developed Sinsabaugh et al., 1992, 1994b, 2000. These models may help circumvent some of the methodological
constraints that have hindered studies of in situ decomposi- tion rates, especially for fine particles.
The purpose of this study was to investigate POM proces- sing in two contrasting ponds. Enzymatic decomposition
models EDM were applied to obtain estimates of in situ processing rates. These models further our understanding of
the decomposition process at the enzyme level, and help document the extent to which the process varies among
systems. At the same time, the study provides some of the first results about decomposition processes at the microbial
community level in aquatic systems in Spain, under contrasting flooding regimes and weather conditions.
2. Materials and methods
2.1. Study sites The study was conducted from March 1998 to April 1999
at Pond del Charco del Toro and Pond del Rio Oro Toro and Oro ponds, located in the southwest coast of Spain. Both
are shallow ponds maximum depth 115 and 120 cm for Toro and Oro, respectively with similar morphometric
and size features, and both are located on the ‘eolian littoral mantle’ Montes et al., 1998. Oro pond is located in a
phytostabilised sand area, whereas Toro pond is located along the northeast margin of an active dune system. Both
have a temporary flooding regime with a tendency towards drying at the end of the warm season July, August. These
ponds are fed by a shallow water table and rainfall that occurs predominantly in the spring and autumn. Toro
pond is located in Don˜ana National Park, while Oro pond is located in Don˜ana Natural Park. Don˜ana ponds are impor-
tant aquatic environments because local differences in groundwater, soils, or vegetation can produce water of vari-
able quality Sacks et al., 1992; Serrano and Toja, 1995. Until now, such heterogeneity has been studied using struc-
tural measures such as zooplankton Galindo et al., 1994, or phytoplankton and aquatic vegetation Lo´pez et al., 1991
but not in terms of the functioning of the system i.e. measuring rates of different processes.
S. Alvarez, M.C. Guerrero Soil Biology Biochemistry 32 2000 1941–1951 1942
Fig. 1. Physico-chemical features of the studied ponds. A Toro. B Oro temperature B; water depth W and conductivity O.
Despite their similarities, Toro and Oro ponds have important differences. Toro pond is surrounded by a pine
Pinus pinea forest and has a littoral belt of Juncus mari- timus
, Scirpus holoschoenus, and S. lacustris. Oro pond is surrounded by a eucalyptus Eucalyptus globulus and E.
camaldulensis plantation, and some of the trees are within
the basin of the pond. These plantations were very abundant during 1960s, and still persist in important areas of the
region. Macrophyte vegetation, although composed of the same species, is more disturbed and much less dense in Oro
pond than in Toro pond. These differences result in impor- tant differences in the inputs and the quality of organic
matter that enters the system, and have an influence over the physico-chemical characteristics of the water.
The area of study is dominated by a Mediterranean climate with relatively mild winters mean temperatures
of 98C during December and January and very dry and hot summers with maximum temperatures .308C. Annual
rainfall is variable. The 1996–1997 hydrological cycle was extremely wet, and ponds reached their maximum depth
values, but the 1997–1998 and 1998–1999 hydrological cycles were dry, with almost no rains, and water levels
decreased continuously, except for some increases during 1999 spring season Fig. 1.
2.2. Experimental design Temperature, pH and conductivity 340-B WTW conduc-
tivity meter were measured in situ. Surface water levels were measured using 2-m long metal rods fixed to the
bottom of the ponds. Polyphenolic concentrations in water and sediments were measured using a modified procedure
for the Folin–Ciocalteu method Box, 1983. Particulate organic matter inputs to the ponds were measured using
litter-traps. Vertical traps consisted of a square structure 0.45 m
2
elevated 0.5 m above ground level with a woven plastic mesh. Lateral traps consisted of a ground level
0.16 m
2
steel frame with a mesh like those used for vertical traps with its opening parallel to the shore and anchored to
stakes. Five traps were placed at each sample site. Collected material was dried 758C, 48 h and weighed.
POM for litter-bag preparation was collected from the study sites in winter 1997. The material was wet-sieved
using 20-cm diameter sieves with mesh sizes of 1, 0.5 and 0.063 mm and dried at 758C for 48 h. Material .1 mm was
designated as coarse particulate organic matter CPOM and material between 0.063–0.5 mm as fine particulate organic
matter FPOM. Only these two fractions were considered, as medium particulate organic matter MPOM 0.5–1 cm
has shown erratic behaviour that prevented the development of enzymatic models in previous studies Sinsabaugh et al.,
1994a; Jackson et al., 1995. Also, the different size ranges of litter should be viewed as separate points along a decom-
position continuum rather than discrete classes.
The dried CPOM was placed into 1-mm mesh fibreglass screening litter-bags, and FPOM was placed into litter-bags
of 0.06-mm mesh. Each CPOM bag received 3 g dry mass DM equivalent to 2.5 g OM in Toro and 2.6 g OM in Oro
organic mass as determined by loss on ignition at 5508C and each FPOM bag received 10 g DM equivalent to 1.3 g
OM in Toro and 0.5 g OM in Oro. In each pond, in the area of maximum initial depth, 45 bags of each size class were
placed along three independent transects and weighed to the bottom. Three independent replicates of each size range
were collected after 7 days to assess rapid leaching of solu- ble material. Subsequent collections were made at one-
month intervals until the end of study except for November 1998. Collected litter-bags were sealed in plastic bags and
transported on ice to the laboratory.
On each sampling site, in situ POM samples were also collected from the same area where bags were placed. Sedi-
ment cores were collected using a 20 cm
2
corer to remove the top 5 cm of the substrate. On each sampling date four
cores were taken from each site. In situ POM samples were also sealed within bags and transported on ice back to the
laboratory.
2.3. Measurement of mass loss and enzyme activities Each collected bag was analysed for mass loss and
enzyme activities. The material in the bag was rinsed in slow running tap water and collected in a 0.063-mm mesh
sieve. Macro-invertebrates present were removed and the recovered POM was divided into three subsamples in
three pre-weighed aluminium pans. One subsample consisting of 70 of recovered material was dried at
758C for 48 h, re-weighed, ashed at 5508C for 3 h, and weighed again to determine moisture and organic matter
content. The other two subsamples were suspended in 100 ml of 50 mM pH 5.0 acetate buffer and 50 mM pH
8.5 tris buffer, respectively. These suspensions were homo- genised Euroturrax T20 dispersion unit.
Each core in situ POM samples was also wet sieved into the same two fractions as the litter-bag POM. Each fraction
was divided into three subsamples as for the confined samples.
Each POM suspension, both from in situ — sediment core — and confined litter-bag samples, was assayed for
the activity of five extracellular enzymes involved, respec- tively, in the degradation of cellulose and hemicellulose —
b -1,4-glucosidase EC 3.2.1.21 and b-xylosidase EC
3.2.2.37; chitin — b-N-acetylglucosaminidase NAGase, EC 3.2.1.30; polyphenolic substances — phenol oxidase
EC 1.10.3.2 and 1.14.18.1; and acquisition of N and P — b
-N-acetylglucosaminidase NAGase, EC 3.2.1.30 and alkaline phosphatase EC 3.1.3.1. The substrates for b-
glucosidase, b-xylosidase, b-NAGase and alkaline phos- phatase were pNP-b-d-glucopyranoside, pNP-b-xylopira-
noside, pNP-b-N-acetylglucosamidine and pNP-phosphate, respectively. The substrate for phenol oxidase was l-3,4-
dihydroxyphenylalanine l-DOPA. All substrates were obtained from Sigma Chemical Company St. Louis, USA.
S. Alvarez, M.C. Guerrero Soil Biology Biochemistry 32 2000 1941–1951 1943
Assays for b-glucosidase, b-xylosidase, b-NAGase and phenol oxidase enzymes were conducted at 208C in pH 5.0
acetate buffer, 50 mM, under previously determined substrate saturating conditions. For alkaline phosphatase,
assays were conducted in pH 8.5 tris buffer 50 mM under previously determined substrate saturating conditions. In all
cases there were three analytical replicates and duplicate controls. A more detailed description of the procedures
used can be found in Sinsabaugh et al. 1994a. Activities for all enzymes were analysed monthly, except for phenol
oxidase, which was analysed every two months.
2.4. Data analysis Data from the litter-bag samples were analysed to
develop enzyme models establishing relationships between cumulative mass loss and cumulative enzyme activities
Jackson et al., 1995. The first step in the analysis is to integrate the enzyme activities over time by multiplying
the mean activity over each interval by the length of the interval. The values obtained are expressed as activity-
day, where activities for all enzymes are expressed in m
mol h
21
g
21
OM. Cumulative activity was calculated as the activity-day summed over all previous sample intervals.
Simple linear regressions are performed relating initial mass loss as a function of cumulative activity-day for each
enzyme and each size class and pond.
Organic matter losses between treatments were compared using t-test
P , 0
: 05
: Enzyme activities among treatments
were compared using Kruskal–Wallis tests, and Mann– Whitney tests were applied for a posteriori pairwise compar-
isons, if appropriate. Relationships between different enzyme activities were determined by calculating Spear-
man’s correlation coefficients Zar, 1996.
3. Results
