Agriculture, Ecosystems and Environment 83 (2001) 177–189

Using Collembola to assess the risks of applying metal-rich sewagesludge to agricultural land in western Scotland

Lorna J. Cole∗, David I. McCracken, Garth N. Foster, Mark N. AitkenConservation and Ecology Department, Environment Division, SAC Auchincruive, Ayr, KA6 5HW, Scotland UK

Received 6 December 1999; received in revised form 5 April 2000; accepted 19 April 2000

Abstract

The recent United Kingdom ban on the disposal of sewage sludge at sea has led to the prediction that land applicationof sludge will become more widespread. The positive aspect of recycling nutrients may, however, be offset by the risk ofcontamination by heavy metals that are frequently present in sludge. The environmental impact of applying metal-rich sewagesludge to agricultural land was, therefore, assessed using Collembola. A combination of pitfall trapping and suction samplingwas used to monitor epigeal/hemiedaphic Collembola on a small plot field trial in the west of Scotland. Four sludge treatmentswere investigated: cadmium-rich sludge, zinc-rich sludge, uncontaminated sludge and a no-sludge control. It was found thatthe abundance ofLepidocyrtus cyaneusTullberg andIsotoma viridisBourlet was significantly lower in plots receivingcadmium-rich sludge than those receiving uncontaminated sludge.Isotoma anglicanaLubbock was not influenced by thepresence of metals in sludge andIsotomurus palustris(Müller) was actually favoured by the application of metal-rich sludge.Other aspects of collembolan ecology, and the efficiency of the two sampling methods, are also discussed. © 2001 ElsevierScience B.V. All rights reserved.

Keywords:Ecotoxicology; Sewage sludge; Heavy metals; Collembola; Agriculture

1. Introduction

Recent estimates suggest that, in the United King-dom, 500,000 t of sewage sludge are applied annuallyto agricultural land (Smith, 1996). However, the Com-mission of the European Communities (CEC) recentban on the disposal of sewage sludge at sea (Commis-sion of the European Communities, 1991) has led tothe prediction that the recycling of sludge to agricul-tural land will become more widespread throughoutEurope (Rund, 1995). Probably, the principal envi-ronmental risk posed by fertilising agricultural land

∗ Corresponding author. Tel.:+44-1292-525-291;fax: +44-1292-525-333.E-mail address:l.cole@au.sac.ac.uk (L.J. Cole).

with sludge lies in the fact that it often contains sig-nificant quantities of heavy metals such as cadmium,zinc, lead and copper (Petruzzelli et al., 1994). Theseelements, collectively known as potentially toxic el-ements (PTEs), are persistent and will accumulatein the upper layers of the soil where they can reachlevels that are potentially toxic to plants and animals(van Straalen et al., 1987).

The CEC’s 1986 Directive onThe protection ofthe environment, and in particular the soil, whensewage sludge is used in agricultural landaddressesthe problem of PTEs in sewage sludge (Commissionof the European Communities, 1986). This Directivecontrols the amount of heavy metals entering the en-vironment through sludge application to agriculturalland by setting maximum metal levels for sludge

0167-8809/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S0167-8809(00)00172-9

178 L.J. Cole et al. / Agriculture, Ecosystems and Environment 83 (2001) 177–189

and soil. However, despite the ample information onhow PTEs in sludge affect micro-organisms (Brookesand McGrath, 1984; Smith, 1996), there is com-paratively little information on how they influenceinvertebrates. Risk assessment studies tend to focuson economically important pests (e.g. Hemiptera:Aphidae), polyphagous predator groups (e.g. Aranaeand Coleoptera) and macro-decomposers (e.g. earth-worms). For example, earthworms and spiders col-lected from plots treated with contaminated sludgewere shown to have elevated metal concentrationsindicating that the metals were in bio-available forms(Benninger-Truax and Taylor, 1993). Green peachaphids (Appelia schwartziBörner) feeding on plantsgrown on contaminated sludge were found to havedecreased fecundity and longevity than those feedingon plants grown on uncontaminated sludge (Pimenteland Warneke, 1989). The metals in sludge are, there-fore, biologically available and have the potential toreduce fitness.

When a pollutant is added to agricultural land,changes in the structure of the microbial communitytend to precede changes in the composition of theplant or invertebrate community. Groups that feed di-rectly on soil fungi and bacteria (e.g. Collembola andAcarina) may, therefore, be more susceptible to metalpollution than predatory or phytophagous groupswhich are further removed from the effects of thepollutant. Previous field studies have indicated thatalthough the addition of metal contaminated sludgedid not adversely affect the abundance of Collem-bola, it influenced the community structure (Lübben,1989; Bruce et al., 1999). Lübben (1989) found thatSminthurinus aureus(Lubbock),Willemia intermediaMills and Isotoma notabilisSchäffer were sensitiveto sewage sludge artificially contaminated with metalsalts (zinc, cadmium, copper, nickel, chromium andlead), whileFolsomia candida(Willem) andMesapho-rura spp. were not. Collembolan communities wouldtherefore appear to be sensitive to metal-contaminatedsludge with species-specific differences in metal sen-sitivity occurring.

Collembola are of agronomic importance not onlyas a consequence of their regulatory role in decompo-sition (Seastedt, 1984), but also because they are animportant source of prey for polyphagous predators(Hopkin, 1997). As Collembola live in close associa-tion with the soil micro-flora and fauna, it is likely that

they will give an earlier indication of ecosystem dis-turbance than predatory groups. Furthermore, Collem-bola are better suited than larger more mobile inverte-brates (e.g. Coleoptera) in small plot agricultural fieldtrials.

In this study, the risks of applying metal-rich sewagesludge to agricultural land were assessed in the fieldusing epigeal Collembola as indicators. A sludge richin zinc, which is an essential micro-nutrient, and asludge rich in cadmium, which is an ecological impu-rity, were investigated on a small-plot field trial. Themetal-rich sludges were derived from sewage treat-ment works with naturally high inputs of each specificmetal. It was considered essential to use naturally con-taminated sludge since this provided a more appro-priate measure of exposure (and, hence, risk) underfield conditions. The use of sludges artificially con-taminated with metal salts was discounted as metalsare more available in such sludges and they thereforedo not represent a natural situation (Smith, 1996). Al-though the use of naturally contaminated sludge meantthat the sludges differed in other respects (see Table 1),these were taken into account in the interpretation ofthe results.

2. Materials and methods

2.1. Study site

The trial site was situated on a well-drained sandyclay loam topsoil overlying a sandy loam subsoil (Eu-ric Cambisol) at SAC Auchincruive, west of Scotland.The topsoil contained 210 g kg−1 clay and 26 g kg−1

organic carbon. The research was conducted as partof a larger scale experiment instigated in 1994. Thelarger experiment consisted of 23 sludge treatmentsestablished across three blocks in a randomised blockdesign (i.e. a total of 69 plots each 6 m×8 m). EpigealCollembola were monitored on four of these sludgetreatments (i.e. a total of 12 plots): no sludge control,uncontaminated sludge, cadmium-rich sludge andzinc-rich sludge (Table 2). The metal-rich sludgeswere derived from sewage treatment works with nat-urally high inputs of the specific metal.

Since 1994, sludge had been applied annuallyto these plots each August in the form of a dewa-tered sludge cake. Following each annual application,

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180 L.J. Cole et al. / Agriculture, Ecosystems and Environment 83 (2001) 177–189

Table 2The four sludge treatments studied at SAC Auchincruive, Scotland, and the maximum metal concentrations set by current CEC legislation(Commission of the European Communities, 1986)a

Treatment Mean soil concentration Mean sludge concentration

Control 85.5 mg Zn kg−1 soil –<0.33 mg Cd kg−1 soil

Uncontaminated sludge 94.8 mg Zn kg−1 soil 725 mg Zn kg−1 sludge0.33 mg Cd kg−1 soil 1.94 mg Cd kg−1 sludge

Zinc-rich sludge 270 mg Zn kg−1 soil 6619 mg Zn kg−1 sludge– 17.2 mg Cd kg−1 sludge

Cadmium-rich sludge 2.2 mg Cd kg−1 soil 48.9 mg Cd kg−1 sludge– 1244 mg Zn kg−1 sludge

CEC maximum concentrations 300 mg Zn kg−1 soil 4000 mg Zn kg−1 sludge3 mg Cd kg−1 soil 40 mg Cd kg−1 sludge

a Mean cadmium and zinc concentrations in sludge and soil at SAC Auchincruive in 1996 and maximum soil and sludge concentrationsset by CEC Directive 86/278/EEC (Commission of the European Communities, 1986).

sludge was incorporated into the soil using small plotequipment and then the plots were sown with Ital-ian ryegrass (Lolium multiflorumLam.) which wascut twice each year. A 1.2-m permanent grass stripsurrounded each plot to prevent soil transfer betweenplots during cultivation. The input of organic matterto the soil was standardised across sludge treatments,and sludge application did not alter soil pH. During1996, the grass was cut on 24 May and 26 July (i.e.prior to the May and August Collembola samplingdates: see below).

2.2. Sampling techniques

Sampling was conducted between April and August1996. A combination of pitfall trapping and suctionsampling was utilised in order to provide an effec-tive sampling regime for epigeal Collembola (Berbierset al., 1989; Frampton, 1994).

Dietrick Vacuum insect nets (D-Vac) have previ-ously been used to collect Collembola by suction(Purvis and Curry, 1978; Frampton, 1988), but canbecome heavy and uncomfortable with prolongeduse. Modified Ryobi RSV3100E sweeper-vac, havebeen found to be as, or more, efficient at samplingCarabidae, Araneae, Staphlinidae, Isopoda and Aphi-didae (Harwood, 1994; MacLeod et al., 1994). Thistechnique was, therefore, adopted in the current studyas a light-weight alternative to sample Collembola.Following the process developed by MacLeod et al.(1994), fine weave (<0.5 mm) nylon/cotton mix voilebags were placed in the sampler nozzle and renewed

after each sample. Suction samples were taken on30 April, 31 May, 22 June and 4 August. For eachsuction sample, an area of 706 cm2 was partitionedoff and extracted for 60 s. On all sampling dates, fivesuch samples were taken randomly from each of the12 plots under investigation. To standardise acrossdates, suction samples were taken on sunny daysbetween 10:00–17:00 h when the grass was dry.

Pitfall traps consisted of plastic beakers (150 mlvolume, diameter 6 cm) containing 20 ml of the preser-vative ethylene glycol. Five pitfalls were inserted ineach plot on 24 April, 27 May, 20 June and 1 Au-gust, and these were retained in situ for seven days.To avoid the ‘digging-in’ effect (Joosse and Kapteijn,1968), the pitfall traps in May, June and August wereinserted into the same holes as the April pitfalls.

Both the suction samples and pitfall traps were pro-cessed by first separating the organic and inorganicmatter by repeated flotation in a saturated salt solution.The organic matter (including Collembola) was thenpassed through two graded sieves (15 mm and 45mm)to separate the Collembola from the larger arthropodsand plant debris.

All Collembola were initially mounted in Hoyer’smedium and identified under a light microscope (mag-nification 400–1000×). After this initial identification,it was found that with the exception of problematicspecimens, most species could be identified as ac-curately under a dissecting microscope. Collembolawere identified using the keys of Gisin (1960), Gough(1977) and Fjellberg (1980) and following the nomen-clature of Fjellberg (1980). As Fjellberg (1980) is a

L.J. Cole et al. / Agriculture, Ecosystems and Environment 83 (2001) 177–189 181

‘splitter’, separating Collembola into species on thebasis of very small differences in morphology (Hop-kin, 1997), the information derived from this studywas maximised.

2.3. Statistical analysis

The five samples collected from each plot on a sam-pling date were pooled to obtain an indication of over-all collembolan community structure for each plot oneach specific sampling date. The pooled data wereused in statistical analyses and the pitfall and suctiondata sets were investigated separately.

Prior to analyses, the variance to mean ratios werecalculated to determine if species abundances werenormally distributed. For the majority of species it wasfound that the variance and means were about equalindicating that the distribution was random (Sokal andRohlf, 1995). Square-root transformation was, there-fore, more appropriate and effective at normalisingthe data than the more common log transformation. Itwas deemed inappropriate to use different transforma-tions for different species, and consequently the abun-dance of all species was square-root transformed. Itwas, however, recognised that even after transforma-tion the variances for some species were still hetero-geneous and, consequently, the significance value was

Table 3Results of ANOVAs and Tukey tests performed on Collembola species obtained by suction sampling at SAC Auchincruivea

Species Date Treatment

F-value Probability Location of difference F-value Probability Location of difference

Total 20.04 <0.001 May and Jun>Apr, May>Aug 5.17 <0.01 U>CIsotoma viridis 20.91 <0.001 Aug>All 16.75 <0.001 All>CdIsotoma notabilis 7.29 <0.001 May>Jun 2.12 N.S. –Isotoma anglicana 129.75 <0.001 May>Apr and Aug>Jun 24.14 <0.001 All>CIsotomurus palustris 86.70 <0.001 May>Jun>Aug and Apr 12.51 <0.001 Zn and Cd>CIsotomurus maculatus 24.80 <0.001 May>Apr and Jun>Aug 4.76 <0.01 U>CIsotoma/Isotomurusjuvenile 56.36 <0.001 May>Jun and Apr>Aug 2.66 N.S. –Lepidocyrtus cyaneus 5.01 <0.01 Aug>Apr 15.77 <0.001 All>CdHeteromurus nitidis 8.12 <0.001 Aug>All 8.12 <0.001 U>CTomocerus longicornis 13.34 <0.001 Aug>Apr and May, Jun>Apr 1.34 N.S. –Sminthurus viridis 4.60 <0.01 Jun>Apr 23.15 <0.001 C>All Cd>U and ZnSminthurinus aureus 27.22 <0.001 May and Jun>Apr>Aug 2.48 N.S. –Sminthurides malmgreni 13.91 <0.001 Apr>All 1.89 N.S. –Ceratophysella denticulata 31.76 <0.001 Apr and May>Jun and Aug 6.08 <0.01 Zn>C

a As no block effect was found for any of the species tested, this information is omitted from the table. The following codes are usedfor treatments: C, control; U, uncontaminated sludge; Zn, zinc-rich sludge; and Cd, cadmium-rich sludge.

set at 0.01 to avoid wrongly rejecting the null hypoth-esis (Day and Quinn, 1989).

Effects of date, treatment and block were testedfor using three-way analyses of variance (withoutreplication). Examination of the data prior to analysisindicated that interaction did not occur and it was,therefore, possible to perform the tests without repli-cation hence enabling block effects to be investigated.For each dataset, separate ANOVAs were performedon the abundance of total Collembola and on fre-quently occurring species (i.e. those occurring in over50% of samples). Tukey multiple comparison testswere then applied to locate significant differencesidentified by ANOVA.

3. Results

Suction sampling collected a total of 122,323 indi-viduals from 22 species and pitfall trapping collected44,745 individuals from 20 species. Of these species,13 occurred frequently enough to allow statisticalanalysis at the species level (Tables 3 and 4). Ju-venile Isotoma and Isotomurusspecies cannot beidentified accurately to species level and were there-fore grouped together and treated as one of the 13species in the analyses. It is interesting to note thatthe following species occurred at too low a frequency

182 L.J. Cole et al. / Agriculture, Ecosystems and Environment 83 (2001) 177–189

Table 4Results of ANOVAs and Tukey tests performed on Collembola species obtained from pitfall trapping at SAC Auchincruivea

Species Date Treatment

F-value Probability Location of difference F-value Probability Location of difference

Total 7.58 <0.001 All>Apr 6.14 <0.01 U>CdIsotoma viridis 12.14 <0.001 Aug>All 8.97 <0.001 U>CdIsotoma notabilis 7.85 <0.001 Apr>All 4.51 <0.01 U>CIsotoma anglicana 62.93 <0.001 May>Jun and Aug>Apr 3.47 N.S. –Isotomurus palustris 25.28 <0.001 All>Apr 6.32 <0.01 All>CIsotomurus maculatus 17.87 <0.001 May and Apr>Aug, May>Jun 8.76 <0.001 U>C and CdIsotoma/Isotomurusjuvenile 31.12 <0.001 All>Aug, May>Apr 12.61 <0.001 All>CLepidocyrtus cyaneus 5.83 <0.01 Aug>Apr and May 10.60 <0.001 C and U>CdHeteromurus nitidis 7.65 <0.001 Aug>Apr and May 4.21 0.01 U>CSminthurus viridis. 0.39 N.S. – 3.99 N.S. –Sminthurinus aureus 23.72 <0.001 May and Jun>Apr, Jun>Aug 1.39 N.S. –Sminthurinus elegans 0.97 N.S. – 1.40 N.S. –Sminthurides pumilis 17.95 <0.001 August>All 0.78 N.S. –Ceratophysella denticulata 1.21 N.S. – 4.56 <0.01 Zn>Cd

a As no block effect was found for any of the species tested, this information is omitted from the table. The following codes are usedfor treatments: C, control; U, uncontaminated sludge; Zn, zinc-rich sludge; and Cd, cadmium-rich sludge.

to merit separate statistical analysis:F. candida, F.fimetarioides(Axelson),I. tigrina (Nicoleti), Entomo-brya nivalis(Linné), Lepidocyrtus lignorum(Fabrici-cus),Pseudosinella decipiensDenis,Tomocerus minor(Lubbock), Dicyrtoma saundersi(Lubbock), Dicyr-toma ornata(Nicolet) andAnurida pygmea(Börner).

Results of the ANOVAs for the suction data arepresented in Table 3 and for the pitfall data inTable 4. While no block effect was found for any ofthe species investigated, effects of treatment and datewere apparent.

3.1. Seasonal variation indicated by suction sampling

The influence of season on collembolan abun-dance was species-specific. The abundance of totalCollembola, Isotomurus/Isotoma juveniles, Isoto-murus palustris (Müller) and Isotomurus maculatusSchaeffer increased from April to May, reaching

Table 5Mean grass height (in cm) in experimental plots for each treatment and sampling date showing standard deviations in brackets

Control Uncontaminated Zinc-rich Cadmium-rich

April 13.0 (±2.02) 31.5 (±3.76) 30.7 (±3.32) 27.1 (±3.54)May 10.9 (±2.74) 10.1 (±2.91) 10.1 (±2.05) 9.2 (±2.19)June 16.7 (±3.18) 43.0 (±8.06) 43.7 (±4.10) 39.2 (±5.14)August 9.1 (±1.61) 7.4 (±1.77) 7.7 (±2.67) 8.5 (±2.87)

their peak in May suction samples, before decreasingagain in August (Table 3).Tomocerus longicornis,Lepidocyrtus cyaneus(Tullberg), Heteromurus ni-tidis (Templeton) andI. viridis (Bourlet) increasedas the summer progressed, occurring in their high-est abundance in August suction samples (Table 3).Ceratophysella denticulata(Bagnall) was more abun-dant in April and May than in June or August andSminthurides malmgreni(Tullberg) occurred almostexclusively in April samples (Table 3).I. anglicanaoccurred in its lowest abundance in June when thegrass was longest, whileSminthurus viridis(Linné)occurred in its highest (Tables 3 and 5).

3.2. Seasonal variation indicated by pitfall trapping

Isotoma viridis, Sminthurides pumilis(Krausbauer),H. nitidis and L. cyaneusoccurred in their highestabundance in August pitfalls (Table 4). With the

L.J. Cole et al. / Agriculture, Ecosystems and Environment 83 (2001) 177–189 183

Fig. 1. Effect of sludge treatment on the abundance of total Collembola,Heteromurus nitidisand Isotomurus maculatuscaught by suctionsampling (showing mean number square-root transformed). Error bars show standard deviations.

exception ofS. pumilis, which did not occur in suctionsamples in sufficient numbers for statistical analysisto be applied, these species were also more abundantin August suction samples. In agreement with thesuction sample results,Isotoma/Isotomurusjuvenilespeaked in May pitfalls (Table 4).Isotoma notabiliswas significantly more abundant in April pitfalls thanon any other sampling date (Table 4). As the higherabundance in April pitfalls was not replicated in suc-tion samples, it is possible that it was a consequenceof an increase in activity due to the digging in effect(Joosse and Kapteijn, 1968).

3.3. Treatment effects indicated by suction sampling

The addition of uncontaminated sludge significantlyincreased the abundance ofH. nitidis, I. maculatusand total Collembola above that of the control plots(Table 3 and Fig. 1). This increase was not significantin plots receiving contaminated sludge (i.e. zinc andcadmium-rich sludge), hence, indicating adverse ef-fects of the metal-rich sludges. Cadmium-rich sludgewas clearly shown to adversely affectL. cyaneusandI. viridis and both species occurred in their lowestabundance in plots receiving this sludge (Table 3 andFig. 2). Isotomurus palustriswas significantly moreabundant in plots receiving contaminated sludge (but

not uncontaminated sludge) than control plots (Table 3and Fig. 3), whileC. denticulatawas significantlymore abundant in plots receiving zinc-rich sludge thancontrol plots (Table 3 and Fig. 4).Isotoma anglicanawas favoured in all plots receiving sludge irrespectiveof metal contamination (Table 3 and Fig. 3).

Sminthurus viridiswas the only species to occur inits highest abundance in control plots, and this specieswas also significantly more abundant in plots receiv-ing cadmium-rich sludge than those receiving uncon-taminated or zinc-rich sludge (Table 3 and Fig. 4).

3.4. Effects of treatment indicated by pitfall trapping

Pitfall trapping also indicated that cadmium-richsludge adversely affectedI. viridis andL. cyaneusanda significantly lower number of both species occurredin pitfalls set in plots receiving cadmium-rich sludgethan those in plots receiving uncontaminated sludge(Table 4 and Fig. 5). Furthermore, the abundance ofL.cyaneusin plots receiving cadmium-rich sludge wassignificantly lower than that of control plots.

Pitfall trapping indicated that numbers ofI. nota-bilis, I. maculatusand H. nitidis were significantlyhigher in plots receiving uncontaminated sludge thancontrol plots, again indicating adverse effects of con-taminated sludge (Table 4 and Fig. 6). Furthermore,

184 L.J. Cole et al. / Agriculture, Ecosystems and Environment 83 (2001) 177–189

Fig. 2. Effect of sludge treatment on the abundance ofIsotoma viridisand Lepidocyrtus cyaneuscaught by suction sampling (showingmean number square-root transformed). Error bars show standard deviations.

the total collembolan abundance and the abundanceof I. maculatuswas significantly lower in pitfalls es-tablished in plots receiving cadmium-rich sludge thanin those receiving uncontaminated sludge (Table 4,Figs. 5 and 6). This difference was not significant inthe suction sampling results (Table 3).

Isotoma/Isotomurusjuveniles andI. palustris hada significantly higher abundance in pitfalls placed inplots receiving sludge than those placed in controlplots (Table 4 and Fig. 7). In agreement with thesuction results,C. denticulataoccurred in its high-

Fig. 3. Effect of sludge treatment on the abundance ofIsotoma anglicanaand Isotomurus palustriscaught by suction sampling (showingmean number square-root transformed). Error bars show standard deviations.

est abundance in pitfalls established in plots receivingzinc-rich sludge (Table 4 and Fig. 7).

4. Discussion

4.1. Seasonal variation in epigeal Collembola

In accordance with findings by other authors, theinfluence of season was species-specific (Joosse,1969; Brussaard et al., 1990). Some species reached

L.J. Cole et al. / Agriculture, Ecosystems and Environment 83 (2001) 177–189 185

Fig. 4. Effect of sludge treatment on the abundance ofSminthurus viridisand Ceratophysella denticulatacaught by suction sampling(showing mean number square-root transformed). Error bars show standard deviations.

their maximum population density in April (e.g. S.malmgreni), others in May (e.g.I. anglicanaandIso-toma/Isotomurusjuveniles) and others in August (e.g.H. nitidis, I. viridis and L. cyaneus). Joosse (1969)suggested that seasonal differences in species abun-dance could be related to humidity. This hypothesiswas not supported for all species, and while some hy-drophilic species (e.g.S. malmgreni) peaked in Aprilas predicted, others (i.e.I. viridis and T. longicor-

Fig. 5. Effect of sludge treatment on the abundance of total Collembola,Isotoma viridisandLepidocyrtus cyaneuscaught by pitfall trapping(showing mean number square-root transformed). Error bars show standard deviations.

nis) peaked in August (Joosse, 1970; Fjellberg, 1980).It would therefore appear that species-specific differ-ences in seasonal abundance could not simply be ex-plained by their humidity preference.

4.2. Effect of treatment on epigeal Collembola

It is well documented that the addition of sewagesludge can promote Collembola, and this is thought

186 L.J. Cole et al. / Agriculture, Ecosystems and Environment 83 (2001) 177–189

Fig. 6. Effect of sludge treatment on the abundance ofHeteromurus nitidis, Isotoma notabilisand Isotomurus maculatuscaught by pitfalltrapping (showing mean number square-root transformed). Error bars show standard deviations.

to be primarily the result of an increase in food sup-ply (Lübben, 1989; Pimentel and Warneke, 1989). Inthis study, a higher abundance of total Collembola,H.nitidis and I. notabilis was found in plots receivinguncontaminated sludge (but not those receiving con-taminated sludge) when compared to control plots. It

Fig. 7. Effect of sludge treatment on the abundance ofIsotomurus palustris, Isotoma/Isotomurus juveniles and Ceratophysella denticulatacaught by pitfall trapping (showing mean number square-root transformed). Error bars show standard deviations.

would therefore appear that while these species ben-efited from the increased nutrients in uncontaminatedsludge plots, this was not the case when the sludgewas contaminated by heavy metals. Furthermore, thespeciesL. cyaneusandI. viridis were both less abun-dant in plots receiving cadmium-rich sludge than in

L.J. Cole et al. / Agriculture, Ecosystems and Environment 83 (2001) 177–189 187

plots receiving uncontaminated sludge, thus indicatingthat these species were particularly sensitive to thecadmium-rich sludge.

It is important to note that the efficiency of severalsampling methods has been found to decrease in longgrass (Greenslade, 1964; Harwood, 1994). In studieswhere treatment influences grass height, the possibilitythat treatment effects may be attributed to changes insampling efficiency must be considered. In this study,the lower abundance ofL. cyaneusand I. viridis inplots receiving cadmium-rich sludge could not simplybe attributed to a reduced sampling efficiency in longergrass, as grass height was actually lower in plots re-ceiving cadmium-rich sludge (Table 5). It is thereforelikely that the observed decrease in these species wasa true effect of treatment. Furthermore, the observedchanges in abundance found by suction sampling werelargely mirrored in the pitfall data, further suggestingthat grass height did not unduly influence sampling ef-ficiency. As additional differences between the sludgesexisted, we cannot however conclude that it was thehigh levels of cadmium in the cadmium-rich sludgethat adversely influenced these species (see Table 1).

Toxicity studies conducted in the laboratory providea useful indication as to what levels of metals couldpotentially effect animals in the field. Smit and vanGestel (1996) found an EC50 value of 185 mg kg−1

soil for the effect of zinc on the population size ofF. candida, while Crommenruijn et al. (1993) foundan EC50-reproduction value in F. candida of 227 mgCd kg−1 soil. From Table 2, it can be seen that whilethe soil concentration of zinc in the study site washigher than that shown to effectF. candidain the lab-oratory, the concentration of cadmium was not. Lab-oratory studies, however, only investigate the directeffects of a contaminant (i.e. effects on growth rate,reproduction and survival) and it is possible that theindirect effects of cadmium were more pronouncedthan those of zinc. Cadmium may indirectly affectCollembola through altering their habitat structureand food (Posthuma et al., 1993). It is possible thatthe lower grass height in the cadmium-rich plots re-sulted in a more variable, and hence less favourable,microclimate (Pimentel and Warneke, 1989). Further-more, species that are able to digest fungal cell walls,where heavy metals are frequently accumulated, areexposed to metal concentrations far in excess of thosefound in the soil (Siepel, 1994).

The fact that total Collembola andI. maculatusappeared to be adversely affected by the applicationof cadmium-rich sludge when sampled by pitfalltrapping, but not when sampled by suction sampling,may indicate sublethal effects of cadmium on mobil-ity. The detoxification of heavy metals is thought tobe energetically costly, and a decrease in glycogenreserves after metal exposure has been found for sev-eral invertebrate species (Richards and Ireland, 1978;Bodar et al., 1990; Reddy and Bhagyalakshmi, 1994).The presence of metals may, therefore, be expected toreduce activity by decreasing the energy available andisopods exposed to heavy metals have been shown tohave reduced activity levels (Hopkin, 1989; Sørensenet al., 1997). Computer-automated video trackingcould provide a quick and effective method of mon-itoring metal pollution in the laboratory (Sørensenet al., 1997) and the present results indicate thatI.maculatamight be a suitable test species.

Sminthurus viridiswas the only species to occur inits highest abundance in control plots. The abundanceof S. viridis was also higher in the plots receivingcadmium rich sludge than in the plots receiving un-contaminated or zinc-rich sludge. It is possible thatthis species was adversely affected by the highergrass yield in the zinc-rich and digested sludge plots,or was sampled less efficiently in long grass. How-ever, asS. viridisoccurred in its highest abundance inJune when the grass was longest, neither explanationis likely. It is therefore suggested thatS. viridis, be-ing phytophagous, did not benefit from the increasein micro-organisms which accompanies sludge ap-plication. The application of sludge can also favourpredatory carabid beetles and this may account forthe lower abundance ofS. viridisobserved in uncon-taminated sludge plots (Larsen et al., 1996). As thisspecies consumes living plant tissue, which generallyhas lower concentrations of heavy metals than fungi(Curry, 1994), it may actually have had a competitiveadvantage over mycophagous Collembola in plotsreceiving cadmium-rich sludge.

4.3. Sampling protocol

While suction samplers provide good quantitativedata on collembolan population density (Johnsonet al., 1957; Frampton, 1994), pitfall traps tend toover-represent the most active species and, hence,

188 L.J. Cole et al. / Agriculture, Ecosystems and Environment 83 (2001) 177–189

give poorer quantitative data (Greenslade, 1964). Pit-fall traps also tend to under-represent plant climbingspecies (e.g.S. viridis), which are well represented insuction samples, but appear to be more effective atcatching strictly surface-active species (Bruce, 1997).In this study, suction sampling and pitfall trappingboth yielded nine significant treatment effects. Thisindicates neither method was superior in assessing theecological impact of sewage sludge. Effects for somespecies were only detected by suction sampling (e.g.S. viridis and I. anglicana) while others were onlydetected by pitfall trapping (e.g.Isotoma/Isotomurusjuveniles andI. notabilis). In agreement with other au-thors, the most effective sampling regime for epigealCollembola would, therefore, appear to be a combina-tion of both pitfall traps and suction samples (Berbierset al., 1989; Frampton, 1994). Limiting resources maymake such a choice unfeasible and when selectingbetween methods, the efficiency of each in collect-ing specific groups of species should be considered.For example, if a treatment predominantly effectsplant-climbing species (e.g. some pesticides), suctionsampling would be the preferred method as it is moreefficient at sampling such species (Bruce, 1997).

5. Conclusion

The addition of uncontaminated sewage sludgeincreased the abundance of total Collembola,H. ni-tidis and I. notabilis in comparison to control plots.However, a similar increase was not observed inplots receiving cadmium and zinc-rich sludges, thusindicating adverse effects of contaminated sludgeon Collembola. Cadmium-rich sludge also reducedthe abundance ofL. cyaneusand I. viridis and bothspecies were found in their lowest abundance in plotsreceiving this sludge. The soil concentration of cad-mium in the experimental plots was below that foundto adversely affect Collembola in laboratory studies,hence suggesting that cadmium was indirectly effect-ing these species. Alternatively, other toxic substancesin the cadmium-rich sludge may have influencedthese species. It is possible that these adverse ef-fects at species level (and the associated alteration ofcommunity structure) could alter the decompositionrate, soil fertility, and the abundance of polyphagouspredators. Further research is therefore required to as-

certain whether any such wider environmental effectsoccur and ultimately to determine if current legisla-tion adequately protects the full range of invertebratesassociated with agricultural land.

Acknowledgements

SAC receives financial support from the ScottishExecutive Rural Affairs Department. This experimentis part of a larger project carried out in collaborationwith ADAS, IACR, MLURI and WRc and we thankall collaborators for their assistance, particularly DrBrian Chambers of ADAS. L.J. Cole was in receipt ofa MacLagen Ph.D. scholarship while undertaking thiswork. We are indebted to Dr Arne Fjellberg for his in-valuable help in Collembola identification. Thanks arealso due to Brian Laird and Mark Cole for their tech-nical assistance and to Dr Geoff Frampton for advice.

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