23
 ACTA BIOLOGICA SLOVENICA 
 LJUBLJANA 2006 Vol. 49, [t. 2: 23–32
Sprejeto (accepted): 2007-01-29
Quantitative analysis of the macroinvertebrate community in the river 
Temenica (SE Slovenia)
Kvantitativna analiza združbe makroinvertebratov v reki Temenici (JV Slovenia)
Mojca PUST, Mihael J. TOMAN
Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111,
SI-1000 Ljubljana, Slovenia; E-mail: mihael.toman@bf.uni-lj.si.
Abstract. Macroinvertebrate community of the river Temenica was investigated in order to 
asses the ecological quality of the stream. Different approaches were used in order to compare 
their usefulness. Surber sampler methodology was used and all together16 quantitative sam-
ples were taken at four stream reaches every three months from October 2003 until July 2004. 
Saprobic and diversity indices showed severe deterioration of water quality longitudinally and 
a strong negative infl uence of the town Trebnje on the ecological state of the river Temenica. 
Multivariate methods DCA and CCA confi rmed those results, but also showed greater differences 
in community structure at sampling site 1 compared to sites 2 and 3 which was not detected 
using the indices. 
Key words: macroinvertebrates, water quality assesment, saprobic index, diversity indices, 
DCA, CCA
Izvleček. Za oceno ekološke kakovosti reke Temenice smo raziskovali združbo makroin-
vertebratov. Uporabili smo različne pristope, da bi primerjali njihovo uporabnost. Uporabljena 
je bila metodologija Surberjevega vzorčevalnika, vzorčili smo na štirih vzorčnih mestih vsake tri 
mesece od oktobra 2004 do julija 2004, skupno smo pobrali 16 kvantitativnih vzorcev. Saprobni 
in diverzitetni indeksi so pokazali resno poslabšanje kakovosti vode po toku navzdol in močan 
negativen vpliv mesta Trebnje na ekološko stanje reke Temenice. Multivariatni metodi DCA in 
CCA sta potrdili te rezultate, pokazali pa sta tudi večje razlike v strukturi združbe na vzorčnem 
mestu 1 v primerjavi z mestoma 2 in 3, ki jih z uporabo indeksov nismo zaznali.
Ključne besede: makroinvertebrati, ocena kakovosti vode, saprobni indeks, diverzitetni 
indeksi, DCA, CCA
Introduction
Macroinvertebrates are extensively used in water quality assessment as they are considered good 
indicators of environmental pollution. There are various methods for assessing water quality which 
have different conceptual basis and may therefore provide different information about the aquatic 
environment. Many authors have compared different methods of summarising responses to pollution 
(CAO et al. 1996, ROSSARO & PIETRANGELO 1993). Water quality indices have been developed as a rou-
tine technique for water monitoring. Saprobic index is often used as a measure of organic pollution 
(URBANIČ 2004) and is also in use by the Slovenian Ministry of the Environment for biomonitoring 
of surface waters. Diversity indices are known to be inaccurate at headwater reaches and were also 
reported to be insensitive to slight and moderate pollution (CAO et al. 1996). However, they can still 
be used as a complementary method. 
24 Acta Biologica Slovenica, 49 (1), 2006
Multivariate methods are increasingly used in biological monitoring of water quality (CAO et 
al. 1996, ROSSARO & PIETRANGELO 1993). Their main advantage is that they can detect more subtile 
changes in community structure than the indices. They can also provide information on the responses 
of taxa to different environmental variables.
The aim of this study was to asses the water quality of the stream Temenica using different methods 
(saprobic index, different diversity indices and multivariate methods DCA and CCA) and to compare 
the effectiveness of those methods.
Description of study sites
Our study area was the fi rst part of the disappearing stream Temenica in the south-eastern part of 
Slovenia. Its length is 25 km and the catchment area comprises 91 km2. 
The upper part of the stream (approx. 5 km) lies in a forested area with little human impact whereas 
the lowland part with meandering watercourse runs through cultivated land and by the town Trebnje, 
which is the largest settlement in the area. Elevations range from 560 m at the source to 260 m at the 
sinking point. Temperatures fl uctuate between 2,2–3  oC (winter) and 14,2–18,2 oC (summer). Stream 
pH lies within the range 8,1–8,7 and conductivity between 504 and 604 μScm–1. Sampling site 1 
was established in the headwaters, site 2 at the beginning of lowland watercourse, and sites 3 and 4 
upstream and downstream from Trebnje. 
Materials and methods
Macroinvertebrates were sampled on four occasions between October 2003 and July 2004 with 
3 month-intervals. At each sampling site 6 random sampling units were taken with a Surber sampler 
(500 cm2 sapling area and 0,5 mm mesh size). Several physical and chemical characteristics (Table 
1) were measured at the same time.
Table 1: Morphometric and physical  characteristics of sampling sites on the river Temenica.
Tabela 1: Morfometrične in fi zikalne značilnosti vzorčnih mest v reki Temenici.
Stream
reach Location
Stream
order*
Distance from
source (km)
Altitude
(m)
Mean
width (m)
Mean
depth (cm)
Maximum
temperature (C)
 1 Pusti Javor 2  2 360 1,2  6 14,2
 2
Stranje pri
Velikem Gabru 3 10 300 3,1 31 14,1
 3 Štefan 3 18 280 7,3 23 16,9
 4 Gorenje Ponikve 3 23 270 5,9 22 18,2
* After Strahler (1952)
All samples were fi xed in 4 % formaldehyde in the fi eld. In the laboratory, organisms were sorted, 
enumerated, identifi ed under a stereomicroscope and stored in 70 % ethyl alcohol. Macroinvertebrates 
were determined at least to the family level (except groups Hydrachnida and Collembola) using the 
keys of BAUERNFEIND & HUMPESCH (2001), BRINKHURST (1971), ELLIOT (1977), GERKEN & STERNBERG 
(1999), GLOER (2002), KARAMAN & PINKSTER (1977), NESEMANN (1997), REYNOLDSON (1987), SCHME-
DTJE & KOHMANN (1992), TACHET (2000), TRONTELJ & SKET (2000), URBANIČ et al. (2003), WARINGER 
& GRAF (1997), ŽIŠKO (2000).
Saprobic index was calculated using saprobic and indicative taxa values after WEGL (1983), MOOG 
(1995) and URBANIČ (2004) (Trichoptera). Number of taxa per sampling unit (S) and diversity indices 
(Shannon-Wiener index H’ = – Σ (p
i * 
ln p
i
), Eveness index E = H’/ln S and Simpson’s index D = 1 – Σ 
25
p
i
2) were calculated using PC-ORD (MCCUNE & MEFFORD 1999). One way ANOVAs were performed 
(using statistical package SPSS) to determine whether the differences in diversity among sampling 
sites were signifi cant. Multivariate methods used in this study included detrended correspondence 
analysis (DCA) and canonical correspondence analysis (CCA). Both were performed using statistical 
package CANOCO. Data on taxa abundances were log transformed. CCA analysis was performed with 
reduced data matrix which included only suffi ciently common taxa (occurrence in at least 2 samples 
or at least 4 individuals present) and 5 statistically signifi cant variables (forward selection).
Results
A total of 76550 individuals, representing 120 taxa were collected (Table 2). Saprobic index (Fig. 
1) indicates strong organic pollution at sampling site 4 where Tubifi cidae and Chironomidae were the 
most abundant. There is an apparent decrease in the value in summertime when Chironomidae (which 
have a lower saprobic value) outnumbered Tubifi cidae. Values of the saprobic index at the other three 
sites were very similar in all seasons and indicate low level of pollution.
Box-plot presentations of taxa number (S) and values of Shannon-Wiener index (H’), Eveness 
index (E) and Simpson’s index (D) are shown in Figure 2. There is a signifi cant decrease in all the 
values at sampling site 4, which confi rms the results of the saprobic index. The number of taxa is 
also lower at site 1, but diversity indices at this site are equal or even higher (E) than at sites 2 and 
3. Using one-way ANOVA we confi rmed satistically signifi cant differences in values S, H’ and E 
among sampling sites.
DCA ordination of 16 samples is shown in Figure 3. The fi rst axis explains 40,5 % of the total 
variance and axis 2 further explains 12,5 %. Samples from each sampling site are clearly separated by 
the fi rst axis which represents the upstream-downstream gradient and presumably also the pollution 
gradient. There is a considerable gap between the samples from site 1 and the other samples which 
indicates signifi cant differences in community structure between the headwaters and the rest of the 
stream. Axis 2 is apparently related to the seasonal changes in community structure.
M. Pust, M. Toman: Quantitative analysis of the macroinvertebrate community in the river ...
Figure 1: Values of saprobic index at different seasons at four sampling sites of theTemenica river.
Slika 1: Vrednosti saprobnega indeksa v različnih letnih časih na štirih vzorčnih mestih v reki Temenici.
26 Acta Biologica Slovenica, 49 (1), 2006
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46
M. Pust, M. Toman: Quantitative analysis of the macroinvertebrate community in the river ...
28 Acta Biologica Slovenica, 49 (1), 2006
With 5 selected environmental variables (Fig. 4a) used in the CCA, we explained 64 % of the 
variance of the taxa-environment relation. Eigenvalues of axes 1 and 2 are 0,38 and 0,28 and display 
42 % of the variance. Axis 1 shows high correlation with stream order (R2 = –0,95), and stream width 
(R2 = –0,74). Taxa which appear on the extreme right side of the biplot (Fig. 4b) therefore prefer 
headwaters of the stream. Axis 2 is mostly correlated with saprobic index (R2 = 0,84) and may thus 
present the gradient of organic pollution. Taxa showing affi nity (in the upper part of the biplot) to this 
factor were mostly found at the sampling site 4.
Figure 2: Box-plots of taxa number/sampling unit (S), Shannon-Wiener index (H’), Eveness index (E) and Sim-
pson’s index (D) in different seasons at four sampling sites of the Temenica river. 
Slika 2: Diagrami kvartilov števila taksonov/vzorčno enoto (S), Shannon-Wienerjevega indeksa (H’), indeksa 
stalnosti (E) in  Simpsonovega indeksa (D) v različnih letnih časih na štirih vzorčnih mestih v reki 
Temenici.
29M. Pust, M. Toman: Quantitative analysis of the macroinvertebrate community in the river ...
Discussion
Macroinvertebrate fauna in the fi rst part of Temenica is relatively diverse with signifi cant differen-
ces in community structure among the sampling sites.  The fourth site, which lies downstream from 
Trebnje (population approx. 3000), is especially dissimilar from the rest due to its high saprobic index, 
low diversity values of indices and the number of taxa. This indicates that the town of Trebnje is a 
major pollutant of the stream. On the other hand, neither the saprobic index nor the diversity indices 
showed signifi cant differences among the other three sampling sites. 
The values of saprobic index at the fourth sampling site are markedly varying between seasons 
as a result of changes in abundance of two most abundant groups – Tubifi cidae (Oligochaeta) and 
Chironomidae (Diptera). There were also signifi cant seasonal differences in diversity. This indicates 
the importance of monitoring over a longer period of time. 
The fi rst DCA axis can be interpreted both as the upstream-downstream gradient as in the study 
of ROSSARO & PIETRANGELO (1993) and the pollution gradient, as suggested by Cao et al. (1996). In 
comparison with the results of saprobic and diversity indices the samples of the sampling site 1 are 
here clearly separated from the other samples while the differences in community structure between 
site 4 and the other sites in much less pronounced than in the case of the indices. The reason for this 
distribution lies in the small number of individuals and different taxonomic structure at site 1, where 
the morphometric parameters (depth, width etc.) are very different from those at the other sampling 
sites. The second DCA axis represents seasonal differences among the samples which are very distinct 
at all four sampling sites.
Group of taxa, found at sampling site 1 (correlated with stream order), is pointing out in the 
CCA biplot, as well as the taxa, characteristic for site 4 (correlation with saprobic index). This is in 
agreement with both the indices and the DCA results.  Although indices did not show the difference 
in community structure between the fi rst and the two next sampling sites, they appear to be better at 
detecting severe pollution (site 4) than multivariate methods. Complementary use of different methods 
is therefore the most reasonable approach in water quality assessment.
Figure 3: DCA ordination of samples.
Slika 3: DCA ordinacija vzorcev.
30 Acta Biologica Slovenica, 49 (1), 2006
Figure 4: F1 X F2 plane of Canonical correspondence analysis (CCA) between 84 taxa and 5 selected environ-
mental variables. See Table 2 for taxa abbreviations.
Slika 4: F1 x F2 ravnina CCA ordinacijskega diagrama med 84 taksoni in 5 izbranimi okoljkimi spremenljivkami. 
Glej Tabelo 2 za okrajšave taksonov.
31
Conclusions
1. High values of saprobic index and low values of diversity indices at sampling site 4 indicate 
that the town Trebnje as the major pollutant of the Temenica river. Differences among other 
three sampling sites are not detectable with the use of indices.
2. Axis 1 of DCA biplot, representing the upstream-downstream gradient, revealed great diffe-
rences in macroinvertebrate community structure between the fi rst and the second sampling 
site, whereas the distinction of the fourth sampling site is much less pronounced. Seasonal 
changes in community structure are distinct at all sampling sites.
3. Stream order and saprobic index were the most prominent factors affecting the longitudinal 
distribution of taxa. Taxa signifi cant for sites 1 and 4 were highly correlated with these two 
factors.
Povzetek
Makroinvertebrati se kot dobri pokazatelji obremenjevanja okolja veliko uporabljajo pri ocenje-
vanju kakovosti voda. Metode, ki se pri tem uporabljajo pa temeljijo na različnih principih in lahko 
dajejo različne informacije o vodnem okolju. Zaradi številnih pomanjkljivosti indeksov, ki se običajno 
uporabljajo za rutinski biomonitoring celinskih voda,  se v vrednotenju kakovosti čedalje pogosteje 
uporabljajo multivariatne metode. S temi lahko zaznamo manjše spremembe v združbi kot z indeksi, 
dajo pa nam lahko tudi informacije o odzivu taksonov na posamezne okoljske spremenljivke. 
V naši raziskavi smo z analizo združbe makroinvertebratov ocenjevali ekološko stanje prvega dela 
ponikalne reke Temenice, pri čemer smo uporabili različne metode, da bi preverili njihovo uporabnost 
pri ocenjevanju kakovosti voda. Kvantitativno vzorčenje s Surberjevim vzorčevalnikom smo izvajali 
vsake tri mesece od oktobra 2003 do julija 2004 na štirih vzorčnih mestih. Vrednosti saprobnega 
indeksa, diverzitetnih indeksov ter število taksonov na vzorčno enoto kažejo na močno poslabšanje 
kakovosti reke Temenice na zadnjem vzorčnem mestu v primerjavi s prvimi tremi in s tem na močan 
negativen vpliv mesta Trebnje na njeno ekološko stanje. Razvrstitev vzorcev po prvi DCA osi potrjuje 
našo domnevo o naraščanju obremenjevanja reke po toku navzdol,  pri čemer pa četrto vzorčno mesto 
v primerjavi z rezultati indeksov ni tako izstopajoče. Večje razlike v združbi makroinvertebratov so 
po tej analizi med prvim vzorčnim mestom in ostalimi tremi. Na vseh vzorčnih mestih so izrazite tudi 
sezonske razlike v strukturi združbe. Najpomembnejši spremenljivki za razporeditev taksonov sta po 
rezultatih CCA red vodotoka in vrednost saprobneega indeksa, kar izpostavi taksone, ki so bili najbolj 
zastopani na prvem in zadnjem vzorčnem mestu.
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