178 L.J. Cole et al. Agriculture, Ecosystems and Environment 83 2001 177–189
and soil. However, despite the ample information on how PTEs in sludge affect micro-organisms Brookes
and McGrath, 1984; Smith, 1996, there is com- paratively little information on how they influence
invertebrates. Risk assessment studies tend to focus on economically important pests e.g. Hemiptera:
Aphidae, polyphagous predator groups e.g. Aranae and Coleoptera and macro-decomposers e.g. earth-
worms. For example, earthworms and spiders col- lected from plots treated with contaminated sludge
were shown to have elevated metal concentrations indicating that the metals were in bio-available forms
Benninger-Truax and Taylor, 1993. Green peach aphids Appelia schwartzi Börner feeding on plants
grown on contaminated sludge were found to have decreased fecundity and longevity than those feeding
on plants grown on uncontaminated sludge Pimentel and Warneke, 1989. The metals in sludge are, there-
fore, biologically available and have the potential to reduce fitness.
When a pollutant is added to agricultural land, changes in the structure of the microbial community
tend to precede changes in the composition of the plant or invertebrate community. Groups that feed di-
rectly on soil fungi and bacteria e.g. Collembola and Acarina may, therefore, be more susceptible to metal
pollution than predatory or phytophagous groups which are further removed from the effects of the
pollutant. Previous field studies have indicated that although the addition of metal contaminated sludge
did not adversely affect the abundance of Collem- bola, it influenced the community structure Lübben,
1989; Bruce et al., 1999. Lübben 1989 found that Sminthurinus aureus Lubbock, Willemia intermedia
Mills and Isotoma notabilis Schäffer were sensitive to sewage sludge artificially contaminated with metal
salts zinc, cadmium, copper, nickel, chromium and lead, while Folsomia candida Willem and Mesapho-
rura spp. were not. Collembolan communities would therefore appear to be sensitive to metal-contaminated
sludge with species-specific differences in metal sen- sitivity occurring.
Collembola are of agronomic importance not only as a consequence of their regulatory role in decompo-
sition Seastedt, 1984, but also because they are an important 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 field
trials.
In this study, the risks of applying metal-rich sewage sludge to agricultural land were assessed in the field
using epigeal Collembola as indicators. A sludge rich in zinc, which is an essential micro-nutrient, and a
sludge rich in cadmium, which is an ecological impu- rity, were investigated on a small-plot field trial. The
metal-rich sludges were derived from sewage treat- ment works with naturally high inputs of each specific
metal. It was considered essential to use naturally con- taminated sludge since this provided a more appro-
priate measure of exposure and, hence, risk under field conditions. The use of sludges artificially con-
taminated with metal salts was discounted as metals are more available in such sludges and they therefore
do not represent a natural situation Smith, 1996. Al- though the use of naturally contaminated sludge meant
that the sludges differed in other respects see Table 1, these were taken into account in the interpretation of
the results.
2. Materials and methods
2.1. Study site The trial site was situated on a well-drained sandy
clay 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 part of a larger scale experiment instigated in 1994. The
larger experiment consisted of 23 sludge treatments established across three blocks in a randomised block
design i.e. a total of 69 plots each 6 m×8 m. Epigeal Collembola were monitored on four of these sludge
treatments i.e. a total of 12 plots: no sludge control, uncontaminated sludge, cadmium-rich sludge and
zinc-rich sludge Table 2. The metal-rich sludges were derived from sewage treatment works with nat-
urally high inputs of the specific metal.
Since 1994, sludge had been applied annually to these plots each August in the form of a dewa-
tered sludge cake. Following each annual application,
L.J. Cole et al. Agriculture, Ecosystems and Environment 83 2001 177–189 179
180 L.J. Cole et al. Agriculture, Ecosystems and Environment 83 2001 177–189
Table 2 The 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
sludge 0.33 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
sludge 3 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 concentrations set by CEC Directive 86278EEC Commission of the European Communities, 1986.
sludge was incorporated into the soil using small plot equipment and then the plots were sown with Ital-
ian ryegrass Lolium multiflorum Lam. which was cut twice each year. A 1.2-m permanent grass strip
surrounded each plot to prevent soil transfer between plots during cultivation. The input of organic matter
to the soil was standardised across sludge treatments, and sludge application did not alter soil pH. During
1996, the grass was cut on 24 May and 26 July i.e. prior to the May and August Collembola sampling
dates: see below.
2.2. Sampling techniques Sampling was conducted between April and August
1996. A combination of pitfall trapping and suction sampling was utilised in order to provide an effec-
tive sampling regime for epigeal Collembola Berbiers et 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 can become heavy and uncomfortable with prolonged
use. Modified Ryobi RSV3100E sweeper-vac, have been found to be as, or more, efficient at sampling
Carabidae, Araneae, Staphlinidae, Isopoda and Aphi- didae Harwood, 1994; MacLeod et al., 1994. This
technique was, therefore, adopted in the current study as a light-weight alternative to sample Collembola.
Following the process developed by MacLeod et al. 1994, fine weave 0.5 mm nyloncotton mix voile
bags were placed in the sampler nozzle and renewed after each sample. Suction samples were taken on
30 April, 31 May, 22 June and 4 August. For each suction sample, an area of 706 cm
2
was partitioned off and extracted for 60 s. On all sampling dates, five
such samples were taken randomly from each of the 12 plots under investigation. To standardise across
dates, suction samples were taken on sunny days between 10:00–17:00 h when the grass was dry.
Pitfall traps consisted of plastic beakers 150 ml volume, diameter 6 cm containing 20 ml of the preser-
vative ethylene glycol. Five pitfalls were inserted in each 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 were inserted into the same holes as the April pitfalls.
Both the suction samples and pitfall traps were pro- cessed by first separating the organic and inorganic
matter by repeated flotation in a saturated salt solution. The organic matter including Collembola was then
passed through two graded sieves 15 mm and 45 mm to separate the Collembola from the larger arthropods
and plant debris.
All Collembola were initially mounted in Hoyer’s medium and identified under a light microscope mag-
nification 400–1000×. After this initial identification, it was found that with the exception of problematic
specimens, most species could be identified as ac- curately under a dissecting microscope. Collembola
were 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 the basis of very small differences in morphology Hop-
kin, 1997, the information derived from this study was 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 on
each specific sampling date. The pooled data were used in statistical analyses and the pitfall and suction
data sets were investigated separately.
Prior to analyses, the variance to mean ratios were calculated to determine if species abundances were
normally distributed. For the majority of species it was found that the variance and means were about equal
indicating that the distribution was random Sokal and Rohlf, 1995. Square-root transformation was, there-
fore, more appropriate and effective at normalising the data than the more common log transformation. It
was deemed inappropriate to use different transforma- tions for different species, and consequently the abun-
dance of all species was square-root transformed. It was, however, recognised that even after transforma-
tion the variances for some species were still hetero- geneous and, consequently, the significance value was
Table 3 Results of ANOVAs and Tukey tests performed on Collembola species obtained by suction sampling at SAC Auchincruive
