Immune response of earthworms (Lumbricus terrestris, Eiseniaandrei and Aporrectodea tuberculata) following in situ soil exposure

to atmospheric deposition from a cement factory

Richard Massicotte,a Pierre Yves Robidoux,b Sebastien Sauve,*c Denis Flipo,a

Michel Fournierd and Bertin Trottiera

aCentre TOXEN, Universite du Quebec a Montreal, P.O. Box 6128 Centre-ville, Montreal,QC, CanadabBiotechnology Research Institute, National Research Council of Canada, 6100 RoyalmountAvenue, Montreal, QC, Canada H4P 2R2cDepartment of Chemistry, Universite de Montreal, P.O. Box 6128 Centre-ville, Montreal,QC, Canada H3C 3J7. E-mail: sebastien.sauve@umontreal.ca; Fax: 514 343-7586;Tel: 514 343-6749dUniversite du Quebec, INRS-Institut Armand Frappier, 245 Hymus, Pointe-Claire, QC,Canada H9R 3G6

Received 18th February 2003, Accepted 15th July 2003

First published as an Advance Article on the web 4th August 2003

In order to reduce their energy costs, many cement plants use fuel product substitutes (old tyres and used oil).

The combustion of these products generates a metal increase (e.g. Cu, Cd, Pb and Zn) in the atmospheric

emissions. After their release, these elements are deposited into the environment and could eventually

accumulate up to concentrations of concern. At the Saint-Laurent cement factory (Joliette, QC, Canada),

maximum deposition of these elements occurs in the direction of prevailing winds (North-East). We evaluated

the potential impact of these depositions upon the immune system of three earthworm species (Lumbricus

terrestris, Eisenia andrei and Aporrectodea tuberculata) exposed in a natural environment. The exposure sites

were 0.5, 1.0, and 2.0 km downwind from the cement factory, along with an upwind reference site. The

immune parameters studied were the cell viability and phagocytic potential of the immune cells (coelomocytes).

For both L. terrestris and E. andrei, after 7 d exposure, none of the measured parameters showed significant

differences among the sites. On the other hand, for the indigenous worm A. tuberculata, in the most exposed

zone (at 0.5 km), we observed an increase in cell viability and phagocytic potential. This increase could possibly

be attributed to physicochemical effects such as the alkaline pH of the soil, or alternatively, it could result from

beneficial effects induced by an increased calcium supply.

Introduction

In order to reduce its energy costs, the Saint-Laurent cementplant (Joliette, QC, Canada) increasingly favours the use ofalternate energy sources namely tyres and spent oils. Given thefairly high temperature in the ovens (y1200 uC), most organicproducts are destroyed with an estimated 99.9999% efficiency.1

The exploitation of those potential energy fuels leads to a slightincrease in the atmospheric release of some trace elements,notably cadmium, copper, lead and zinc.1,2 A previousdispersion model study indicated that trace element depositionoccurred primarily in the axis of prevailing winds.3,4 Indepen-dent determinations of atmospheric emissions,1,2 atmosphericdeposition using collectors,5,6 and soil analyses6 concluded thatthe cement plant emissions contribute to an overall increase inthe environmental burden of various trace elements.7 A gradualdecrease in soil pH and conductivity is observed up to 5 kmfrom the plant. These results indicate that the plant isimpacting the surrounding soils.It is well recognized that total soil metal content is a poor

predictor of bioavailability, more refined indices beingnecessary to assess toxicological effects and tissue metalaccumulation.7,8 Giroux et al.9 used a standard agronomicsoil testing extraction (Mehlich-3) to derive plant toxicitythresholds for various trace elements. The extraction data forthe sites exposed to the atmospheric deposition show that therecommended thresholds are not exceeded. Chabot and

Simard6 attributed this low extractability to the neutralalkaline soil character and the very low resulting chemicalavailability. Since the atmospheric metallic burden is definitelyincreased, despite no evidence of higher availability usingchemical extractions, it is pertinent to evaluate the potentialecotoxicological impacts of the cement dust deposition onbiological organisms. In earthworm toxicity testing, theorganisms are in close contact with soil and can be used toevaluate bioavailability without requiring the use of chemicalextraction procedures.10 In addition, the earthworm isrepresentative of soil fauna and recognized to be a relativelysensitive indicator.11,12

In soils, earthworms constitute 60–80% of the animalbiomass13 and play a critical ecological role.14,15 For example,it is estimated that under favourable conditions, earthwormscan move up to 18 tons of soil per acre per year.16 Beingcontinuously exposed to the soil through direct dermal contactand through ingestion, earthworms are therefore in closecontact with exogenous dusts.The immune system parameters may be used as a sensitive

sub-lethal endpoint to assess toxicity of atmospheric depositionto earthworms. The immune system of earthworms iscomposed mainly of coelomocytes, i.e. cells found within thefluids in the worm coelomic cavity.17 These cells are analogousto macrophages found in the immune system of vertebrates.18

It has been demonstrated that many chemicals, includingvarious trace elements, can adversely affect the immune

774 J. Environ. Monit., 2003, 5, 774–779 DOI: 10.1039/b301956j

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system.19 Suppression of the phagocytic activity or reduction ofcoelomocyte viability compromise an animal’s ability tosequester and kill microorganisms and further reduces itsresistance to infection.19 The immunodeficiency of exposedspecies is interpreted as an indication of toxic effects ofenvironmental contaminants.19,20 At the lower concentrations,we observe an increase in the phagocytic activity and viabilitywhich we interpret as a positive stimulation, a phenomenoncalled hormesis, such as described by Calabrese and Baldwin21

and often observed with immunotoxicological tests.12,19

Given that tolerance to different contaminants variesaccording to species,22–24,25 we have chosen to compare thecell viability and phagocytic potential of three earthwormspecies (Lumbricus terrestris, Eisenia andrei and Aporrectodeatuberculata). The objective is to evaluate, in situ, if theatmospheric emission from cement plants burning fuelproducts substitutes can potentially produce toxic effects tothe earthworms.

Experimental

Animals

Adult L. terrestris (Oligochaeta) worms were purchased fromAppats Saint-Gabriel (Saint-Gabriel de Brandon, QC,Canada). The worms were maintained at 10 uC, in the dark,in a peat-rich worm media (Magic Products Inc., Ahmerst Jct,WI. USA). Worms were fed weekly with worm food (MagicProducts Inc., Ahmerst Jct, WI. USA). Before beginning theexperimentation, the worms were acclimated to the laboratoryconditions for at least two weeks.26

Earthworms E. andrei were obtained from a culture ofspecimens originally purchased from Carolina BiologicalSupply (Burlington, NC, USA). They were maintained at20 uC but otherwise subjected to the same conditions asdescribed for L. terrestris.The A. tuberculata worms were harvested from the study site

in the first week of September 1999. The worms wereindividually collected at a depth of approximately 15–20 cmfrom the surface.

Exposure

The in situ exposure was carried out in netted mesocosmcompartments (15 6 15 6 30 cm).27,28 Those mesocosms wereburied in sandy soils (reference and 0.5 km) and clay type(1 and 2 km) soils at exactly the same sites used for thecollection of atmospheric deposition samples.28 The referencesite was located at 10 km upwind of the factory and the exposedsites were 0.5, 1.0, and 2.0 km downwind of the factory (Fig. 1).Chabot and Simard6 have previously demonstrated significantdifferences between the chemical characteristics of cultured and

non-cultured parts of the fields (e.g. field borders). To eliminatethat source of variability, all exposures were done in theuncultivated field borders. The soil was then collected down toa depth of about 20 cm and transferred into the nettedcompartment. The mesocosms were installed in the hole, 30 630 cm and 20 cm depth. Given the drought period at the end ofAugust 1999, and the relative dryness of the soils, the waterholding capacity (WHC) was estimated in a soil subsample. Forthis estimation, a soil sample volume similar to that of themesocosms is taken next to the site and a graduated cylinder isused to add distilled water until saturation. This volume is thenused to estimate how much water corresponds to about 40% ofthe water holding capacity for the sandy soils (0.5 km andcontrol) and 20% in the case of the clay soils (1.0 km and2.0 km). Five E. andrei and five L. terrestris individuals weredeposited onto the surface of the mesocosm which was thensealed using Velcro bands, to prevent the worms from escaping.The total number of worms was a function of the volume of themesocosm. Four mesocosms were installed at each site, for atotal of 16. The A. tuberculata individuals were indigenousworms naturally exposed to the atmospheric depositions.Groups of 10 worms were picked up directly from each site.

Earthworm cell preparation

Coelomocytes were obtained using an adaptation of theelectrical extrusion method.20,29 Each worm was pressed lightlyfrom the middle to the posterior end, to extrude coelomiccavity content. The worm was then rinsed with a cold salinesolution (0.85 mg NaCl mL21 at 10 uC). Then, the worm wasinserted into a disposable polyethylene tube containing 3 ml ofBasal Salt Solution (BSS). The BSS contains 1.5 mM NaCl,4.8 mMKCl, 1.1 mMMgSO4?7H2O, 0.4 mMKH2PO4, 0.3 mMNa2PO4?7H2O, 3.8 mM CaCl2 and 4.2 mM NaHCO3 adjustedto pH 7.3 with HNO3 or NaOH.26 The liquid medium was thensubjected to a 6 V current (lantern battery) for 35–40 s usingaluminium wires. The worm was removed from the solutionand rinsed with BSS; the liquid extracts were combined and thevolume brought to 15 ml. The suspension was then centrifugedat 150 g for 10 min at 4 uC.26 Cell concentrations aredetermined by diluting 50 ml of cell suspension with 50 ml of0.4% trypan blue (Gibco, Grand Island, N.Y. USA) using aNeubauer haemocytometer. The presence of enough viablecells (approximately 106) was then confirmed using an opticalmicroscope.

Phagocytosis and cell viability

The recovered cells are incubated in the BSS solution with1.2 mm fluorescent latex beads (Molecular Probe, Eugene, OR)using a bead : cell ratio of 100 : 1 to favour optimalphagocytosis conditions.26 The cells are then incubated for 18 hat 15 uC. To remove free beads that were not phagocyted, thesuspension is layered over a 3% bovine serum albumin solutionand centrifuged at 150 g for 5 minutes.12,26 The free beads areretained in the liquid fraction whereas the cells are collected inthe pellet. The cells are then resuspended in hematal(Fisher Scientific, Ottawa, Ontario). The viability, the propor-tion of phagocytically active cells and the mean number ofbeads phagocyted per cell is measured by a fluorescencemethod12,25,26 using flow cytometry with a FACScan Instru-ment (Becton-Dickinson, San Jose, CA). The number of beadsengulfed by a cell induces an increase of the cells’ fluorescence.Total fluorescence is electronically converted into a number ofbeads phagocyted. Cell viability is measured using a propidiumiodide marker (PI) (Sigma, St-Louis, MO, USA). The cellviability is defined as membrane permeability to propidiumiodide.12 The dead cells are permeable to PI and therefore havedifferent fluorescence properties relative to live cells which arecapable of extruding the PI dye. The fluorescence emission isFig. 1 Diagram of exposure and sampling sites

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determined at 520 nm using an air-cooled argon laser providingan excitation at 488 nm. Coelomocyte populations were definedbased on their forward scatter (FSC) and side scatter (SSC)properties.12,25,26

Determination of body burdens

For the determination of body burden of some metals (Cd, Cu,Pb and Zn) we used atomic absorption spectrometry (ARLGBC 906 AA). Before measurements, tissues were adequatelyprocessed to convert metals to a suitable chemical form and todecompose organic materials. The worms were kept in a dryingoven at 30 uC for five days and triplicate analyses wereperformed on the dried tissues. The digestion was performedusing y0.5000 g of dried tissues with 5.00 ml of concentratednitric acid and heating in a closed vessel using a pressure-controlled microwave heating system (on a MDS-2100instrument, Matthews, NC, USA). The tissue analyses wereroutinely controlled for quality assurance using a bovinestandard reference material (NIST-bovine liver #1577a).

Statistical analyses

Phagocytosis, viability, and body burden results are quitevariable and given the non-normal distribution of the data,statistical analyses were first performed using a non-parametricKruskal-Wallis test with significance concluded at P ¡ 0.05.We also used an analysis of variance (ANOVA) to comparemedian body burden values among the various sites.

Results and discussion

Let us first underline the fact that the drought conditionsprevailing during the in situ mesocosm exposure inducedmortality in the exposed worms. This fact resulted in a differentreplicate number ‘‘n’’ for L. terrestris and E. andrei with respectto A. tuberculata. For each site, we initially used 5 L. terrestrisand 5 E. andrei.

Cement industry emissions and atmospheric deposition

According to the literature, the main cement plant emissionscome from the stacks and the major chemical elements thereofare calcium, cadmium, copper, lead, and zinc (Table 1). Theconcentration of metals in these emissions varies according tothe type of fuel used as energy source (Table 1). Such emissionscan potentially lead to a deposition in the environment asevidenced from the results shown in Fig. 2. The atmosphericmetal deposition is corroborated by the results of Chabot andSimard upon soil analysis.6 It is interesting to note that thetotal metal concentration decreases as one goes away fromstacks (Fig. 2). Based on these results we hypothesized that theresponse of the immune system (cell viability and phagocytosis)to the contamination could vary with the distance from thesource.

Immune system response

The purpose of studying this parameter was to determine if thecontaminant exposure reduced the cell defence system in termsof cell survival and concomitant phagocytic activity.

Cell viability

The results for the indigenous A. tuberculata from the 0.5 kmdownwind site are significantly higher than the values from thecontrol and from the other sites (Fig. 3). Cell viability ofL. terrestris shows a tendency to increase with increasingdistance from the source (Fig. 3). The median values for cellviability of E. andrei show no statistically significant differencesbetween control and exposed sites (Fig. 3).

Phagocytosis

For A. tuberculata, phagocytosis was significantly higher at0.5 km from the cement factory (Fig. 4). The phagocytosisfunction for L. terrestris is altered at 0.5 km and 2 km from thesource when compared with the control and the 1 km site(Fig. 4). On the other hand, we observed no statisticallysignificant differences between the control and treatments inthe number of phagocyted beads per cell for E. andrei (Fig. 4).The results show differences in immunotoxicological

response between the three earthworm species. These differ-ences may be attributed to the specific physiological char-acteristics of each species. The presence of calciferous glandscontributes to considerable differences among species in both

Table 1 Environmental release of chemical elements by a cement plantas a function of the energy source used

Elements

Demers 19943

(mg Nm23)Duchesne et al. 19971

(mg Nm23)

Old tyres (meanof 2 trials) Control

PCP-treatedwood Control

Calcium 10422.0 11804.0Cadmium 2.3 — 1.10 1.02Copper 10.8 3.9 3.61 2.62Lead 366.5 120.8 18.10 14.20Zinc 2260.0 185.1 8.60 7.00aNm3: A cubic meter of gas in normalized conditions: 101.3 kPa and25 uC. bPCP: pentachlorophenol.

Fig. 2 Calcium and total metals (Pb 1 Zn 1 Cu 1 Cd) concentrationsin atmospheric depositions as a of function of distance from the cementplant.49

Fig. 3 Effect of distance from the cement plant on cellular viability ofthree worm species (median ¡ SD; n ~ 5 for L. terrestris and E. fetidaand n ~ 10 for A. tuberculata).

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the degree of gland development and the secretion output.30

These glands have three main functions: elimination of excesscalcium absorbed from the diet, production of secretions toneutralize humic acids in the material consumed and thirdly,the fixation of part of the metabolic carbon dioxide to facilitaterespiration under anaerobic conditions.30 Some studies showthat the metabolism of Ca has important functions regardingthe body burdens of heavy metals.31–33 These metabolicdifferences can have an impact on the body burdens ofmetals.33,34 The differences in the metabolism of Ca are not theonly factor influencing the metal burdens. The metal accumu-lation also depends on the interaction of these soil-dwelling,soil-eating animals with local environmental factors, such as:soil Ca concentration, total soil metal concentrations, soilorganic matter and pH.35,36 It is also clear that soil liming andacidification modulate the bioavailability of metals as well asthe feeding activities and habits of the worms.37

The Ca body burden shows a significant decrease forL. terrestris (Fig. 5) and the body burdens of the sum of metalsfor E. andrei and L. terrestris are variable but tend to decreaseas a function of the distance from the source (Fig. 6).Concentration in metals can be correlated with the atmosphericdeposition that also varies with the distance (Fig. 2).

Aporrectodea tuberculata

Earthworms collected at 0.5 km downwind from the sourceshowed a significantly different immune response potentiallyattributable to the deposition of dust from the cement plant.The viability of coelomocytes and their phagocytic potential

are statistically increased relative to controls (Figs. 3 and 4).Given the known increase in the soil burden of trace elements, apositive stimulation of the immune system was not, a priori,anticipated.5 Fig. 6 shows that the total metal concentrations(cadmium, copper, lead and zinc) were higher than controlsand those metals released in the atmosphere by cement plantshave previously been shown to be potentially toxic to thegrowth, survival and reproduction of earthworms.23,25,37,38

Hypothetically, at 0.5 km, the indigenous earthworm popula-tion has adapted its Ca metabolism to the local conditions, asmentioned by Morgan and Morgan.35 The population ofindigenous earthworms can thus possibly be geneticallyadapted to the selection pressures exerted by toxic metals.In their study, Chabot and Simard6 observed that calcium

concentrations and pH were significantly higher in the site mostexposed to the atmospheric deposition. They interpreted theselast two observations as potential factors reducing thebioavailability of the contaminants. Soil physico-chemicalconditions can indeed drastically contribute to reduce the metalsolubility and modulate their bioavailability.39 The high pHvalues measured in some of the soils are thus expected tominimize the toxicity of the soil metals and therefore reduce theeffects on earthworms.40–42 For example, a soil containing280 mg Pb kg21 yielded about 0.1 mM of Pb l21 at pH 7, butupon soil acidification to pH 4, the same soil yielded about10 mM Pb l21 in solution.43 The same soil submitted to a threepH-unit acidification showed a hundred-fold increase in thesoil solution concentration of Pb. Earthworms are also knownto be sensitive to soil pH, with soil acidity aggravating metaluptake and toxicity.34,44,45

The combination of both factors, high pH and geneticadaptation, may explain, at least partially, the results obtainedwith A. tuberculata at 0.5 km from the source.

Eisenia andrei

E. andrei was not affected by the short field exposureconditions used in this study (7 days). Considering the lowbioavailability of metals and the steady-state concentrations inthe worm tissues,46 toxic effects could perhaps be observableonly after a longer exposure period. Based on the lethalityendpoint, E. andrei is considered a representative earthwormspecies.22 In some cases, this soil organism has been observed tobe sensitive to metals such as Cd but the response dependsheavily on the exposure conditions.11 However, for manychemicals, E. andrei is considered as a relatively resistantspecies.46

Lumbricus terrestris

The immunological response of L. terrestris is different fromthat of the other two species. From the lethality endpoint,L. terrestris is recognized as sensitive to metals.11,33 This species

Fig. 4 Phagocytic response of three worm species (median ¡ SD; n ~5 for L. terrestris and E. fetida and n ~ 10 for A. tuberculata) as a offunction of distance from the cement plant.

Fig. 5 Ca body burden of three worm species as a function of distancefrom the cement plant (median ¡ SD; n ~ 5 for L. terrestris andE. andrei and n ~ 10 for A. tuberculata).

Fig. 6 Total metal body burdens (sum of Pb1 Zn1 Cu1 Cd) of threeworm species as a of function of distance from the cement plant.

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sensitivity may be partly attributed to the complexity of itscalciferous gland system.47

Given that the alkaline pH reduces the solubility andbioavailability of metals and decreases their toxicity, thesefactors would contribute to reduce the negative effectsobserved on the immunological system of the other twoworm species at 0.5 km from the source. This interpretation ofour observations is also consistent with the significantly highertissue Ca contents found in L. terrestris (Fig. 5). These effectson the immune parameters are observed further away from thesource. The worms used in our study do not seem geneticallyadapted to the heavy selection pressures exerted by toxic metalsat this site (0.5 km). On the other hand, the soil at 2 kmcontains a slightly elevated Cu level that Chabot and Simardhave attributed to earlier land application of swine slurry.6 Aconcurrent lowering of soil pH (6.4) at this site (2 km) alsocontributes to increasing metal bioavailability (Zn and Cu).The sublethal suppressive immunological effects of Cu and Znon the cell-mediated immune system have been well establishedfor annelids.25,48 Results from the site at 2 km seem to indicatethat phagocytosis function is altered in comparison with theother sites (Fig. 4) but not the cell viability (Fig. 3). Theseresults are concordant with previous in vitro observations bySauve et al. showing that the immune function of coelomocytesexposed to trace elements is impaired before the onset of celldeath.25

By comparing the atmospheric deposition (Fig. 2), bodyburden and cell viability for L. terrestris (Figs. 3, 5 and 6) onecan observe that Ca and to a lesser extent metal concentrationsin both atmospheric depositions and worm tissues decreasewith distance from the cement plant (0.5 to 2.0 km) whereas thecell viability tends to increase.

Control site

In general, the reference site showed a sub-lethal response(Figs. 3, 4, 5 and 6) except possibly for the indigenous worm,A. tuberculata, (Figs. 3 and 4). The control site is not subjectedto the influence of the atmospheric deposition from the cementwork.3,4 Like the other three sites, the control site is located inan agricultural environment, without any industrial source ofmetals nearby. It is however possible that different agriculturalpractices could have had an influence on the properties of thesoil (e.g., fertilizer and liming practices). This also highlightsthe difficulty to find a control environment which exactlymimics the exposure sites and is totally devoid of impacts.

Conclusion

This study showed the immune response of three species ofearthworms after an in situ exposure to cement dust deposition.Immune response varied significantly across the worm speciestested. The use of indigenous worm allowed the observations ofresults which could arise from a long-term exposure. Theresults showed an increase of the cell viability and thephagocytic response of A. tuberculata in the maximal deposi-tion site (0.5 km). However, the cement kiln dust did notsignificantly affect the immune cell viability and the phago-cytric response of L. terrestris and E. andrei at that site. Thisincrease could possibly be attributed to physicochemical effectssuch as the alkaline pH of the soil, or alternatively, it couldresult from beneficial effects induced by an increased calciumsupply. Also, Ca and metal body burdens were higher atexposed sites compared to the reference, but decreased as afunction of distance from the cement plant. Future studies withL. terrestris using longer exposure periods, more representativeof chronic exposure are warranted. The use of an indigenousspecies could be used in parallel for comparative purposes anda better representativity of this particular environment.Furthermore, the cement plant will eventually stop its

operations. It would therefore be advisable to continue theenvironmental monitoring since the calcareous inputs wouldthen decrease and the atmospheric acidic depositions andnitrogen fertilizers could acidify the soil so that the metals thatare now present in the soils but inert could again becomeavailable.

Acknowledgements

The authors wish to extend their gratitude to Comite de suivienvironnemental Lanaudiere (COSE Lanaudiere), the TOXENResearch Center and the CIRTOX for their financial support.The authors thank Mr Pierre Cayer for excellent technicalassistance. We also thank Dr John W. Reynolds for his help inidentifying the field-collected species, Aporrectodea tuberculata.

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