Metal uptake from soils and soil–sediment mixtures by larvae of Tenebrio molitor (L.) (Coleoptera)

Ecotoxicology and Environmental Safety 54 (2003) 277–289

Metal uptake from soils and soil–sediment mixtures by larvae ofTenebrio molitor (L.) (Coleoptera)

Martina Vijver, Tjalling Jager, Leo Posthuma, and Willie Peijnenburg�

Laboratory of Ecological Risk Assessment, National Institute of Public Health and the Environment, P.O. Box 1, BA 3720, Bilthoven, Netherlands

Received 10 October 2001; accepted 7 June 2002

Abstract

Bioassays were performed to evaluate the impact of soil characteristics on Cd, Cu, Pb, and Zn uptake by larvae of Tenebrio

molitor. Metal accumulation was determined in 13 natural field soils, one metal-spiked field soil, four soil–sediment mixtures, and

Cd- or Zn-spiked OECD artificial soil. Statistical analyses were used to investigate covariation of accumulation patterns with

various soil metal pools and soil properties. Body concentrations of Cu and Zn in Zn-spiked OECD soils, field soils, and

soil–sediment mixtures mostly remained constant. Considerable variation was noted for all Cd and Pb steady-state body

concentrations among field soils and soil–sediment mixtures. For the spiked field soil and in the Cd-spiked OECD soil, body

concentrations increased almost linearly with time. For the nonessential metals Cd and Pb, larval body concentrations correlated

mainly to the total metal pool of the soil. Cd uptake at similar total Cd concentrations was within the same range among spiked

OECD soils, field soils, and mixtures. A comparison of the findings with studies on other soil-inhabiting species shows that metal

uptake patterns depend on metal type, soil type, and exposed species. It is suggested that soil organisms can be categorized according

to gross divergence in ecophysiological characteristics, determined by, for instance, (non)permeability of the outer integument.

These characteristics appear as similarities among multivariate functions as derived for the beetle.

r 2002 Elsevier Science (USA). All rights reserved.

Keywords: Tenebrio molitor; Metal; Accumulation; Bioavailability; Risk assessment; Metal uptake; Kinetics; Exposure; Exposure route; Pore water

hypothesis

1. Introduction

Ecotoxicological risk assessment aims at quantifyingpotential adverse effects of exposure on biota. Atpresent, risk assessment for metals in soils is usuallyperformed on the basis of total concentrations. This isdisputable since metals are taken up by organisms froma complex system of equilibria between a variety ofmetal species and soil and sediment constituents(Posthuma et al., 1998). Metal accumulation andtoxicity depend on the metal fraction that can actuallybe taken up by biota. New insights into the issue ofmetal bioavailability show that dissolved metal fractionsin the pore water are of major importance for uptake byvarious biota (Allen, 2002). The so-called ‘‘pore waterhypothesis’’ states that exposure to contaminants occursmainly through the solution phase, or indirectly by

phases that are in equilibrium with the pore water. Theimportance of the pore water for uptake of chemicalshas been demonstrated for oligochaete species (L^kkeand Van Gestel, 1998; Peijnenburg et al., 1999a, b) andsoil microbes (McGrath, 2002; Plette et al., 1999). Inaddition, a recent study of Oste et al. (2001) demon-strates that competing ions such as Ca2+ and H+ affectuptake of the free metal ion by earthworms. Besidesthe faunal examples referred to, plants are also knownto accumulate metals from the pore water (Smolderset al., 1997).Most species used in metal uptake and toxicity

experiments for soils and sediments are species with awater-permeable integument, so-called soft-bodied or-ganisms. When focusing on different species, it hasbecome evident that metal bioavailability for at leastsome soil- or sediment-inhabiting species is not easilypredicted with the pore water hypothesis. Only a fewstudies have focused on metal uptake by terrestrialspecies for which the integument is not evidently

�Corresponding author. Fax: +31-30-2744413.

E-mail address: [email protected] (W. Peijnenburg).

0147-6513/03/$ - see front matter r 2002 Elsevier Science (USA). All rights reserved.

doi:10.1016/S0147-6513(02)00027-1

permeable to water. Recent examples of such researchinclude exposure of the springtail Folsomia candida

to contaminated soils. Studies in which exposureroutes and ecotoxicological effects of Cu (Pedersenet al., 1999) and metal accumulation by F. candida fromsoils (Vijver et al., 2001) were investigated indeedconfirm the nonadherence of F. candida to the porewater hypothesis.Studies on metal accumulation in soil–sediment

mixtures have been conducted even less frequently.Nevertheless, metal-loaded sediments are an importantenvironmental management topic in many countries.Often, polluted sediments are deposited on agriculturalfields for water management purposes. Followingdeposition, the sediment material is mixed with theunderlying soil mechanically, where after aging andweathering processes, the metal speciation andhence metal bioavailability are significantly changed.The quality of these mixtures is disputable, and adverseecotoxicological effects on biota cannot be ruled out.For setting soil quality criteria, it is desirable that a

variety of organisms are studied, acknowledging theexistence of organisms for which uptake via the porewater is not obvious. In this article, metal accumulationfrom field soils and soil–sediment mixture by larvae ofthe beetle Tenebrio molitor is described. This tenebrionidlarva was chosen as a representative of soil-dwellingorganisms that are restricted mainly to the upper part ofthe soil. The larvae feed on all types of organic matter.T. molitor (Coleoptera) is morphologically differentfrom species used in earlier studies in which metalbioavailability was quantified. Tenebrionid larvae havea considerably high osmotic regulation due to a hardwax-coated cuticle to protect them against water loss(Zachariassen et al., 1987a, b). This contrasts withearthworms, which have a water-permeable epidermis(Laverack, 1963), and springtails such as F. candida,which have a cuticle and a ventral tube for maintainingtheir water balance (Hopkin, 1997). It is likely that thesemorphological differences imply that the differentuptake routes for metals that can be imagined differwith respect to their relative importance to total uptake.Investigating species having dissimilar physiologies anddescribing species-specific uptake characteristics invarious soils constitute the fifth step in a conceptualframework developed to enable prediction of thebioavailability of metals from soils for environmentalmanagement purposes, as stated by Peijnenburg et al.(1997).The aim of this study is to assess the relationship

between soil characteristics and metal accumulation bythe soil-dwelling larvae of T. molitor. Uptake kinetics ofCd, Cu, Pb, and Zn are reported, and estimated steady-state concentrations are correlated to soil metal poolsand soil characteristics. The working hypothesis is thatmetal uptake can best be correlated to the metal pool in

solution for these hard-bodied larvae, as was observedin, e.g., oligochaetes. Differences in metal accumulationby beetle larvae were investigated after exposure inspiked OECD artificial soil, metal spiked field soil,naturally contaminated field soils, and soil–sedimentmixtures. This variety of substrates was chosen to reflecta variety of exposure situations and test procedures. Asynthesis of the experimental results is presented and theconsequences for risk assessment are discussed.

2. Material and methods

2.1. OECD soil, field soils, and soil–sediment mixtures

2.1.1. OECD soil

Artificial soil was prepared according to OECDGuideline 207 (OECD, 1984). Cd or Zn was addedindividually in a geometric series to the OECD soil asaqueous stock solutions of chloride salts. The totalmetal concentrations of the spiked OECD soils rangedbetween 1.5� 10�3 and 0.49mmol Cd/kg dry soil, or0.16 and 9.08mmol Zn/kg dry soil. Soil pH in bothspiked OECD soils, measured after 0.01M CaCl2extraction, was approximately 6, and varied less than 1unit among the different treatments. The range of spikedmetal concentrations was based on the median values incontaminated Dutch field soils sampled by Peijnenburget al. (2000). The OECD soils were brought to 40% oftheir water holding capacity using a 0.002M Ca(NO3)2solution, to simulate the average ionic strength in porewater from Dutch field soils (Peijnenburg et al., 2000).Modified artificial soil (with and without metals) wasstored in a sealed container for 1 month at +41C, toequilibrate the system prior to bioassays. Aqua regia(HCl:HNO3) digestion and 0.01M CaCl2 extractionwere carried out to measure actual metal concentrationsin the exposure soils. Extractable metal concentrationsand pH were determined at the beginning and at the endof the experiments, but no significant effects of eithertime or exposure time or exposure of the beetle larvaewere observed.

2.1.2. Field soils

Fourteen Dutch field soils were selected as adiversified sub-set of soils sampled by Peijnenburg et al.(2000). Principal component analysis (PCA) was used tomaximize the variation among soil properties and metalpools of the soils selected. Hence, soil characteristics aswell as metal contamination levels differed stronglyamong the selected soils. The soil characteristics aregiven in Table 1. Details on soil codes, sample sites,treatments, analyses, characterization, and the concen-trations of the different metal pools are given byPeijnenburg et al. (2000). Soil AQ is an uncontaminatednatural field soil to which salts of As, Cd, Cr, Cu, Ni,

M. Vijver et al. / Ecotoxicology and Environmental Safety 54 (2003) 277–289278

Pb, and Zn were added, after which the solid was storedfor more than 2 years at 41C.

2.1.3. Soil–sediment mixtures

Four mixtures of field soils and sediments wereprepared in the laboratory for the uptake experiments.The soil–sediment mixtures were treated the same wayas the natural soils, and their major characteristics aftermixing are given in Table 1.

2.2. Tenebrio molitor

Beetle larvae (8–10 weeks old) used in the bioassayswere in their second stage of molting. The organismswere kept in plastic containers with lids perforated withair holes. The bottom of the containers was filled with asubstrate of bran and a chunk of carrot. Experimentalconditions were maintained according to ISO/TC190/SC4/WG2 (ISO, 1988), with a modification of thetemperature to 17721C.

2.3. Exposure of larvae

For each sampling time and soil combination, fiveplastic jars (15mL) were filled with 8.5 g of moistenedsoil. One larva was transferred to each jar. Animals wereexposed under climatized conditions and were not fedduring the experiment. In the Cd-spiked OECD soil,larvae were harvested after 0, 1, 3, 7, 14, and 21 days of

exposure, and in the Zn-spiked OECD soil, after 0, 1, 3,7, 14, and 21 days of exposure. Larvae exposed to fieldsoils were sampled as in the Zn experiment, except forthe longest exposure period, which was 17 days for fieldsoils. The organisms exposed to the soil–sedimentmixtures were sampled after 0, 1, 3, 7, and 14 days ofexposure. Every day the jars were inspected for moltedlarvae. Exuviae were collected and analyzed.During the exposure period, animals showing adverse

effects (visual inspection on immobility and survival)were discarded from the analyses, since such effectsmight bias the metal accumulation by the larvae.Healthy organisms were collected on the chosensampling date and subsequently freeze-dried for at least72 h. Thereafter they were weighed, and metal contentswere determined.

2.4. Analyses

The lyophilized larvae were digested in a 7:1HNO3:HClO4 mixture. Cd, Cu, and Pb concentrationsin the digests were determined using graphite furnaceAAS. Zn analyses were carried out using flame AAS.Dolt-2 (certified by the Community Bureau of Refer-ence, BCR, Brussels, Belgium) and tobacco leaves(certified by the Instytut Chemii i Techniki J)adrowej,Warsaw, Poland) were used as biological referencematerials. No systematic correction was applied to thebody residue analyses, since recovery was always

Table 1

Some characteristic soil properties and metal pools of the soils and soil–sediment mixtures used in this study

Soil properties Metal pools

Soil pH LOI550(%)

LOI900(%)

Silt OM (%) DOC

(mmol/L)

[Cu]cc [Cu]pw [Zn]cc [Zn]pw [Cd]cc [Cd]pw [Pb]cc [Pb]pw

B 5.60 16.1 1.15 19.5 14.96 7.25 0.912 0.73 6.581 8.59 0.124 0.012 — 0.027

C 5.07 14.4 1.16 14.3 10.33 5.45 3.738 3.12 28.26 2.00 0.226 0.009 0.055 0.117

E 6.65 6.59 0.87 25.2 5.24 2.54 0.861 1.37 756.6 62.9 3.023 0.153 0.139 0.029

F 7.38 7.59 3.18 40.6 8.73 3.52 0.870 1.02 11.87 1.00 0.192 0.008 — 0.006

G 7.24 6.05 5.89 30.3 6.76 2.52 1.719 1.64 5.731 0.60 0.236 0.011 — —

I 3.81 5.21 0.14 2.04 4.81 12.3 0.388 1.44 73.59 22.5 2.294 0.234 5.321 0.206

K 4.49 0.38 0.11 0.26 — 5.28 0.099 1.39 3.381 5.79 0.019 0.036 63.23 4.375

O 6.09 4.88 0.50 5.48 4.69 4.74 1.182 2.03 82.49 16.0 3.794 0.267 — 0.008

R 7.36 4.46 2.62 10.6 4.66 3.68 1.524 2.05 2.503 1.69 0.099 0.024 — 0.030

S 7.08 15.9 1.88 36.0 16.8 2.23 1.220 0.97 21.22 1.10 0.972 0.022 — 0.014

AF 7.30 1.20 3.05 5.70 1.89 2.33 0.188 1.08 — 0.40 0.003 0.004 — 0.016

AQ 4.45 6.70 0.30 3.40 6.40 22.6 3.611 1.34 1851 355.0 10.891 1.586 1.427 0.363

AS 7.21 4.72 1.94 3.60 5.01 2.87 0.511 0.28 — 0.40 0.005 0.001 0.078 0.128

AW 6.33 6.79 1.80 1.78 12.7 1.32 2.509 0.65 6404 838 28.93 1.340 5.510 0.250

MWAA 7.38 16.4 3.01 44.9 16.7 3.42 12.80 0.51 19.93 6.81 0.601 0.050 0.662 0.002

MDEV 7.07 15.8 7.43 23.3 7.82 3.53 4.350 0.56 123.8 9.16 0.054 0.007 0.155 0.005

MAPE 6.79 33.6 1.57 11.6 18.0 1.87 18.70 0.45 35.30 8.17 0.162 0.013 0.401 0.006

MEPE 6.79 2.51 1.22 21.0 5.61 4.55 4.411 0.57 372.6 43.7 1.600 0.094 0.098 0.008

Note: All soil codes beginning with M are soil-sediment mixtures. pH was determined in a 0.01M CaCl2 extract, LOI550 is loss-on-ignition at 5501C

(organic fraction), LOI900 is loss-on-ignition at 9001C (inorganic fraction), silt is the fraction of particles with a diameter between 2 and 38mm, OM is

organic matter calculated from the total carbon content, DOC is dissolved organic carbon determined with a Dohrmann DC-190 TOC analyzer. cc

refers to 0.01M CaCl2-extractable metal concentration (mmol/kg), and pw to metal concentration in pore water (mmol/L). —, not determined.

Further details available on request.

M. Vijver et al. / Ecotoxicology and Environmental Safety 54 (2003) 277–289 279

between 90% and 105%. At least three individualorganisms and their separate exuviae were analyzed ateach exposure time, to obtain insight into biologicalvariation among the individuals.Ingestion of soil particles by organisms or adherence

of soil particles to the integument can influencemeasured internal metal concentrations. Three methodswere used to check for the absence of soil particles in oron the lyophilized animal bodies:

* Dissection of freeze-dried organisms and subsequentvisual inspection.

* Visual inspection for the presence of soil particles inthe digests.

* Ashing of the lyophilized animals at 5001C, since allorganic matter (the animals’ bodies) will be destroyedat this temperature and the silicate of soil particleswill be left behind.

2.5. Data handling

Several larvae molted during the experiment. Moltedand nonmolted larvae were used in the accumulationmodeling, as no consistent differences in internal metalconcentrations between nonmolted and molted animalswithin a treatment were found.The metal concentrations of molted organisms (Cib)

and their skin (Cis) were taken together as a weightedaverage:

Cc ¼Cis�Weights þ Cib�Weightb

Weights�Weightb; ð1Þ

where Cc is the metal concentration in the larvae (mmol/kg dry wt), Weights the weight of exuvium (kg), Weightbthe weight of organism (kg), Cis the metal concentrationin the exuvium (mmol/kg dry wt), and Cib the metalconcentration in the molted organisms (mmol/kg drywt). The mean of the individual total body concentra-tions per time treatment was used for further calculationand modeling.Metal accumulation in the larvae was modeled

according to a one-compartment model. The modelingoffered the possibility of analyzing the dynamic changein body concentration in the larvae over time. In thisway, steady-state concentrations Cc(ss) among soilscould be calculated, even when steady-state was not yetreached. The general equation of a one-compartmentmodel is

CcðtÞ ¼ Ccð0Þe�k2t þ CcðssÞð1� e�k2tÞ; ð2Þwhere t is the time (days), CcðtÞ the metal concentrationin the larvae at time t (mmol/kg dry wt), and k2 theelimination rate constant (day�1), Ccð0Þ is the initialconcentration in the larvae, which is a measured valuethat was present in the animal before exposure and isassumed to participate in all equilibration processeswithin the organism. GraphPad Prism 2.01 (1996) was

used to fit the model to the data for each soil and eachmetal separately. The model was fitted to the averagevalues of the time treatments, weighted for the numberof separate observations contributing to this average. Ifsteady state was reached in the duration of theexperiment, the calculated steady-state levels werecompared with the mean of the measured levels after14 and 28 days of exposure.Unfortunately, the calculated elimination rate con-

stants, k2; had to be excluded from further data analysis,owing to large standard errors caused by identificationproblems of the model. Steady-state levels could beestimated more reliably; the time required to reach 95%of the steady-state level (Tss) can be derived by means ofthe equation

Tss ¼1

k2ln 20 1� Ccð0Þ

CcðssÞ

� �� ; ð3Þ

which is obtained from Eq. (2). To quantify the impactof soil characteristics on metal accumulation, thecalculated steady-state levels of the metals were corre-lated to a number of hypothetically bioavailable metalfractions, as well as to soil properties. The followingpools (total metal concentrations) were imagined to be(partially) bioavailable for uptake: (1) the soil solidphase (mmol/kg dry wt), (2) the pore water (mmol/L),and (3) the 0.01M CaCl2-extractable metal concentra-tion (mmol/kg dry wt).Monovariate and stepwise multivariate regression was

used to obtain the correlations. The multivariatefunctions took the form

log CcðssÞ ¼ a logðAÞ7b log ðBÞ7cy; ð4Þ

where A and B are the descriptors, i.e., different metalpools and soil properties; and a; b; and c are thecoefficients. The significant descriptors were arranged indecreasing order of importance. Descriptors that do notexplain a significant part of the variation (P40:005)were not incorporated into the formulas. It should benoted that multivariate expressions as developed in thisstudy do not necessarily provide information onmechanistic influences of soil variables on organisms’metal uptake. The number of descriptors in the formulaswas limited by the condition that the ratio of the numberof data points vs the number of variables should behigher than 4 (Hermens et al., 1995).

3. Results

3.1. General observations in OECD soils

All beetle larvae were recaptured alive from the Cdexperiment. In the Zn-spiked OECD soil, 3 of a total of150 organisms were found dead after 21 days ofexposure, independent of exposure treatment. In the


Cd and Zn experiment 31% and 35% of the organismshad molted during the exposure, respectively. Moltingoccurred earlier in time in the higher-exposure concen-trations, but the frequency of molting was only onceduring the experiment in all treatments. Organismsincreased significantly in fresh weight during theexperiments. However, only negligible increases in dryweight were found.

3.2. Update characteristics in OECD soils

Body Cd concentrations ranged from below detectionlimit (0.01 mmol Cd/kg dry wt) to 3.2 mmol/kg dry wt, ascompared with total Cd concentrations of up to0.49mmol/kg dry soil. The general observation wasthat body Cd concentrations of the larvae increasedlinearly with increasing exposure concentrations. Thesame trend was found when expressing exposure on thebasis of CaCl2-extractable metal pools. Body concentra-tions of zinc were largely unaffected by external Znconcentrations, as only a marginal increase in internalzinc residues at higher exposure was seen. The organ-isms were apparently able to maintain their internalconcentration at a fixed level of 22647156 mmol Zn/kgdry wt at all external concentrations and throughout allexposure time employed.

3.3. General observations in soils and soil–sediment

mixtures

Forty percent of the larvae had molted once duringthe experiment in both soils and soil–sediment mixtures.Mortality during the 17-day exposure period in fieldsoils was negligible; only two species died during thecourse of the whole experiment. No changes in dry bodyweight were observed. In the soil–sediment mixtures, alarge fraction of the larvae showed adverse effects after10 days of exposure. The percentage of mortality in themixtures was 45% after 14 days of exposure. Therefore,the experiments were terminated at this moment.

Organisms showing adverse effects either in soil or insoil–sediment mixtures were excluded from the chemicalanalyses.

3.4. Uptake characteristics in soils and mixtures

Organisms and their exuviae were analyzed sepa-rately. The metal concentration in the exuviae wassimilar or lower than body concentrations and theweight of the exuviae was much lower than the bodyweights. This means that in all cases, the concentrationof the exuvium had a low impact on the total bodyconcentration. Hence, exuvation did not form animportant elimination route for metals. There was noevidence to suggest that soil particles were ingested oradhered to the body. Potential bias to the metalmeasurements resulting from metals sorbed to soilparticles was therefore assumed negligible in theaccumulation calculations.The metal accumulation patterns by the larvae could

be addressed using a one-compartment model. In caseswhen elimination is apparently negligible, this results inan apparently linear uptake with exposure time. Uptakepatterns of a metal were of similar shape in all soils andmixtures, but differed with respect to the level ofequilibrium and the speed at which this level wasreached. As an illustration, Fig. 1 shows the typicalmetal uptake patterns observed, i.e., uptake accordingto a one-compartment model with apparent elimination(left) and uptake according to a one-compartmentmodel whereby elimination is approximately zero (linearuptake, right). In general, the uptake patterns hadsimilar variability in shape compared with earlierfindings for Oligochaeta and Collembola (Peijnenburget al., 1999b; Vijver et al., 2001). Differences in uptakepatterns between essential and nonessential elements aredisplayed in Fig. 2, where steady-state levels of the fourmetals are plotted against external total metal concen-trations for the 14 soils and the four soil–sedimentmixtures included in this research. Data on organisms

Fig. 1. Typical appearances of T. molitor accumulation patterns over time: fitting of the one compartment with high (left) and low (right) elimination

rate constants, respectively.


that displayed linear uptake could not be plotted in thefigure, as was the case for data obtained for soils havingexternal concentrations below the detection limit.Fig. 2 shows a variation of 2 or 3 orders of magnitude

of the internal steady-sate levels for the nonessentialmetals Cd and Pb. Body residues of the essential metals2 Cu and Zn varied less, although the external soilconcentrations differed strongly, as did most of the soilproperties (Table 1). Addition of dredged sedimentmaterial to soils did not affect the patterns of metaluptake by larvae of T. molitor.

3.5. Essential metals

The steady-state levels of Cu in the organisms werefixed in most soils. In one soil (AW) and in one mixture(MAPE) the larvae showed linear uptake of Cu, andlarvae exposed in soil AS had elevated body Cu

concentrations. Data on Cu for one soil (S) were lostdue to analytical problems.Larvae accumulated Zn to a fixed level from most

soils. Significantly higher steady-state levels of Zncompared with the average fixed levels in other soilswere found in two soils (AW, AQ) and two soil–sediment mixtures (MAPE, MEPE). Linear uptake wasdemonstrated in three soils (E, F, O). However, internalconcentrations of larvae sampled in soils F and O, after17 days of exposure, did not differ significantly from thefixed levels in the other soils. The measured internal Cuand Zn steady-state concentrations in the different soilsand mixtures are given in Table 2. As can be seen fromTable 2, steady-state levels of Cu and Zn varied onlyto a small extent. Therefore, it was not useful tocorrelate internal levels of Cu and Zn to soil properties.The average internal concentrations in T. molitor were:375765 mmol Cu/kg dry wt and 32467653 mmol Zn/kg

Fig. 2. Estimated (log-transformed) steady-state concentrations in T. molitor (in mmol/kg dry wt) vs log-transformed external metal concentrations

of Cu, Zn, Cd, and Pb (in mmol/kg dry wt). External concentrations below the detection limit are not given in the figure. Open squares refer to

mixtures of sediment and soil; closed squares refer to field soils only.


dry wt. In some soils, linear and significantly higheruptake of Cu and Zn was observed. These soils were notincluded in the data set used for calculating the averagesas presented. Internal Cu concentrations in the larvaeexposed to the metal-spiked field soil (AQ) were similarto the values given above, but for Zn a higher internallevel (5000 mmol/kg) was measured. Initial Zn bodyconcentration was 25157159 mmol/kg dry wt.

3.6. Nonessential metals

For all soils and soil–sediment mixtures, the larvaeaccumulated Cd above the initial body concentrations,which equaled 0.7 mmol/kg dry wt (Table 3). As can beseen from Table 3, linear uptake occurred in four soils,including the spiked field soil (AQ). In four cases, the Cdsteady-state conditions were not reached within themaximum exposure duration.Body concentrations of Pb in the larvae were

significantly higher following exposure to field soilsand soil–sediment mixtures, as compared with the initialPb concentrations, which equaled 0.3 mmol/kg dry wt

(Table 4). In four field soils and in the spiked field soil,linear uptake of Pb by the larvae was observed. In fivecases, the Pb steady-state levels were not reached withinthe duration of exposure.The soil metal concentrations of the nonessential

metals (Cd and Pb) varied considerably among thedifferent exposure soils. Steady-state levels of Cd and Pbwere correlated to a number of potentially bioavailablemetal fractions, as well as to physical soil properties.This was done using mono- and multivariate regressionanalyses. The parameters explaining most of thevariance in organism body metal concentration afterexposure in field soils are listed in Table 5. Table 6contains the formulas for the steady-state concentra-tions of Cd and Pb after exposure to the various soilsand soil–sediment mixtures.In the monovariate approach, the total Cd pool was

the descriptor that explained most of the variance inbody residues, after exposure to soils (Table 5) and afterinclusion of the data obtained for the soil–sedimentmixtures (Table 6). Multivariate regression analyses(Table 5) revealed that the total Cd pool combined withthe organic fraction (loss-on-ignition at 5501C, LOI550)and the CaCl2-extractable fraction modulated by pHand LOI550 explain the observed Cd levels in the animalsequally well (both at P ¼ 0:001). It should be noted thatthese two multivariate regressions can be considered asdifferent expressions of the same metal pool, since thetotal metal pool can be statistically substituted with theCaCl2-extractable pool and pH due to correlationsbetween these variables. The correlations among thevarious Cd pools for the soils and mixtures included inthe regression analyses given in Tables 5 and 6 arepresented in Table 7. Similar multivariate formulas werederived for the data set that included both soils and soil–sediment mixtures (Table 6). Here the variation of Cdbody residues among the soils and mixtures was alsoexplained well by the pore water pool matched with pH(Po0:001). This was in contrast to the formulasobtained for soils only (Table 5), where the pore waterpool was not identified as a significant descriptor.The monovariate regression equations that were

obtained for describing the Pb body concentrations insoils (Table 5) and in soil and soil–sediment mixtures(Table 6) demonstrated that the total Pb concentrationwas the descriptor explaining most of the variance ininternal Pb concentration. No other descriptors thatsignificantly (P40:005) described the variance of thedata could be identified. There were no substantialdifferences between the regressions obtained for fieldsoils only and those including soil–sediment mixtures.Soil properties (silt in this case) did contribute sig-nificantly to the description of steady-state Pb concen-trations, but did not substantially improve theregression value. This suggests that the total Pb poolin itself describes the main part of the uptake variance.

Table 2

Measured steady-state concentrations of Cu and Zn in T. molitor

(Cc(ss)) and exposure concentrations in field soils and soil–sediment

mixtures

Soila [Cu] [Zn]

Total

(mmol/kg)Cc(ss)

(mmol/kg)Total

(mmol/kg)Cc(ss)

(mmol/kg)

B 430 237 1520 2500

C 1650 419 1920 2885

E 410 367 47700 Lin 8530b

F 480 325 12600 Lin 3916b

G 750 423 9140 3238

I 50 319 230 2913

K 10 354 110 2569

O 310 315 2280 Lin 3230b

R 540 402 50600 3416

S 2130 Lostc 20000 3645

AF 40 382 310 2885

AQd 210 365 690 5000e

AS 260 529e 770 2794

AW 5130 Lin 1758b 115700 Lin 19480b

MWAA 2450 393 160 3506

MDEV 540 426e 230 3747

MAPE 3000 Lin 512b 30 4856e

MEPE 380 371 230 6101

aFor soil codes see Peijnenburg et al. (2000) or Table 1.bLin=linear uptake, further increase in Cc(ss) observed. The values

given are the internal concentration found in the last sampling

treatment (after 17 and 14 days of exposure for soils and mixtures,

respectively).cSteady-state levels could not be determined due to analytical

failures.dSpiked field soil.eModeled steady-state levels are given, because no equilibrium was

reached at the maximum duration of exposure.

Note: All soil codes beginning with M are soil–sediment mixtures.


4. Discussion

Patterns of metal uptake did not differ significantlybetween field soils and soil–sediment mixtures for the T.

molitor larvae. In most soils, organisms can keep theirinternal body concentrations of essential metals on afixed level. In some soils, these internal concentrationwere significantly elevated, indicating that the organismswere not able to maintain their internal fixed metalconcentrations in the face of increasing exposureconcentrations and the soil properties of the soilsstudied.The 0.01M CaCl2-extractable Cd pool and the total

amount of Cd in the soil solid phase explained thevariance in steady-state levels equally well. In the case ofPb, the total metal pool was the sole descriptor ofrelevance. This suggests that uptake of nonessentialmetals by T. molitor larvae is associated primarily withthe total metal pools of the soil. In some soils, linearuptake of Cd or Pb by larvae was observed. The steady-state concentrations of Cd and Pb in these soils could beestimated by means of the formulas obtained with theother soils. The modeled concentrations were comparedwith the measured internal concentration on the lastsampling time. For Cd (Table 3) the measured bodyconcentrations were below the calculated maximum

steady-state concentrations. This does not invalidate theformulas obtained with the other soils, and thereforethese formulas are useful in predicting steady-stateconcentrations of the larvae. It should be noted thatthe Cd concentrations of soil AW did not fit in thedomain of the formulas obtained, and accumulation ofCd from this soil by the larvae cannot be estimated bythe formulas obtained with the other soils. For Pb, bodyresidues for two soils were below the maximum steady-state concentrations derived. In three soils, the measuredbody concentrations of Pb were higher than thepredicted steady-state concentrations. Therefore, thesesoils did not fit the statistical descriptions. Furtherresearch is required to understand the deviatingaccumulation from these soils. Appearance of toxiceffects as the reason for disrupted accumulation patternscould be excluded, since linear uptake occurred indifferent soils for Cd and Pb. Soil AW is the exception inthis respect.

4.1. From statistical relationships to mechanistic uptake

routes

Our approach was not aimed at unraveling theunderlying uptake mechanisms for T. molitor.The empirical relationships found do not necessarily

Table 3

Parameters of Cd uptake by Tenebrio molitor in various soils and soil–sediment mixtures

Soila Total [Cd] (mmol/kg) Time 95% Cc(ss)b (Days) Calculated Cc(ss)

c (mmol/kg) R2 Derived Cc(ss)d

Min–Mean–Max

B 2.99 0.5 1.54 0.90 —

C 3.85 0.5 0.90 0.74 —

E 72.44 417 Lin 10.4e 0.97 6.90–16.77–40.76

F 23.99 417 Lin 5.06e 0.99 2.93–7.11–17.28

G 42.95 2.6 7.88 0.95 —

I 3.13 0.5 1.36 0.99 —

K od.l.f 2 1.24 0.97 —

O 53.58 17 25.36 0.98 —

R 14.22 10 10.26 0.93 —

S 164.06 6 15.54 0.97 —

AF 0.79 0.5 1.19 0.92 —

AQg 2.95 417 Lin 7.59e 0.99 3.46–8.41–20.4

AS 0.84 5 1.33 0.99 —

AW 188.80 417 Lin 71.2e 0.98 13.42–32.62–79.24

MWAA 143.55 10 21.00 0.94 —

MDEV 77.80 5 2.94 0.85 —

MAPE 21.04 7 2.04 0.97 —

MEPE 69.98 7 19.95 0.95 —

aFor soil codes see Peijnenburg et al. (2000) or Table 1.bTime required to reach 95% of the steady-state level.cCalculated steady-state concentration (Eq. (2)).dSteady-state concentrations in the organisms, derived by means of the formulas obtained with the other soils in the training set. The minimum

and maximum values are calculated as 1.96� SD (=confidence limits).eLin=linear uptake, further increase in Cc(ss) observed. The values given are the internal concentration found in the last sampling treatment (after

17 and 14 days of exposure for soils and mixtures, respectively).fDetection limit (od.l.)=0.0018 mmol Cd/kg dry wt.gSpiked field soil.

Note: All soil codes beginning with M are soil-sediment mixtures.


provide clues for the actual uptake routes. Hypotheti-cally, four metal uptake routes (or combinations ofthem) may be envisaged:

1. Uptake via drinking (including indirect uptake frompore water, possibly after modification of the porewater in the gut and thus depending on gutconditions).

2. Uptake via the skin (directly via pore water).3. Soil ingestion (indirect uptake via solid phases,

possibly following modification by gut conditions).4. Ingestion of food (indirect uptake via solid phases,

possibly following modification by gut conditions).

Uptake via the skin can be excluded, since the larvaehave a water-impermeable integument and wax coating.Soil ingestion may be excluded as a significant uptakeroute given the negative outcome of the three indepen-dent checks for the presence of soil particles in thelarvae. Uptake via ingested food (e.g., hyphae, organicmatter) is the most likely uptake route, as accumulationwas best described by total metal concentrations. Theobservation of a lack of correlation between steady-statelevels and pore water metal pools or pore water-relatedproperties indicates that indirect metal uptake from porewater has a low quantitative importance.

4.2. Synthesis of findings for risk assessment purposes

The consequences for risk assessment can be discussedfor Cd and Pb, since body concentrations of thesemetals could be described by external properties. Everyspecies is unique with respect to its suite of exposureroutes and kinetics of metal uptake. Risk assessmentprocedures should take this immense variation intoaccount, since toxic effects are likely dependent both onconcentrations in soil and on soil and organismcharacteristics. For pragmatic use in risk assessment,however, it is obvious that the number of species testedfor adverse effects should be limited, and a pragmaticclassification of organisms seems necessary. Janssen(1991), who presented groupings of soil arthropods withrespect to their cadmium turnover, gave an example ofsuch a classification. This classification is based onfeeding habit, Cd assimilation, and elimination. It ishypothesized that assimilation and elimination can belinked to the physiology of species (Walker, 1987).Moreover, a different physiology of animals results indifferent uptake patterns of metals by organisms.Looking at the current experimental design, subgroupswould be defined by species that respond similarly in aquantitative sense to soil characteristics and metal poolsidentified by the multivariate descriptive statistics. At

Table 4

Parameters of Pb uptake by Tenebrio molitor in various soils and soil–sediment mixtures

Soila Total [Pb] (mmol/kg) Time 95% Cc(ss)b (Days) Calculated Cc(ss)

c (mmol/kg) R2 Derived Cc(ss)d

Min–Mean–Max

B 180 5 6.64 0.92 —

C 310 7 24.86 0.87 —

E 4290 7 197.1 0.96 —

F 470 4 25.51 0.94 —

G od.l.e 5 26.29 0.91 —

I 150 417 Lin 34.4f 0.96 4.74–11.52–27.99

K 260 417 Lin 877.3f 0.93 7.86–19.1–46.40

O 120 6 9.25 0.94 —

R 440 6 72.24 0.92 —

S 1830 4 79.58 0.92 —

AF 30 3 3.15 0.87 —

AQg 230 417 Lin 49.41f 0.99 15.17–36.86–89.55

AS 1790 417 Lin 200.4f 0.99 40.0–97.15–236.0

AW 7110 417 Lin 1154f 0.99 130.9–318.1–772.9

MWAA 2190 6 169.40 0.96 —

MDEV 5070 6 114.2 0.87 —

MAPE 4350 14 796.6 0.99 —

MEPE 3700 5 167.7 0.93 —

aFor soil codes see Peijnenburg et al. (2000) or Table 1.bTime required to reach 95% of the steady-state level.cCalculated steady-state concentration (Eq. (2)).dSteady-state concentrations in the organisms, derived by means of the formulas obtained with the other soils in the training set. The minimum

and maximum values are calculated as 1.96� SD (=confidence limits).eLin=linear uptake, further increase in Cc(ss) observed. The values given are the internal concentration found in the last sampling treatment (after

17 and 14 days of exposure for soils and mixtures, respectively).fDetection limit (od.l.)=0.019 mmol Pb/kg dry wt.gSpiked field soil.

Note: All soil codes beginning with M are soil–sediment mixtures.


least two subgroups of soil-inhabiting species can yet bedistinguished on this gross basis, given the availableliterature data:

(A) Species for which direct or indirect uptake via thepore water is, in a quantitative sense, the dominantmode of uptake. Examples include microbes(McGrath, 2002; Plette et al., 1999), plants(McLaughlin, 2002), and soft-bodied oligochaetespecies like earthworms (Oste et al., 2001; Peijnen-burg, 2002) and enchytraeids (Peijnenburg et al.,1999a).

(B) Species for which total metal pools are dominantfor describing metal uptake. Examples include thelarvae of T. molitor, and F. candida (Vijver et al.,2001).

This classification addresses the similarities in de-scriptors, not focusing on the internal metal concentra-tions reached, and the sensitivity of species. In the scopeof the conceptual framework for environmental man-agement purposes (Peijnenburg et al., 1997), additionalspecies with dissimilar morphology/physiology shouldbe studied and classified to confirm the classification andits usefulness as a generalized concept in risk assessment.

4.3. Extrapolation of metal uptake from spiked soils to

field soils

Toxicity data for metals in soil are typically derived inspiked OECD artificial soil under laboratory settings.Extrapolation of data obtained in spiked soils to fieldsoils is a key issue in the derivation of soil qualitycriteria for natural ecosystems (Smit, 1997). Extrapola-tion should quantify the impact of differences inbioavailability among spiked soils and field soils,in combination with the impact of typical differencesin mode of application of metals (typically leading todifferences in metal sorption). Cd uptake by beetlelarvae exposed to spiked OECD soils, field soils, andsoil–sediment mixtures vs the external total Cd concen-tration is displayed in Fig. 3. As can be deduced fromFig. 3, the magnitude of Cd uptake at similar total metalconcentrations was in the same range for spiked OECDsoils, field soils, and mixtures. These findings indicatesimilarity in accumulation patterns among spiked andnonspiked soils. Assuming that toxicity is related tointernal body concentrations, these results imply thattoxicity data for this species obtained in OECD soil maybe used directly in risk assessment for natural soils. Incontrast, Vijver et al. (2001) found that, despite similar

Table 5

Monovariate and multivariate expressions describing the steady-state concentrations of Cd and Pb in T. molitor after exposure in field soils only

Normalizationa Formula obtainedb Statisticsc

Cadmium

Monovariate

Total logCc (ss)=0.61 log[Cd]s R2 ¼ 0:77; n ¼ 9; F ¼ 28:5; P ¼ 0:001; se ¼ 0:27

CaCl2 — n.s.d

Pw — n.s.d

Soil properties — No descriptors identified

Multivariate

Total logCc(ss)=0.33+0.71 log[Cd]s�0.52 logLOI550 R2 ¼ 0:86; n ¼ 9; F ¼ 25:0; P ¼ 0:001; se ¼ 0:21

CaCl2 logCc(ss)=�0.98+0.58 log ½CdCaCl2+0.38 pH�0.48 logLOI550 R2 ¼ 0:91; n ¼ 10; F ¼ 30:0; P ¼ 0:001; se ¼ 0:17

Pw — n.s.d

Soil properties — n.s.d

Lead

Monovariate

Total logCc(ss)=�0.81+0.86 log[Pb]s R2 ¼ 0:88; n ¼ 8; F ¼ 52:2; Po0:001; se ¼ 0:21

CaCl2 — n.s.d

Pw — n.s.d


Multivariate

Total Same as monovariate regression

CaCl2 — n.s.d

Pw — n.s.d


aTotal=total metal concentration determined by aqua regia destruction (mmol/kg); CaCl2=0.01M CaCl2-extractable concentration (mmol/kg);Pw=pore water concentration (mmol/L).

bSignificant characteristics are arranged according to decreasing importance; LOI550 is loss-on-ignition at 5501C (organic fraction); pH was

determined in 0.01M CaCl2 extracts.c se, standard error of estimate.dNot significant at the P ¼ 0:005 significance level.


uptake patterns among the soils, metal uptake by thespringtail F. candida in Cd-spiked OECD artificial soils(stored for 1 month) differed by more than a factor of1000 compared with Cd uptake in nonspiked field soilsat similar metal loadings. In terms of risk assessmentthis clearly shows that extrapolation of accumulationdata between soils needs to be carried out on a species-specific basis.

5. Conclusions

The present research focused on the statisticaldescription of metal uptake patterns for larvae of thebeetle T. molitor. From the multivariate regressions

obtained in the present and previous studies, it can bededuced that metal uptake by soil-dwelling organisms ismetal, soil, and species specific.For essential elements, biotic factors (homeostasis

mechanisms) are dominant over the impact ofexternal metal pools and soil properties. Uptake ofnonessential metals is associated with abiotic factors,such as total metal pools and metal binding phases ofthe soils.Uptake patterns in soil–sediment mixtures could be

described using a similar approach as for field soils only.Internal concentrations of larvae exposed to Cd-spikedOECD soil overlapped with the internal Cd concentra-tions found in the experiments performed in field soilsand soil–sediment mixtures having similar external Cd

Table 6

Monovariate and multivariate expressions describing the steady-state concentrations of Cd and Pb in T. molitor after exposure in field soils and

soil–sediment mixtures

Normalizationa Formula obtainedb Statisticsc

Cadmium

Monovariate

Total logCc(ss)=�0.01+0.56 log[Cd]s R2 ¼ 0:65; n ¼ 13; F ¼ 23:7; Po0:001; se ¼ 0:32

CaCl2 — n.s.d

Pw — n.s.d


Multivariate

Total logCc(ss)=0.38+0.67 log [Cd]s�0.59 logLOI550 R2 ¼ 0:81; n ¼ 13; F ¼ 26:3; Po0:001; se ¼ 0:24

CaCl2 logCc(ss)=�1.02+0.38 pH�0.43 logLOI550+0.59 log ½CdCaCl2 R2 ¼ 0:91; n ¼ 14; F ¼ 44:7; Po0:001; se ¼ 0:17

Pw logCc(ss)=�0.73+0.75 log[Cd]pw�0.41 pH R2 ¼ 0:86; n ¼ 14; F ¼ 40:5; Po0:001; se ¼ 0:21


Lead

Monovariate

Total logCc(ss)=�0.88+0.90 log[Pb]s R2 ¼ 0:86; n ¼ 12; F ¼ 66:3; Po0:001; se ¼ 0:27

CaCl2 — n.s.d

Pw — n.s.d


Multivariate

Total logCc(ss)=�0.65+1.03 log[Pb]s�0.50 log silt R2 ¼ 0:88; n ¼ 12; F ¼ 39:6; Po0:001; se ¼ 0:25

CaCl2 — n.s.d

Pw — n.s.d


aTotal=total metal concentration determined by aqua regia destruction (mmol/kg); Pw=pore water concentration (mmol/L); CaCl2=0.01M

CaCl2-extractable concentration (mmol/kg).bSignificant characteristics are arranged according to decreasing importance; LOI550 is loss-on-ignition at 5501C (organic fraction); Silts the

fraction of particles with a diameter between 2 and 38 mm; pH was determined in 0.01M CaCl2 extracts.c se, standard error of estimate.dNot significant at the P ¼ 0:005 significant level.

Table 7

Statistical relationships between the various Cd pools in the soils and mixtures included in the regression analyses given in Tables 5 and 6

Exposure soils Formula obtained Statistics

Field soils log½Cds¼ 0:77 log½CdCaCl2 þ 0:48 pH R2 ¼ 0:94; n ¼ 10; F ¼ 57:3; Po0:001

Field soils and mixtures log½Cds¼ 0:74 log½CdCaCl2 þ 0:53 pH R2 ¼ 0:85; n ¼ 14; F ¼ 30:5; Po0:001

Field soils and mixtures log½Cds¼ 0:81 log½Cdpw þ 0:48 pH R2 ¼ 0:61; n ¼ 14; F ¼ 8:74; Po0:003


concentrations. It should be noted that this is not ageneral observation for all animals, and extrapolationbetween data collected form OECD experiments andfrom field soils might nevertheless require a species-specific correction.The prediction that metal uptake can be best

correlated to the metal pool in the pore water is notconsidered valid for the larvae tested in this study assolid soil phases are the most important variables whendescribing metal concentrations in the larvae. For thepragmatic use of metal risk assessment procedures, i.e.,to correct for soil-type influences on bioavailability,organisms probably can be divided into classes ofspecies with grossly similar uptake characteristics. Onegroup of organisms can be distinguished by apparentlydominant pore water-mediated uptake, while metaluptake by another group of soil-dwelling organisms isshown to be related mainly to total metal pools in thesoil. For the general purpose of a well-balanced riskassessment, more species with a suite of ecophysiologicalcharacteristics should be studied.

Acknowledgments

We thank Rens van Veen, Arthur de Groot, and RobBaerselman for their participation in the project, and weare grateful to S. Oosterwaal (Ermelo) for donating thebeetle larvae.

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Metal uptake from soils and soil–sediment mixtures by larvae of Tenebrio molitor (L.) (Coleoptera)

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