PRIMARY RESEARCH PAPER

Spatial distribution patterns of the hyporheic invertebratecommunities in a polluted river in Romania

Oana Teodora Moldovan • Erika Levei • Constantin Marin • Manuela Banciu •

Horia Leonard Banciu • Claudia Pavelescu • Traian Brad • Mirela Cımpean •

Ioana Meleg • Sanda Iepure • Ioan Povara

Received: 1 June 2010 / Revised: 12 February 2011 / Accepted: 26 February 2011 / Published online: 13 March 2011

� Springer Science+Business Media B.V. 2011

Abstract The purpose of this study was to examine

the sensitivity, in a field situation, of the hyporheic fauna

to pollution by heavy metals and also to test the use of

oxidative stress enzymes produced by this fauna as a

sensitive indicator of oxidative stress generated by

chemical contamination. This was done by surveying

the patterns of distribution, structure, and composition

of hyporheic invertebrate communities in one of the

most polluted rivers in Romania. Twelve permanent

sampling stations with differing water qualities were

established along a 180 km transect of the Aries River.

Data on hyporheic invertebrate abundance and richness,

chemistry of the surface and hyporheic water and

interstitial suspended particles were analyzed via mul-

tifactorial analyses. In the downstream, more polluted

stations, epigean species were less abundant and

hyporheic communities, especially macrocrustaceans

and oligochetes, became dominant. The higher levels of

hyporheic invertebrate biodiversity in the moderately

polluted stations compared to highly polluted, and the

increase of the number of some hyporheos (especially

macrocrustaceans) in the moderately polluted stations,

suggested that the hyporheic fauna was more tolerant of

heavy metal pollution than the surface water fauna of the

area. However, the different richness and abundance of

hyporheic fauna in sites of similar water chemistry

suggested that additional factors, such as sediment

structure are shaping the spatial distribution of hypor-

heic fauna. Strong correlations between superoxide

dismutase (SOD) activity in pooled tissues extracts and

some chemical parameters suggest that oxidative stress

enzymes may prove to be sensitive indicators of

chemical pollution in hyporheic zones.

Keywords Interstitial fauna � Crustacea � Heavy

metals � Oxidative stress enzymes � Aries River

Introduction

The surface water/groundwater interface of river

alluvia, the interstitial or the hyporheic zone (Creuze

Handling editor: Stuart Anthony Halse

O. T. Moldovan (&) � T. Brad � I. Meleg � S. Iepure

‘‘Emil Racovitza’’ Institute of Speleology,

Romanian Academy, Clinicilor 5, 400006 Cluj-Napoca,

Romania

e-mail: oanamol35@yahoo.com

M. Banciu � H. L. Banciu � M. Cımpean

Faculty of Biology and Geology, ‘‘Babes-Bolyai’’

University, Cluj-Napoca, Romania

C. Pavelescu

Romanian Waters National Administration, Somes Tisa

Division, Cluj-Napoca, Romania

E. Levei

National Institute of Research and Development for

Optoelectronics, Research Institute for Analytical

Instrumentation, Bucharest, Romania

C. Marin � I. Povara

‘‘Emil Racovitza’’ Institute of Speleology, Romanian

Academy, Bucuresti, Romania

123

Hydrobiologia (2011) 669:63–82

DOI 10.1007/s10750-011-0651-2

des Chatelliers et al., 1994), is a spatially fluctuating

ecotone zone of high diversity and variability.

Horizontal and vertical gradients of oxygen, temper-

ature, light, and other environmental parameters

define this ecotone, which is complex to study and

the physical boundaries of which are difficult to

define (Boulton et al., 1998). The hyporheic zone acts

both as source and sink for nutrients, potentially

regulating biotic productivity (Moser et al., 2003),

and has an impact on the fate and transport of mining-

derived pollutants, influencing both surface water and

groundwater (Bourg & Bertin, 1993). The hyporheic

zone can be viewed as an ‘‘archive’’ of past episodes

of pollutants passing through its sediments, and it can

also function as a barrier preventing or retarding the

movement of pollutants between surface and ground-

water (Fuller & Harvey, 2000). Accordingly, the

study of the hyporheic zone benefits from a multi-

disciplinary approach that takes into account hydro-

logical, hydrogeological, chemical, microbiological,

and faunal variables.

It has been suggested that groundwater fauna are

sensitive indicators of aquifer contamination, including

contamination with heavy metals (e.g., Malard et al.,

1994, 1996; Notenboom et al., 1994; Boulton, 2000a).

Although the impact of contaminants on groundwater

invertebrates has been extensively investigated (review

in Notenboom et al., 1994; Mosslacher & Notenboom,

1999), the results are somewhat contradictory. Hypor-

heic animals seem to be less sensitive than their epigean

relatives, at least for groups such as typical hyporheic

isopods and amphipods (Plenet & Gibert, 1994; Plenet,

1995; Plenet et al., 1996; Plenet, 1999; Williams &

Fulthorpe, 2003). However, more recently, El Adnani

et al. (2007) found that hyporheic fauna may show both

qualitative and quantitative changes downstream from

metal sulfide tailings containing Pb, Zn and Cu (see also

Pyle & Mirza, 2007).

Perhaps a more useful indicators of groundwater

contamination are the oxidative stress enzymes

generated by aquatic invertebrates in response to

chemical contamination and/or abnormal physical

parameters of their surroundings (Cantu-Medellın

et al., 2009). High intracellular concentration of

reactive oxygen species (ROS), such as glutathione

S-transferase, glutathione peroxidase, glutathione

reductase, catalase, and superoxide dismutase

(SOD), may provide an estimate of the impact of

heavy metal pollution on the metabolic status of

aquatic macroinvertebrates such as bivalves (Geret

et al., 2002; Manduzio et al., 2004; Cravo et al.,

2009) or microinvertebrates such as cladocerans and

copepods (Fan et al., 2009; Wong et al., 2010).

In this study, we examined the sensitivity of

hyporheic Crustacea to pollution and compared the

utility of using hyporheic invertebrate composition

and oxidative stress enzymes as indicators of pollu-

tion. Our study site was one of the most polluted rives

in Romania, as a result of mining-related water

contamination. We assessed the impact of pollution

on the spatial distribution patterns of hyporheic

invertebrates. Pollution impact was investigated

using invertebrate abundance and species richness,

with emphasis on hyporheic Crustacea, by empha-

sizing the changes in biological hyporheic commu-

nities in each station in correlation to chemical

composition of both interstitial water and sediments.

The most polluted sites, presumably located in the

middle part of the studied transect, were expected to

have less species and fewer individuals than the less

polluted locations, although the extent of decline

should be dependent on the sensitivity of hyporheic

invertebrates to pollution, with some studies predict-

ing minimal impact (see above). Two hypotheses

were tested along the studied longitudinal transect.

The ecological and evolutionary implications of

our findings in the context of the exchanges between

surface and hyporheic communities are discussed,

emphasizing the importance of habitat heterogeneity

and biological interactions.

Materials and methods

Study area

The study area was in Transylvania, Romania, in the

so-called ‘Golden Quadrilateral’, where important

gold resources of Europe are concentrated. The

mining activities in the region have led to extensive

and high levels of pollution of the Aries River, one of

the main rivers in north-western Romania (Fig. 1).

While most of the pollution of the Aries River is from

leakages and unchecked discharges of the non-treated

acid waste-water, other pollution sources include

waste dumps and settlings from mines, sewage waters

from towns along the river and pesticides and

fertilisers from agricultural activities (Forray, 2001).

64 Hydrobiologia (2011) 669:63–82

123

Survey planning

Twelve permanent stations (Table 1) were estab-

lished along a 180 km longitudinal transect of the

eastward flowing river (Fig. 1). Three of the stations

(1–3) were selected upstream of Baia de Aries, the

locality known as the most polluted with heavy

metals along the Aries River. The locations of the

stations were chosen according to accessibility to

the river, state of conservation of alluvia (many of the

sandy beaches are exploited by industry) and by the

presence of fauna, as established in preliminary

samplings. The sampling stations were located in

the unconsolidated detritic deposits of the floodplain,

and interstitial waters of the flood plain are prone to

pollution from surface waters during flooding peri-

ods. The lithology and granulometry of alluvia

deposits is non-homogenous, being a mixture of fine

sands, oozy sands, gravels and boulders, without clear

vertical distribution and highly dynamic through

time.

Standpipes of galvanized iron 20 mm in diameter,

with perforations at 1.3–1.4 m depth, were perma-

nently fixed in the alluvia at distances of 1–5 m from

the river. A manual diaphragm pump was installed

and water was pumped according to the technique

described by Bou (1974). Through our method of

pumping, 0.3 m3 of water was extracted from sedi-

ments. In each sample, very fine sand, rich organic

matter and some sawdust were collected. From each

sampling station two replicate samples for fauna and

interstitial and surface water were taken monthly

from February 2008 to March 2009 (a total of 168

samples). Three samples (March, July and October

2008) were taken from each sampling station for the

chemical analysis of suspended particles.

Analysis of fauna and of physico-chemical

parameters

From each sample the first 20 l of water was filtered

through a planktonic net of 160 lm and fixed in 96�ethanol or 4% formaldehyde. Collected invertebrates

were sorted into major taxonomical groups with the

adults subsequently sorted to species level for most

taxa. The identification of individuals was performed

according to the specific methods for each group.

Invertebrates were also assigned to one of the

Fig. 1 Aries River basin with its localization in north-western Romania. Sampling stations and input of pollutants along the studied

sector are marked on the map

Hydrobiologia (2011) 669:63–82 65

123

following ecological classifications: (1) typical hy-

porheos restricted to groundwater and rarely occur-

ring near the surface; (2) epigeans that are usually

restricted to benthos; and (3) transitionals that can be

found in the above mentioned habitats, but rarely

occurring in deep groundwater.

Temperature (�C), pH, and electrical conductivity

(EC) (lS/cm) of surface and interstitial waters were

measured on site using Multiparameter Combo

Hanna Instruments HI 98129. Oxygen concentration

in water was not measured because of the errors

introduced by the pumping method.

For chemical analysis, surface and interstitial

waters were collected in 1-l acid-washed polyethyl-

ene bottles. During the transportation to the labora-

tory, the samples were kept at 4�C.

The concentrations of Al, Cr, Ni, Cu, Zn, As, Sr,

Cd, Pb, Na, Mg, K, Ca, Mn, Fe, Ti, Cl-, NO2-,

NO3-, SO4

2- were analyzed and the chemical

oxygen demand (COD) was measured. For total

heavy metal concentration, 100 ml unfiltered water

samples were digested on a sand bath with 5 ml

Ultrapur� 60% Nitric acid. After cooling, the solution

was filtered through 0.45-lm pore-diameter filters

and made up to 100 ml with ultrapure water.

Depending on their concentration, metals were

quantified using Inductively Coupled Plasma Optical

Emission Spectrometry (ICP-OES OPTIMA

5300DV, Perkin-Elmer, USA) or Inductively Cou-

pled Plasma Mass Spectrometry (ICP-MS ELAN

DRC II, Perkin-Elmer, USA).

Anions measurement was taken from samples

filtered on 0.45 lm pore-diameter cellulose nitrate

membrane filters by ion chromatography (761Com-

pact IC System, Metrohm, Switzerland).

A COD test was used as an indirect measure of the

amount of organic compounds in sampled water. The

method is based on the oxidation in strong acid

Table 1 General

information about the

sampling stations along the

Aries River

No. Station Distance from

headwaters

(km)

River

side

Distance

from

water (m)

UTM

coordinates

Relief

1 Scarisoara 1 21.5 Left 7.80 34 T 0643874 Base of the inferior

terrace5146460

2 Scarisoara 2 26.45 Left 2.50 34 T 0646710 High river meadow

5145965

3 Vadu Motilor 44.27 Left 10.50 34 T 0652692 Low river meadow

5139174

4 Baia de Aries 81.70 Left 3.50 34 T 0675486 High river meadow

5139031

5 Sartas 84.56 Left 1.20 34 T 0676858 Base of the inferior

terrace5140089

6 Brazesti 1 87.04 Left 2.50 34 T 0677960 Minor river bed

5140621

7 Brazesti 2 89.56 Right 4.50 34 T 0679404 Minor river bed

5141915

8 Salciua de Jos 101.42 Left 6.15 34 T 0687603 Minor river bed

5142005

9 Lunca Aries 109.54 Left 12.30 34 T 0688565 High river meadow

5145950

10 Vidolm 117.29 Right 13.15 34 T 0692634 Low river meadow

5150484

11 Buru 1 129.00 Left 5.00 34 T 0700074 Low river meadow

5153340

12 Buru 2 129.92 Left 5.55 34 T 0700886 Terrace

5153629

66 Hydrobiologia (2011) 669:63–82

123

Ta

ble

2C

hem

istr

yo

fin

ters

titi

alw

ater

s(I

),su

rfac

ew

ater

s(S

),an

dS

PM

(P)

inth

e1

2m

on

ito

red

stat

ion

s

Sta

tio

ns

I Al

(mg

/l)

Cr

(mg

/l)

Ni

(mg

/l)

Cu

(mg

/l)

Zn

(mg

/l)

Sr

(mg

/l)

Pb

(mg

/l)

Na

(mg

/l)

Mg

(mg

/l)

K (mg

/l)

Ca

(mg

/l)

Mn

(mg

/l)

Fe

(mg

/l)

Ti

(mg

/l)

Cl-

(mg

/l)

NO

2-

(mg

/l)

NO

3-

(mg

/l)

SO

42-

(mg

/l)

CO

D

(mg

/l)

10

.1\

0.0

1\

0.0

10

.10

.40

.3\

0.0

19

3.7

11

.81

2.2

92

.72

.00

.4\

0.0

12

10

.30

.12

.71

3.9

9.8

21

.04

.53

.23

2.4

39

.16

6.4

69

.44

5.2

6.0

4.7

31

.21

.82

.30

.21

04

.11

.54

.07

.75

.0

31

.33

.11

.81

4.6

17

.92

6.6

29

.65

6.1

7.1

5.4

42

.61

.92

.60

.21

32

.01

.44

.49

.49

.3

41

.02

.11

.51

7.5

21

.13

7.7

38

.25

7.2

6.6

4.6

50

.21

.72

.00

.11

47

.60

.83

.71

2.9

12

.5

51

.41

.91

.41

6.0

19

.43

4.7

34

.95

2.7

7.3

4.6

62

.61

.82

.80

.21

33

.80

.73

.28

0.9

23

.2

62

.80

.80

.67

.39

.41

6.0

15

.93

4.1

9.2

4.9

88

.52

.96

.00

.27

6.4

0.3

6.5

23

7.3

51

.4

72

.40

.90

.77

.99

.81

7.5

17

.53

0.7

8.0

4.1

68

.73

.25

.00

.27

2.8

0.3

5.1

15

2.7

37

.6

83

.00

.60

.55

.87

.31

2.8

12

.82

4.9

9.4

4.2

76

.03

.76

.20

.25

6.5

0.3

6.4

18

1.1

61

.0

93

.10

.40

.33

.74

.78

.38

.22

0.4

10

.13

.86

7.3

4.0

6.5

0.2

42

.10

.27

.71

32

.05

5.7

10

3.0

0.3

0.3

3.2

3.9

7.2

7.0

16

.11

0.6

3.2

66

.33

.76

.30

.23

3.5

0.3

6.8

20

3.0

56

.7

11

2.9

0.3

0.2

2.3

2.8

5.2

5.1

14

.01

0.7

3.0

69

.82

.45

.90

.22

7.2

0.3

7.0

21

6.1

57

.3

12

2.2

0.2

0.2

1.8

2.1

3.9

3.8

10

.98

.72

.86

3.0

1.8

4.4

0.1

23

.40

.48

.82

32

.23

7.1

Sta

tio

ns

S Al

(mg

/l)

Cr

(mg

/l)

Ni

(mg

/l)

Cu

(mg

/l)

Zn

(mg

/l)

Sr

(mg

/l)

Pb

(mg

/l)

Na

(mg

/l)

Mg

(mg

/l)

K (mg

/l)

Ca (mg/l)

Mn

(mg

/l)

Fe

(mg

/l)

Ti

(mg

/l)

Cl

(mg

/l)

NO

2

(mg

/l)

NO

3

(mg

/l)

SO

4

(mg

/l)

CO

D

10

.1\

0.0

1\

0.0

1\

0.0

10

.10

.1\

0.0

18

.23

.31

.83

2.2

0.3

0.3

0.1

6.5

0.2

2.6

6.0

8.6

20

.1\

0.0

1\

0.0

1\

0.0

10

.10

.0\

0.0

13

.62

.70

.72

7.0

\0

.01

0.2

\0

.01

5.6

0.4

2.3

5.7

4.0

30

.1\

0.0

1\

0.0

1\

0.0

10

.10

.0\

0.0

14

.94

.70

.92

8.8

\0

.01

0.3

\0

.01

5.1

0.2

1.9

6.1

4.3

41

.5\

0.0

1\

0.0

10

.20

.20

.1\

0.0

15

.06

.11

.93

8.8

0.4

0.9

0.1

5.0

0.2

2.0

47

.12

.9

51

.4\

0.0

1\

0.0

10

.10

.20

.1\

0.0

14

.96

.92

.14

3.3

1.0

0.7

0.1

5.9

0.2

2.2

45

.94

.2

61

.8\

0.0

1\

0.0

10

.20

.20

.1\

0.0

17

.96

.22

.24

4.1

0.8

1.2

8.0

5.3

0.6

2.1

43

.73

.2

70

.9\

0.0

1\

0.0

10

.50

.50

.1\

0.0

15

.56

.72

.06

2.0

0.3

0.9

0.1

4.9

0.2

2.4

41

.74

.1

81

.3\

0.0

1\

0.0

10

.10

.20

.1\

0.0

14

.75

.41

.93

8.5

0.4

0.9

0.1

6.1

0.4

2.5

46

.32

.3

90

.9\

0.0

1\

0.0

10

.10

.20

.1\

0.0

14

.55

.71

.84

2.1

0.4

0.7

0.1

6.0

0.4

2.4

63

.25

.1

10

0.9

\0

.01

\0

.01

0.1

0.2

0.1

\0

.01

4.8

6.0

2.0

45

.10

.41

.00

.15

.50

.32

.14

3.1

3.4

11

0.5

\0

.01

\0

.01

0.1

0.1

0.1

\0

.01

5.1

6.2

2.1

41

.40

.20

.70

.17

.60

.74

.34

3.5

6.1

12

0.4

\0

.01

\0

.01

0.1

0.2

0.1

\0

.01

4.7

6.0

1.9

42

.00

.30

.50

.15

.20

.42

.54

4.6

3.8

Hydrobiologia (2011) 669:63–82 67

123

solution with a known excess of potassium perman-

ganate (KMnO4), titration of the remaining unre-

duced KMnO4 with sodium oxalate solution and

calculation of the consumed KMnO4.

A direct method of measuring the concentrations

of Ba, Mn, Cr, Ni, Cu, Zn, Cd, Pb, Sb, and As

adsorbed on the surface of particles in suspension in

interstitial water was used. Separation of suspended

particulate matter (SPM) was realized in situ. When

collected, samples were filtered by means of a

Chromatography Research Supplies filtering system,

provided with a manual Nalgene vacuum pump. Pre-

weighed (Kern 770-60) MF-Millipore Membrane of

mixed cellulose esters of 0.45 lm porosity and

47 mm diameter were used for filtering. The mem-

branes were dried at room temperature and then

weighed to the nearest 0.01 mg to determine the mass

of SPM in the known volume of filtered water. The

filters were then digested at room temperature for

48 h with 5.0 ml of 1:1 solution Ultrapur� 60%

Nitric acid and Ultrapur� 30% Hydrochloric acid. A

2 ml aliquot of the digest solution (not including any

visible solid) was diluted to 5 ml with ultra-pure

water and analyzed by atomic absorption spectros-

copy (AAS). This process was repeated for duplicate

unused filters and the average elemental content in

the filter blanks was subtracted from the sample

results. The Mn, Cu, and Zn concentrations for the

digested SPM were determined by means of standard

flame-atomic absorption spectrometry methods and

Ba, Cr, Ni, Cd, Pb, Sb and As concentrations by

standard electrothermal-AAS methods. The determi-

nations were carried out with a Perkin-Elmer atomic

absorption spectrometer model AAnalyst 700. The

calibration lines were traced using solutions prepared

from standard solutions CertiPUR�. The methods

accuracy, precision and sensitivity have been tested

by using the Perkin-Elmer groundwater and waste-

water pollution control certified reference materials.

Determination of superoxide dismutase (SOD)

activity

Biological samples for the assay of SOD activity

were collected at four different sampling stations (2,

3, 8, and 12) in May and November 2008 (Table 3).

Biological samples were prepared for SOD analysis

according to the method described by de Oliveira

et al. (2005). Organisms (all individuals found at thatTa

ble

2co

nti

nu

ed

Sta

tio

ns

P Ba

(mm

ol/

kg

)

Mn

(mm

ol/

kg

)

Cr

(mm

ol/

kg

)

Ni

(mm

ol/

kg

)

Cu

(mm

ol/

kg

)

Zn

(mm

ol/

kg

)

Pb

(mm

ol/

kg

)

As

(mm

ol/

kg

)

Sb

(mm

ol/

kg

)

13

.83

.40

.20

.21

.70

.60

.20

.30

.1

22

.11

2.1

0.7

0.3

1.0

1.9

0.2

0.1

0.1

39

.22

2.7

1.7

0.5

0.0

78

.31

.90

.00

.0

41

.61

.51

.00

.50

.71

.51

.50

.30

.2

50

.91

.50

.70

.80

.80

.50

.60

.20

.1

61

.11

3.5

1.0

0.6

2.9

2.8

0.2

0.8

0.1

71

.21

.60

.80

.70

.70

.40

.20

.10

.1

81

.41

.71

.20

.81

.60

.50

.30

.20

.7

90

.71

2.2

0.4

0.5

0.6

4.9

0.2

1.5

0.0

10

1.7

1.4

0.4

0.4

0.8

0.4

0.2

0.1

0.1

11

1.1

2.5

0.5

0.4

0.9

0.5

0.8

0.2

0.1

12

0.8

20

.40

.60

.52

.31

2.9

0.3

0.2

0.0

68 Hydrobiologia (2011) 669:63–82

123

Ta

ble

3N

um

ber

of

ind

ivid

ual

so

fea

chfa

un

ag

rou

p(a

)an

do

fth

eh

yp

orh

eic

Cru

stac

eaan

dO

lig

och

aeta

(b)

inth

e1

2st

atio

ns

on

the

Ari

esR

iver

(su

b-a

du

lts

no

tin

clu

ded

inb

)

Sta

tion

Gro

up

Rot-

ifer

a

Tar

di-

gra

da

Nem

-

atoda

Oli

go-

chae

ta

Aca

riG

as-

tropoda

Cycl

op-

oid

a

Har

pac

-

tico

ida

Ost

ra-

coda

Am

ph-

ipoda

Isopoda

Bat

hy-

nel

lace

a

Inse

ct

larv

ae

R Indiv

idual

s

R Gro

ups

a 124

019

66

10

062

65

76

01

030

353

9

20

08

24

19

141

943

26

414

012

597

10

30

123

26

34

28

29

31

25

230

76

242

12

40

03

17

00

20

10

01

226

6

50

083

96

00

67

00

043

10

245

6

60

15

90

0294

24

00

00

315

6

70

04

76

22

0561

450

52

610

36

1388

10

80

02

81

10

31

082

03

15

3218

8

90

05

21

30

259

20

14

15

46

16

5404

10

10

00

117

20

174

216

18

33

82

273

10

11

00

06

60

92

22

45

55

127

9

12

00

328

80

95

247

25

52

12

7279

10

RIn

div

idual

s24

2156

467

105

29

1646

144

360

142

1194

110

88

4467

Sta

tion

Spec

ies

Nip

harg

us

transy

l-va

nic

us

Nip

harg

us

rom

anic

us

Bogid

-ie

lla

sp.

Bath

y-nel

lasp

.

Mic

ro-

charo

nsp

.

Pro

ase

llus

sp.

Aca

nth

o-

cycl

ops

sp.

Aca

nth

o-

cycl

ops

b.

bis

-aet

osu

s

Dia

cy-

clops

clandes

-ti

nus

Ela

phoid

-el

lael

aphoid

es

Para

ste-

noca

ris

sp.

1

Para

ste-

noca

ris

sp.

2

Tri

cho-

dri

lus

sp.

R Indi-

vid

ual

s

R Spec

ies

b 10

00

01

00

00

21

00

527

3

228

00

0412

10

01

11

15

450

8

30

50

19

00

20

00

00

17

4

40

00

10

00

02

00

08

11

3

50

00

43

00

00

10

05

18

67

4

60

00

00

00

00

00

01

11

716

37

03

601

90

00

00

022

688

6

80

00

13

30

00

00

00

50

66

3

92

13

016

42

40

10

10

07

12

107

9

10

018

08

33

021

42

20

02

14

140

8

11

03

05

50

00

00

00

922

4

12

619

112

50

00

00

00

01

89

6

RIn

div

idual

s52

95

1102

1156

14

21

54

722

115

145

1685

Hydrobiologia (2011) 669:63–82 69

123

time in the station) from an entire sample were

lysed in 0.5 M sucrose, buffered at pH 7.4 with

20 mM Tris–HCl (7 ml/g of tissue), at 4�C. Phen-

ylmethanesulfonyl fluoride (PMSF), as protease

inhibitor, was prepared as 100 mM stock solution in

isopropanol. PMSF was added to the lysis buffer

(10 ll/ml of buffer). Samples were homogenized in

lysis buffer using a Potter-Teflon homogenizer. The

homogenates were incubated for 30 min on ice and

centrifuged at 10,000g, 4�C, for 10 min. The

supernatants were collected and the total protein

content of the lysates was determined colorimetri-

cally using Coomassie Brilliant Blue G-250 (Sigma)

as a dye (Bradford, 1976) and bovine serum albumin

as a standard.

The SOD activity in lysates was determined

indirectly by using a SOD Assay Kit (Biochemika,

Sigma-Aldrich) to measure inhibition of the reduction

of highly water-soluble tetrazolium salt, WST-1

(2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophe-

nyl)-2H-tetrazolium, monosodium salt) by superoxide

anion (O2-) produced by xanthine oxidase activity.

Upon reduction of WST-1 with the superoxide anion,

a water-soluble formazan dye was developed that

could be measured colorimetrically at 450 nm. The

rate of the reduction with O2- is linearly related to

the inhibition of xanthine oxidase activity by SOD

activity. As the absorbance at 450 nm is proportional

to the amount of superoxide anion, the SOD activity

as an inhibition activity can be quantified by

measuring the decrease in the color development at

450 nm with a reference wavelength of 630 nm

(Ukeda et al., 2002). The assay was performed in

96-well plates by using a Stat Fax 2100 Microplate

Reader (Awareness Technology, Palm City, FL). For

each sample, SOD activity was determined for the

same amount of protein (10 lg). Blanks and samples

were prepared according to the manufacturer’s

instructions. The activity of SOD was expressed as

a rate of inhibition of xanthine oxidase activity. SOD

activity for each experiment was determined in

triplicate.

Statistical analysis

The log-transformated data used for analyses corre-

spond to four categories: physico-chemical charac-

teristics of interstitial waters, metal concentrations,

and COD of interstitial waters, metal concentrations

in sediments and abundance of hyporheic Crustacea

species. Statistical analyses, such as multiple factor

analysis (MFA), principal component analysis (PCA)

and Mantel tests were performed using XLSTAT

Version 2010. The MFA is a synthesis of principal

component analysis and multiple correspondence

analysis and allows the study of the relationships

between the observations, the variables, and tables

(Pages, 2004). Mantel tests were used to compare the

chemistry of surface and interstitial waters in the 12

stations, by using Spearman correlation and 10,000

permutations.

Superoxide dismutase activity for each experimen-

tal group was determined in triplicate. The final

results represent mean ± SD of three measurements.

For statistical analysis, a value of P \ 0.05 was

considered significant. The differences between the

SOD activities in different samples at the same

season were analyzed by one-way ANOVA with

Bonferroni correction for multiple comparisons using

GraphPad Prism v.4.02 for Windows, GraphPad

Software (San Diego, CA).

Results

Water and SPM chemical features

Temperature and pH of interstitial and surface water

were similar at each sampling station. Water pH was

generally lower (0.5 pH units) at stations situated

downstream of the Baia de Aries mining area

(Table 2).

Heavy metal concentrations of interstitial waters,

especially Cu, Pb, Zn, and Fe, varied among different

stations (Table 2), with high levels recorded at

Stations 2, 4, 5, and 6. Heavy metal concentrations

in interstitial waters were much higher in these

stations than in the corresponding surface water

samples. At the other stations, heavy metal concen-

trations were only slightly higher in interstitial waters

and not significantly different from levels in surface

waters.

A PCA of the composition of heavy metals (Cr,

Ni, Cu, Zn, Pb, and Mn) in surface and interstitial

waters and SPM (Fig. 2) showed a clear separation of

SPM samples along the first axis (72% of the

variation). Interstitial and surface samples are sepa-

rated along the second axis (10%) with very low

70 Hydrobiologia (2011) 669:63–82

123

significance in the analysis. Although there is no legal

regulation concerning the levels of heavy metals in

the SPM, all obtained values were high compared to

other water quality standards.

Abundance, species richness, and spatial

distribution of invertebrates

A total of 4,363 individuals from 14 invertebrate

groups were recorded from the hyporheic zone of the

Aries River: Rotifera, Tardigrada, Nematoda, Oligo-

chaeta, Acari, Gastropoda, Cyclopoida, Harpactico-

ida, Ostracoda, Amphipoda, Isopoda, Bathynellacea,

Collembola and Insecta larvae (Table 3). Rotifera,

Tardigrada, and Gastropoda were the most rarely

collected, occurring in only one or two samples.

Collembola, a group not associated with the hypor-

heic zone was present in almost all the samples. The

most abundant hyporheic taxa were (in order of

numerical dominance) Cyclopoida, Isopoda, Oligo-

chaeta, and Ostracoda (Fig. 3). All typical hyporheic

species belong to the Crustacea, and they were used

in further analyses at the species level.

The analysis of the distribution of hyporheic fauna

along the studied longitudinal transect of the Aries

River shows that the highest diversity (of taxonom-

ical groups) was found at Station 3 and the lowest

from Stations 4, 5, and 6. The highest numbers of

individuals (all groups) were collected from Station 7

(1,388 individuals from 260 l of water), followed by

Stations 2 (597 individuals from 260 l of water) and 9

(404 individuals), while the lowest number of indi-

viduals was recorded from Stations 4 (26 individuals)

and 11 (127 individuals). Station 2 was dominated by

Isopoda, Station 9 by Cyclopoida, and both groups

were well represented in Station 7. A PCA of

sampling stations using invertebrate abundance

revealed some patterns of community composition

along the river (Fig. 4), with 53% of variance

explained by the first two axes in the PCA. Stations

1 and 3 are separated along the first axis (28%) from

Stations 9, 10, 12. None of these stations appeared to

have interstitial water affected by heavy metal

pollution. This axis is positively defined mainly by

Oligochaeta and Harpacticoida, and negatively by

Bathynellacea. Along the second axis (25%), Stations

2 and 7, defined by Isopoda, Amphipoda and

Cyclopoida, are separated from Stations 5 and 8;

Stations 4, 6, and 11 to form a group along the second

axis.

As a general trend, the number of faunal groups

decreased in the most polluted stations (4, 5, and 6),

while the total number of individuals (all groups)

decreased from up-stream to down-stream stations,

with a drastic increase in Station 7 (Fig. 5a; Table 3).

A trend towards higher number of species and

lower number of individuals of typical hyporheic

crustaceans was found for downstream stations

Fig. 2 PCA on the chemical composition in Pb, Cr, Ni, Cu, Zn, and Mn of surface (s), interstitial (i) waters and SPM (p) in the 12

stations (1–12); surface and interstitial waters are gathered on the left side of the graph, along the first axis

Hydrobiologia (2011) 669:63–82 71

123

(Fig. 5b; Table 4). The local distribution of Crustacea

species was variable. For example, highly polluted

Stations 4 and 5 supported a very low diversity of

Crustacea. Station 5 was dominated by a cosmopol-

itan epigean species, Paracyclops fimbriatus. Down-

stream stations were dominated by the transitional

Diacyclops languidoides, with more than 200 indi-

viduals in August in Station 8. Of the typical

hyporheic cyclopoids, two species of Acanthocyclops

were well represented in Station 10. Harpacticoida

was present only in Stations 1, 2, 9, and 10, where

hyporheic species were best represented (Paraste-

nocaris sp., Bryocamptus tauricus, Elaphoidella

elaphoides). All the macrocrustaceans collected are

typical hyporheic species. The dominant isopod was a

species of Microcharon, an interstitial genus, which

was abundant in Stations 2 and 7 (with more than 250

individuals counted in a single sample at Station 7,

December 2008) (Fig. 6), Less abundant were species

of Niphargus (Amphipoda), Bogidiella (Amphipoda),

Fig. 3 Ranking of the

taxonomical groups based

on their abundance

cumulated in all 12

sampling stations

Fig. 4 PCA of the

relationships between

stations (1–12) according to

common fauna groups

72 Hydrobiologia (2011) 669:63–82

123

Bathynella (Bathynellacea) and Proasellus (Isopoda).

Except for N. transylvanicus (relatively abundant

especially in Station 2), all other macrocrustaceans

were recorded in higher numbers downstream of

Station 4, particularly from Station 5 (Bathynella sp.)

and Station 7 (N. romanicus).

The other well-represented group was Oligochae-

ta, with the dominant species belonging to the

typically interstitial genus Trichodrilus. It was most

abundant downstream of Station 4, reaching maxi-

mum numbers in Stations 7 and 8 (Table 3b). The

other identified oligochete species were recorded in

low numbers.

Correlation analyses between invertebrate

communities and water/sediment quality

The general MFA combines results given by the

PCAs on physico-chemical features, on chemistry of

the water, on chemistry of the SPM and on typical

hyporheic species of Crustacea. In terms of interstitial

water, Station 1 was well-separated from the other

stations in the PCA (Fig. 7a) by high values of

conductivity. Stations 2, 3, 10, and 11 were positively

correlated with pH along the first axis (50%), while

Stations 4, 5, and 6 were negatively correlated with

pH. Station 7 was isolated along the second axis

Fig. 5 a Spatial

distributions of fauna

groups (squares) and

number of individuals

(circles) (a) and of

hyporheic Crustacea species

(squares) and number of

individuals (circles) (b);

polynomial trendlines and

equations are also

represented

Hydrobiologia (2011) 669:63–82 73

123

Table 4 List of fauna (major groups and species) found in the

hyporheic of the Aries River

Taxa E T H

Tardigrada

Oligochaeta

Amphichaeta sp.

Aulodrilus limnobius

Aulodrilus sp.

Cernosvitoviella sp. *

Cognettia glandulosa

Eiseniella tetraedra *

Fridericia bisetosa *

Haplotaxis gordioides *

Lumbriculus variegatus *

Nais communis *

Pristina aequiseta foreli *

Pristinella jenkinae *

Rhyacodrilus coccineus *

Stylodrilus heringianus *

Trichodrilus sp. *

Acari

Hydrachnidia

Athienemanniidae

Phreatohydracarusmosticus

*

Aturidae

Lethaxona sp. *

Hygrobatidae

Atractides cf latipalpis *

Atractides sp.

Lebertiidae

Lebertia insignis *

Momoniidae

Momonisia phreatica *

Stygomononia latipes *

Sperchontidae

Sperchon glandulosus *

Torrenticolidae

Torrenticola amplexa *

Halacarida

Gastropoda

Amphipoda

Bogidiella sp. *

Niphargus romanicus *

N. transsylvanicus *

Ostracoda

Bathynellacea

Table 4 continued

Taxa E T H

Bathynella sp. *

Cyclopoida

Acanthocyclops sp. *

A. balcanicus bisaetosus *

A. venustus *

A. vernalis *

Diacyclops sp.

D. bisetosus *

D. languidus *

D. clandestinus ssp. *

D. languidoides hypnicola *

Eucyclops s. serrulatus *

Megacyclops viridis *

Paracyclops fimbriatus *

Harpacticoida

Atheyella crassa *

Bryocamptus cuspidatus *

B. dacicus *

B. tauricus *

B. weberi cf. tauricus *

Elaphoidella elaphoides *

Moraria aff. alpina

Paracamptus schmeili

Parastenocaris sp. 1 *

Parastenocaris sp. 2 *

Isopoda

Microcharon sp. *

Proasellus sp. *

Collembolla *

Insecta (larvae) *

Coleoptera

Scirtidae

Diptera

Athericidae

Ceratopogonidae

Chironomidae

Limoniidae

Tabanidae

Ephemeroptera

Baetidae

Heptageniidae

Plecoptera

Leuctridae

Trichoptera

74 Hydrobiologia (2011) 669:63–82

123

(35%) and characterized by temperature. The other

stations were defined by two or all three of the

physico-chemical features. In terms of interstitial

water chemistry (Fig. 7b), Stations 4, 5, 6, and 8 were

separated from all other stations except 1 along the

first axis (34.5%). Station 1 was separated along the

second axis (21.5%) by high levels of Cl, Na, and K.

In terms of hyporheic crustacean composition

(Fig. 7c), Stations 2 and 7 defined by amphipods,

isopods, and harpacticoids were separated along the

first axis (27%) from Stations 4 and 5. Stations 9 and

10 form another group defined on the second axis

(23%) by cyclopoids, while all other stations were

grouped by the presence of Bogidiella sp. and

Elaphoidella elaphoides. In the PCA of SPM chem-

istry (Fig. 7d), only Stations 3, 4, and 8 had a distinct

position. Stations 3 and 4 are grouped along the first

Fig. 6 a Spatial

distribution of hyporheic

crustaceans identified along

the longitudinal transect of

the Aries River; b without

the dominant isopod

Microcharon sp.

Table 4 continued

Taxa E T H

Brachycentridae

Glossosomatidae

E epigean (surface), T transitional, H hyporheic

Hydrobiologia (2011) 669:63–82 75

123

axis (42%) because of high content of Cr, Pb, Zn, and

Ba; Station 8 belongs to the second axis (22%) by the

content of Sb. In the overall MFA, along the first axis

(23%), Stations 4, 5, 6, and 8 were separated from

Stations 2, 3, and 12. For all these stations, the

chemical features of water and SPM had strong

impact on their position on the plot. Station 1 was

separated from Stations 7, 9, 10, and 11 along the

second axis (Fig. 7e), with lower significance in the

analysis (17%). Station 1 was defined by fauna and

physico-chemical features, while Stations 7, 9, and 10

were defined by fauna composition.

Indirect measurement of SOD activity

in biological samples

Statistical analysis of SOD activity in biological

samples from different sampling sites collected

during May 2008, showed that only the sample from

Station 12 differed statistically. SOD activity was two

to three times higher in samples from Stations 2, 3,

and 8 than Station 12 (P \ 0.03) (Table 5). SOD

activity in samples collected during May was most

strongly correlated with COD (Spearman correlation

coefficient [ 0.9) (Fig. 8a). The normal value of

COD is lower than 3 mg O2/dm3, according to SR

EN ISO 8467/2001.

From the biological samples collected during

November 2008, the activity of SOD in the organisms

isolated from Station 2, was significantly higher than

the SOD activity in the other samples (P \ 0.001).

SOD activity was also significantly different

(P \ 0.001) from one season to another. During late

spring (May), the SOD activity was much higher than

those recorded in organisms sampled during the fall

season (November). For the latter, the strongest

positive correlation was with lead concentration

(Spearman correlation coefficient [ 0.9) (Fig. 8b).

Lead concentration only exceeded the acceptable

level (10 lg/dm3, according to SR ISO 17294-2/

2003) at Station 2.

Discussion

Water quality

Significant differences between the heavy metal

content of surface and interstitial waters were evident

at only a few stations (2, 4, 5, and 6) along the studied

longitudinal transect of the Aries River. At the other

stations, interstitial water was only slightly higher in

heavy metals than the surface water, but richer in

nitrites and nitrates due to the biogeochemical

processes that occur there (Brunke & Gonser, 1997;

Hinkle et al., 2001). Pollution of surface waters

probably occurs in pulses, and past episodes of

pollution can be detected by analyzing the SPM

chemistry. Much higher concentrations of heavy

metals are being carried adsorbed on the SPM active

surface, as compared to concentrations of the corre-

sponding dissolved ions which are carried in the

aqueous solution (Morel, 1983; Munk et al., 2002).

The SPM values differed from one sampling site to

another, suggesting distinct recharge mechanisms for

each specific sampling site.

Spatial patterns of fauna distribution and impact

of heavy metals

This study of the interstitial biodiversity and impact

of heavy metal pollution along the longitudinal

transect revealed spatial patterns of fauna distribution

illustrating the complexity and patchiness of the

hyporheic zone, even at depths over one meter. The

Aries River hyporheic fauna was diverse, with 14

invertebrate groups identified, compared to other

rivers of Transylvania, such as the Somes and the

Table 5 SOD activities in biological samples collected during May and November 2008

Month 2008 SOD activity (% inhibition of xanthin oxidase activity)

Station 2 Station 3 Station 8 Station 12

May 43.792 ± 5.673 33.026 ± 2.151 45.995 ± 7.711 13.431 ± 2.390

November 24.911 ± 2.802 5.272 ± 1.054 3.322 ± 2.916 6.372 ± 4.963

The results represent the mean of three independent measurements ± standard deviation (SD)

76 Hydrobiologia (2011) 669:63–82

123

Cris (10 and 14 groups, respectively) (Moldovan

et al., 2005). Most of the sampled individuals were

Crustacea, especially the co-dominant Cyclopo-

ida and Isopoda, belonging to both the micro- and

macro-interstitial faunas. Harpacticoida was abun-

dant only in clean waters, where hyporheic and

interstitial species were best represented. The mac-

rocrustaceans are all groundwater species, some of

Fig. 7 PCAs on observations (stations 1–12) and the physical

and chemical features (a), on interstitial water chemistry (b),

on hyporheic crustaceans (c), and on SPM chemistry (d); the

variable space of the total MFA (e) and the total MFA (f);

T temperature, EC electrical conductivity, P physico-chemical

features, C interstitial water chemistry, F hyporheic Crustacea,

S SPM chemistry

Hydrobiologia (2011) 669:63–82 77

123

which are better adapted to narrow interstices due to

their finer shape, while the larger species can exploit

larger spaces. Another abundant group was Oligo-

chaeta with the main representative being a common

hyporheic genus, Trichodrilus. These dominant

groups were recorded from different periods or/and

stations, a pattern previously reported for other rivers

(Danielopol et al., 1997).

Using multiple sampling stations with differing

water qualities enabled the structure of biological

communities in sites with natural and polluted waters

to be compared. Station 1 was not affected by heavy

metal pollution due to its geographical position

upstream of the main pollutant inflow. However,

Station 1 (with high concentrations of Na, K, and Cl)

revealed the effects of another human impact: salt

used during winter on a nearby road. There were

downstream stations (Stations 9, 10 and 12) whose

communities were not affected by heavy metal

pollution due to water dilution. Species richness and

abundance of individuals were also variable among

the unpolluted stations.

In the most polluted stations (Stations 4 and 11),

fewer individuals were collected during the study

compared with more than 1,300 individuals from

moderately polluted Station 7, downstream of the

polluted area. In some samples, polluted stations

almost completely lacked fauna. Macrocrustaceans,

however, were dominant in the moderately polluted

stations, such as Stations 2 and 7. Such lack of

sensitivity to sediment toxicity has been reported for

other benthic macroinvertebrates, which are more

sensitive to water pH (Van Damme et al., 2008).

The highest numbers of hyporheic Crustacea (both

individuals and species) were found in the moderately

polluted stations (Stations 2 and 7) and less polluted,

downstream stations (Stations 9, 10, and 12). The

most polluted stations had both fewer faunal groups

and typical hyporheic Crustacea (Stations 4, 5, and

6). The highest number of individuals was found in

moderately polluted stations (Stations 2 and 7)

followed by the unpolluted stations (Stations 3, 9,

10, and 12).

The ‘‘intermediate disturbance hypothesis’’

(Connell, 1978) states that intermediate levels of

disturbance will lead to the greatest species diversity.

In an undisturbed or rarely disturbed environment,

the most competitively dominant species will take

over, whereas in a highly disturbed environment, only

species that can cope with the disturbance will

survive. Between these two extremes, both types of

species populate the same place, thus enhancing the

species diversity. This theory may be applied to our

study of the Aries River, where the moderately

disturbed stations (Stations 2, 7, and 9), supported the

highest diversity and number of individuals.

The general spatial pattern of occurrence of

invertebrates, and also the pattern of hyporheic

crustaceans, can be explained by water chemistry.

However, neither interstitial water nor SPM chemis-

try could explain the differences between stations

with similar level of pollution and there appear to be

additional factors affecting species occurrence. One

of these is the heterogeneous grain size distribution in

the Aries alluvia, which is beneficial for hyporheic

fauna (Bretschko, 1991; Schmid-Araya, 1998; Chafiq

Fig. 8 Evaluation of the correlation between SOD activity and the two chemical parameters for different sampling stations:

correlation is positive between COD and SOD activity in samples collected during May 2008 (a) and during November 2008 (b)

78 Hydrobiologia (2011) 669:63–82

123

et al., 1999). Different authors have emphasized

sediments as a significant factor in the interactions

between biotic and abiotic elements of surface and

interstitial waters (Hendricks & White, 1995; Brunke

& Gonser, 1997; Dole-Olivier, 1998). Others high-

light the importance of organic matter (Griebler &

Mosslacher, 2003; Datry et al., 2005; Hahn &

Matzke, 2005), or bacteria (Brown et al., 2003;

Griebler & Mosslacher, 2003; Febria et al., 2009) in

shaping the biological communities of groundwater

and hyporheic zone.

Typical interstitial communities are composed of

fauna with different degrees of adaptations to micro-

habitats and to various (micro-) environmental needs.

The presence of extremely tiny (Parastenocaris sp.)

and large crustaceans (isopods and amphipods) in the

same sample emphasizes the heterogeneity of the

hyporheic habitat. Local impacts, organic matter

input, sediment structure, and many other features

of the physical–chemical-biological microcosm will

favor some species and some assemblages of species

(see review in Bork et al., 2009).

Ecological and evolutionary implications

The general axiom in groundwater ecology is that

typical hyporheic animals have low ecological toler-

ance (Dole-Olivier et al., 2009), and can survive only in

relatively stable habitats. Our results from the Aries

River show that colonization of different levels of the

hyporheic zone is not necessarily a result of stressful

conditions or climate, such as heavy metal pollution,

but exists more because of lower levels of stress. The

stressing condition can be, in this case, competition

from surface species or from other hyporheic repre-

sentatives. In the Aries River interstitial, heavy metal

pollution is the limiting factor for surface benthic and

for hyporheic species sensitive to pollution, but will

indirectly ‘promote’ the development of populations of

pollution-resistant species. Macrocrustaceans and

some microcrustaceans (harpacticoids) in intermediate

stations may have developed larger populations due to

a lack of competition for resources from benthic

surface species or from other hyporheos.

Originating from epigean organisms with wide

ecological tolerance and retaining plasticity for

vertical and horizontal migrations in a highly patchy

and heterogeneous three-dimensional biotope,

groundwater species are opportunistic in their attempt

to colonize lower levels of the interstitial habitat

(Rouch & Danielopol, 1987). In stressful conditions,

species with higher adaptability, resistance or mobil-

ity will flourish. Reduction of competition and

predation will give opportunistic or ‘neutral’ species

the possibility to develop larger populations.

The interstitial habitat is an ecotone with a contin-

uous interplay between surface and subsurface

communities colonizing each vertical layer of a three-

dimensional environment (Boulton, 2000b; Hakenkamp

& Palmer, 2000). The balance may favor epigean or

hyporheic species, depending on water quality and

microbiota, because the interstitial habitat is extremely

dynamic in space and time (Datry et al., 2005; Hahn,

2006), and interspecific competition is intense.

For the Aries River surface waters it has been found

that the number of individuals and species of benthic

macroinvertebrate groups decrease drastically in

response to pollution (Momeu et al., 2007). However,

hyporheic fauna flourish in the moderately polluted

locations. The low metabolic rates of hyporheic

species versus intense metabolism of surface species

(e.g. Culver, 1982; Danielopol et al., 1994; Mosslacher

& Creuze des Chatelliers, 1996; Malard & Hervant,

1999) is one of the factors favouring hyporheic species

and enabling them to resist episodes of water pollution

for longer periods. In such situations, the hyporheic

fauna will also invade those superficial layers depleted

of surface fauna.

In general terms, the hyporheic fauna is more

tolerant of heavy metal pollution than animals of the

same taxonomical groups in surface water. The

hyporheic Crustacea of the Aries River appeared to

be insensitive to heavy metals, probably until

concentrations reach lethal levels. Plenet (1995)

described a similar situation, with no relationship

between hyporheic amphipod abundance and metal

levels suggesting that these taxa could not be used as

biomonitors for metal contamination. Macrocrusta-

ceans are resistant to heavy metal pollution and can

survive and develop larger populations by lack of

competition and predation from surface representa-

tives which are normally present in the upper layers

of the interstitial habitat.

SOD activity significance

The indirect measurements of SOD activity in

organisms collected from various sampling sites

Hydrobiologia (2011) 669:63–82 79

123

along Aries River have provided an interesting

insight on the response of a whole interstitial

invertebrate community to an environmental chal-

lenge from one particular, or a group, of environ-

mental factors. The assay of SOD activity in

biological samples has the advantages of low cost,

speed and the need of a very small amount of

biomass. The strong correlations found between SOD

activity in pooled tissues extract and some chemical

parameters, such as Pb concentration and COD,

suggesting that the oxidative stress enzymes may be

sensitive indicators of chemical pollution in intersti-

tial zones and could be used in assessing and

monitoring pollution. The values of the SOD activity

are congruent to those obtained for the distribution of

hyporheic communities along the studied transect.

Stations with both fewer fauna groups and species

have higher SOD activity. Until present, experiments

were undertaken only on whole communities and the

method is refined by using Oligochaeta (Rusu et al.,

2010). Further investigations will focus on macro-

crustacean species or other taxa sensitive to heavy

metal pollution, though difficulties arise from the

need to collect a sufficient number of individuals

from different stations in the same period of time.

Final remarks

Heavy metals associated with mining can pollute

surface streams, groundwater and alluvial aquifers,

which in turn will impact on both the hydrological

exchange and biological activity of the hyporheic

zone (Hancock, 2002). The reduction of both the

number of invertebrate groups and crustacean species

in the polluted sectors of the Aries River supports

this. However, water chemistry was not the only

determinant of hyporheic biodiversity, as was also

emphasized by Dole-Olivier et al. (2009). There are

factors related to hydraulic conductivity, availability

of food resources, physical habitat fragmentation, and

biological interactions that can be also important.

The presence of high biodiversity levels in the

moderately polluted stations compared to strongly

polluted stations, or even cleaner waters, and the

increase of the number of some hyporheos (especially

macrocrustaceans and oligochetes) in the intermedi-

ate stations suggests that factors other than just water

chemistry influence their spatial distribution. On the

Aries River, such changes over a relatively short

longitudinal transect, may be explained by (1) higher

tolerance of some hyporheic species compared to

surface fauna in response to heavy metal pollution;

(2) biotic interactions between surface and subterra-

nean species; and (3) the physical habitat fragmen-

tation given by extreme diverse granulometry of the

Aries alluvia.

Further research into the effects of pollution and

other environmental impacts on interstitial habitats

needs to focus both on the identification of hyporheic

species (such as micro-crustaceans) and of oxidative

stress enzymes that can be used as indicators of

chemical pollution in interstitial zones.

Acknowledgments We are grateful to Frank Fiers for useful

discussions and suggestions, to Geza Rajka, Akos Nagy and

Nimrod Nemeth for help with samplings, to Vlad Paul for insect

larvae identification, and to Mihai Terente for the map. Claire

Stephens was very helpful in improving the content and the

English of the manuscript. Stuart Halse, Koen Martens and two

anonymous reviewers made valuable remarks improving

considerably the manuscript quality. This study was funded

through the grants 31_032/2007 (CNMP, Ministry of Education,

Research and Innovation, Romania) and SYNTHESYS BE-TAF

4681 (European Union-funded Integrated Activities grant).

References

Bork, J., S. E. Berkhoff, S. Bork & H. J. Hahn, 2009. Using

subsurface metazoan fauna to indicate groundwater–sur-

face water interactions in the Nakdong River floodplain.

South Korea Hydrogeology Journal 17: 61–75.

Bou, C., 1974. Recherches sur les eaux souterraines. 25—

Methodes de recolte dans les eaux souterraines interstiti-

elles. Annales de Speleologie 29: 611–619.

Boulton, A. J., 2000a. River ecosystem health down under:

assessing ecological condition in riverine groundwater

zones in Australia. Ecosystem Health 6: 118–198.

Boulton, A. J., 2000b. The subsurface macrofauna. In Jones, J.

B. & P. J. Mulholland (eds), Streams and Ground Waters.

Academic Press, San Diego: 337–361.

Boulton, A. J., S. Findlay, P. Marmonier, E. H. Stanley & H.

M. Valett, 1998. The functional significance of the hyp-

orheic zone in streams and rivers. Annual Review of

Ecology, Evolution, and Systematics 29: 59–81.

Bourg, A. C. M. & C. Bertin, 1993. Biogeochemical processes

during the infiltration of river water into an alluvial

aquifer. Environmental Science & Technology 27:

661–666.

Bradford, M. M., 1976. A rapid and sensitive method for the

quantitation of microgram quantities of protein utilizing

the principle of protein-dye binding. Analytical Bio-

chemistry 72: 248–254.

Bretschko, G., 1991. Bedsediments, groundwater and

stream limnology. Verhandlungen der Internationalen

80 Hydrobiologia (2011) 669:63–82

123

Vereinigung fur Theoretische und Angewandte Limnologie

24: 1957–1960.

Brown, R. J., S. D. Rundle, T. H. Hutchinson, T. D. Williams

& M. B. Jones, 2003. Small-scale detritus-invertebrate

interactions: Influence of detrital biofilm composition on

development and reproduction in a meiofaunal copepod.

Archiv fur Hydrobiologie 157: 117–129.

Brunke, M. & T. Gonser, 1997. The ecological significance of

exchange processes between rivers and groundwater.

Freshwater Biology 37: 1–33.

Cantu-Medellın, N., N. O. Olguın-Monroy, L. C. Mendez-

Rodrıguez & T. Zenteno-Savın, 2009. Antioxidant

enzymes and heavy metal levels in tissues of the black

chocolate clam Megapitaria squalida in Bahıa de La Paz,

Mexico. Archives of Environmental Contamination and

Toxicology 56: 60–66.

Chafiq, M., J. Gibert & C. Claret, 1999. Interactions among

sediments, organic matter, and microbial activity in the

hyporheic zone of an intermittent stream. Canadian

Journal of Fisheries and Aquatic Sciences 56(3): 487–495.

Connell, J. H., 1978. Diversity in tropical rain forests and coral

reefs. Science 199: 1302–1310.

Cravo, A., B. Lopes, A. Serafim, R. Company, L. Barreira, T.

Gomes & M. J. Bebianno, 2009. A multibiomarker

approach in Mytilus galloprovincialis to assess environ-

mental quality. Journal of Environmental Monitoring

11(9): 1673–1686.

Creuze des Chatelliers, M., D. Poinsart & J.-P. Bravard, 1994.

Geomorphology of alluvial groundwater ecosystems. In

Gibert, J., D. L. Danielopol & J. A. Stanford (eds),

Groundwater Ecology. Academic Press, San Diego:

158–185.

Culver, D. C., 1982. Cave Life: Evolution and Ecology. Har-

vard University Press, Cambridge.

Danielopol, D. L., M. Creuze des Chatteliers, F. Mosslacher, P.

Pospisil & R. Popa, 1994. Adaption of Crustacea to inter-

stitial habitats: a practical agenda for ecological studies. In

Gibert, J., D. L. Danielopol & J. A. Stanford (eds),

Groundwater Ecology. Academic, New York: 218–243.

Danielopol, D. L., R. Rouch, P. Pospisil, P. Torreiter & F.

Moßlacher, 1997. Ecotonal animal assemblages; their

interest for groundwater studies. In Gibert, J., J. Mathieu

& F. Fournier (eds), Groundwater/Surface Water Eco-

tones: Biological and Hydrological Interactions and

Management Options. Cambridge University Press,

Cambridge: 11–20.

Datry, T., F. Malard & J. Gibert, 2005. Response of inverte-

brate assemblages to increased groundwater recharge rates

in a phreatic aquifer. Journal of the North American

Benthological Society 24: 461–477.

de Oliveira, U. O., A. S. da Rosa Araujo, A. Bello0-Klein, R.

S. M. da Silvaa & L. C. Kucharski, 2005. Effects of

environmental anoxia and different periods of reoxygen-

ation on oxidative balance in gills of the estuarine crab

Chasmagnathus granulate. Comparative Biochemistry

and Physiology Part B 140: 51–57.

Dole-Olivier, M.-J., 1998. Surface water-groundwater

exchanges in three dimensions on a backwater of the

Rhone River. Freshwater Biology 40: 93–109.

Dole-Olivier, M.-J., F. Malard, D. Martin, T. Leferbure &

J. Gibert, 2009. Relationships between environmental

variables and groundwater biodiversity at the regional

scale. Freshwater Biology 54: 797–813.

El Adnani, M., A. Ait Boughrous, M. Y. Khebiza, A. El

Gharmali, M. L. Sbai, A. S. Errouane, L. Loukili Idrissi &

A. Nejmeddine, 2007. Impact of mining wastes on the

physicochemical and biological characteristics of

groundwater in a mining area in Marrakech (Morocco).

Environmental Technology 28: 71–82.

Fan, W. H., G. Tang, C. M. Zhao, Y. Duan & R. Zhang, 2009.

Metal accumulation and biomarker responses in Daphniamagna following cadmium and zinc exposure. Environ-

mental Toxicology & Chemistry 28(2): 305–310.

Febria, C. M., R. R. Fulthorpe & D. D. Williams, 2009.

Characterizing seasonal changes in physicochemistry and

bacterial community composition in hyporheic sediments.

Hydrobiologia. doi:10.1007/s10750-009-9882-x.

Forray, F. L., 2001. Aplicarea analizei factoriale ın studiul

poluarii raului Aries (Muntii Apuseni). Studia Universi-

tatis Babes-Bolyai (Geologia) 46: 47–58.

Fuller, C. C. & J. W. Harvey, 2000. Reactive uptake of trace

metals in the hyporheic zone of a mining-contaminated

stream, Pinal Creek, Arizona. Environmental Science &

Technology 34: 1150–1155.

Geret, F., A. Serafim, L. Barreira & M. J. Bebianno, 2002.

Effect of cadmium on antioxidant enzyme activities and

lipid peroxidation in the gills of the clam Ruditapes de-cussates. Biomarkers 7(3): 242–256.

Griebler, C. & F. Mosslacher, 2003. Grundwasser-eine oko-

systemische Betrachtung. In Griebler, C. & F. Mosslacher

(eds), Grundwasserokologie. UTB-Facultas Verlag, Wien:

253–310.

Hahn, H. J., 2006. The GW-fauna-index: a first approach to a

quantitative ecological assessment of groundwater habi-

tats. Limnologica 36: 119–137.

Hahn, H. J. & D. Matzke, 2005. A comparison of stygofauna

communities inside and outside groundwater bores. Lim-

nologica 35: 31–44.

Hakenkamp, C. C. & M. A. Palmer, 2000. The ecology of

hyporheic meiofauna. In Jones, J. B. & P. J. Mulholland

(eds), Streams and Ground Waters. Academic, San Diego:

307–335.

Hancock, P. J., 2002. Human impacts on the stream-ground-

water exchange zone. Environmental Management 29(6):

763–781.

Hendricks, S. P. & D. S. White, 1995. Seasonal biogeochem-

ical patterns in surface water, subsurface hyporheic, and

riparian ground water in a temperate stream ecosystem.

Archiv fur Hydrobiologie 134: 459–490.

Hinkle, S., J. Duff, F. Triska, A. Laenen, E. Gates, K. Bencala,

D. Wentz & S. Silva, 2001. Linking hyporheic flow and

nitrogen cycling near the Willamette River—a large river

in Oregon, USA. Journal of Hydrology 244: 157–180.

Malard, F. & F. Hervant, 1999. Oxygen supply and the adaptations

of animals in groundwater. Freshwater Biology 41: 1–30.

Malard, F., J.-L. Reygrobellet, J. Mathieu & M. Lafont, 1994.

The use of invertebrate communities to describe ground-

water flow and contaminant transport in a fractured rock

aquifer. Archiv fur Hydrobiologie 131(1): 93–110.

Malard, F., S. Plenet & J. Gibert, 1996. The use of inverte-

brates in ground water monitoring: a rising research field.

Ground Water Monitoring and Remediation 16: 103–113.

Hydrobiologia (2011) 669:63–82 81

123

Manduzio, H., T. Monsinjon, C. Galap, F. Leboulenger &

B. Rocher, 2004. Seasonal variations in antioxidant

defences in blue mussels Mytilus edulis collected from a

polluted area: major contributions in gills of an inducible

isoform of Cu/Zn-superoxide dismutase and of glutathi-

one S-transferase. Aquatic Toxicology 70(1): 83–93.

Moldovan, O., Iepure, S. & A. Persoiu, 2005. Biodiversity and

protection of Romanian karst areas: the example of

interstitial fauna. In Stevanovic, Z. & P. Milanovic (eds),

Water Resources and Environmental Problems in Karst.

Proceedings International Conference & Field Seminar,

Beograd & Kotor, 13–19 September 2005: 831–836.

Momeu, L., K. W. Battes, F. Pricope, A. Avram, K. P. Battes,

M. Cımpean, D. Ureche & I. Stoica, 2007. Preliminary

data on algal, macroinvertebrate and fish communities

from the Aries catchment area, Transylvania, Romania.

Studia Universitatis Babes-Bolyai (Biologıa) 52: 25–36.

Morel, F. M. M., 1983. Principles of Aquatic Chemistry.

Wiley, New York.

Moser, D. P., J. K. Fredrickson, D. R. Geist, E. V. Arntzen, A.

D. Peacock & S.-M. W. Li, 2003. Biogeochemical pro-

cesses and microbial characteristics across groundwater-

surface water boundaries of the Hanford Reach of the

Colombia River. Environmental Science & Technology

37: 5127–5134.

Mosslacher, F. & J. Notenboom, 1999. Groundwater biomon-

itoring. In Gerhardt, A. (ed.), Biomonitoring of Polluted

Water. Environmental Science Forum 96: 119–140.

Mosslacher, F. & M. Creuze des Chatelliers, 1996. Physio-

logical and behavioural adaptations of an epigean and a

hyporheic dwelling population of Asellus aquaticus (L.)

(Crustacea, Isopoda). Archiv fur Hydrobiologie 138:

187–198.

Munk, L., G. Faure, D. Pride & J. Bigham, 2002. Sorption of

trace metals to an aluminum precipitate in a stream

receiving acid rock-drainage; Snake River, Summit

County, Colorado. Applied Geochemistry 17: 421–430.

Notenboom, J., S. Plenet & M.-J. Turquin, 1994. Groundwater

contamination and its impact on groundwater animals and

ecosystems. In Gibert, J., D. L. Danielopol & J. A. Stan-

ford (eds), Groundwater Ecology. Academic Press, San

Diego: 477–504.

Pages, J., 2004. Multiple factor analysis: main features and

application to sensory data. Revista Colombiana de Est-

adistica 27: 1–26.

Plenet, S., 1995. Freshwater amphipods as biomonitors of

metal pollution in surface and interstitial aquatic systems.

Freshwater Biology 33: 127–137.

Plenet, S., 1999. Metal accumulation by an epigean and a

hypogean freshwater amphipod: considerations for water

quality assessment. Water Environment Research 71:

1298–1309.

Plenet, S. & J. Gibert, 1994. Invertebrate community responses

to physical and chemical factors at the river/aquifer

interaction zone. I. Upstream from the city of Lyon. Ar-

chiv fur Hydrobiologie 132: 165–189.

Plenet, S., H. Hugueny & J. Gibert, 1996. Invertebrate com-

munity responses to physical and chemical factors at the

river/aquifer interaction zone. II. Downstream from the

city of Lyon. Archiv fur Hydrobiologie 136: 65–88.

Pyle, G. G. & R. S. Mirza, 2007. Copper-impaired chemo-

sensory function and behavior in aquatic animals. Human

Ecological Risk Assessment 13: 492–505.

Rouch, R. & D. Danielopol, 1987. L’origine de la faune

aquatique souterraine, entre le paradigme du refuge et le

modele de la colonization active. Stygologia 3: 345–372.

Rusu, M., H. L. Banciu, M. Banciu, T. Brad & O. T. Moldovan,

2010. Oxidative stress enzymes as biomarkers of heavy

metal pollution in interstitial invertebrates. Studia Uni-

versitatis Babes-Bolyai 2: 61–66.

Schmid-Araya, J. M., 1998. Small-sized invertebrates in a

gravel stream: community structure and variability of

benthic rotifers. Freshwater Biology 39: 25–39.

Ukeda, H., T. Shimamura, M. Tsubouchi, Y. Harada, Y. Nakai

& M. Sawamura, 2002. Spectrophotometric assay ofsuperoxide anion formed in Maillard reaction based on

highly water-soluble tetrazolium salt. Analytical Sciences

18: 1151–1154.

Van Damme, P. A., C. Hamel, A. Ayala & L. Bervoets, 2008.

Macroinvertebrate community response to acid mine

drainage in rivers of the High Andes (Bolivia). Environ-

mental Pollution 156: 1061–1068.

Williams, D. D. & R. R. Fulthorpe, 2003. Using invertebrate

and microbial communities to assess the condition of the

hyporheic zone of a river subject to 80 years of contam-

ination by chlorobenzenes. Canadian Journal of Zoology

8: 789–802.

Wong, S. W., P. T. Leung, A. B. Djurisic & K. M. Leung,

2010. Toxicities of nano zinc oxide to five marine

organisms: influences of aggregate size and ion solubility.

Analytical and Bioanalytical Chemistry 396(2): 609–618.

82 Hydrobiologia (2011) 669:63–82

123