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Project no. 003956

Project acronym NOMIRACLE

Project title Novel Methods for Integrated Risk Assessment ofCumulative Stressors in Europe

Instrument IP

Thematic Priority 1.1.6.3, ‘Global Change and Ecosystems’Topic VII.1.1.a, ‘Development of risk assessmentmethodologies’

Start date of project: 1 November 2005 Duration: 5 years

Organisation name of lead contractor for this deliverable: NERI

Revision: 2. revision

Project co-funded by the European Commission within the Sixth Framework Programme (2002-2006)Dissemination Level

PU Public XPP Restricted to other programme participants (including the Commission Services)RE Restricted to a group specified by the consortium (including the Commission Services)CO Confidential, only for members of the consortium (including the Commission Services)

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Authors and their organisation:

Irina Lehmann and Gunnar Wichmann, UFZ, Partner 3Albert Duschl, USALZ, Partner 17Almut Gerhardt and Cornelia Kienle, LimCo, Partner 24Volker Scheil, Silke Müller and Heinz-R. Köhler, EKUT, Partner 11Susanna Loureiro and Amadeu Soares, UAVR, Partner 13Jan Kammenga, WU, Partner 12Ryszard Laskowski and Paulina Kramarz, UJAG, Partner 10Anne-Mette Bindesbøl and Martin Holmstrup (editor), NERI, Partner 1

Deliverable no:D.3.2.1

Nature:Report

Disseminationlevel: PU

Date of delivery:August 31, 2005

Status: First version Date of publishing:August 31

Reviewed by (period and name):19-26 August, 2005 by Dave Spurgeon

Deliverable reference number and title:

D.3.2.1. Report describing baseline responses to allergenic, pathogenic andnatural stressors for the various cell lines/test organisms as dose-responserelationships.

Due date of deliverable: August 31 Actual submission date: August 31, 2005

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Contents

1 Introduction...............................................................................................................................5

2 Report describing baseline responses to allergenic, pathogenic and natural stressors for the

various cell lines/test organisms as dose-response relationships.....................................................6

3 Baseline responses of human reporter gene cells to the immune stressor TNF-α....................9

4 Baseline response of Zebrafish larvae (Danio rerio) to a natural stressor (Anoxia) in the

Multispecies freshwater Biomonitor (MFB) .................................................................................13

5 Baseline responses of the zebrafish (Danio rerio) to a natural stressor (temperature) during

prolonged embryo tests..................................................................................................................19

6 Effects of natural stressors on Daphnia Magna......................................................................23

7 Effects of temperature and nickelchloride on life-history traits of the free-living soil

nematode Caenorhabditis elegans.................................................................................................25

8 Preliminary results of range-finding experiments for environmental stressors for P.

oblongopunctatus (Coleoptera: Carabidae) ...................................................................................29

9 Baseline studies of natural stressors: Establishment of survival of subzero temperatures in

earthworms ....................................................................................................................................32

10 Baseline studies of natural stressors: Establishment of survival of drought in Collembola ...34

11 Summary and future research in WP 3.2 ................................................................................36

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1 IntroductionThis work package is key to the aims of NOMIRACLE as it will place the classical empirical mixture toxicityanalysis in the context of the diverse environmental conditions that exist across Europe thereby unifying the fields oftoxicology and stress biology. The environmental and population specific factors considered are selected in theprioritisation and scooping process undertaken in RP1. Scenarios selected will be relevant for the systems understudy. For instance, the influence of pathogens/allergens may be most relevant for human health, the influence ofdrought most relevant for soil organisms, anoxia for aquatic organisms, diet quality for all scenarios and so on.Biological systems used for these mixed stressor studies will overlap closely with those used in WP 3.1 allowing usto establish response conservation for single and later multiple stressors.The approach to identifying the interactive effects of chemical and environmental stressors will mirror that used forcontaminant mixtures in WP 3.1. First the effects of the single prioritised environmental stressor on cells andorganism systems will be described. These responses will be collated thereby providing spin-off informationconcerning the limits of the tolerance of cells, tissues and individuals to environmental variation and change. Oncewe have established the effects of single environmental factors the effects of these in combination with chemicalstaken from the prioritisation exercise in WP 1.2 will be investigated. The same experimental design and dataanalysis approach will be used in the multiple stressor studies as used for the contaminant mixtures investigated inWP 3.1. Interactions will be modelled initially assuming independent action, although, as the data modellingapproach can be used without a priori knowledge of the interaction mechanism, this assumption can be validated foreach multiple stressor experiment. For both analyses, likelihood analysis will be applied in order to identifydeviations (absolute synergism/antagonism, effect level and ratio dependent) from the underlying model. Whenclear and consistent interactions between environmental and chemical stress factors are identified these responseswill be catalogued for subsequent analysis of underlying toxicokinetic and molecular mechanisms in WP 3.3 and3.4. This data will be collated and stored, thereby providing essential information for cumulative risk assessment forvulnerable populations. The ultimate goal of WP 3.2 is to have a broad understanding of the effects of relevantcumulative stressor combinations (chemicals combined with environmental stress) on a wide range of organisms andhuman immuno-responses relevant to communities in Europe.

The contents of this report is not a detailed review of existing information, but a description of initial experimentsperformed with aim to set out standards for the coming investigations of the combined effects of natural andchemical stressors in WP 3.2. This focus on the development of “Novel Methods” is in accordance with the overallgoal of NOMIRACLE. Thus, the focus of the present deliverable is on method development and scenarioidentification.

In this report we group the reports of the individual partners according to exposure scenario. First we report onscenarios for human health (Section 2 and 3), then on scenarios for freshwater organisms (Section 4-6), and lastlythe scenarios for terrestrial invertebrates (Section 7-10).

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2 Report describing baseline responses to allergenic, pathogenic and natural stressors

for the various cell lines/test organisms as dose-response relationshipsIrina Lehmann and Gunnar Wichmann, UFZ Centre for Environmental Research, Partner 3

Introduction

Partner 3 (UFZ) investigates the influence of environmental chemicals on responses of human primary immune cellsto allergenic and pathogenic stressors. Human peripheral blood mononuclear cells (PBMC) of healthy blood donorsare used as indicator cells. Lipopolysaccharide (LPS) was selected as an example for a pathogenic stressor. LPS,a cell wall component of Gram-negative bacteria, is a common bacterial endotoxin and known as a potent inducer ofinflammatory reactions. Birch pollen (BP) allergen extract (AE) was selected as an example for an allergenicstressor. BP is a common allergen and known as a potent inducer of allergic sensitization and, following re-contactof sensitized individuals to its allergenic components, triggers allergic reactions of the immediate type.

Results

LPS induces strong activation signals and among others the production of the pro-inflammatory cytokines tumour-necrosis-factor-α (TNF-α) and interleukin-6 (IL-6). The dose-dependent cytokine production of human PBMC afterstimulation with LPS is shown in Figure 2.1. Since the application of those chemicals which are planned to beincluded in future investigations to the experimental system in most cases requires the use of solvents, LPS indifferent concentrations was applied in medium alone (without any solvent), and in medium containing eithermethanol (0.05% v/v) or dimethyl sulfoxide (0.05% v/v).

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Figure 2.1. Dose-dependent cytokine production of human PBMC after 20 hours stimulation with LPS (Escherichia coli O55:B5).Following separation by density gradient centrifugation, PBMC were seeded into flat-bottomed 96-well plates (final density 1 x 106 PBMC/ml).Media without any solvent (white bars), with 0.05% v/v methanol (grey bars) or 0.05% v/v dimethyl sulfoxide (dark grey bars) were used for theculture of PBMC. At 20 hours of incubation, 180 µl of cell-culture supernatant were withdrawn and transferred into deep well plates for storageat –20°C until cytokine measurements in ELISA (OptEIA™, BD Biosciences).The bars represent the median and upper (75.) and lower (25.)percentile of six tested donors. Each of the LPS concentrations was analyzed in triplicate and the respective medium control fivefold.

LPS concentrations between 1 and 10000 pg/ml were tested. A dose-dependent induction of TNF-α and IL-6production by LPS was observed. Reproducible strong cytokine signals were found after application of LPSconcentrations of 100 pg/ml and above, maximum responses after application of 1000 pg/ml and above (in the caseof TNF-α). We decided to use 100 pg/ml LPS in future analyses of immunomodulatory effects of chemicals.Two reasons argue for this decision. At first, 100 pg/ml LPS trigger a significant induction of pro-inflammatorysignals. At second, the application of a suboptimal stimulus opens the possibility to detect not only inhibitory butalso stimulatory effects (in particular in the case of TNF-α production). The effects of the preferred solvents, DMSO

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and methanol, in concentration of 0.05% v/v in culture in each case were insignificant, but showed, especially in thecase of IL-6, a slightly diminishing effect (compare Figure 2.1). Moreover, none of the LPS concentrations andsolvents caused significant effects of the MTT metabolizing activity (results not shown). Therefore, it can beconcluded that both the used solvent and LPS in the applied concentrations exerted no cytotoxic effects on humanPBMC.

As mentioned above, Birch pollen (BP) allergen extract (AE) was selected as an example for an allergenicstressor. BP is a common allergen and known as a potent inducer of allergic sensitization and, following re-contactof sensitized individuals to its allergenic components, triggers allergic reactions of the immediate type.Analogous the to above described baseline experimental settings for the description of LPS responses BP-AE wasapplied to human PBMC in different concentrations in medium alone (without any solvent), and in mediumcontaining either methanol (0.05% v/v) or dimethyl sulfoxide (0.05% v/v).

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Figure 2.2. Dose-dependent cytokine production, proliferation and IgE production of human PBMC in response to birch pollen allergen extract.The cytokines IL-10 and IFN-γ were measured in culture supernatants after 20 hours incubation. MTT metabolization as measure forvitality/proliferation as well as IgE production of human PBMC were analysed after 7 days of stimulation. Following separation by densitygradient centrifugation, PBMC were seeded into flat-bottomed 48-well plates (final density 2 x 106 PBMC/ml). Media without any solvent (whitebars), with 0.05% v/v methanol (grey bars) or 0.05% v/v dimethyl sulfoxide (dark grey bars) were used for the culture of PBMC. At 20 hours and7 days of incubation, respectively, cell-culture supernatant were withdrawn for cytokine measurements or measurement of anti-birch pollenallergen extract-specific IgE in ELISA. The remaining PBMC were used for the measurement of vitality/proliferation in the MTT bioassay. Thebars represent the median and upper (75.) and lower (25.) percentile of six tested donors. Each of the BP-AE concentrations and the respectivemedium control was analyzed in quadruplicate.

BP-AE stimulates only allergen-specific T-cells and therefore induces comparatively weak cytokine production (in

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the lower range of pg/ml). Consequently, the response to stimulation is more affected by the individual response ofthe respective donor due to its individual susceptibility and therefore more heterogenic in general. This mightexplain the higher variability of the observed effects on cytokine and IgE production in response to BP-AE. Butdespite such limitations, a quantification of the BP-AE induced immune responses and consequently also theinfluence of various noxes on their strength is possible.In the first 20 hours, BP-AE induces the production of significant amounts of interleukin-10 (IL-10) and alsointerferon-γ (IFN-γ). The dose-dependent cytokine production of human PBMC after stimulation with BP-AE isshown in the upper part of Figure 2.2. Effects of the solvents methanol and dimethyl sulfoxide on IL-10 and IFN-γproduction appeared to be insignificant. After 7 days, contrary to this, a slightly diminished proliferation of PBMCcultured in medium containing dimethyl sulfoxide compared to those cultured in medium alone or in mediumcontaining methanol was observed. Nevertheless, all BP-AE concentrations applied in dimethyl sulfoxide containingmedium were also found to induce proliferation.Concerning the induction of allergen specific IgE antibodies no clear dose-response relationship was observed interms of the applied allergen concentration. Difficulties in the detection of the really very low levels of allergenspecific IgE antibodies in the culture supernatants could explain this outcome. Trying to avoid such effects probablyto face especially in the lower range of BP-AE concentrations, we decided to choose stimulation with 500 PNU ofBP-AE in the investigations planned for the future.Even if the allergen specific assay is difficult to handle and the results obtained depend strongly on the individualsusceptibility of the respective donor this assay may give some first evidence for allergy promoting effects ofchemical compounds. All other so far used testing strategies require animal experiments. The in vitro assayestablished in our laboratory can be used as a first screening assay to search for compounds with allergy mediatingeffects. The confirmation of positive results will occur using a mouse model (provided by partner 17: USALZ).

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3 Baseline responses of human reporter gene cells to the immune stressor TNF-αAlbert Duschl, USALZ, University of Salzburg, Partner 17

Introduction

USALZ, partner 17, will be testing single compounds as well as mixtures of chemicals either with or without astressor on stable human reporter cell lines for WP 3.2 and 3.1, respectively. These reporter cell lines can be used toshow immune modulation by several substances because the reporter genes are under the control of cytokinepromoters. Ten stable cell lines have previously been created during the 5FP project MAAPHRI (see Table 3.1) andthose will be used for NOMIRACLE. As part of NOMIRACLE approximately five new reporter cell lines will begenerated and are estimated to be ready in January 2006.

Generation of new stable cell linesThe cytokine promoter of interest is cloned into the pGL3neo plasmid upstream of a reporter gene encoding for theenzyme luciferase. Furthermore, the vector encodes for ampicillin as well as for neomycin resistance (see Figure3.1). Human cell lines are transfected with the promoter reporter construct and grown in a medium containing G418for neomycin selection. Thus, a pool of stably transfected cells is created. In order to generate a stable clone from asingle cell, multiple rounds of single cell selection are applied. This process will take approximately three to fourmonths until a new cell line is available for screening tests.

Mechanism of the test system: Luciferase-assayThe stable reporter cell lines can be used to show the induction (e.g. by a chemical, stressor or pathogen) of acytokine promoter of interest. Activation of the promoter leads to increased expression of luciferase. After 24 hours

pGL3 + IL-6 Promoter6816 bp

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Figure 3.1.pGl3neo / IL-6 promoter reporter plasmid.The human IL-6 promoter (blue) is clonedinto the pGl3neo vector, upstream of thereporter gene luciferase (red, “LUC”).Furthermore, the vector encodes forresistances for antibiotics (green, “NeoTK”and “AmpR”), a poly A-tail (green “PolyA”)and a replication origin (yellow,“RepOrigin2”).

Promoter host cell lineIL-4 Jurkat

IFN-γ (short) JurkatTGF-β1 Jurkat

A549TNF-α Jurkat

Eotaxin-1 HEK293IL-8 A549

HelaNF-kB A549

Jurkat

Table 3.1.Reporter cell lines:This table shows the reporter cell lines which arepresently available.

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exposure, cells are lysed, luciferin (substrate of luciferase) is added, and luminescence (emission of light) ismeasured. The light intensity depends on the quantity of luciferase that is expressed in the cells.The basic expression of the reporter gene depends on where it integrates into the genome of the cell. Some of ourclones have high constitutive expression of luciferase and may therefore be used to show suppression of thepromoter.

Selection of natural stressorsBacterial infections are natural stressors for the immune system. Bacterial lipopolysaccharides (LPS) are among thebacterial factors, which are recognized by immune cells and induce a signalling cascade in those cells. As aresponse, the immune cells secrete high amounts of the cytokine TNF-α (tumor necrosis factor-α) which is thebody’s own stressor.Cell systems consist of a single cell type and therefore cannot fulfil all the complex interactions of the many celltypes of the immune system. This is the reason why some of our stable cell lines do not show a reaction in thepresence of LPS. By replacing LPS with TNF-α, we simulate a bacterial stress situation. TNF-α is suitable for allcell types included in the study and will be used by UFZ (Partner 3) as well.

Data and responsiveness of cell linesA549-based stable cell lines:As a demonstration of the baseline responses which are obtained in our cell line systems, Figure 3.2a showsinduction of the interleukin-8 (IL-8) promoter in A549 (human epithelial lung cells) with TNF-α. Uninduced cells(0) demonstrate the background. With increasing concentration of the inducer (here TNF-α), the signal alsoincreases until it reaches a plateau phase. It will have to be discussed among the partners of WP 3.2 and particularlythose working on human health which amount of stressor should be used. EC50 for each cell type or a fixed dose forall, are both possible solutions. We would prefer using EC50.

Induction of the IL-8/A549 reporter cell line with TNFa

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Figure 3.2a:Induction of the IL-8 promoter / A549 reporter cellline with TNF-α for 24 hours.The light signal increases with increasing TNF-αconcentration and reaches a plateau. RLU = relativelight units

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Induction of the NFkB/A549 reporter cell line with TNFa

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Jurkat-based cell lines:In Figure 3.3, induction of the TGF-β (transforming growth factor-β) promoter in Jurkat (human T-lymphocytes)cells is shown. Stimulation of T-cells via TCR (T-cell receptor) and CD28 (a cell surface molecule), can bemimicked by PMA/ionomycin. Upon this stimulation two major pathways are activated: the Ca2+-dependentpathway and the MAPkinase pathway, resulting in the transcription of a set of genes necessary for an appropriateimmune response. The data demonstrate that the same promoter may be regulated by three completely differentfactors, including a natural stressor (LPS).

Induction of the TGFß/Jurkat reporter cell line w ith diverse inducers

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Figure 3.4 shows the induction of the TNF-α promoter in Jurkat cells with PMA/ionomycin, IL-4, IL-10 or IFN-γ(interferon-γ). As a proof of principle the synergistic effects of PMA/ionomycin and IL-4 is indicated. Other effectsof mixed inducers are shown as well (PMA/Ionomycin and IL-10 or IFN-γ).

Induction of the TNFa promoter in Jurkat cells with diverse inducers

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Figure 3.2b:Induction of the NFkB / A549 reporter cell line withTNF-α for 24 hours.The light signal increases with increasing TNF-αconcentration and reaches a plateau. RLU = relativelight units

Figure 3.3.Induction of the TGF-β promoter / Jurkat reportercell line with diverse inducers for 24 hours.TGF-β is produced in vivo by T-cells as a responseto bacterial infection and can be induced in thereporter cell line by either PMA/Ionomycin, TGF-βitself (3ng/ml) or LPS (100ng/ml).ui = uninduced; P/I = PMA/Ionomycin; RLU =relative light units

Figure 3.4.Induction of the TNF-α promoter in transientlytransfected Jurkat cells with diverse inducers for 24hours.The TNF-α promoter in Jurkat cells can be inducedby either PMA/Ionomycin, IL-4 (50ng/ml), IL-10(10ng/ml) or IFN-γ (20ng/ml). If the cells areinduced with the combination of PMA/Ionomycinand IL-4, synergism appears. This effect is not seenfor IL-10 or IFN-γ in combination withPMA/Ionomycin.ui = uninduced; P/I = PMA/Ionomycin; RLU =relative light units

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Conclusion and recommendations for future work

These experiments demonstrate the principles of our test system and show reliability, sensitivity and specificity.Depending on the growth properties of the particular reporter cell line (some grow slower than others), eachexperiment can be repeated within 3 days, including seeding of the cells, induction and measurements. Theconcentration range of the inducers is comparable between the particular reporter cell lines, and responses tostressors look generally like those shown in Figs. 3.2a and b. Variation between individual tests is unproblematic, sothe test system is robust in this respect and results are reproducible. Technical problems are mostly due to cellculture problems, which can be handled by routine procedures. In toxicity tests we anticipate that solvents may be asource of problems, and volatile compounds will be particularly problematic.For WP 3.1 we will test single chemicals and mixtures. For WP3.2 the same experiments will be done combinedwith TNF-α as the stressor. The work involved is directly correlated with the number of data points, so within theWP it will have to be decided whether to produce more extensive data sets for fewer substances, or the reverse.Either is feasible.

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4 Baseline response of Zebrafish larvae (Danio rerio) to a natural stressor (Anoxia) in the

Multispecies freshwater Biomonitor (MFB)

Almut Gerhardt and Cornelia Kienle, LimCo International, Partner 24

Introduction

The Zebrafish, Danio rerio is a widely used test species representative of freshwater fish. Whereas this species intraditional ecotoxicological testing is provided with optimal temperature, oxygen and food conditions, we will herereport on the performance of D. rerio larvae under sub-optimal oxygen conditions. Anoxia is a frequently occurringnatural stressor in the field.

Materials and Methods

Adult zebrafish of both sexes were kept in the laboratory in aerated and filtered aquaria with a minimum of 1 litrewater per fish on the average. Culture conditions were 26+1°C at a 12:12 hour light:dark cycle. The adult fish werefed twice per day with dry flake food and frozen crustaceans or midge larvae, respectively.The eggs used in the tests were collected using spawn traps which had been placed at the bottom of each aquariumthe evening before spawning was required.In the morning, the spawn traps were removed from the aquaria, the eggs sieved and cleaned under flowingfreshwater and put in petri dishes. Embryos and larvae were kept in glass petri dishes in OECD standard test water,which had been aerated for 12 hours before use with an aquarium pump. The petri dishes were placed into a climatechamber and the embryos and larvae were kept at 26+1°C at a 12:12 hour light:dark cycle until two days afterhatching.Two to four hours after fertilization the eggs were sorted and distributed over several petri dishes with test water. Anappropriate amount of water (~1/3 of the volume) was changed every day. After 24 hours the eggs were put intonew petri dishes with fresh OECD standard test water. Every day the constitution of the larvae was checked under abinocular and malformed embryos and larvae were removed.Test vessels were 4 litre aquaria, with a test volume of 1.5 litre, of which 2 were put into a surrounding black basinwith water and two heating sticks, to keep the temperature constant at 26+1°C. Oxygen poor water was made byaerating with gaseous nitrogen for an appropriate time (~ 30-40 min) in the test vessels via an aerating stone.Only healthy larvae were used for the test. The larvae were put into the chambers and the air bubbles were removedwith a Pasteur pipette. After an acclimatisation time of 10 min the measurement was started. Behaviour wasrecorded constantly for 2 hours in intervals of 10 min and for a duration of 4 min. The test aquaria were sealed witha PP-foil.The tests were conducted like described in the SOP for Danio rerio larval screening and behaviour.

Method optimization

For the following tests an optimisation of the test method will be used. As surrounding vessel for the tests withanoxia a glass aquarium (60*30*30 cm) with appropriate holes for putting the cables of the measuring chambersthrough and for the aerating and venting tube will be used. A glass plate will serve as lid for the vessel. Thedenseness of the construction will be guaranteed through sealing tape, which is used at all openings and put over oraround them. In Figure 4.1 the experimental set-up is shown.

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Figure 4.1. Experimental set-up for testing anoxia in zebrafish

Test vessels will also be 4 litre aquaria or PE-vessels (208*208*64 mm) with a test volume of 2 l, of which two willbe put into the surrounding chamber. Oxygen poor water will be made like described above. Also a nitrogenatmosphere will be kept over the test vessels in the surrounding aquaria in order to avoid oxygen diffusion into thewater from the air above. With this construction it should be possible to keep the oxygen content constant for thetest period.

Results

Fig. 4.2 shows movement patterns of zebrafish larvae in oxygen saturated control water and in water with 20%oxygen saturation.

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Figure 4.2. Movement pattern and FFT histogram of two day old zebrafish larvae in the Multispecies FreshwaterBiomonitor after one hour of measurement (above: Larva in oxygen saturated water; below: Larva in oxygen poorwater).

From this picture a decreased movement of the larva in water with low oxygen content after one hour ofmeasurements can be seen.

In the following figures the development of the activity over time is displayed.

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Figure 4.3. Activity of 2 day old larvae of Danio rerio over a time period of two hours in oxygen saturatedwater (n=11), median+sd

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Activity decreased with exposure time and standard deviations increased (Fig. 4.3).Under oxygen depletion stress (O2-content: 20-30% of saturation) activity decreased much faster (Fig. 4.4).

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Figure 4.4. Activity of 2 day old larvae of Danio rerio over a time period of two hours in water with an oxygencontent of ~20% (n=12), median+sd

Fig. 4.5 shows control recordings during the first hour of exposure on different days. Test-to-test variation in controlactivity levels is low, thus demonstrating a robust and reliable test system.

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Figure 4.5: Activity of 2 day old larvae of Danio rerio in five control measurements (n=66-72) (median+sd) inthe first hour of measurement

There were only few significant differences, such as: 13.04.05 and 01.07.05 (p<0,05), 22.04.05 and 15.04.05(p<0,01), and 22.04.05 compared with 01.07.05 (p<0,01) (Friedmann-Test, Statistica 5.0).

Fig. 4.6 shows control recordings during the second hour of exposure on different days.

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Figure 4.6: Activity of 2 day old larvae of Danio rerio in five control measurements (n=44-84) (median+sd) inthe second hour of measurement

Variability in behaviour is much higher during the second hour of monitoring (Table 4.1).

Table 4.1. Statistical analysis of the behavioural data in Danio rerio during the first (Tab. 1) and second hour ofmonitoring (Tab. 2)

Comparing locomotion of larvae with 100% oxygen and with 20 % oxygen we find significant differences in thefirst hour as well as in the second hour of exposure (p<0.05 (1. h); p<0.001 (2. h)). 50% of the larvae exposed tooxygen depletion stress (20% saturation) died after the test.

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5 Baseline responses of the zebrafish (Danio rerio) to a natural stressor (temperature)

during prolonged embryo testsVolker Scheil, Silke Müller and Heinz-R. Köhler, Eberhard Karls Universität, Tübingen, Partner 11

Introduction

The Zebrafish, Danio rerio is a widely used test species representative of freshwater fish. Whereas this species intraditional ecotoxicological testing is provided with optimal temperature, oxygen and food conditions, we will herereport on the performance of D. rerio larvae under sub-optimal temperature conditions. High or low temperature is afrequently occurring natural stressor in the field.

Material and methods

Adult zebrafish (Danio rerio) of both sexes were kept in the laboratory in aerated and filtered aquaria with aminimum of 1 litre water per fish on the average. Culture conditions were 26 ± 1°C at a 12:12 hour light: dark cycle.The adult fish were fed twice per day with dry flake food and frozen Tubifex or midge larvae, respectively.The eggs used in the tests were collected using spawn traps which had been placed at the bottom of each aquariumthe evening before spawning was required. Fish keeping was the same for all tests.Prolonged embryo tests were conducted at three different water temperatures, including the standard temperature26°C, a lower temperature (23°C), and a higher temperature (33.5°C).The test procedure (except of water temperature and time points of observations) and the investigated endpoints inall experiments were the same as described in the “Standard Operational Procedure (SOP) for testing chemicals,using a prolonged embryo test on zebrafish (Danio rerio)”. As this SOP is designed for the standard watertemperature of 26°C, the time points for endpoint investigations had to be adjusted to the different developmentrates at higher or lower water temperature. Tests at 23°C were prolonged to 120h after fertilization, tests at 33.5°Cwere shortened to 72h after fertilization, and tests at 26°C lasted 96h as described in the SOP. Because of this, ratheridentical development stages than the same time points are compared in the following. Those endpoints, which arelisted in the SOP but not mentioned below, were not affected by temperature variability.For every temperature, 3 repeated experiments with 4 replica were performed. In total, 360 individuals have beenexamined.

Results and discussion

Since the data for the three repeated experiments at a given temperature did not differ significantly, data werepooled for every temperature.During the tests, mortality was below 10% at 26°C and 33.5°C. Experiments conducted at 23°C led to asignificantly higher mortality compared to the higher temperatures (p<0.05, Wilcoxon-test) as shown in Fig. 5.1.The higher mortality of the zebrafish embryos kept at 23°C is a result of reduced hatching success (see Fig. 5.4),indicating that mortality occurred while the embryos were still located inside the egg but should have hatchedalready, according to their development stage.

20

0%10%20%30%40%50%60%70%80%90%

100%

23°C 26°C 33.5°C

mor

talit

y **

Figure 5.1. Mortality during the tests at three different temperatures, means of 3 repeated experiments, ± sd, *:p<0.05.

A second endpoint affected by temperature was the occurrence of yolk sac oedema. This parameter also was onlyfound to be elevated by the lower temperature of 23°C. The occurrence of yolk sac oedema was significantly higherin the zebrafish embryos kept at this temperature in comparison with 33.5°C (p<0.01, Wilcoxon-test). Due to a highstandard deviation, occurrence of yolk sac oedema at 26°C was neither significantly different to the percentagesrecorded for 23°C nor for 33.5°C but, indeed, very low.

0%

5%

10%

15%

20%

25%

30%

23°C (72h) 26°C (60h) 33.5°C (48h)

yolk

sac

oed

ema

**

Figure 5.2. Percentage of yolk sac oedema during the tests at three different temperatures (time point of observationgiven in parentheses), means of 3 repeated experiments, ± sd, **: p<0.01.

21

0

50

100

150

200

250

300

23°C (60h) 26°C (48h) 33.5°C (36h)

hear

tbea

ts /

min

.

**

**

***

Figure 5.3. Heartbeats per minute of zebrafish embryos at three different temperatures (time point of observationgiven in parenthesis), means of 3 repeated experiments, **: 0.001<p<0.01, ***: p<0.001.

Heartbeat rates were the lowest at 23°C, higher at 26°C (p<0.01, Wilcoxon-test) and the highest at 33.5°C (p<0.01,compared with 26°C and p<0.001, compared with 23°C, respectively, Wilcoxon-test), following the reactionrate/temperature rule (Fig. 5.3).

The last investigated endpoint, which was affected by temperature, was the hatching time. Due to the fasterdevelopment of the embryos kept under higher temperature the larvae hatched earlier in higher water temperatures.Because of high mortality in the embryos kept at 23°C, the hatching success was relatively low in this group (Fig.5.4).

0%10%20%30%40%50%60%70%80%90%

100%

48 60 72 84 96 108 120

hours after fertilization

hatc

hed

larv

ae

23°C26°C33.5°C

Figure 5.4. Hatched larvae in % of the initial stock of eggs per temperature, means ± sd.

Standard Operational Procedure for the prolonged embryo test on zebrafish (Danio rerio)

AnimalsAdult zebrafish (Danio rerio) of both sexes are kept in the laboratory in aerated and filtered aquaria with a minimumof 1 litre water per fish. Culture conditions are 26 ± 1°C at a 12:12 hour light:dark cycle. The adult fish are fed twiceper day with dry flake food and different frozen live food, respectively.

22

The eggs used in the test are collected using spawn traps which have been placed at the bottom of each aquarium theevening before spawning is required.

ExposureThe water for the exposure of the eggs/embryos is prepared according to ISO-Standard 7346/3, containing 294mg/LCaCl2, 123,25mg/L MgSO4, 64.75mg/L NaHCO3 and 5.75mg/L KCL, dissolved in aqua bidest. The water is aeratedto oxygen saturation before addition of the test substance.

Experimental designDifferent concentrations of the test substance are dissolved in the exposure water. The test is performed using theseconcentrations of the test substance, and a negative control containing pure exposure water. If a solubilising agent isused, a control containing this agent in the respective concentration is necessary.The evening before spawning is required, spawn traps covered with stainless steel mesh are placed in the aquaria. Aspawning substrate is placed into the spawn traps. 30 minutes after the light is turned on, the spawn traps areremoved and the eggs are collected. All eggs are transferred immediately into glass petri dishes containing thedifferent test solutions. Then the unfertilized eggs are removed, and the fertilized eggs are placed into new glasspetri dishes (10 embryos per petri dish, 3 dishes per concentration) containing the respective test solutions. The testis performed in climate chambers at a 12:12 hour light:dark cycle, water temperature is maintained at 26 ± 1°C, thepetri dishes are covered to avoid evaporation. Embryo development is observed using a binocular at specified timepoints (see Table 5.1) during the next 96h. If different temperature conditions are tested, the time points have to beadjusted, according to faster or slower development of the embryos.

Table 5.1:Investigated endpoints during egg development (modified after OECD 210 and DIN 38415-6)Endpoint 8h 12h 24h 48h 60h 72h 84h 96h (Time points at 26°C)

Coagulated eggs / dead * (1) *(2) *(3) *(4) *(5) *(6) *(7) *(8)

No epiboly (70%) *(9)Incomplete gastrulation *(10)Exogastrulated embryo *(11)No formation of somites *(12)No detachment of tail *(13)No spontaneous contraction *(14)No formation of the eye *(15)No heart beat *(16)No circulation *(17)Heart rate *(18)No otolith formation *(19)No melanocyte formation *(20)Yolk sac endema *(21)Eye / brain defects *(22)Total number of malformations *(23) *(24) *(25) *(26)Number of hatched embryos *(27) *(28) *(29) *(30)Edema (heart and head) *(31) *(32) *(33)Eye defects *(34) *(35) *(36)Tail deformities *(37) *(38) *(39)Fin blistering *(40) *(41) *(42)Weak pigmentation *(43) *(44) *(45)Helical bodies *(46) *(47) *(48)Spiral nervous system *(49) *(50) *(51)

* The number in parentheses (1) … (51) are coding for the respective parameters observed at different time points.

23

6 Effects of natural stressors on Daphnia MagnaSusanna Loureiro and Amadeu Soares, Universidade de Aveiro, UAVR, Partner 13

Introduction

Daphnids are considered eurythermic animals, tolerating a wide range of temperatures. However, more extremetemperature fluctuations occur frequently in natural freshwater habitats that may potentially have an impact in theperformance of daphnids. Temperature influences the physiological processes of daphnids e.g. by alteringrespiratory processes, feeding activity and water column migration. From literature, a LT50 (lethal temperature)value of 35ºC was observed after 24h and heat shock temperatures of 38ºC induced a quick death after 15 min [1].Daphnids are also believed to react behaviourally to thermal gradients of as little as 1ºC, showing avoidancebehaviours at 27ºC and 11ºC. Regarding other natural stressors, different levels of oxygen will also be tested. Foodquality is considered at least as important as food quantity in terms of the influence on both fecundity andpopulation growth responses of cladocerans.

High and low temperature

The immobilization bioassay with Daphnia magna was used to evaluate lethality of organisms exposed to extremetemperatures (higher and low than the control temperature of 20ºC). ASTM media were left for 2 hours fortemperature acclimation and only afterwards daphnids were added. According to bibliography, our results showedLTD50 values of 34ºC (for 24h) and 31ºC (for 48h) (for higher temperatures) and 4.1ºC (48h, for lowertemperatures). These LT50 values were obtained based on real medium temperatures.Chronic tests will now be carried out and the effects of low and high temperatures on reproduction effort and growthwill be evaluated.

Anoxia

Regarding other natural stressors, different levels of oxygen will also be tested. The experiment setup is now beingdeveloped and tested for controlled conditions (Fig.6.1). Although being a very active species, D. magna is able totolerate oxygen depletion, surviving up to 1day without oxygen [2]. Even though some previous studies from 1997stated that daphnids heart beat ceased just after 2h of anoxia.

GAS

12I. Gas (N2 or N2 and O2 mixture) pump into the vessel (1);II. Simultaneously, O2 is released from the test medium (2);III. After O2 levels are stable (PO2 sensor in (2)), the gas flow

is blocked (1);IV. Daphnids are introduced in the vessel through (2);V. The vessel is sealed blocking (1) and (2).

GAS

GAS

12I. Gas (N2 or N2 and O2 mixture) pump into the vessel (1);II. Simultaneously, O2 is released from the test medium (2);III. After O2 levels are stable (PO2 sensor in (2)), the gas flow

is blocked (1);IV. Daphnids are introduced in the vessel through (2);V. The vessel is sealed blocking (1) and (2).

Figure 6.1.- Setup for anoxia experiments.

Anoxia can lead to important effects on populations and life history parameters because they can influencecirculatory processes, metabolic rate, and haemoglobin synthesis. This haemoglobin synthesis can be easilyobserved by a change in daphnids colour [3].

Food Quality

Food quality is considered at least as important as food quantity in terms of the influence on both fecundity andpopulation growth responses of cladocerans. In future studies diet quality will be tested, concerning the importanceof nitrogen (N), carbon (C) and phosphour (P) contents in the daily diet. Immobilization, growth and reproductionparameters will be evaluated [4].

24

Due to the late start of the project activities at UAVR, experiments are currently being performed. Results are notyet available.

Bibliography

1. Kinvivuori LA, Lahdes EO. 1996. How to measure the thermal death of Daphnia? A comparison ofdifferent heat tests and effects of heat injury. J. therm. Biol. 21:305-311.

2. Paul RJ, Colmorgen M, Pirow R, Chen Y-H, Ming-Cheng T. 1998. Systematic and metabolic responses inDaphnia magna to anoxia. Comparative Biochemistry and Physiology Part A 120:519-530.

3. Wiggins PR, Frappell PB. 2002. Behavioural thermoregulation in Daphnia carinata from different depthsof a natural water body: influence of environmental oxygen levels and temperature. Comparative Biochemistry andPhysiology Part A 133:771-780.

4. Sterner RW. 1993. Daphnia growth on varying quality of Scenedesmus: mineral limitation of zooplankton.Ecology 74:2351-2360.

25

7 Effects of temperature and nickelchloride on life-history traits of the free-living soil

nematode Caenorhabditis elegansJan Kammenga, Wageningen University, Partner 12

Introduction

Caenorhabditis elegans is widely used in ecotoxicological testing. The species is especially interesting for theNOMIRACLE project because the complete genome has been sequenced and microarray chips are available for C.elegans. Normally, C. elegans is cultured at 24°C, and tests are routinely run at this temperature. However, this isnot a relevant temperature for most temperate soil conditions, where temperatures are typically only 15°C and belowduring most of the year. It is therefore interesting to investigate the influence of lower culture temperatures on theoutcome of toxicity tests.

Material and Methods

Nickel chloride hexahydrate (NiCl2·6H2O) were obtained from Sigma Chemical CO., US and dissolved in distilledwater with a stock concentration of 50 mg/mL which was kept at 4°C.

Culture of nematodesC. elegans, wild type strain N2, was used. Worms were maintained on NGM agar plates (60-mm diameter) andpopulations stage synchronized. For experiments worms were transferred to multi-well dishes (12 well) with E. coliOP50 as a food source. The standard NGM agar consisted of: 0.032 M KCl, 0.051 M NaCl (Merck, Germany), 2.5%Bacto-peptone (Becton, Dickinson & Co, France), 0.17% Bacto-agar (Becton, Dickinson & Co, France), 0.01%cholesterol (Sigma, Germany), 0.1 M CaCl2, and 0.1 M MgSO4.

Table 7.1. Toxicity assay showing temperatures and concentrations of NiCl2

12°C 24°C 12°C 24°Ccontrol control control control5mg/L 10mg/L 5mg/L 10mg/L

10mg/L 20mg/L 10mg/L 20mg/L15mg/L 30mg/L 15mg/L 30mg/L20mg/L 40mg/L 20mg/L 40mg/LPl

ates

6m

m Ø

25mg/L 50mg/L Sync

hron

izat

ion

Mul

ti-di

shes

25mg/L 50mg/L

The metal salt was dissolved in distilled water and mixed with agar before the solidification. The followingconcentrations, in 12°C, 5, 10, 15, 20, 25 mg NiCl2 /L agar, and in 24°C, 10, 20, 30, 40, 50 mg/L agar. Bothtemperatures had his respective concentration control without metal.

SynchronizationStage synchronized populations were started by transferring adult animals to a plate, allowing them to lay eggs for 4hrs, and then removing them, leave the eggs behind into the agar with different concentrations of NiCl2 for thestudy. These eggs were then allowed to hatch and the resulting animals allowed to grow. Populations, which were togrow for more days before measuring, were transferred to multi-dishes as necessary to prevent exhaustion of thebacterial food supply and with the corresponding concentrations. All ages are recorded from the time at which theoriginating eggs are laid.

Results

The body size and survival of the nematode Caenorhabditis elegans have been studied at 12°C and 24°C at differentNiCl2 concentrations with Escherichia coli as a food source. Temperature influenced both traits as well as toxicity.Body growth rate was slower but size of adults was larger at the lower temperature (Fig. 7.1 to 7.4). Nematodesurvival was higher at 12°C. The LC50 was 45 mg/L and 58 mg/L at 12°C and 24°C respectively. The EC50 was 24and 46 mg/L at 12°C and 24°C respectively. Overall, the toxicity of NiCl2 increased at the lower temperature. This

26

was quite unexpected because usually toxicity increases at higher temperatures. We believe our assay is robustbecause it is based on a widely used standardized culturing and exposure system.

% SURVIVAL 24 C

0

10

2030

40

50

60

7080

90

100

0 2 4 6 8 10 12 14 16 18

DAYS

%

Control 10mg/l 20mg/l 30mg/l 40mg/l 50mg/l

Figure 7.1. Survival of C. elegans at 24°C

% SURIVAL 12 C

0

20

40

60

80

100

0 5 10 15 20 25 30 35

DAYS

%


Figure 7.2. Survival of C. elegans at 12°C

27

Body growth at 24 C

0

20

40

60

80

100

120

140

0 10 20 30 40 50 60 70 80 90 100Hours

Av

vol l

engt

h un

its

control 10mg/l 20mg/l 30mg/l 40mg/l 50mg/l

Figure 7.3. Body growth of C. elegans at 24°C

Body growth at 12

0

20

40

60

80

100

120

140

0 50 100 150 200 250hours

Avvollengthunits


Figure 7.4. Body growth of C. elegans at 12°C

28

N2

Figure 7.5. Overview of setup in C. elegans experiments

12°C

24°C

C 2510 15 205 C 10 20 30 5040

5 nematodesper dish

during 4 hours

Synchronization

All concentrations

5 nematodesper dish

during 4 hours

All concentrations

Eggs counted

Pictures taken

Survival observed

Eggs counted

Pictures taken

Survival observed

DATAANALYSIS

Different NiCl2

concentration tested

29

8 Preliminary results of range-finding experiments for environmental stressors for P.

oblongopunctatus (Coleoptera: Carabidae)Ryszard Laskowski and Paulina Kramarz, Jagiellonian University, Partner 10

Introduction

Carabid beetles represent an interesting group of species for ecotoxicological studies and risk assessment for anumber of reasons. They represent a carnivorous guild and are, thus, potentially exposed to biomagnificationproblems. They are important pest-control species and because of that they need special care in environmentalrisk assessment. They are relatively abundant in most terrestrial ecosystems and can be easily collected for bothfield and laboratory studies. They are relatively large specimen so they are suitable for monitoring purposes aswell as for toxicokinetic studies. In this workpackage, important natural stressors such as temperature and wateravailability will be investigated in combination with chemical stressors. We will focus our studies ontemperature and water availability because these particular parameters have a large influence on the fitness ofcarabids.

Development of the culture and test methods for Pterostichus oblongopunctatus

Different species of field-collected carabid beetles (C. auronitens, P. versicolor, P. melanarius, P.oblongopunctatus) were collected in September 2004 to establish a laboratory culture satisfactory forecotoxicological tests. The most successful results were obtained for P. oblongopunctatus, and this species hasbeen chosen as a test organism for final tests in the NOMIRACLE project. During the first months of the projectthe detailed protocol on how to culture P. oblongopunctatus has been developed and different sources of food,food quality, feeding frequency, test boxes, photo periods, air humidity, etc. were checked to standardizemethods (for details see SOP WP 3.2 UJAG Car).

Preliminary tests to determine ranges of temperature and water holding capacity (WHC) to be used infinal tests for adult and larval stages of P. oblongopunctatus

The experiments were conducted in climatic chambers (relative humidity 75%; photoperiod L:D 16:8). Both theadult beetles and the larvae were placed in vials with wet peat (pH 4.5-5.0) and were fed three times a week. The48 pairs of adult beetles collected from the field on the same day were cultured at different temperatures (10, 20,30oC ) and soil moistures (20, 40, 60, 80, 100% WHC) and their reproduction and survival were observed. Eachpair of beetles was kept in a separate plastic box and the eggs laid by females were picked out three times a weekand placed in 24-well tissue culture plate for hatching. Plates were checked every day for hatched larvae (SOPWP 3.2 UJAG Car).The tests on the larval stage were conducted at the same temperatures and soil moistures as used for adults, andwere run in ten replicates – one individual per vial. The larvae were checked after 1, 2, 3, 4, 5, 7, 10 and 14 daysand three times a week thereafter until the first pupae were observed when the cultures were check on a dailybasis again.

Influence of temperature and soil moisture on reproduction of P. oblongopunctatus – results after a one-month reproduction period

With the same food source, feeding frequency, and culturing conditions but at different temperatures (10, 20,30oC) the egg production (the mean number of eggs laid per day) after one-month period of reproduction showedminimum at 30oC and was much higher at 20 and 10oC. The larval hatching success, measured as a rate ofoffspring hatched from eggs laid, was the highest at 20oC (Table 8.1).The soil moisture (20, 40, 60, 80, 100 % WHC) influenced the number of eggs laid per day, which generallyincreased with increasing moisture. Although the egg production was lower in 80% WHC than in 60 and 100%,the hatchability of the eggs laid was the highest in 80% WHC (Table 8.2).

Influence of the temperature and water content capacity on the larval survival – results after the firstmonth of tests

Because at 20 and 40% WHC it was impossible to prepare a hole for larvae in the peat which is necessary for 1-2-day old larvae for successful development, two separate experiments with soil moisture were conducted: thefirst with 1-day old larvae only, and the second with 10-days old larvae.

30

Most of tests involving the larval stage are still in progress, and the mortality during development time isobserved every two days. The first results showed that neither the 1-2-day-old larvae nor the 10-days-old oneswere able to survive at 30oC (Table 8.3). The development time, from hatching of larvae from an egg to hatchingof immature individuals from pupae, appears to be shortest at 20oC – the first pupae were observed only at thistemperature so far, and the larvae are more vital than at 10oC.Results from tests with different soil moisture indicate that the mortality of 1-2-day-old larvae used in theexperiment with different % of WHC was the highest in 20% and 40% WHC - most of the larvae died during thefirst week of the experiment (data not shown). As the experiment has not been finished yet, no differencebetween 60, 80 and 100% WHC can be stated at the moment

Conclusions

Carabids are rather difficult to breed for long-term experiments, mostly because of high level of cannibalism andthe resulting necessity to culture single larvae in separate culture boxes/vials. They are also very sensitive tofood quality, and so, special care must be taken when developing culturing conditions. As the development timeis relatively long (the whole life cycle takes at least half a year), it was not possible so far to check the resultsobtained for their robustness and repeatability.

Tables

Table 8.1. Mean values of number of eggs per female, daily egg production and larvae hatching rate (mean ±standard deviation and a median value) of P. oblongopunctatus at different temperatures; N - number of pairs ofadult beetles.

Temperature [oC] N Number of eggs perfemale

Daily eggproduction

Larvae hatchingrate

18.83 ± 4.83 1.45 ± 0.37 a 0.54 ± 0.35 a10 6 18.00 1.38 0.6816.33 ± 8.19 1.26 ± 0.63 a 0.76 ± 0.44 a

20 6 17.50 1.35 0.821.67 ± 1.63 0.13 ± 0.13 c 0.32 ± 0.39 a

30 6 1.50 0.12 0.13a,b,c: p<0,05 Tukey’s Paired Comparison Procedure. Means in the same column followed by thesame letter are not significantly different.

Table 8.2. Mean values of number of eggs laid per female, daily egg production and larvae hatching rate (mean± standard deviation and median values) of P. oblongopunctatus in the soil of different moisture (% WHC), N-number of pairs of adult beetles.soil moisture [%

WHC]Number of pair Number of eggs per

femaleDaily eggproduction

Larvae hatching rate

9.07 ± 3.30 0.76 ± 0.27 a 0.00 a,c

20 6 10.50 0.88 0.0013.83 ± 12.12 1.15 ± 1.01 a 0.21 ± 0.22 a

40 6 14.50 1.21 0.1917.33 ± 8.16 1.44 ± 0.68 a 0.57 ± 0.44 a

60 6 18.50 1.54 0.8016.50 ± 8.12 1.38 ± 0.68 a 0.78 ± 0.22 a,b

80 6 15.50 1.29 0.8819.67 ± 10.31 1.64 ± 0.86 a 0.52 ± 0.62 a

100 6 17.50 1.46 0.40a,b,c: p<0,05 Tukey’s Paired Comparison Procedure. Means in the same column followed by thesame letter are not significantly different.

31

Table 8.3. Influence of temperature on survival of larvae (mean ± standard deviation and median); N-number ofreplicates.

Temperature [oC] N Survival of larvae [day] 1-day old larvae 10-days old larvae

10 10 * *

20 10 * *

30 10 10.6 ± 4.5810

23 ± 11.229

* All larvae alive after one month of experiment

32

9 Baseline studies of natural stressors: Establishment of survival of subzero

temperatures in earthworms

Anne-Mette Bindesbøl and Martin Holmstrup, National Environmental Research Institute, NERI, Partner 1

Introduction

In natural environments it is not unusual for an organism to be exposed to several stressful factors, both physicaland chemical, at the same time. These could include climatic stress or the exposure to chemicals ofanthropogenic origin. In traditional ecotoxicological studies organisms are usually exposed to a single chemicalat increasing concentrations, while factors such as temperature and moisture content are held at a constantoptimum. These traditional laboratory tests can therefore lead to an underestimate of the toxicity of the chemicalin natural environments where the organism will periodically encounter several physical and chemical factorssimultaneously. In the context of the current concerns over global climate changes it is important to study theinteractions between the toxic chemicals present in the environment and the climatic events likely to alter speciesbiogeography. One of the climatic factors important in defining the geographical limits of ectotherm species isthe winter temperature regime and many ectotherm species will at some time in their life span be exposed tosubzero temperatures that may cause freezing of their body fluids. The earthworm Dendrobaena octaedra isfreeze tolerant, but mortality may arise if temperatures are too low. It is therefore important to investigate ifchemical stress will increase mortality from freezing. Here we report on the survival of subzero temperatures inD. octaedra, and describe a test system to be used in future investigations of WP 3.2.

AnimalsAnimals used in this study were sub-adult and adult individuals of Dendrobaena octaedra collected from thefield and since kept in culture at 15ºC in the soil described below on a diet of cow dung. Animals used forexperimentation were approximately 3 months old and had a fresh weight of between 100 and 320 mg.

SoilTopsoil from an ecologically farmed Danish pea field (Foulum, Viborg) was used for the experiment. The soilwas a loamy sand consisting of 35 % coarse sand, 45 % fine sand, 9.4 % silt, 8.9 % clay and 1.7 % organicmatter. The pH was approximately 6.8. Prior to use the soil was dried for 24 hours at 80ºC and sieved through a2-mm mesh. Water content of the soil was adjusted to 20 % of dry weight.

Experimental designTemperature was varied in a dose-response design with 5 temperatures.Animals were acclimated at 2ºC prior to exposure to the experimental temperatures. There were 10-15 worms ineach group depending on the treatment. Each worm was weighed and kept individually in a small container with75 g of soil (wet weight) and 4 g of cow dung (wet weight) mixed into the soil. The cow-dung feed wasproduced by adding 400 ml demineralised water to 150 g dried and finely ground cow-dung. The mean bodymass was equal between treatments. All containers were covered with lids with equal ventilation. After 4 weeksat 2ºC. With the exception of the controls (+2 °C) the remaining were placed in 8 ml tubes along with a fewgrams of the appropriate substrate. In each lid 2 small needle holes were made for ventilation. The worms wereexposed to -2, -4, -6 and -8ºC in a freezer cabinet (WTB Binder Labortechnik, Tuttlingen, Germany). The freezercabinet was programmed to lower the temperature from 0ºC to -8ºC at 0.042ºC/h. When the temperature reachedapproximately -1.5ºC a small ice crystal was added to each tube to initiate freezing.As the temperature fell the animals were removed to separate chambers at their intended experimentaltemperature. The animals remained at their experimental temperature for different periods such that each groupremained at subzero temperatures for equal time (Fig 9.1). Prior to the assessment of survival rate, thetemperature was raised from 0°C to 2°C and the worms allowed 24 hours to thaw. The earthworms wereconsidered to have survived if there was a reaction to tactile stimuli, normal locomotor activity and if no signs offreezing damage were visible.

33

Figure 9.1. Temperature protocol in the freeze tolerance experiments.

Results

So far, populations from Greenland and Finland have been tested in order to select a population with sufficientfreeze tolerance. Both these populations have a considerable freeze tolerance with good survival even down to–8°C (Fig 9.2). A Danish population is currently under study.

Figure 9.2. Survival of freezing (internal ice formation) in D. octaedra from Finland and Greenland.

2 0 -2 -4 -6 -80

20

40

60

80

100

Finland population Greenland population

Surv

ival

(%)

Temperature (°C)

34

10 Baseline studies of natural stressors: Establishment of survival of drought in

Collembola

Martin Holmstrup, National Environmental Research Institute NERI, Partner 1

Introduction

One of the most important environmental factors determining the performance of soil-dwelling Collembola,and soil fauna in general, is the availability of water in the soil. This has long been realised, and theecophysiology of desiccation tolerance and water balance in Collembola has been rigorously studied.

Plants are typically able to absorb soil water until the soil water potential reaches a value known as thepermanent wilting percentage. At this stage, suction pressure of the soil is -15 bar, equivalent to a relativehumidity of the soil air of approximately 98.9% RH. This should be compared with the osmolality ofcollembolan body fluids, which is typically 300 mOsm, equivalent to a RH of 99.4%, or a water potential ofapproximately -8 bar. Soil-dwelling Collembola are therefore likely to meet water potentials during summerdrought that will cause a loss of body water. It is important to develop a realistic experimental droughtexposure for the study of soil-dwelling Collembola because even extreme drought will seldom produce soil airhumidities lower than 96% RH. If any physiological mechanisms involved in the drought tolerance of soil-dwelling Collembola are to be studied, it is necessary that the experimental desiccation levels be adjusted tosimulate the conditions that the animals are likely to meet in the field. Here we present an appropriate droughtexposure scenario that will be used in investigations of the soil dwelling collembolan Folsomia candida.

Methods

Test animalsThe springtail species Folsomia candida was used for the experiments. They are permanently cultured at theDanish National Environmental Research Institute (NERI) (Silkeborg, Denmark) at 20 ± 1°C with a 12:12-hlight:dark photoperiod. The animals are kept in Petri Dishes containing a 5 mm layer of water-saturatedcharcoal/plaster of Paris mixture (1:8) and fed a diet of dried Baker’s yeast. In this study animals were furtherstandardized to ensure that all individuals were of the same age at the outset of experimentation. 16-19 daysold animals were used.

Drought exposureThe animals were exposed to six relative air humidities (RH) between 97 and 100%. This range is chosen sinceit represents realistic soil pore air humidities occurring during summer drought. Ten animals, previouslyexposed to the chemical, were placed in a plastic sample vial measuring 4 cm in height and 2 cm in diameter.The vial was placed in the center of a 200 mL plastic cup containing approximately 30 mL of an unsaturatedNaCl solution. The plastic cup was sealed with Parafilm M® and a tightly fitting plastic lid. By using a NaClsolution it is possible to control the RH in the drought chamber. The concentrations of the NaCl solution werecalculated using the formula RH = 55.56 / (55.56 + Osm) * 100, where Osm is the osmolality of the NaClsolution. The air in the drought chambers rapidly equilibrates with the NaCl solution according to Raoult´slaw. The drought chambers were incubated at 20±1°C with a 12:12 h photoperiod for seven days and thenrehydrated for two days at 100% RH by replacing the NaCl-solution with demineralized water. Subsequentlythe mortality was estimated. Animals were scored as dead if they were not able to walk more or lesscoordinated when gently touched with a preparation needle.

Results

Three independent experiments were conducted to determine the baseline tolerance to drought in F. candida.Basically the same result in terms of LRH50 was obtained (the Relative humidity causing 50% mortality).LRH50 values were close to 97.5% RH in all three cases (Fig 9.1), which is consistent with previousobservations. It is therefore concluded that applied drought exposure system is robust and giving comparableresults in independent experiments.

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Figure 8.1. Survival of drought in Folsomia candida after exposure to various drought intensities during7 days. Values are shown as mean ± SD (n = 4). Each replicate consisted of adult animals.

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11 Summary and future research in WP 3.2Baseline studies of selected natural stressors have been carried out, and test systems to quantify the effects on thevarious test organisms have now been developed. For human health issues WP 3.2 partners have selected apathogenic stressor (Lipopolysaccharide of Gram-negative bacteria) and an allergenic stressor (Birch pollenallergen extract). The effects of these natural stressors associated with human health has been quantified usinghuman peripheral blood mononuclear cells and human reporter cell lines as indicators for such effects. These testsystems have shown reliability, sensitivity and specificity.

For freshwater organisms, two major natural stressors, i.e. anoxia and suboptimal temperature, have beeninvestigated in fish embryos and larvae. Further, work on daphnids is in good progress. These baseline studieshave shown that reproducible results can be produced in well-functioning and established test systems.

Within terrestrial organisms, work has been carried out to investigate the response to extreme and suboptimaltemperatures and suboptimal water availability. Such dose-response relationships have been established forcarabid beetles, earthworms, Collembola and nematodes. Also for these test systems, methods are now availableand established in the respective laboratories of the NOMIRACLE partners.

It is concluded that WP 3.2 with the present deliverable is largely on schedule and ready to enter the secondphase of the first 18-month plan. During the coming 6 months work shall focus on initial experiments to validatethe applicability of the chemical mixture assessment model for use in assessing the impacts of relevantcombinations of cumulative stressors (Deliverable 3.2.2, due 31 February).

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