You are on page 1of 7

Ecotoxicology DOI 10.

1007/s10646-013-1137-y

The use of elements as a substitute for biomass in toxicokinetic studies in small organisms
Nina Cedergreen • Peter E. Holm • Helle Marcussen

Accepted: 25 September 2013 Ó Springer Science+Business Media New York 2013

Abstract Determining pollutant concentrations in the tissues of experimental test organisms is necessary for understanding uptake and excretion mechanisms of toxicants. Using small organisms can make the determination of organism biomass inaccurate. We here propose the use of selected tissue element contents as a proxy for tissue biomass. Forty different elements were determined in tissues of the two worm species Enchytraeus crypticus and Caenorhabditis elegans derived from cultures exposed to combinations of varying temperatures and sublethal concentrations of Cu and Cd. Three criteria were used to select good biomass indicators: The element concentration must (1) be present in concentrations above the limit of quantification of the analytical method, (2) must be stable and (3) must not be affected by the treatment. If the organisms are believed to have significant amounts of soil in their gut, the element must also be present at higher concentrations in the tissue compared to the soil. The three elements K, Mg and P all lived up to the first three criteria for both worm species, showing correlation coefficients between element content and tissue biomass of 0.97, 0.96 and 0.97 (n = 25) and 0.997, 0.998 and 0.992 (n = 10) for K, Mg and P in the E. crypticus and C. elegans, respectively. Only P would be an appropriate biomass indicator for organisms with a soil gut uptake assuming the tissue concentrations in soil eating organisms are similar to those measured in the present study. Using Mg as a biomass indicator on a verification dataset of Cu and Cd uptake in E. crypticus, compared to giving Cu and Cd content per individual organism, decreased the coefficient of variation from 31 ± 21 to 21 ± 17 % and from 34 ± 22 to
N. Cedergreen (&) Á P. E. Holm Á H. Marcussen Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederksberg, Denmark e-mail: ncf@life.ku.dk

9.3 ± 6.4 % for tissue Cu and Cd, respectively. We therefore conclude that the use of an element as a biomass indicator can reduce tissue concentration variability. Keywords Biomass indicator Á Uptake kinetic Á Metals Á Variability Á Dry weight

Introduction Determining pollutant concentrations in the tissues of experimental test organisms is necessary for understanding uptake and excretion mechanisms under different toxicant exposures and environmental conditions. Such determinations require sampling of several organisms over time for each treatment. Hence, small test organisms such as springtails, enchytraeids, mites and woodlice are often used for terrestrial test, while daphnids or gammarids are often preferred in aquatic experiments on toxicokinetics (Vijver et al. 2001; Spurgeon and Hopkin 1999; Ashauer et al. 2007; Heugens et al. 2003; Kramarz 1999; Lagisz et al. 2005). The most common way to express internal pollutant contents is to use concentrations per biomass fresh or dry weight (Vijver et al. 2001; Kramarz 1999; Lagisz et al. 2005), but also content per number of organisms have been used (Spurgeon and Hopkin 1999). For soil and sedimenteating animals fresh and dry weight measurements carries the risk of overestimating biomass, as the soil content of the gut will be included in the mass determination. In addition the error in weight determination due to gut content are likely to be very variable between individuals as gut content can vary considerably (Piearce 1978). Letting, for example, earthworms empty their gut in a non-soil environment prior to sampling (Lock et al. 2000; Spurgeon and Hopkin 1999), can introduce a bias, as they might also

123

000 per treatment) were collected. After 24 days large numbers of adult worms (100–1. 1 mL 1 M MgSO4 per L MilliQ water. When the synchronized cultures had reached the L4 larval stage. if any elements. respectively (Fountain and Hopkin 2001). will decompose any organic molecules that one could use as a measure of biomass. The worms were kept in LUFA 2. 1980). Mg and P content in the agar was 25. 5 g NaCl. added as CdCl2 stock in MilliQ water). To test the correlation between biomass and element content in differentially stressed worms the following experiments were carried out: Samples of worms exposed to sublethal concentrations of Cu (150 mg added Cu kg-1 dry soil.000 individuals per plate. excrete the toxicant during the depuration time of typically 12–48 h. and water holding capacity of 55 ± 4 %. 18 and 25 °C). however. are very reliant on accurate and consistent measures of biomass.5 °C in darkness on plates of a modified bacteriological agar [nematode growth medium (NGM) (Brenner 1974)] and fed Escherichia coli.5 M NaOH. cation exchange capacity of 10 ± 0. Stitt and Schulze 1994). were reared from cultures originally established with worms obtained by the terrestrial ecotoxicology group at Vrije University. Three plates per replicate and three replicates per treatment were used. the digestion of the sample. Age synchronization of the cultures was done by washing gravid adults of culture plates with autoclaved M9 media (3 g KH2PO4. in cases where biomass estimates are difficult to make (Schroeder et al. Cedergreen et al. In this study the tissue content of 40 elements were tested and evaluated as possible substitutes for tissue dry weight in worms exposed to combinations of variable temperatures and sublethal concentrations of copper (Cu) and cadmium (Cd). Alfa Aesar) to prevent the test animals from reproducing (Gandhi et al.5 %). After seven days. The aim of this study was therefore to investigate. requires very accurate balances and consistent methods for the error in weight determination to be tolerable. with masses around 40 lg dry weight and 10 lg fresh weight per specimen. elegans. the element concentration in the soil should ideally be much smaller than that of the tissue in order to avoid the bias mentioned for weighing worms with gut soil contents. H2O to 1 L and then bleaching them with a 0. necessary to release all internal metal. 1 and 25 mM. a precise measure of biomass is of the utmost importance. These where then spread thinly on OP50 inoculated agar plates. We aimed to identify elements to be used as biomass indicators that lived up to the following criteria: (1) the content of the biomass indicator is well above the limit of quantification (LOQ) for the amount of biomass proposed to be available (2) there is a strong correlation between the total amount of biomass indicator and the tissue dry weight and (3) this correlation (where the slope gives the tissue concentration of the biomass indicator). For quantification of the biomass of fungi in ecological studies. In addition. Germany) and were fed weekly with a mixture of autoclaved oat grains and fish food (protein content 33. such as uptake and elimination rates. or agar spiked with 2 mg Cu L-1 and 0.5 cmol(?) kg-1. 1 % sodium hypochlorite solution to obtain liquid cultures containing eggs (http://www. where tissue weights and soils weights cannot be distinguished. and left to hatch at 21 °C. The K. for example. Amsterdam. E. The cultures were maintained by transferring a chunk of agar from the existing culture plates to freshly prepared NGM agar plates each week. Speyer. which would not be affected by a total digestion. of the uracil deficient strain OP50. 6 g Na2HPO4.1 ± 0. Materials and methods Test organisms The enchytraeids. 2006.pdf). C. added as CuCl2 stock in MilliQ water). As the determination of toxicokinetic parameters. could act as robust biomass indicators for the worms Enchytraeus crypticus and Caenorhabditis elegans. or no metals were raised at three different temperatures (11. the internal contaminant content is sometimes given per protein content or nucleic acid equivalent. Weighing very small animals like springtails and small worms such as nematodes. the worms were gently washed off the agar plates with MilliQ water into a 15 mL centrifuge tube. and Cd (40 mg added Cd kg-1. is independent of external stressors such as changes in temperatures or pollutant level. All plates contained 50 lM of 5-fluorodeoxyuridine (FUdR) (98 %.5 ± 1.4 %. frozen and freeze dried. elegans Genetics Centre) were cultivated at 21 °C ± 0. rinsed. The LUFA 2.und Forschungsanstalt (LUFA). organic carbon content of 2.N.2 soil is a loamy sand soil with a pH of 5. of the N2 Bristol strain (obtained from the C. The nematodes. The plates were either clean agar. the fungi specific membrane component ergosterol has been used as a biomass indicator (Charcosset and Chauvet 2001). if the gut content of soil is believed to be significant. When measuring organic contaminants or various metabolites.1 (CaCl2). they were suspended in M9 media and spread on test plates (90 mm diameter).1 mg Cd L-1 from CuCl2 and CdCl2 stocks in MilliQ water.wormbook.2 standard soil (Landwirtschaftliche Untersuchungs. with approximately 1. The plates were checked daily and when necessary a few drops of concentrated OP50 suspension was added to the plate and spread with an inoculation hoop.org/chapters/www_strainmaintain/ strainmaintain. They were gently shaken to wash off excess bacteria and spun down for two minutes at 500 rpm 123 . crypticus. If the contaminant is a bioactive metal.

Ho. it was concluded that accurate dry weight determination was not possible for the worms. Data treatment All elements with any samples below LOQ were discarded as potential indicators of biomass together with Cu and Cd. Manchester. respectively. K. elegans and with a two-way ANOVA for E. elegans. Cu.Elements as a substitute for biomass (21. Yb and Zn) were determined by inductively coupled plasma mass spectrometry (ICP-MS) (Agilent 7500C. External calibration was applied and drift within 10 % was corrected for by recalibration. Cadmium was determined by graphite furnace atomic absorption spectrometry (GFAAS) using Zeeman-effect background correction (Perkin Elmer 5100 AAS. SigmaAldrich) in a microwave assisted system operated at 350 W for 20 min with a 10 min ramp and 450 W for 30 min with a 5 min ramp (Multiwave 3000. Co.a. the following analysis was carried out for the verification dataset. To test for treatment effects. The supernatant was removed with a pipette and approximately 10 mL of MilliQ water was added. with the aim of finding the most stable tissue element concentration. grown as described above. Chemical analyses To establish the correlation between the elements and the biomass. The content of other elements than Cu and Cd and the variance of the data when expressed per worm versus when expressed per Mg content.5 g) (Sigma. the element concentration for the three elements with the smallest CV was tested with a one-way ANOVA for C. HNO3 (Baker Instra-Analyzed. Fe. 2013). As the worm dry weight calculated after extracting the weight of the eppendorf tube was negative in approximately 30 % of the samples. Gd. Determined concentrations of Mg. p. the worms were gently shaken. To further investigate. Ce. HGA-600 graphite furnace) applying a 0.1 % Pd and 0.250 mL conc. Dy. Cu. crypticus and C. Ba. crypticus grown at three temperatures over 24 days. Ni. Mo. J. internal Cu and Cd concentrations were given per worm. Sm. to test the consequences of using Mg as a biomass indicator. Mg was used as a proxy for biomass in a study of uptake kinetics of Cu and Cd in E. assuming Mg can be used as a proxy for biomass. Y. whether the selected biomass indicator Mg could be used in studies of Cu and Cd in enchytraeids experiments where the total sample mass is too low to be determined by weighting. As. Then the element concentration per dry weight (DW) was calculated and the variance given as the coefficient of variation (CV) (standard deviation/average) was compared between elements. Blanks and bovine liver standard reference material NIST 1577c (National Institute of Standards and Technology (NIST)) were included in each digestion for quality assurance. Mg. Nd. 123 . Cr. Two times five worms were taken out at each sampling time into oven dried preweighed eppendorph tubes. crypticus using temperature and metal treatment as the two independent variables. digestion of 1-20 mg worm were carried out using 0. Se and Mo in the digested NIST 1577c standard were within ± 10 % of the certified range and the concentration of the other certified elements in the reference material were below the limit of quantification. Co. Lu. Baker) and 0. 2013.T. Zn. The determined element concentrations in digestion blanks were all below the detection limit or less than 1 % of the sample concentrations. which formed part of the treatment. The digestion was done for 25 and 12 samples of E. They were dried at 80 °C and re-weighed on a five-decimal balance (Sartorious MC210S). Pr.2 % Mg modifier (Merck). Limit of detection and quantification were calculated as three and ten times the standard deviation of at least 8 replicate analyses of the calibration blank. Sc. Tb. The Cu and Cd uptake kinetics. Finally. Cs. Hence. Verification dataset To verify the use of an element as a biomass indicator. Be. has not been previously published. La. The concentrations of Cu or Cd were determined by graphite furnace atomic absorption spectrometry using Zeemaneffect background correction (Perkin Elmer 5100 AAS. Al. P. Mn. Uptake kinetics based on these data were compared to those based on Cu and Cd per lg sample Mg. Pb. UK) equipped with an octopole reaction system (ORS). HGA-600 graphite furnace). Centrifuge 2K15). Eu. Er. Tm. spun down and the procedure was repeated another two times after which the nematodes were frozen and freeze dried. Se. Sb. These results show that the digestion and analytical procedure in general is accurate and reproducible. Agilent Technologies. Li. The dataset comprised 52 and 47 measurements of Cu and Cd tissue concentrations. rotor 64MG5. Five worms per sample were digested with the same digestion procedure and quality assurance as described above. which adhered to the inside of the eppendorf tubes. their dependence on growth temperatures and Cu and Cd availability in the soil and the resulting toxicity have been published in Cedergreen et al. Magnesium concentrations were determined by flame atomic absorption spectrometry. the CV of Cu and Cd per Mg content was compared to that of Cu and Cd per worm for the verification dataset (Cedergreen et al.125 mL 30 % H2O2 (puriss.. V. K. Worm dry weight was later calculated from the element contents to be in the range of 1–2 mg per worm. Th. The concentrations of 40 elements (Ag. Anton Paar GmbH). U.

whereas for the nematodes it was P.380 fold the LOQ for E. U and Y. Nd. while in nematodes they were 0.2 ± 0.504 ± 681 1.97 for K. 0. 1b).0 16 ± 6 4. in the case of organisms within this size range. A good element indicator of biomass for small organisms must be present in concentrations which allow quantification with a reasonable accuracy. these elements were present in 188–813 fold and 67–1. A good biomass indicator must. For the enchytraeids. 0. crypticus (n = 25) Element K Co P Mg Zn U Ca Pb V Ba Sr Mn Fe Al Li Th Pr Y La Ce Nd Sb Ni Cr Min:LOQ ratio 28 12 456 187 45 2 15 19 99 31 79 570 813 809 10 2 5 18 16 34 3 1 6 8 Element conc. crypticus and C.5 ± 0. Ho. with the exception of K for the enchytraeids which were number 11 on the list with a minimum concentration to LOQ ratio of 28. Cu and Cd. Dy. Results and discussion Enchytraeus crypticus samples had concentrations below the LOQ for the following elements: Ag.998 and 0. but as it is present in lower concentrations. Be.153 ± 382 4. elegans). For the lowest biomasses used in this study (2. as expected.997. 2).2 CV % 20 21 22 26 28 28 33 36 36 37 39 42 44 45 46 50 53 53 53 55 55 57 76 80 Data are sorted with increasing CV for the element concentration the nematodes (Tables 1. Cedergreen et al. 1a). showed. Mg and P (n = 10). the correlation coefficients were 0. Mo.N. Th.4 1. Se.29 ± 0. it is important to ensure that the biomass indicators are not prone to covariate with the treatments.29 ± 0. Se. (lg g-1) 4. Al. elegans. In addition all three elements were within the top five candidates with regards to the ratio between the minimum concentration and the LOQ. As. The Pearson correlation coefficients between these elements and biomasses were 0. Cs. The relatively poor correlation between elements and biomass for the nematodes was primarily due to two outliers (Fig. Hence.9 ± 3.6 0. Mg and P (n = 12). This means that the minimum biomass that can be quantified by use of a biomass indicator within their LOQ would be in the range of 10–45 lg DW for the enchytraeids and 4–85 lg DW for the nematodes. With such high correlation coefficients. Such biomasses are in the very low range of what is accurately quantifiable using even the most sensitive balances. Lu. using one of these elements as a biomass indicator. If these were removed.73.97. Different metal stressors are known to affect the uptake and excretion of other elements (Goyer 1997). Mg and P). Tm and Yb. Mg and P. In the presented example. Sm. Tb. depending on the choice of element. Gd.37 ± 0.8 3.7 0. the five elements present in the highest quantity relative to what could be determined were Fe.308 ± 854 0. Hence.46 1. will be very minor. Er.08 1. For C. Eu.11 4.36 ± 0.8 mg DW for E. Mn.96 and 0.5 5.86 ± 0.53 ± 0.06 2. the ratio of the observed smallest element concentration determined in a sample of 1–20 mg relative to the LOQ of that element was used.1 ± 1. its detection may not always be possible in studies with limited amount of biomass. also be very stable and unaffected by the growth conditions of the organisms in question. Pr. Sb. crypticus and 1.85. 123 .94 for K.6 ± 2. the CV of the element to biomass ratio (tissue concentration) was B26 % for the enchytraeids and B29 % for Table 1 A measure of the sample minimum element concentration (Min) relative to limit of quantification (LOQ) is given together with the average element concentration given per dry weight ± SD and the corresponding CV for E. To get a measure of the quantity of each element in digested samples relative to what is quantifiable by the analytical method.992 for K.022 543 ± 139 82 ± 23 0. Or it could provide a suitable alternative in the laboratories that do not have access to balances that can weigh such small biomasses accurately. Al and K (Table 1 and 2). Li. Co was also very stable.93 and 0. Mg and P measured on biomasses in the range of 1–20 mg DW. Mn. were excluded for further investigation. the three elements that were most stable biomass indicators across treatments for both enchytraeids (Table 1) and nematodes (Table 2) were K.18 0.7 55 ± 23 855 ± 376 1. if any. 0.15 0.4 ± 1.5 ± 1.72 and 0. or other stressors. For the enchytraeids. respectively.2 ± 1.73 2.9 mg DW for C. as this could cause a bias in the final results. The two-way ANOVA on the element to biomass ratio made on the enchytraeid data. if working with pollutant stressors. 0. could possibly provide a more accurate estimate of biomass compared to what can be obtained using a balance. For K.10 ± 0. For the nematodes. Mg and P in the enchytraeids (n = 25) (Fig. Mg. These elements together with the two amended treatment metals/elements: Cu and Cd. however.71 for K.722 ± 1. an ANOVA analysis on the element to biomass ratio of the three treatments: control. elegans the elements with samples below the LOQ were the same as for the enchytraeids with the addition of: La. no significant difference between treatments (p = 0. P and Mg. treatment effects.

57 0. the enchytraeids because reviews of enchytraeids feeding habits suggest that the small worms feed selectively on decomposed plant material and microorganisms and that the ingestion of large amounts of inorganic soil is unlikely (O’Connor 1967. Considering the fine correlations between the contents of selected elements and the biomass. 9 and 0. for such a case.39 ± 0.1).01 2. 18 and 25 °C) (p = 0.34 0. Correlation coefficients are given in the text. because they grew on agar. Cd (circles) or without metals (triangles).033 ± 3. broken lines (P) or dotted lines (K).50 ± 0.80).255 0.85 2.03 ± 0.641 ± 5. show the importance of checking for treatment effects to be sure to identify if potential outliers could be treatment related.05 ± 0.60 ± 0.98 ± 0. The nematodes.001). For the 28 and 21 combinations of temperature and time where two or more samples were sampled in the Cu and Cd uptake experiments. Finally using Mg as a biomass indicator for studying uptake kinetics of Cu and Cd. the element concentration in the soil should ideally be much lower than that of the tissue concentration in order to avoid a bias from the soil gut content. Performing a one-way ANOVA on treatment effects using a post hoc Tukey test showed that there was no difference between Cu and Cd treated worms for any of the elements. 1 Correlations between the tissue content of the three elements: Mg (black symbols).743 1.04 0. however. respectively.2 ± 2. The analyses showed significant interactions for all elements (p \ 0.6 179 ± 224 CV % 29 29 29 30 32 32 35 41 42 46 59 85 96 106 114 125 Data are sorted with increasing CV for the element concentration however. The example shows the importance of including the relationship between soil and tissue content in cases where soil gut content might be of significance.38 and 0.946 ± 565 17. The correlations are given as full lines (Mg).27 % of the K. showed a significant effect of treatment (control. this would mean that 11.002). K. but for P (p = 0. Cu and Cd treatment) (p \ 0. as opposed to using the Cu and Cd content per worm. the CV % decreased from in average 31 ± 21 to 21 ± 17 % for internal Cu concentrations and from 34 ± 22 to Fig. showed a much smaller variation between replicates. Assuming that in average 10 % of the weight of for example an earthworm consisted of soil and that the earthworm had a tissue mineral composition of a nematode (Table 1). elegans (n = 12) Element K Mg P Mn Fe Zn Sr Co Ca Th V Ce Cr Ni Ba Al Min:LOQ ratio 67 572 1.43 mg g-1 soil (Sposito 1999).Elements as a substitute for biomass Table 2 A measure of the sample minimum element concentration (Min) relative to Limit of Quantification (LOQ) is given together with the average element concentration given per dry weight ± SD and the corresponding CV for C. Mg and P are present in soils in average concentration of around 15. and corresponding low CV of element concentrations.957 ± 1.81 ± 0.23 0. They do. but that two high control values of the 11 °C made the control treatment significantly higher than the metal treated samples. Mg and P. (lg g-1) 13. Hence. but not for temperature for K and Mg (11. Neither nematodes nor the enchytraeids are believed to have a gut content affecting the weight measurements. The outlier samples marked with an asterisk are excluded from the correlations 123 .380 133 63 33 25 1 38 3 5 1 1 1 3 100 Element conc. we do not consider the ANOVA results problematic in the sense that it should disregard the use of the elements as biomass indicators. The three elements. only P would be suitable as a biomass indicator. respectively came from the gut soil content.23 0. 34 and 0. Rombke 2003). For organisms where the gut content of soil elements could be of quantitative importance. P (grey symbols) and K (open symbols) as a function of dry weight of the enchytraeid Enchytraeus crypticus (a) and the nematode Caenorhabditis elegans (b) for worms treated with either Cu (squares).154 14 ± 4 102 ± 33 69 ± 22 0.

when using the biomass indicator Mg rather than giving the tissue concentrations per individual. App Environ Microbiol 67:2051–2055 Fountain MT. Mg. Alvarez et al. CVs in the 30 % range and up to [100 % is. The ground beetle. Broerse et al. 2011. Environ Toxicol Chem 25:3230–3237 Amorim MJB. University of Copenhagen. Spurgeon D (2013) Low temperatures enhance the toxicity of Cu and Cd to Enchytraeus crypticus through different mechanisms. Genetics 77:71–94 Broerse M. Using the correlation between Mg and biomass established in this study (R2 = 0. we suggest that laboratory specific element to dry weight correlations are made for the organisms used in a particular study. leading to more accurate and reproducible estimates of uptake and depuration kinetics of metals and organic pollutants in small organisms. Marcussen H. Kramarz P. Spurgeon and Hopkin 1999. Rombke J. Arch Environ Contam Toxicol 48:484–489 Lock K. 1. Bull Environ Contam Toxicol 63:531–537 Lagisz M. aging populations of Caenorhabditis Elegans. Nørhave N. Redondo EM. Hendriks AJ. Conflict of interest of interest. Environ Toxicol Chem 32:2274–2283 Charcosset JY. Brown CD (2007) Modeling combined effects of pulsed exposure to carbaryl and chlorpyrifos on Gammarus pulex. The kinetic curves for Cd are shown in Fig. Environ Sci Technol 41:5535–5541 Brenner S (1974) Genetics of Caenorhabditis Elegans. (2013) 9. De Coen WM (2000) Multivariate test designs to asses the influence of zinc and cadmium bioavailability in 123 . Data is described with a two parameter saturation kinetic model. 2012).e-bx) where a is proportional to the slope of the curves while b denotes its maximum. Ecotoxicol Environ Saf 48:275–286 Gandhi S.3 ± 6.). Cedergreen et al. Soares AMVM (2005) Avoidance behaviour of Enchytraeus albidus: effects of benomyl. not an uncommon range (Amorim et al. Kraak MHS. Loureiro S. Jager T. Creyghton R. Fig. Gravato CS. Svendsen C. Lock et al. Soares AMV (2011) Toxicity and Bioaccumulation of Phenanthrene in Enchytraeus Albidus (Oligochaeta: Enchytraeidae). The work was supported by a grant given by the Department of Basic Sciences and Environment at the Faculty of Life Science. Environ Sci Technol 37:2145–2151 Kramarz P (1999) Dynamics of accumulation and decontamination of cadmium and zinc in carnivorous invertebrates. 2000. Raosanadi D (1980) A simple method for maintaining large. Until the generality of element to dry weight ratios across genotypes and between different laboratories are verified. Nørhave for giving us access to the nematode data and to help with the metal analyses and Birgitte Boje Rasmussen for carrying out GFAAS and ICP-MS analyses. Data is from Cedergreen et al. Hopkin SP (2001) Continuous monitoring of Folsomia candida (Insecta: Collembola) in a metal exposure test. phenmedipham and different soil types. is most likely a reflection of the worms varying slightly in size depending on growth temperature (pers obs.) originating from metal-contaminated and reference areas. relative to each other. Van Gestel CA (2012) Cadmium affects toxicokinetics of pyrene in the collembolan Folsomia candida. The authors declare that they have no conflict References Alvarez OA. Mech Ageing Dev 12:137–150 Goyer RA (1997) Toxic and essential metal interactions. Poecilus cupreus L. Oorsprong H.4 % for internal Cd concentrations. The reason for the slight shift in the three temperature dependent uptake kinetic curves. 2005.96) makes it easy to convert the Mg to biomass. 2006. Janssen CR. 2. We therefore suggest that in many cases the use of an element as a biomass indicator can reduce that variability. The variation between replicates is markedly decreased using the biomass indicator. Annu Rev Nutr 17:37–50 Heugens EHW. y = a(1 . Acknowledgments We wish to thank Nils J. Boxall ABA. Ecotoxicology 21:795–802 Cedergreen N. Nielsen K. Jager T. carbendazim. Santelli J. Chemosphere 59:501–510 Amorim MJ. Niklinska M (2005) Metal kinetics and respiration rates in F-1 generation of carabid beetles (Pterostichus oblongopunctatus F. from a visual assessment of uptake kinetic curves.N. Oliveira E. Mitchell DH. however. 2 Uptake kinetics of Cd in Enchytraeus crypticus at three different temperatures given either as Cd content per individual (a) or as Cd per Mg content (b). Teixeira AS. Stiles JW. Johansson HKL. Van Gestel CA. Environ Toxicol Chem 30:967–972 Ashauer R. Kammenga JE (2006) Physiological modes of action of toxic chemicals in the nematode Acrobeloides nanus. Chauvet E (2001) Effect of culture conditions on ergosterol as an indicator of biomass in the aquatic hyphomycetes. Guilhermino LC. van Straalen NM. Admiraal W (2003) Temperature-dependent effects of cadmium on Daphnia magna: accumulation versus sensitivity.

Granzow M. pp 213–257 Piearce TG (1978) Gut content of some lumbricid earthworms. Jager T. Salowsky R. Oxford University Press. Environ Toxicol Chem 19:2666–2671 O’Connor FB (1967) The Enchytraeidae. Environ Toxicol Chem 20:712–720 123 . Stocker S. Hopkin SP (1999) Comparisons of metal accumulation and excretion kinetics in earthworms (Eisenia fetida) exposed to contaminated field and laboratory soils. Lightfoot S. Oxford Spurgeon DJ. Posthuma L. 1st edn. vol 8.Elements as a substitute for biomass soils on the toxicity to Enchytraeus albidus. Leiber M. Raw F (eds) Soil biology. Gassmann M. Pedobiologia 18:153–157 Rombke J (2003) Ecotoxicological laboratory tests with enchytraeids: a review. Peijnenburg W (2001) Impact of metal pools and soil properties on metal accumulation in Folsomia candida (Collembola). London. Schulze D (1994) Does Rubisco control the rate of photosynthesis and plant growth? An exercise in molecular ecophysiology. Menzel W. Ragg T (2006) The RIN: an RNA integrity number for assigning integrity values to RNA measurements. Mueller O. In: Burges A. Plant Cell Environ 17:465–487 Vijver M. BMC Mol Biol 7:3 Sposito G (1999) The Chemistry of Soils. Academic Press. App Soil Ecol 11:227–243 Stitt M. Pedobiologia 47:607–616 Schroeder A.