

1.50 0.44 3.45

1.72 0.44 3.95

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

Marino et al. 1992

125

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

508 Spain Da. octaedra

509 Spain Ap. caliginosa

510 Spain L. friendi

511 Spain D. madeirensis

512 Spain Ap. caliginosa

513 Spain L. friendi

514 USA

515 USA

6.0

2.0

1173 1.37 0.66 2.08 1.00 0.66 1.52 1.14 0.66 1.73 3.42 0.66 5.18 1.17 0.66 1.77 0.85 0.66 1.29 1.40 0.29 4.83 1.57 0.29 5.41

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HClO4+HNO3

HCl+HNO3

HCl+HNO3

Marino et al. 1992

Efroymson et al. 1996

126

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

516 USA

517 USA

518 USA

519 USA

520 USA

521 USA

522 USA

523 USA

524 USA

6.8

7.6

6.8

6.9

6.9

7.0

1.9 7.6 833 13.70 0.31 44.19 3.20 0.54 5.93 6850 5.00 0.78 6.41 3870 2.40 1.00 2.40 5570 1.32 1.10 1.20 3180 2.50 1.20 2.08

2.3

6.7

3.0

1363 6.93 1.34 5.17

2718 11.90 4.50 2.64

3275 12.20 10.33 1.18

HCl+HNO3

HCl+HNO3

HCl+HNO3

HCl+HNO3

HCl+HNO3

HCl+HNO3

HCl+HNO3

HCl+HNO3

HCl+HNO3

Efroymson et al. 1996

127

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

525 USA

526 USA

527 USA

528 Germany L. rubellus field 3 3.8 0.01 M CaCl2 1:2.5 silt loam

529 Germany L. terrestris field 3 4.8 0.01 M CaCl2 1:2.5 silt loam

530 Germany Ap. caliginosa field 3 4.8 0.01 M CaCl2 1:2.5 silt loam

531 Germany L. terrestris field 3 4.8 0.01 M CaCl2 1:2.5 silt loam

532 Germany Ap. caliginosa field 3 4.8 0.01 M CaCl2 1:2.5 silt loam

533 Germany L. rubellus field 3 3.8 0.01 M CaCl2 1:2.5 silt loam

6.9

3.2

3423 12.80 10.50 1.22

28400 4.05 16.00 0.25

40600 24.40 30.00 0.81 18.85 0.50 37.70 119.32 1.90 62.80 84.17 1.90 44.30 57.90 1.50 38.60 68.70 1.50 45.80 23.52 1.20 19.60

HCl+HNO3

HCl+HNO3

HCl+HNO3

Efroymson et al. 1996

Emmerling et al. 1997

128

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

534 Germany L. terrestris field 3 4.8 0.01 M CaCl2 1:2.5 silt loam

535 Germany Ap. caliginosa field 3 4.8 0.01 M CaCl2 1:2.5 silt loam

536 Germany L. terrestris field 3 4.8 0.01 M CaCl2 1:2.5 silt loam

537 Germany Ap. caliginosa field 3 4.8 0.01 M CaCl2 1:2.5 silt loam

538 Germany L. rubellus field 3 3.7 0.01 M CaCl2 1:2.5

539 Germany L. terrestris field 3 5.0 0.01 M CaCl2 1:2.5 silt loam

540 Germany Ap. caliginosa field 3 5.0 0.01 M CaCl2 1:2.5 silt loam

541 Germany L. terrestris field 3 5.0 0.01 M CaCl2 1:2.5 silt loam

66.22 1.10 60.20

94.49 1.10 85.90

37.35 0.90 41.50

31.05 0.90 34.50

10.14 0.30 33.80

16.50 0.60 27.50

13.14 0.60 21.90

11.28 0.80 14.10

Emmerling et al. 1997

129

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

542 Germany Ap. caliginosa field 3 5.0 0.01 M CaCl2 1:2.5 silt loam

543 Germany L. rubellus field 3 3.7 0.01 M CaCl2 1:2.5

544 Germany L. terrestris field 3 5.0 0.01 M CaCl2 1:2.5 silt loam

545 Germany Ap. caliginosa field 3 5.0 0.01 M CaCl2 1:2.5 silt loam

546 Germany Ap. caliginosa field 3 5.0 0.01 M CaCl2 1:2.5 silt loam

547 USA L. rubellus field 0 7.6

548 USA L. rubellus field 0 7.6

549 USA L. rubellus field 0 7.7

6.5

6.1

6.5

20.32 0.80 25.40

8.16 0.30 27.20

14.56 0.70 20.80

17.85 0.70 25.50

17.90 0.50 35.80

59.00 13.00 4.54

72.00 14.00 5.14

91.00 15.00 6.07

HCl+HNO3

HCl+HNO3

HCl+HNO3

Emmerling et al. 1997

Beyer and Stafford 1993

130

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

550 USA Ela. tetraedra field 0 7.9

551 USA L. rubellus field 0 7.7

552 USA Ap. trapezoides field 0 7.9

553 USA Ap. trapezoides field 0 8.0

554 USA field 0 7.9

555 USA field 0 8.0

556 USA field 0 7.6

557 USA field 0 7.6

558 USA Ap. tuberculata field 0 7.9

5.3

6.5

6.0

2.0

12.3

11.0

14.8

12.2

4.8

36.00 14.00 2.57

26.00 4.90 5.31

10.00 4.60 2.17

16.00 3.50 4.57

52.00 11.00 4.73

23.00 10.00 2.30

85.00 31.00 2.74

85.00 32.00 2.66

15.00 2.60 5.77

HCl+HNO3

HCl+HNO3

HCl+HNO3

HCl+HNO3

HCl+HNO3

HCl+HNO3

HCl+HNO3

HCl+HNO3

HCl+HNO3

Beyer and Stafford 1993

131

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

559 USA Ap. tuberculata field 0 8.1

560 UK L. terrestris field 4 6.5 H2O 5.6

561 UK L. terrestris field 4 6.5 H2O 5.6

562 UK L. terrestris field 4 6.5 H2O 5.6

563 UK L. terrestris field 4 6.5 H2O 5.6

564 UK L. terrestris field 4 6.5 H2O 5.6

565 UK L. terrestris field 4 6.5 H 2O 4.1

566 UK L. terrestris field 4 6.5 H 2O 4.1

5.1

24.00 3.60 6.67

117.00 14.70 7.96

123.00 14.70 8.37

80.00 14.70 5.44

223.00 14.70 15.17

164.00 14.70 11.16

78.00 6.50 12.00

144.00 6.50 22.15

HCl+HNO3 mine mine mine mine mine mine mine

Beyer and Stafford 1993

Morgan and Morgan 1992

132

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

567 UK L. terrestris field 4 6.5 H2O 4.1

568 UK L. terrestris field 4 6.5 H2O 4.1

569 UK L. terrestris field 4 6.5 H 2O 4.1

570 UK L. terrestris field 4 6.8 H2O 5.0

571 UK L. terrestris field 4 6.8 H2O 5.0

572 UK L. terrestris field 4 6.8 H 2O 5.0

573 UK L. terrestris field 4 6.8 H 2O 5.0

574 UK L. terrestris field 4 6.8 H2O 5.0

575 UK L. terrestris field 4 6.7 H2O 6.5

76.00 6.50 11.69

135.00 6.50 20.77

145.00 6.50 22.31

44.00 10.50 4.19

57.00 10.50 5.43

102.00 10.50 9.71

229.00 10.50 21.81

261.00 10.50 24.86

147.00 17.00 8.65

mine

mine

mine

mine

mine

mine

mine

mine

mine

Morgan and Morgan 1992

133

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

576 UK L. terrestris field 4 6.7 H2O 6.5

577 UK L. terrestris field 4 5.0 H2O 2.9

578 UK L. terrestris field 4 5.0 H2O 2.9

579 UK L. terrestris field 4 5.0 H2O 2.9

580 UK Ds. rubidus field 3 6.0 H2O 1:2

581 UK Ds. rubidus field 3 5.5 H2O 1:2

582 UK Ds. rubidus field 3 6.5 H2O 1:2

583 France Eia. fetida lab 7.3 H2O 2.0

584 France Eia. fetida lab 7.3 H2O 11.6

344.00 17.00 20.24

46.00 2.70 17.04

214.00 2.70 79.26

122.00 2.70 45.19

30.01 0.90 33.38

12.03 1.24 9.73

1190.08 350.04 3.40

27.00 13.00 2.08 24

56.00 89.00 0.63 24

HNO3 mine mine mine mine

HNO3 mine

HNO3 mine smelter smelter

Morgan and Morgan 1992

Morgan et al. 2002

Grelle and Descamps 1998

134

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

585 France Eia. fetida lab 7.5 H2O 7.1

586 France Eia. fetida lab 6.6 H2O 8.2

587 France Eia. fetida lab 7.3 H2O 2.0

588 France Eia. fetida lab 7.3 H 2O 11.6

589 France Eia. fetida lab 7.5 H 2O 7.1

590 France Eia. fetida lab 6.6 H 2O 8.2

591 UK L. rubellus field 4 6.2 1 M KCl 32 186

592 UK L. rubellus field 4 5.9 1 M KCl 30 174

593 UK L. rubellus field 4 6.0 1 M KCl 37 215

594 USA Eia. fetida lab 0.25 7.0 1 M KCl 16 4.6 18.1 2750

74.00 29.00 2.55 24

58.00 24.00 2.42 24

34.00 13.00 2.62 31

60.00 89.00 0.67 31

92.00 29.00 3.17 31

66.00 24.00 2.75 31

730.67 100.04 7.30

948.74 89.93 10.55

125.90 79.81 1.58

7.72 22.70 0.34

HNO3 smelter smelter smelter smelter smelter smelter mine mine mine mine

Grelle and Descamps 1998

Marino et al. 1999

Maenpaa et al. 2002

135

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

595 USA Eia. fetida lab 0.25 5.5 1 M KCl 16 4.6 18.1 2750 6.01 23.10 0.26

596 USA Eia. fetida lab 0.25 6.0 1 M KCl 16 4.6 18.1 2750 2.74 21.10 0.13

597 USA Eia. fetida lab 0.25 6.4 1 M KCl 16 4.6 18.1 2750 6.33 22.60 0.28

598 USA Eia. fetida lab 0.25 6.2 1 M KCl 16 4.6 18.1 2750 3.12 22.30 0.14

599 USA Eia. fetida lab 0.25 6.8 1 M KCl 14 6.3 38 4050 6.34 26.40 0.24

600 USA Eia. fetida lab 0.25 6.0 1 M KCl 14 6.3 38 4050 7.05 24.30 0.29

601 USA Eia. fetida lab 0.25 6.0 1 M KCl 14 6.3 38 4050 6.05 24.20 0.25

HNO3 mine P2O5 amended

HNO3 mine P2O5 amended

HNO3 mine KH2PO4 amended

HNO3 mine KH2PO4 amended

HNO3 mine

HNO3 mine P2O5 amended

HNO3 mine P2O5 amended

Maenpaa et al. 2002

136

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

602 USA Eia. fetida lab 0.25 6.6 1 M KCl 14 6.3 38 4050 9.83 28.90 0.34

603 USA Eia. fetida lab 0.25 6.1 1 M KCl 14 6.3 38 4050 7.48 25.80 0.29

604 Netherlands Ap. caliginosa field 7.1 1 M KCl 30 5.8 clay loam 26.3

605 Netherlands Ap. caliginosa field 7.0 1 M KCl 30 6.7 clay loam 24.5

606 Netherlands Ap. caliginosa field 6.9 1 M KCl 30 8.4 clay loam 25.1

607 Netherlands Ap. caliginosa field 6.6 1 M KCl 10 2.8 sandy loam 9.4

608 Netherlands Ap. caliginosa field 7.0 1 M KCl 10 4.0 sandy loam 10.5

12.70 1.03 12.30

14.20 1.33 10.70

16.10 1.42 11.30

15.00 0.57 26.20

22.40 0.74 30.40

HNO3 mine KH2PO4 amended

HNO3 mine KH2PO4 amended

Maenpaa et al. 2002

Ma 1982

137

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

609 Netherlands Ap. caliginosa field 7.0 1 M KCl 10 4.9 sandy loam 12.3

610 Netherlands Ap. caliginosa field 5.3 1 M KCl 40 6.9 clay loam 26.4

611 Netherlands Ap. caliginosa field 5.8 1 M KCl 40 9.2 clay loam 28.7

612 Netherlands Ap. caliginosa field 5.9 1 M KCl 40 9.7 clay loam 28.7

613 Netherlands Ap. caliginosa field 4.7 1 M KCl 12.4 sand 20.5

614 Netherlands Ap. caliginosa field 5.2 1 M KCl 11.2 sand 19.2

615 Netherlands Ap. caliginosa field 5.8 1 M KCl 13.6 sand 18.3

616 Netherlands Ap. caliginosa field 5.4 1 M KCl 6.4 sand 13.5

17.60 1.01 17.50

16.80 1.10 15.30

20.90 1.54 13.60

31.80 2.00 15.90

25.80 0.23 112.30

34.40 0.56 60.90

33.30 0.82 40.50

20.90 0.26 79.50

Ma 1982

138

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

617 Netherlands Ap. caliginosa field 5.4 1 M KCl 7.4 sand 12.7

618 Netherlands Ap. caliginosa field 5.7 1 M KCl 8.1 sand 23.2

619 Netherlands Ap. caliginosa field 4.8 1 M KCl 2.8 5.3

620 Netherlands Ap. caliginosa field 5.5 1 M KCl 3.7 6.1

621 Netherlands Ap. caliginosa field 6.0 1 M KCl 4.3 7.1

622 Netherlands Ap. caliginosa field 7.1 1 M KCl 30 5.8 clay loam 26.3

623 Netherlands Ap. caliginosa field 7.0 1 M KCl 30 6.7 clay loam 24.5

624 Netherlands Ap. caliginosa field 6.9 1 M KCl 30 8.4 clay loam 25.1

24.30 0.57 42.60

25.00 0.81 30.70

30.60 0.22 140.00

31.80 0.54 59.40

25.00 0.62 40.50

13.80 0.99 13.90

11.20 1.40 8.00

13.10 1.54 8.50

Ma 1982

139

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

625 Netherlands Ap. caliginosa field 6.6 1 M KCl 10 2.8 sandy loam 9.4

626 Netherlands Ap. caliginosa field 7.0 1 M KCl 10 4.0 sandy loam 10.5

627 Netherlands Ap. caliginosa field 7.0 1 M KCl 10 4.9 sandy loam 12.3

628 Netherlands Ap. caliginosa field 5.3 1 M KCl 40 6.9 clay loam 26.4

629 Netherlands Ap. caliginosa field 5.8 1 M KCl 40 9.2 clay loam 28.7

630 Netherlands Ap. caliginosa field 5.9 1 M KCl 40 9.7 clay loam 28.7

631 Netherlands Ap. caliginosa field 4.7 1 M KCl 12.4 sand 20.5

632 Netherlands Ap. caliginosa field 5.2 1 M KCl 11.2 sand 19.2

11.20 0.60 18.60

13.10 0.74 17.60

12.00 1.03 11.70

13.50 1.10 12.30

10.80 1.59 6.80

19.10 2.15 8.90

27.70 0.23 120.00

23.90 0.57 42.10

Ma 1982

140

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

633 Netherlands Ap. caliginosa field 5.8 1 M KCl 13.6 sand 18.3

634 Netherlands Ap. caliginosa field 5.4 1 M KCl 6.4 sand 13.5

635 Netherlands Ap. caliginosa field 5.4 1 M KCl 7.4 sand 12.7

636 Netherlands Ap. caliginosa field 5.7 1 M KCl 8.1 sand 23.2

637 Netherlands Ap. caliginosa field 4.8 1 M KCl 2.8 5.3

638 Netherlands Ap. caliginosa field 5.5 1 M KCl 3.7 6.1

639 Netherlands Ap. caliginosa field 6.0 1 M KCl 4.3 7.1

640 Netherlands Eia. andrei lab 0.5 5.7 0.01 M CaCl2 1:10 22.4

20.90 0.80 26.10

20.20 0.26 77.00

23.20 0.56 41.80

22.40 0.81 27.70

34.40 0.22 156.40

23.20 0.54 43.30

19.10 0.64 29.70

5.40 0.37 14.70 21 HNO3

Ma 1982

Janssen et al. 1997

141

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

641 Netherlands Eia. andrei lab 0.5 5.4 0.01 M CaCl2 1:10 11.2

642 Netherlands Eia. andrei lab 0.5 7.2 0.01 M CaCl2 1:10 3.2

643 Netherlands Eia. andrei lab 0.5 6.5 0.01 M CaCl2 1:10 5.0

644 Netherlands Eia. andrei lab 0.5 6.5 0.01 M CaCl2 1:10 8.9

645 Netherlands Eia. andrei lab 0.5 6.8 0.01 M CaCl2 1:10 10.0

646 Netherlands Eia. andrei lab 0.5 3.5 0.01 M CaCl2 1:10 2.8

647 Netherlands Eia. andrei lab 0.5 3.0 0.01 M CaCl2 1:10 4.0

648 Netherlands Eia. andrei lab 0.5 5.0 0.01 M CaCl2 1:10 6.3

649 Netherlands Eia. andrei lab 0.5 3.8 0.01 M CaCl2 1:10 3.6

7.42 0.36 20.60 21 HNO3

16.07 2.64 6.10 21 HNO3

24.96 7.34 3.40 21 HNO3

23.16 16.54 1.40 21 HNO3

29.11 10.78 2.70 21 HNO3

12.14 1.52 8.00 21 HNO3

10.90 1.05 10.40 21 HNO3

173.00 49.43 3.50 21 HNO3

4.38 0.12 35.90 21 HNO3

Janssen et al. 1997

142

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

650 Netherlands Eia. andrei lab 0.5 3.8 0.01 M CaCl2 1:10 2.5

651 Netherlands Eia. andrei lab 0.5 3.9 0.01 M CaCl2 1:10 2.0

652 Netherlands Eia. andrei lab 0.5 5.3 0.01 M CaCl2 1:10 5.0

653 Netherlands Eia. andrei lab 0.5 6.8 0.01 M CaCl2 1:10 3.2

654 Netherlands Eia. andrei lab 0.5 6.7 0.01 M CaCl2 1:10 12.6

655 Netherlands Eia. andrei lab 0.5 6.9 0.01 M CaCl2 1:10 7.9

656 Netherlands Eia. andrei lab 0.5 7.0 0.01 M CaCl2 1:10 5.6

657 Netherlands Eia. andrei lab 0.5 7.0 0.01 M CaCl2 1:10 6.3

8.21 0.04 202.80 21 HNO3

8.09 0.04 201.60 21 HNO3

109.49 14.04 7.80 21 HNO3

7.19 6.00 1.20 21 HNO3

11.58 0.92 12.60 21 HNO3

25.07 5.01 5.00 21 HNO3

25.74 7.35 3.50 21 HNO3

32.26 16.13 2.00 21 HNO3

Janssen et al. 1997

143

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

658 USA Eia. andrei lab 1 4.1 H2O 1:1 0.6 loam 6.7

659 USA Eia. andrei lab 0.5 7.8 H2O 1:1 6.0 loam 30.5

660 USA Eia. andrei lab 0.5 4.8 H 2O 1:1 3.8 9.8

661 USA Eia. andrei lab 0.5 5.5 H2O 1:1 1.6 14.6

662 USA Eia. andrei lab 0.5 4.7 H2O 1:1 2.4 loam 3.3

663 USA Eia. andrei lab 0.5 3.9 H2O 1:1 2.4 loam 4.6

664 USA Eia. andrei lab 0.5 6.5 H2O 1:1 3.2 loam 16.3

665 USA Eia. andrei lab 0.5 4.4 H2O 1:1 2.4 4.8

666 USA Eia. andrei lab 0.5 4.7 H2O 1:1 2.9 14.0

541.15 269.88 2.01 28

440.66 306.10 1.44 28

597.57 312.38 1.91 28

449.89 314.65 1.43 28

908.33 300.48 3.02 28

77.43 288.03 0.27 28

466.26 292.55 1.59 28

764.90 314.58 2.43 28

538.18 303.30 1.77 28

yes

yes

yes

yes

yes Bradham 2003

yes

yes

yes

yes

144

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

667 USA Eia. andrei lab 1 6.8 H2O 1:1 4.0 32.4

668 USA Eia. andrei lab 1 7.6 H2O 1:1 3.0 loam 16.5

669 USA Eia. andrei lab 1 7.5 H2O 1:1 1.1 loam 11.7

670 USA Eia. andrei lab 1 6.0 H2O 1:1 5.2 28.3

671 USA Eia. andrei lab 1 5.7 H2O 1:1 4.0 27.5

672 USA Eia. andrei lab 1 3.9 H2O 1:1 1.7 loam 3.0

673 USA Eia. andrei lab 1 4.4 H2O 1:1 3.8 silt 10.7

674 USA Eia. andrei lab 1 5.6 H 2O 1:1 1.6 silt 12.5

675 USA Eia. andrei lab 1 5.2 H2O 1:1 1.8 sand 4.4

676 USA Eia. andrei lab 1 5.1 H2O 1:1 1.0 sand 3.4

339.06 325.83 1.04 28

318.50 290.75 1.10 28

330.73 308.68 1.07 28

705.27 290.48 2.43 28

383.10 305.58 1.25 28

365.93 294.60 1.24 28

673.76 284.95 2.36 28

866.81 306.00 2.83 28

846.21 308.30 2.74 28

641.46 324.15 1.98 28

yes

yes

yes

yes

yes

yes

yes

yes

yes

yes

Bradham 2003

145

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

677 USA Eia. andrei lab 1 6.7 H2O 1:1 2.2 22.3

678 USA Eia. andrei lab 1 7.0 H2O 1:1 4.8 29.4

679 USA Eia. andrei lab 1 6.5 H2O 1:1 2.5 27.6

680 USA Eia. andrei lab 1 4.2 H 2O 1:1 0.6 loam 6.7

681 USA Eia. andrei lab 1 7.8 H 2O 1:1 6.0 loam 30.5

682 USA Eia. andrei lab 1 4.8 H 2O 1:1 3.8 9.8

683 USA Eia. andrei lab 1 5.6 H 2O 1:1 1.6 14.6

684 USA Eia. andrei lab 1 4.6 H 2O 1:1 2.4 loam 3.3

685 USA Eia. andrei lab 1 3.8 H2O 1:1 2.4 loam 4.6

686 USA Eia. andrei lab 1 6.5 H2O 1:1 3.2 loam 16.3

433.56 278.35 1.56 28

496.45 313.90 1.58 28

444.08 308.80 1.44 28

526.72 41.79 12.61 28

172.84 41.78 4.14 28

321.48 48.47 6.63 28

251.44 46.44 5.41 28

324.82 46.06 7.05 28

148.09 44.39 3.34 28

198.10 47.97 4.13 28

yes

yes

yes

yes

yes

yes

yes

yes

yes

yes

Bradham 2003

146

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

687 USA Eia. andrei lab 1 4.4 H2O 1:1 2.4 4.8

688 USA Eia. andrei lab 1 4.8 H2O 1:1 2.9 14.0

689 USA Eia. andrei lab 1 7.0 H2O 1:1 4.0 32.4

690 USA Eia. andrei lab 1 7.3 H 2O 1:1 3.0 loam 16.5

691 USA Eia. andrei lab 1 7.7 H 2O 1:1 1.1 loam 11.7

692 USA Eia. andrei lab 1 6.5 H 2O 1:1 5.2 28.3

693 USA Eia. andrei lab 1 6.1 H2O 1:1 4.0 27.5

694 USA Eia. andrei lab 1 3.9 H2O 1:1 1.7 loam 3.0

695 USA Eia. andrei lab 1 4.6 H2O 1:1 3.8 silt 10.7

696 USA Eia. andrei lab 1 5.7 H2O 1:1 1.6 silt 12.5

332.95 53.23 6.26 28

285.50 48.48 5.89 28

134.65 50.50 2.67 28

150.29 45.22 3.32 28

85.83 47.34 1.81 28

168.71 49.88 3.38 28

195.46 48.27 4.05 28

71.29 48.25 1.48 28

265.79 46.08 5.77 28

719.08 46.63 15.42 28

yes

yes

yes

yes

yes Bradham 2003

yes

yes

yes

yes

yes

147

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

697 USA Eia. andrei lab 1 5.5 H2O 1:1 1.8 sand 4.4

698 USA Eia. andrei lab 1 5.4 H2O 1:1 1.0 sand 3.4

699 USA Eia. andrei lab 1 6.7 H2O 1:1 2.2 22.3

700 USA Eia. andrei lab 1 6.9 H2O 1:1 4.8 29.4

701 USA Eia. andrei lab 1 6.8 H2O 1:1 2.5 27.6

702 USA L. terrestris field 0 6.2

703 USA L. terrestris field 6.2

704 USA L. terrestris field 0 6.2

4.1 silt loam

4.1 silt loam

4.1 silt loam

787.92 53.85 14.63 28

1193.04 40.20 29.68 28

240.69 43.94 5.48 28

266.67 50.03 5.33 28

231.45 53.88 4.30 28

57.20 26.82 2.13

17.75 26.82 0.66

5.50 1.08 5.09

sludge-amended yes yes yes Bradham 2003 yes yes

sludge-amended

cattle manure amended

Wade et al. 1982

148

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

705 USA L. terrestris field 6.2

706 Finland Ap. caliginosa lab 7 4.8 0.1 M KCl 1:25 17.8

707 Finland Ap. caliginosa lab 7 5.7 0.1 M KCl 1:25 18.9

708 Finland Ap. caliginosa lab 7 6.4 0.1 M KCl 1:25 18.2

709 Finland Ap. caliginosa lab 7 5.1 0.1 M KCl 1:25 16.2

710 Finland Ap. caliginosa lab 7 5.8 0.1 M KCl 1:25 16.0

711 Finland Ap. caliginosa lab 7 6.5 0.1 M KCl 1:25 17.3

4.1 silt loam

2360 3.30 1.08 3.06 31.72 3.30 9.61 35 yes HNO3+HClO4 Cd(NO3)2 cattle manure amended yes Wade et al. 1982

2370 13.67 3.40 4.02 35 yes HNO3+HClO4 Cd(NO3)2 yes

2310 8.20 3.00 2.73 35 yes HNO3+HClO4 Cd(NO3)2 yes

2200 239.06 36.30 6.59 35 yes HNO3+HClO4 Cd(NO3)2 yes

2410 168.75 37.20 4.54 35 yes HNO3+HClO4 Cd(NO3)2 yes

2490 60.94 38.70 1.57 35 yes HNO3+HClO4 Cd(NO3)2 yes

Peramaki et al. 1992

149

Observation No. Study Location Species Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

712 Finland Ap. caliginosa lab 7 4.3 0.1 M KCl 1:25 4.2

713 Finland Ap. caliginosa lab 7 6.4 0.1 M KCl 1:25 4.9

714 Finland Ap. caliginosa lab 7 4.3 0.1 M KCl 1:25 4.7

715 Finland Ap. caliginosa lab 7 5.1 0.1 M KCl 1:25 5.1

716 Finland Ap. caliginosa lab 7 6.1 0.1 M KCl 1:25 5.0

717 Netherlands L. rubellus field 2 6.9 CaCl2 20.5 11.4

718 Netherlands Ds. rubidus field 2 6.9 CaCl2 20.5 11.4

719 Netherlands Ap. caliginosa field 2 6.9 CaCl2 20.5 11.4

1400 831.25 40.40 20.58 160 yes HNO3+HClO4 Cd(NO3)2 yes

1580 1006.25 42.60 23.62 160 yes HNO3+HClO4 Cd(NO3)2 yes

9260 1378.13 41.50 33.21 230 yes HNO3+HClO4 Cd(NO3)2 yes Peramaki et al. 1992

8920 896.88 43.70 20.52 230 yes HNO3+HClO4 Cd(NO3)2 yes

10280 875.00 45.20 19.36 230 yes HNO3+HClO4 Cd(NO3)2 yes Hobbelen et al. 2004 51.00 11.70 4.36 142.00 11.70 12.14 82.00 11.70 7.01

150

Observation No. Study Location Species

720 Netherlands L. rubellus

721 Netherlands Ds. rubidus

722 Spain Al. ch lo ro ti ca

723 Spain N. caliginosus

724 Spain Al. chlorotica

725 Spain N. caliginosus

726 Spain Al. chlorotica

727 Spain N. caliginosus

Experiment Type Depuration Time day pH pH Measurement Method % clay % OM Soil Texture CEC cmol/kg Soil Ca mg/kg dry wt Worm Cd mg/kg dry wt Soil Cd mg/kg dry wt BAF Exposure Time day Preexposed Soil Extraction Method Source Term Special Material Cd Spiked Aging Time day Reference

field 2 6.9 CaCl2 35.1 18.8

field 2 6.9 CaCl2 35.1 18.8

field 5 8.3 H 2O 31.6 1.4 5.9 27600

field 5 8.3 H 2O 31.6 1.4 5.9 27600 6.40 0.20 32.00

field 5 7.9 H2O 28.8 1.7 6.0 50800 3.60 0.30 12.00

field 5 7.9 H2O 28.8 1.7 6.0 50800 3.70 0.30 12.33

field 5 7.5 H2O 28.1 2.2 6.4 51600 8.70 0.30 29.00

field 5 7.5 H2O 28.1 2.2 6.4 51600 6.90 0.30 23.00

44.00 19.30 2.28

106.00 19.30 5.49

4.30 0.20 21.50

HNO3+HCl mine

HNO3+HCl mine

HNO3+HCl mine sludge-amended

HNO3+HCl mine sludge-amended

HNO3+HCl mine sludge-amended

HNO3+HCl mine sludge-amended

Hobbelen et al. 2004

Barrera et al. 2001

151

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Carter, A. 1983. Cadmium, copper, and zinc in soil animals and their food in a red clover system. Can. J. Zool. 61: 2751-2757. Conder, J. M., L. D. Seals, and R. P. Lanno. 2002. Method for determining toxicologically relevant cadmium residues in the earthworm Eisenia fetida. Chemosphere 49: 1-7. Corp, N. and A. J. Morgan. 1991. Accumulation of heavy metals from polluted soils by the earthworm, Lumbricus rubellus: can laboratory exposure of control worms reduce biomonitoring problems? Environ. Pollut. 74: 39-52. Cotter-Howells, J., J. M. Charnock, C. Winters, P. Kille, J. C . Fry, and A. J. Morgan. 2005. Metal compartmentation and speciation in a soil sentinel: The earthworm, Dendrodrilus rubidus. Environ. Sci. Technol. 39: 7731-7740. Czarnowska, K., and K. Jopkiewicz. 1978. Heavy metals in earthworms as an index of soil contamination. Polish J. Soil Sci. 11: 57-62. Dai J., T. Becquerb, J. Rouillerc, G. Reversata, F. Bernhard-Reversata, J. Nahmania, and P. Lavelle. 2004. Heavy metal accumulation by two earthworm species and its relationship to total and DTPA-extractable metals in soils. Soil Biology & Biochemistry 36: 9198. Diercxsens, P. And D. de Weck, N. Borsinger, B. Rosset, and J. Tarradellas. 1985. Earthworm contamination by PCBs and heavy metals. Chemosphere. 14: 511-522. Efroymson, R., B. L. Jackson, D. S. Jones, B. E. Sample, G. W. Suter II and C. J. E. Welsh. 1996. Waste Area Grouping 2 Phase I Task Data Report and White Oak Creek Watershed Screening Ecological Risk Assessment. ORNL/ER-366. Oak Ridge National Laboratory, Oak Ridge, TN. Emmerling, C., K. Krause, and D. Schroder. 1997. The use of earthworms in monitoring soil pollution by heavy metals. Z. Pflanzenernahr. Bodenk. 160: 33-39. Gish, C. D., and R. E. Christensen. 1973. Cadmium, nickel, lead, and zinc in earthworms from roadside soil. Environ. Sci. Technol. 7: 1060-1062. Grelle, C., and M. Descamps. 1998. Heavy metal accumulation by Eisenia fetida and its effects on glutathione-S-transferase activity. Pedobiologia 42 (4): 289-297. Hankard, P. K., C. Svendsen, J. Wright, C. Wienberg, S. K. Fishwick, D. J. Spurgeon, and
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J. M. Weeks. 2004. Biological assessment of contaminated land using earthworm biomarkers in support of chemical analysis. Science of the Total Environment 330: 920. Hobbelen, P. H. F., J. E. Koolhaas, and C. A. M. van Gestel. 2004. Risk assessment of heavy metal pollution for detritivores in floodplain soils in the Biesbosch, the Netherlands, taking bioavailability into account. Environmental Pollution 129: 409-419. Hobbelen, P. H. F., J. E. Koolhaas, and C. A. M. van Gestel. 2006. Effects of heavy metals on the litter consumption by the earthworm Lumbricus rubellus in field soils. Pedobiologia 50: 51-60. Homa, J., M. Niklinska, and B. Plytycz. 2003. Effect of heavy metals on coelomocytes of the earthworm Allolobophora chlorotica. Pedobiologia 47: 640645. Hsu M. J., K. Selvaraj, and G. Agoramoorthy. 2005. Taiwan's industrial heavy metal pollution threatens terrestrial biota. Environmental Pollution 143(2): 327-334. Ireland, M. P. 1979. Metal accumulation by the earthworms Lumbricus rubellus, Dendrobaena venata, and Eiseniella tetraedra living in heavy metal polluted sites. Environ. Poll. 19: 201-206. Janssen, R. P. T., L. Posthuma, R. Baerselman, H. A. Den Hollander, R. P. M. Van Veen, and W. J. G. M Peijnenburg. 1997. Equilibrium partitioning of heavy metals in Dutch field soils. II. Prediction of metal accumulation in earthworms. Environmental Toxicology and Chemistry 16(12): 24792488. Kennette, D., W. Hendershot, A. Tomlin, and S. Sauv. 2002. Uptake of trace metals by the earthworm Lumbricus terrestris L. in urban contaminated soils. Applied Soil Ecology 19: 191198. Laszczyca, P., M. Augustyniak, A. Babczynska, K. Bednarska, A. Kafel, P. L. Migula, G. Wilczek, and I. Witas. 2004. Profiles of enzymatic activity in earthworms from zinc, lead and cadmium polluted areas near Olkusz (Poland). Environ. Int. 30: 901-910. Liu, X., C. Hu, and S. Zhang. 2005. Effects of earthworm activity on fertility and heavy metal bioavailability in sewage sludge. Environment International 31: 874 879. Lock, K. and C. R. Janssen. 2001. Cadmium toxicity for terrestrial invertebrates: Taking
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soil parameters affecting bioavailability into account. Ecotoxicology 10: 315-322. Ma, W.-C. 1982. The influence of soil properties and worm-related factors on the concentration of heavy metals in earthworms. Pedobiologia 24: 109-119. Ma, W.-C. 1987. Heavy metal accumulation in the mole, Talpa euorpa, and earthworms as an indicator of metal bioavailability in terrestrial environments. Bull. Environ. Contam. Toxicol. 39: 933-938. Maenpaa, K. A., J. V. K. Kukkonen, and M. J. Lydy. 2002. Remediation of heavy metalcontaminated soils using phosphorus: Evaluation of bioavailability using an earthworm bioassay. Arch. Environ. Contam. Toxicol. 43: 389398. Marino, F., A. Ligero, and D. J. Diaz Cosin. 1992. Heavy metals and earthworms on the border of a road next to Santiago (Galicia, Northwest of Spain). Initial results. Soil Biol. Biochem. 24: 1705-1709. Marino, F. and A. J. Morgan. 1999a. The time-course of metal (Ca, Cd, Cu, Pb, Zn) accumulation from a contaminated soil by three populations of the earthworm, Lumbricus rubellus. Applied Soil Ecology 12: 169-177. Marino, F. and A. J. Morgan. 1999b. Equilibrated body metal concentrations in laboratory exposed earthworms: can they be used to screen candidate metaladapted populations? Applied Soil Ecology 12: 179-189. Marino, F, S. R. Sturzenbaum, P. Kille, and A. J. Morgan. 1998. CuCd interactions in earthworms maintained in laboratory microcosms: the examination of a putative copper paradox. Comparative Biochemistry and Physiology Part C 120: 217223. Marino F., C. Winters, and A. J. Morgan. 1999. Heat shock protein (hsp60, hsp70, hsp90) expression in earthworms exposed to metal stressors in the field and laboratory. Pedobiologia 43 (6): 615-624. Marinussen M. P. J. C, S. E. A. T. M Van der Zee, F. A. M. De Haan, L. M. Bouwman, and M. Hefting. 1997. Heavy metal (copper, lead, and zinc) accumulation and excretion by the earthworm, Dendrobaena veneta. J. Environ. Qual. 26: 278284. Morgan, J. E., and A. J. Morgan. 1990. The distribution of cadmium, copper, lead, zinc, and calcium in the tissues of the earthworm Lumbricus rubellus sampled from one uncontaminated and four polluted soils. Oecologia. 84: 559-566.

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Appendix C. Equations Used for Normalizing pH Values Measured Using Different Soil: Solution Ratios

Extractant H2O 0.01 M CaCl2 1 M KCl

Regression function pH (1:5) = 1.0789 pH (1:1) - 0.2395 pH (1:5) = 1.0387 pH (1:2) - 0.1175 pH (1:5) = 0.9519 pH (1:10) + 0.1691 pH (1:5) = 0.9999 pH (1:2) + 0.1324 pH (1:5) = 0.9969 pH (1:10) - 0.1174 pH (1:5) = 1.0220 pH (1:2) 0.0055 pH (1:5) = 0.9774 pH (1:10) - 0.0105

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