Chemosphere 112 (2014) 9–17

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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Deformities of chironomid larvae and heavy metal pollution:

From laboratory to field studies
A. Di Veroli, F. Santoro, M. Pallottini, R. Selvaggi, F. Scardazza, D. Cappelletti, E. Goretti ⇑
Dipartimento di Chimica, Biologia e Biotecnologie, Università degli Studi di Perugia, Via Elce Di Sotto, 06123 Perugia, Italy

h i g h l i g h t s

 We used chironomid deformities as toxicity endpoint in a stream contaminated.

 A relationship between sediment metals and deformities in a field study was observed.
 Sediment metals play a pivotal role on the deformities, manly on the severe ones.
 Deformities can be used as bioassessment of metal polluted freshwater ecosystems.

a r t i c l e i n f o a b s t r a c t

Article history: Mouthpart deformities of Chironomus riparius larvae (Diptera) have been investigated to evaluate the
Received 18 July 2013 toxic effects of contamination by heavy metals in the Genna Stream (Central Italy), situated in an area
Received in revised form 12 March 2014 subjected to intensive swine farms (40 000 heads). The livestock farming (fertirrigation) contributes to
Accepted 16 March 2014
metal pollution of the Genna Stream with an increase of copper, zinc, cadmium, chromium and nickel
in the sediments of the downstream stations. The incidence of mentum deformities was very high at
Handling Editor: S. Jobling all sampling stations, about 56%. The highest values of deformities were found in the intermediate river
reach (St. 3: 65%) and in March (66%), mainly due to an increase in severe deformities. The high incidence
Keywords: of severe deformities (30%) is attributed to the high pollution level by heavy metals in the sediments, in
Mentum deformities particular to copper and zinc, which showed the highest average value at St. 3 and in March. This field
Chironomus riparius study reflected the relationships between sediment metal concentrations and chironomid mouthpart
Sediment toxicity deformities, previously observed in laboratory tests, and highlighted these deformities as toxicity
Heavy metals endpoints. This feature paves the way for their use as an effective tool in freshwater bioassessment
Livestock breeding monitoring programs to evaluate the toxic effects of metal contamination in freshwater ecosystems.
Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction interactions, mobility, biological availability and potential toxicity

Rivers are prone to pollution since they serve as conveyors for
dissolved and particulate matter, and sediments in particular play
an important role in the accumulation and transport of nutrients,
metals, and other contaminants through river systems (Orkun
et al., 2011).
Stream sediments can act as metal reservoirs, with the primary
exchange modes being adsorption or precipitation, and the
sediment-associated metals can be released into the water column
and/or accumulate in plants and animals, thus entering the food
web (Gibbs, 1977; Jain and Sharma, 2001; Filgueiras et al., 2002;
Orkun et al., 2011). Moreover, in sediments, heavy metals can be
present in a number of chemical forms, exhibiting different
⇑ Corresponding author. Tel./fax: +39 075 5855733.
E-mail addresses: enzo.goretti@unipg.it, benthos@unipg.it (E. Goretti).
(Arnason and Fletcher, 2003; Singh et al., 2005; Liu et al., 2009).
Zootechnical farms are an important source of heavy metals,
which can be conveyed in the river systems through punctual
(irregular emptying) or diffused (fertilizer irrigation) drains.
In recent years particular attention has been given to the opti-
mal concentration of metals used as oligoelements in animal food,
on one hand to fully meet the nutritional needs of the animals and
on the other hand mitigating the adverse effects of the current lev-
els of some metals on human health and the environment (Cottrill,
2003; Mantovi and Bonazzi, 2004). In particular, copper and zinc
(Eckel et al., 2003), essential oligoelements for the maintenance
of various physiological processes in animals, are used in high
doses because of their generally very low assimilation efficiency.
Due to improper disposal of farm effluents, a substantial part of
the metals contained in animal food may end up in the receiving
water bodies (Mantovi and Bonazzi, 2004). Aquatic organisms

http://dx.doi.org/10.1016/j.chemosphere.2014.03.053
0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.
10 A. Di Veroli et al. / Chemosphere 112 (2014) 9–17
can bioaccumulate, biomagnificate or biotransfer these metals
reaching concentrations high enough to cause harmful effects
(Naimo, 1995; Liu et al., 2009). Consequently, aquatic biota, espe-
cially insect larvae may serve as sentinel organisms in freshwater
biomonitoring programs (Hare and Tessier, 1998; Nahmani and
Rossi, 2003; Santoro et al., 2009). Moreover, many insect taxa are
strongly related to the sediment, making them useful bioindicators
of sediment quality, representing local environmental conditions.
Most species of the large insect family Chironomidae live in
freshwater environments. Among these, the species Chironomus
riparius Meigen, 1804 is a common and widely distributed organ-
ism, present in high densities also under eutrophic conditions. It
is often used as a key organism in biotic indexes, in environmental
assessing programs and in toxicity tests (Ristola et al., 1999; De
Haas et al., 2004; OECD, 2004; León Paumen et al., 2008; Di Veroli
et al., 2012b). Growth and emergence are used as toxicity end-
points in these kinds of protocols, but also morphological deformi-
ties (i.e. mouthparts) of these larvae are becoming an interesting
endpoint for freshwater ecosystems assessments. Chironomids
deformation analysis is applicable under both field and laboratory
conditions, so deformities screening offers an attractive biological
instrument to detect and monitor toxicants in sediments
(Hamilton and Saether, 1971; Warwick, 1988; Canfield et al.,
1994; Madden et al., 1995; Watts and Pascoe, 1996).
Previously, we studied the relationship malformations and
heavy metal concentrations in sediments (Di Veroli et al., 2012b)
in laboratory tests with C. riparius larvae. Specifically, we focused
on the induction of mouthparts deformities by mixtures of metals
(Cr, Ni, Cu, Zn, Cd and Pb). This study pointed out significant corre-
lations between metal concentrations in the sediments and
deformities.
Also, we demonstrated significant correlations between metals
concentration in sediments and deformities, only when larvae
Fig. 1. Map of the study area and location of the sampling stations.
were exposed to a known matrix of sediment artificially contami-
nated with respect to natural sediments, by including a complex
unknown toxic mixture. In fact, in the natural sediments toxicants
may have a long lasting persistence and interact in different com-
binations with newly introduced elements (Marking, 1985).
The aim of the present study was to extend the above approach
to the field, by evaluating the incidence of mouthpart deformities
of C. riparius larvae as a measure of the toxicity of environmental
pollutants, in particular heavy metals, in a water course draining
an area subjected to intensive livestock activity.
2. Materials and methods
2.1. Study area
The Genna Stream originates near the city of Perugia (Umbria)
and 22.8 km further south it enters the Nestore River, which is a
tributary of the Tiber River (Central Italy). The hydraulic regime
of the Genna Stream is strongly influenced by seasonal rainfall
(ARPA Umbria, 2010).
The sampling (2010–2012) was conducted at four stations lo-
cated along the entire stream, ranging from 242 m a.s.l. (St. 1) to
199 m a.s.l. (St. 4). The last station is located about 0.7 km before
the confluence with the Nestore River and about 2 km downstream
from the wastewater treatment plant (biodigester) from the swine
farms (40 000 heads, at full capacity) (Fig. 1). Since summer 2009, a
large number of swine farms in the catchment area of the Genna
Stream no longer employed the sewage treatment plant which
was shutdown, because of management decisions. Currently
farmers store the wastes in lagoons next to the farms, to then dis-
pose them through agricultural fertirrigation on soils surrounding
the catchment area of the Genna Stream without treatment. This
 A. Di Veroli et al. / Chemosphere 112 (2014) 9–17
situation is increasing the pollution load carried to the receiving
stream.
2.2. Sampling campaign
A preliminary survey was carried out (May 2010 – February
2011) at Station 4 to analyze the presence and consistency of larval
deformities of C. riparius. The actual sampling campaign covered
the entire stream and consisted of three seasonal samplings for
each station (St. 1–4) from March to October 2012. Several repli-
cates were performed to dispose of a significant number of larvae
(IV instar) of C. riparius (about 300–500 specimens for sampling
site/season). Water samples were collected in 500 ml polyethylene
bottles (5 sub-samples of water, 100 ml each), and then stored in a
refrigerator at 5 °C until analysis. Chironomid larvae samples were
collected with a dragnet equipped with a 335-lm mesh. The sam-
ples were transported to the laboratory for in vivo sorting, and then
fixed in 70% alcohol. Sediment samples were also taken for heavy
metals determination. Samplings were carried out with a hand
dredge and interested the superficial layer of the bottom sediments
(3–5 cm). The samples (500 g) were preserved in pirex glass bottles
(5 sub-samples of the sediment, about 100 g each) and kept refrig-
erated at 18 °C (MATT and APAT, 2005).
2.3. Chemical and physico-chemical parameters of surface water
The following physico-chemical water-parameters were mea-
sured in situ: water temperature, dissolved oxygen, pH and con-
ductivity. COD (chemical oxygen demand) determination was
performed by colorimetric method. The concentrations of anion
and cation species were determined in the laboratory by
Suppressed Ion Chromatography after filtration of the samples
with cellulose filters (0.2 lm) (Selvaggi et al., 2010).
2.4. Mouthpart deformities of chironomid larvae
Chironomus riparius (Diptera, Chironomidae) is non-biting
midge widely used in monitoring programs and in ecotoxicity test-
ing (Ristola et al., 1999; De Haas et al., 2004; León Paumen et al.,
2008; Di Veroli et al., 2012a,b); standard OECD guidelines for sed-
iment toxicity are also available for this species (OECD, 2004).
Chironomid larvae live in close contact with freshwater sediments
and develop in four instars. In this study we investigated
morphological deformities of the mentum of chironomid larvae
belonging to the last instar (IV instar). Each specimen was classi-
fied according to a morphological response protocol (modified
from Janssens de Bisthoven et al., 1998; Di Veroli et al., 2010): class
1 (CL. 1 – specimens without morphological deformity); class 2 (CL.
2 – specimens with weak deformity); class 3 (CL. 3 – specimens
with strong deformity).
2.5. Heavy metals analysis
Concentrations of heavy metals (Cr, Ni, Cu, Zn, Cd and Pb) in
sediments, larval tissues and water samples were determined by
Inductively Coupled Plasma Optical Emission Spectrometry
(ICP-OES Ultima 2, HORIBA Scientific, instrumental detection limit:
0.14–1.58 lg L 1) equipped with ultrasonic nebulizer (CETAC
Technologies, U-5000AT). Commercially produced (ICP multi-
element standard solution IV CertiPURÒ, VWR Merck Chemicals
and Reagents) standard solutions (1000 mg L 1) in nitric acid were
used to prepare appropriate elemental calibration standards.
The water samples were acidified with ultrapure nitric acid
(Fluka, TraceSELECTÒ, for trace analysis P69%); while an acid
digestion was performed on the sediment (MATT and APAT,
2005) and on the larval tissue samples (Türkmen and Ciminli,
11
2007). Each measure of the metals in sediments, in larval tissues
and in water was based on two replicates.
The sediment samples were air-dried, disaggregated using a
mortar and pestle to pass through a 2 mm mesh sieve, dried at
105 °C for 24 h and digested as following: 15 mL concentrated
ultrapure nitric acid (Fluka, TraceSELECTÒ, for trace analysis
P69%) were added to the sample (2.0 g) and heated to 160 °C for
1 h, using an aluminum block heating digester (VELP Scientific,
Inc.); then, the vessel was cooled to room temperature and
10 mL ultrapure concentrated hydrochloric acid (Fluka,
TraceSELECTÒ, for trace analysis P37%) were added and the flask
was heated to 160 °C for 1 h. The mixture was cooled, filtered
(Whatman Grade No. 42, particle retention 2.5 lm) and diluted
with ultrapure water to 50 mL.
Chironomus riparius larvae were washed with double-distilled
water and frozen at 18 °C in glass containers.
Then, about 300/500 larvae per station were taken and thawed
for removing the head capsule to analyze the mouthpart deformi-
ties. The remaining parts of the larval bodies were pooled and
placed in an oven at 105 °C until reaching a constant weight. Diges-
tion of larval tissues was carried out using acid digestion with
ultrapure concentrated nitric acid: the samples (0.5 g) were heated
to 160 °C with HNO3 (2.5 ml) for 1 h, using an aluminum block
heating digester (VELP Scientific, Inc.); then vessels were cooled
to room temperature, and then re-heated with nitric acid 1 M
(2 ml) until almost dried. After digestion, samples were filtered
(Whatman Grade No. 42, particle retention 2.5 lm) and ultrapure
water was added to reach the desired volume (25 ml).
Therefore, metal concentrations were determined in pooled
chironomid larvae samples, while deformities were checked per
single individual.
2.6. Statistical analysis
The observed differences of the deformities incidences were
explored with Chi-square test (v2).
Principal component analysis (PCA), as ordination technique,
was used to establish associations among the sampling sites and
the heavy metals (in sediments, larval tissues and water).
Pearson correlation was employed to reveal the degree of asso-
ciation between the metal contamination (based on the PC
extracted from the PCA analysis), and the incidence of deformities
(arcsin transformed percent values) at the sampling sites.
These analyses were performed for the year 2012 campaign at
Stations 1–4, and were not performed for the 2010 exploratory
campaign, because of the reduced set of data (monitoring of heavy
metals only in the sediments and deformities at Station 4).
The statistical treatment was performed with the R software
package (2.7.2) (R Development Core Team, 2008).
3. Results
The preliminary survey of the most impacted sampling site
(Station 4: 2010–2011) at the Genna Stream showed a condition
of strong alteration of the environment, particularly for the chem-
ical and physical–chemical parameters of the water, indicative of a
strong organic pollution, and of the high concentrations of heavy
metals in the sediments and the high incidence of malformations
for C. riparius larvae (IV instar). The actual sampling campaign
(Stations 1–4: year 2012) confirmed this state of degradation for
the entire stream (Tables 1–3). The results of the survey are
discussed below.
The chemical and physical–chemical parameters of the water
samples showed that the environmental condition of the Genna
Stream was affected by strong pollution, as evidenced by high
 12 A. Di Veroli et al. / Chemosphere 112 (2014) 9–17

Table 2
Incidence of morphological deformity classes of C. riparius larvae from the stations (St.
1

125.16
118.27

113.82

125.91
144.02

102.85

109.76
106.21

107.17

118.01
104.02

103.04

109.60
106.00
99.89

83.63

97.95
84.88
mg L
Ca2+

1–4; year 2012) of the Genna Stream (CL. 1 – specimens without any morphological
deformity, CL. 2 – specimens with weak deformity, CL. 3 – specimens with strong

–
deformity) and from St. 4 (years 2010–2011), during the explorative sampling; ‘‘–’’,
1
Chemical and physico-chemical parameters of the surface water in at the sampling stations (St. 1–4; year 2012) of the Genna Stream and of St. 4 (years 2010–2011), during the explorative sampling; ‘‘–’’, dry stream.

dry stream.
15.88

12.48
16.55
13.85

12.59

12.12
11.66
13.53
12.44

11.69
13.41
11.22

11.83
13.25
11.61
10.49
Mg2+
mg L

8.57

9.75
–

–
St Date CL. 1 CL. 2 CL. 3 Tot. %CL. (2 + 3) %CL. 3
1

4 June 2010 245 42 23 310 20.97 7.42

16.53
11.64
11.87
13.34

12.96
13.16
11.69

13.74

12.44
12.03

10.52

13.07
mg L
3.89

6.83
7.94

6.23

8.96
9.01

4 August 2010 162 60 71 293 44.71 24.23

K+

4 October 2010 88 67 153 308 71.43 49.68

4 February 2011 113 69 128 310 63.55 41.29
1

33.65
58.29
68.23
48.87
52.26

66.42

72.26
77.58

75.78
54.93

68.32
59.77
78.32

72.93
77.50

77.04
73.01

80.70
Tot. 608 238 375 1221 50.20 30.71
mg L
Na+

1 March 2012 – – – – – –
–

1 June 2012 146 93 64 303 51.82 21.12

1

1 October 2012 – – – – – –
mg L
<0.5
<0.5
<0.5
<0.5
<0.5

<0.5

<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
2 March 2012 195 148 155 498 60.84 31.12
Li+

2 June 2012 174 94 26 294 40.82 8.84

2 October 2012 147 105 54 306 51.96 17.65
1

17.66

11.99
11.56
14.46

12.32
13.35
14.25
11.53
10.93

13.04
mg L
2.55

7.59
5.86

4.39

9.91
8.41

Tot. 516 347 235 1098 53.01 21.40

5.30
8.02
NH+4

3 March 2012 110 112 271 493 77.69 54.97

3 June 2012 175 88 45 308 43.18 14.61
1

25.46
66.81
37.55

25.85
11.46

17.43
31.10

40.23

10.77
mg L

3 October 2012 106 71 128 305 65.25 41.97

5.74

5.66
2.13

6.19
3.32
6.23

8.99
0.77

2.80
NO3

Tot. 391 271 444 1106 64.65 40.14

–

4 March 2012 203 155 140 498 59.24 28.11

1

4 June 2012 133 100 77 310 57.10 24.84

mg L
1.29
3.91

1.58

2.21
1.74
1.85

2.44
1.01

2.90

0.68

0.88
<0.1

<0.1

<0.1

<0.1
<0.1

<0.1
<0.1
NO2

4 October 2012 127 90 92 309 58.90 29.77

–

Tot. 463 345 309 1117 58.55 27.66

1

51.16
45.97

14.39

45.63
52.64
47.99
48.75

49.27
41.17
31.59
42.19

41.96
46.70
56.60

30.93
43.30

52.09
50.10
mg L
SO24

–
1

values of conductivity, COD, nitrogen (particularly ammonium)

mg L

3.92
5.48

3.43

2.42

4.78
7.84

6.56
2.83
6.31

4.87
8.18
5.95
6.33
7.05

6.06
5.07
0.90
PO34

n.d.

and low values of D.O. In March and October 2012, St. 1 was dry
–

(Table 1).
1

In total 4875 larvae of C. riparius (IV instar) (Table 2) were sam-

mg L
<0.1
<0.1
<0.1
<0.1
<0.1

<0.1

<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1
<0.1

pled during the whole investigation. The incidence of mentum

Br

deformities (CL. 2 + 3) was generally high, about 56.2% (29.5% in

1

113.14

125.43
105.54

110.16
105.49

106.36

105.15
104.70

CL. 3) with higher values at Station 3 (64.7%), mainly because of

71.76
95.96
68.48
69.24

93.19

77.71

95.91
93.33
85.32
40.77
mg L

the higher incidence of strong deformities (40.1% in CL. 3). In year

Cl

2012, weak deformities (CL. 2) showed, with the exception of Sta-

1

tion 3 (24.5%), constant values around 31%.

mg L
3.43

6.94

2.86
0.65

0.43

0.44

0.32

0.24
0.51

0.23
0.44
0.33
0.75
0.61
0.56
0.20
0.20

0.60

June presented the lowest incidence of deformities (CL. 2 + 3) at

–

–
F

all stations, while March the highest one, with average values
mg L 1
C.O.D.

47.33

41.67

46.17
42.50
19.50

64.50

25.50

67.50
44.50
26.50
16.00
31.00
13.00
17.00

61.00

35.00
80.00

respectively of 48.2% (17.4% in CL. 3) and of 65.9% (38.1% in CL.

8.00

3). The Chi-square test (year 2012: stations 1–4) showed that the
–

observed differences were statistically significant (v2 = 327.56,

1
mg L

p < 0.01) among normal (CL. 1), weakly (CL. 2) and strongly (CL.
6.48

3.67

3.73
8.50
4.30
4.90
8.20

1.20

1.30
2.10
2.80
5.10
2.20
3.70

4.80
1.90
4.50
5.00
D.O.

3) deformed specimens.
During the year 2012, the mean concentrations of Cd, Cr and Ni
8.16
7.73
7.88
8.16
7.98

8.17
7.61
7.73
7.84
7.88
7.77
7.92
7.86
7.97
7.75
7.97
7.20

7.90
pH

in the sediments were higher at St. 4, Zn and Cu at St. 3, and Pb at

–

St. 2. In larval tissues the highest average concentrations were de-

1
lS cm

tected at St. 4 for all metals except for Ni (St. 3). In the water the
Cond.

1022

1018
771
783
881
815
813

955

962

969
994
888
739
996

918
880

909

700

average highest metal concentrations were found at St. 2 except

–

for Zn (St. 4).

Wat. T.

In sediments the mean concentrations of Cd, Zn and Cu were

14.25

16.33
21.40
15.20

12.70

18.10
17.10
11.60
22.70
16.10
16.80

23.60
15.30
15.00

20.30

20.50

10.10
5.40

higher in March, Cr and Ni in October and Pb in June. In larval tis-

°C

sues the highest average concentrations were found in June for all
Depth

metals except for Zn (October). In water the average highest metal

0.38

0.15

0.35
0.32

0.23
0.35

0.32
0.40
0.50
0.30
0.30

0.30
0.30

0.20
0.20
0.30

0.30
0.30
m

concentrations were detected in June.

TEC (Threshold Effect Concentration) and PEC (Probable Effect
Bed width

Concentration) reference values were used to predict sediment

6.75

5.17

4.67

6.17
3.50

5.50

4.50
4.50

6.50
7.00
8.00
6.00
6.00

5.00
5.00

5.00

6.00
6.00

toxicity by heavy metals, accordingly to the SQGs (Sediment

m

Quality Guidelines) (Mac Donald et al., 2000).

The concentrations of Cd registered in the sediments of the
February 2011

October 2012

October 2012

October 2012

October 2012
October 2010
August 2010

March 2012

March 2012

March 2012

March 2012

Genna Stream, were always higher than the TEC reference value,
June 2012

June 2012

June 2012

June 2012
May 2010

except in June 2010 (explorative sampling) at St. 4. Cd exceeded

Mean

Mean

Mean

Mean
Date

the PEC value only in October 2010 (explorative sampling) at St.

4. Pb exceeded the TEC value in August and October 2010 at St. 4
Table 1

and in June 2012 at St. 1 and 2. Ni concentrations exceeded the

St

4
4
4
4

1
1
1
2
2
2

3
3
3

4
4
4

TEC value except in March and June 2012 at St. 2. The values of
Table 3a
Heavy metals (cadmium, lead, zinc, chromium, nickel and copper) in sediments, water and larval tissues of Genna Stream stations (St. 1–4; year 2012) and of St. 4 (years 2010–2011), during the explorative sampling; ‘‘–’’, dry stream.
TEC (Threshold Effect Concentration), PEC (Probable Effect Concentration) from Mac Donald et al. (2000).
1 1 1
a) Date Sediments (mg kg ) Larval tissues (mg kg ) Water (lg L )
St Cd Pb Zn Cr Ni Cu Cd Pb Zn Cr Ni Cu Cd Pb Zn Cr Ni Cu
4 June 2010 0.75 19.43 71.37 19.47 34.95 14.70
4 August 2010 1.30 49.38 79.31 16.47 33.19 27.18
4 October 2010 5.66 36.18 109.60 26.57 38.03 27.14

A. Di Veroli et al. / Chemosphere 112 (2014) 9–17

4 February 2011 1.89 28.24 90.80 16.83 35.72 23.35
Mean 2.40 33.31 87.77 19.84 35.47 23.09
1 March 2012 – – – – – – – – – – – – – – – – – –
1 June 2012 3.69 53.81 90.60 22.88 31.71 40.44 1.42 2.72 278.20 10.83 36.81 17.51 10.28 6.23 24.71 4.75 14.84 0.14
1 October 2012 – – – – – – – – – – – – – – – – – –
2 March 2012 2.96 25.68 68.12 14.22 18.55 24.84 0.52 1.48 274.55 7.86 5.74 20.38 8.98 5.33 106.26 4.38 9.80 5.49
2 June 2012 2.49 47.95 72.14 16.44 21.66 24.64 1.07 1.59 213.27 5.72 26.52 15.75 10.33 15.99 136.84 7.56 15.85 31.24
2 October 2012 3.05 31.57 81.86 29.78 22.83 24.88 0.92 2.58 289.72 4.38 5.44 11.88 10.23 5.37 44.38 4.63 10.51 0.65
Mean 2.84 35.07 74.04 20.15 21.01 24.79 0.84 1.88 259.18 5.99 12.57 16.00 9.85 8.90 95.83 5.52 12.05 12.46
3 March 2012 3.66 23.80 95.85 22.52 24.26 40.53 0.41 1.10 232.63 2.24 2.00 15.04 8.86 4.55 26.46 4.50 10.24 3.49
3 June 2012 2.24 33.73 91.55 16.60 22.76 34.16 1.49 5.73 216.64 7.93 34.03 26.89 10.02 12.85 35.22 5.28 11.22 2.04
3 October 2012 2.37 21.84 75.95 15.28 23.69 20.60 1.25 6.78 228.93 6.26 10.07 33.46 9.86 4.91 42.55 4.76 11.55 0.57
Mean 2.76 26.46 87.78 18.13 23.57 31.77 1.05 4.54 226.06 5.48 15.37 25.13 9.58 7.44 34.75 4.85 11.00 2.03
4 March 2012 3.89 22.31 102.91 25.65 30.55 28.34 0.36 1.73 225.57 2.56 2.74 16.16 8.86 4.14 23.46 4.59 10.38 6.01
4 June 2012 4.37 25.99 62.78 26.17 35.64 19.85 2.27 10.53 363.89 11.95 16.65 34.71 10.14 9.25 242.34 4.96 12.34 0.14
4 October 2012 3.84 28.92 78.92 36.02 42.90 22.95 1.43 4.91 387.58 8.26 11.09 25.41 9.84 5.59 45.62 5.02 12.47 1.23
Mean 4.03 25.74 81.54 29.28 36.36 23.71 1.35 5.73 325.68 7.59 10.16 25.43 9.61 6.33 103.81 4.86 11.73 2.46
TEC 0.99 35.8 121 43.4 22.7 31.6
PEC 4.98 128 459 111 48.6 149

13
14 A. Di Veroli et al. / Chemosphere 112 (2014) 9–17

Table 3b BSAF decreased, as follows: St. 2: Zn > Cu > Ni > Cr > Cd > Pb; St.
BSAFs (Biota to Sediment Accumulation Factor: ratio between metal in tissues and in 3: Zn > Cu > Ni > Cd > Cr > Pb; St. 4: Zn > Cu > Cd > Ni > Cr > Pb; as
the sediments) in C. riparius larvae; ‘‘–’’, dry stream.
far as the seasons were concerned, BSAF decreased, as follows:
b) BSAF Cd Pb Zn Cr Ni Cu June: Zn > Cu, Ni > Cd > Cr > Pb; March: Zn > Cu > Cr > Ni > Cd > Pb;
1 March 2012 – – – – – – October: Zn > Cu > Cd > Ni > Cr > Pb (Table 3b).
1 June 2012 0.38 0.1 3.1 0.5 1.2 0.43 PCA carried out on metals in sediments, water and larval tissues
1 October 2012 – – – – – – allowed the level of contamination due to heavy metals to be ana-
2 March 2012 0.2 0.1 4.0 0.6 0.3 0.8 lyzed at the stations of the Genna Stream over the year 2012.
2 June 2012 0.4 0.0 3.0 0.3 1.2 0.6 Dim 1 (33.80%) showed a seasonal gradient, where March and
2 October 2012 0.3 0.1 3.5 0.1 0.2 0.5
Mean 0.30 0.06 3.51 0.35 0.59 0.65
June were placed on the left and right side of the axis respectively.
In March, the stations were mainly associated to heavy metals in
3 March 2012 0.1 0.0 2.4 0.1 0.1 0.4
3 June 2012 0.7 0.2 2.4 0.5 1.5 0.8
the sediments, in particular Zn and Cu, whereas in June the stations
3 October 2012 0.5 0.3 3.0 0.4 0.4 1.6 were associated to metals in water and larval tissues. In October,
Mean 0.44 0.18 2.60 0.33 0.67 0.93 the stations were in an intermediate position.
4 March 2012 0.09 0.08 2.19 0.10 0.09 0.57 PCA in Dim 2 (27.80%) showed a gradient partially reflecting the
4 June 2012 0.52 0.41 5.80 0.46 0.47 1.75 longitudinal profile of the Genna Stream over each seasons, where
4 October 2012 0.37 0.17 4.91 0.23 0.26 1.11 downstream station (St. 4) was placed on the upside of Dim 2 and
Mean 0.33 0.22 4.30 0.26 0.27 1.14
mainly associated to Cd, Ni and Cr in the sediments and to Zn, Pb
and Cu in larval tissues. The remaining stations (in particular at
June) were placed on the downside of Dim 2 and mainly affected
Cu exceeded the TEC in June 2012 at St. 1 and in March and June by metals Cu, Cr, Pb, Ni in water, and by Pb in the sediments and
2012 at St. 3. Zn and Cr never exceed the TEC values (Table 3a). by Ni in larval tissues (Fig. 2a–b).
Eventually, in year 2012, the mean concentration of heavy met- No significant correlations between PC (Dim 1 and 2), extracted
als in the sediments for each station never exceeded the PEC from PCA analysis, and the morphological deformity classes of
threshold, while the TEC threshold was exceeded for Cd at all the C. riparius (angular transformation was applied to percent values)
stations, for Ni at St. 3 and 4, and for Cu at St. 3. nor between single heavy metals in different compartments
The BSAFs (Biota to Sediment Accumulation Factor: ratio be-
tween metal concentration in tissues and sediments) in C. riparius
larvae were >1 for Zn (all samples), for Ni (June 2012, St. 1–3) and Table 4a
for Cu (October 2012, St. 3 and 4; June 2012, St. 4). The BSAF for Correlation between the degree of contamination by heavy metals, based on the PC
Genna Stream was metal-specific, with Zn and Cu always showing extracted from the PCA analysis, and the incidence of deformities (CL.) (arcsin
transformed percent values); Dim 1 and Dim 2, first two axes of PCA; r, Pearson’s
the highest accumulation factors and Pb always at the lower limit correlation coefficient; p, probability value.
of the series. Specifically, as far as the stations were concerned,
Heavy metals: all toxicity
CPA Dim Deformity r p
(A) 4Jun Dim 1 CL. 2 0.368 0.295
4Oct Dim 1 CL. 3 0.542 0.105
2

4Mar 3Oct Dim 1 CL. 2+3 0.570 0.086

Dim2 (27.80%)

2Oct Dim 2 CL. 2 0.055 0.880

Dim 2 CL. 3 0.504 0.138
0

3Mar 2Mar
Dim 2 CL. 2+3 0.522 0.122
1Jun
-2

3Jun
-4

2Jun Table 4b
Correlation between the degree of contamination by single heavy metals in different
compartments (sediments, larval tissues and water) and the incidence of deformities
-4 -2 0 2 4
(CL.) (arcsin transformed percent values); r, Pearson’s correlation coefficient; p,
Dim1 (33.80%) probability value.

Heavy metals: Sediments Larval tissues Water

(B) Cd(s) Zn(t)
Pb(t) Deformity r p r p r p
Ni(s)
0.5

Cr(s)
Cd CL. 2 0.208 0.564 0.234 0.515 0.436 0.208
Cu(t) Cd(t)
Cd CL. 3 0.293 0.411 0.346 0.327 0.570 0.030
Dim2 (27.80%)

Cr(t) Cd CL. 2+3 0.387 0.270 0.390 0.265 0.701 0.024

Zn(w) Pb CL. 2 0.401 0.251 0.001 0.998 0.298 0.403
0.0

Pb CL. 3 0.658 0.039 0.026 0.944 0.735 0.015

Zn(s) Pb CL. 2+3 0.657 0.039 0.082 0.822 0.756 0.011
Cd(w) Zn CL. 2 0.231 0.521 0.323 0.363 0.380 0.279
Cu(s) Ni(t)
Zn CL. 3 0.191 0.597 0.036 0.922 0.263 0.463
-0.5

Ni(w)
Pb(s)
Zn CL. 2+3 0.176 0.626 0.080 0.826 0.204 0.086
Cu(w)
Pb(w) Cr CL. 2 0.329 0.353 0.290 0.416 0.258 0.472
Cr(w)
Cr CL. 3 0.041 0.911 0.315 0.375 0.643 0.045
-0.5 0.0 0.5 Cr CL. 2+3 0.116 0.749 0.359 0.308 0.639 0.086
Ni CL. 2 0.153 0.673 0.222 0.538 0.277 0.438
Dim1 (33.80%) Ni CL. 3 0.072 0.842 0.638 0.047 0.553 0.086
Ni CL. 2+3 0.080 0.826 0.712 0.021 0.564 0.090
Fig. 2. PCA (first two axes) carried out on heavy metals (Cr, Ni, Cu, Zn, Cd and Pb) in Cu CL. 2 0.284 0.426 0.271 0.448 0.215 0.551
different compartments: sediments (s), water (w), and larval tissues (t), at the Cu CL. 3 0.163 0.653 0.150 0.680 0.477 0.163
sampling stations of Genna Stream over the year 2012; (A), sampling sites; (B), Cu CL. 2+3 0.151 0.677 0.048 0.895 0.439 0.204
heavy metals in different compartments.
 A. Di Veroli et al. / Chemosphere 112 (2014) 9–17
(sediments, larval tissues and water) and deformities were observed
(Table 4).
4. Discussion
In the present paper we characterized the heavy metal contam-
ination of the Genna stream, and we demonstrated that mouthpart
deformities of C. riparius larvae are the endpoint of toxicity, after
investigating the relationships between such deformities and
metal contamination in natural conditions.
The contamination of the stream appeared to be related to the
to discharges of the South-west zone of the city of Perugia (about
150 000 inhabitants). In addition, towards the valley part of the
catchment of the Genna Stream is subjected to intensive swine
farms (about 80 farms), which contribute (via agricultural fertirri-
gation) to metal pollution (except lead) from St. 2 to the confluence
with the Nestore River (St. 4), thus explaining the increase of zinc
and copper at the St. 3, and of cadmium, chromium, nickel and zinc
at St. 4.
Mouthpart deformities of C. riparius larvae observed in the pres-
ent work, resulted an efficient endpoint of toxicity assessment in
the Genna Stream, because more than the half (56%) of the exam-
ined C. riparius larvae (about 5000) were deformed (CL. 2 + 3). This
result reflects the high level of heavy metals contamination in the
stream (sediments, in particular), and confirms previous laboratory
studies, in which mouthpart deformities were demonstrated to be
actual bioindicators of environmental stress (Martinez et al., 2002;
Di Veroli et al., 2012b).
By contrast, the natural background incidence of deformities,
calculated in chironomid populations that inhabit non-contami-
nated environments or with a modest pollution load, was esti-
mated below 10% (Wiederholm, 1984; Warwick, 1985; Madden
et al., 1992; Bird, 1994; Di Veroli et al., 2012a).
Despite the large number of studies on chironomids deformi-
ties, clear dose–response relationships were established for only
a few chemical substances, in particular heavy metals (Kosawalt
and Knight, 1987; Vermeulen et al., 2000). In experimental
conditions, the deformities of chironomids were induced by expo-
sure to single metals, such as cadmium, copper, lead and zinc
(Van de Guchte and van Urk, 1989; Michailova and Belcheva,
1990; Bird et al., 1995; Janssens de Bisthoven et al., 2001;
Martinez et al., 2003) and by exposure to metal mixture (Di
Veroli et al., 2012b).
In the present study, among heavy metals examined in natural
conditions in different compartments (sediments, larval tissues
and water), the metals in the sediments, particularly copper and
zinc, showed an association with the stations sampled in March,
where deformities (CL. 2 + 3: 66%) were prevalent, in particular,
the severe ones (CL. 3: 38%). In addition, in this month, the mean
concentrations of Cu and Zn in the sediments were higher (respec-
tively 31.2 and 89.0 mg kg 1) than in the remaining months. Con-
versely, the heavy metals in water (a very dynamic compartment)
and in larval tissues showed an association with the stations sam-
pled in June, month in which the deformities (CL. 2 + 3: 48%) were
not prevalent, because the severe ones were reduced (CL. 3: 17%).
Therefore, deformities reflected mainly the heavy metal contami-
nation in sediments more than larval tissue and/or water
contamination.
These results of the effects of heavy metals examined in differ-
ent compartments (sediments, larval tissues and water) are in
agreement with previous investigations carried out under
controlled conditions, where significant correlations between
deformities and metal concentrations have been observed only
for sediments artificially contaminated, but not for larval tissues
(Di Veroli et al., 2012b). In particular, laboratory tests indicated a
15
direct dependence of the severity of deformities with the heavy
metal concentrations in the sediments.
The concentrations of metals in larval tissues of C. riparius are
not correlated with those in sediments and with deformities, most
likely because the physiological and metabolic functions and the
environmental conditions influenced the bio-availability of the
metals, likewise observed in many macroinvertebrate species
(Bervoets et al., 1997; Besser et al., 2008; Desrosiers et al., 2008;
Faria et al., 2008; Péry et al., 2008).
Bioaccumulation processes in C. riparius were fairly homoge-
neous along the Genna Stream as shown by similar trend of BSAF
for the six heavy metals in different stations and months. In partic-
ular, zinc and copper (essential micro-elements, toxic only in case
they exceed a particular threshold) were always the metals with
the highest rate of bioaccumulation, while lead was the lowest.
Notwithstanding the high incidence of deformities detected in
the Genna Stream (in particular the severe ones, CL. 3: about
30%), the correlation between heavy metals in sediments and
deformities was not significant. It seems acceptable that the ab-
sence of such a correlation could be due to a mixture of toxicants,
including mineral and organic compounds, which are responsible
for the incidence of the larval deformities of C. riparius in the Genna
Stream (Di Veroli et al., 2012b).
Our results suggest that the major cause of morphological
deformities could be due to the synergistic effect of a mixture of
toxicants, more than of a single contaminant, in agreement with
previous studies carried out in natural and experimental condi-
tions (Vermeulen, 1995; Martinez et al., 2003; Di Veroli et al.,
2010, 2012a,b).
5. Conclusions
This field study confirms that the high heavy metal concentra-
tions (particularly copper and zinc) in the sediments play a pivotal
role on the morphological deformities, manly on the severe ones,
observed in chironomid larvae.
These data are in agreement with previous experimental tests
and highlight that chironomid mouthpart deformities represent
an effective endpoint for freshwater bioassessment monitoring
programs to evaluate the toxic effects of contaminants, such as
heavy metal.
Chironomus riparius, a tolerant species able to survive at chem-
ically contaminated sites, successfully colonizes freshwater sys-
tems that receive high loads of organic matter. On this account,
this species represents a valid bioindicator to be used in particu-
larly polluted environments, such as those affected by wastewater
of the livestock farming.
The assessment of heavy metals, among the most common
environmental pollutants, in river sediments can be of great
importance for human and environmental health (Graney and
Eriksen, 2004; Singh et al., 2005; Liu et al., 2009). In fact, the eco-
logical impact of metals can be crucial because of their toxicity
(each of them has different toxic effects), persistence, and non-
degradability in the environment (Luoma, 1983; De Souza et al.,
1986; Lacerda et al., 1992; Allen and Hansen, 1996).
Finally, the present study highlights the importance to evaluate
the toxic effects of contamination in freshwater ecosystems by
carrying on, in parallel, investigations with the help of laboratory
bioassays, for a better comprehension of the effects of the environ-
ment pollution on the biota.
Acknowledgments
The authors thank the entire team of the laboratory, in particu-
lar Dr. A. Fabrizi, Dr. F. Luchetti, Dr. V. D’Allestro and Dr. R.M. Pel-
16 A. Di Veroli et al. / Chemosphere 112 (2014) 9–17

legrino for supporting the research program during field investiga- Janssens De Bisthoven, L., Postma, J., Vermeulen, A., Goemans, G., Ollevier, F., 2001.
Morphological deformities in Chironomus riparius Meigen larvae after exposure
tions. A special thanks goes to Prof. E. Gaino for her valuable sug-
to cadmium over several generations. Water, Air, Soil Pollut. 129, 167–179.
gestions. Finally, we express our gratitude to the Foundation Kosawalt, P., Knight, A.W., 1987. Chronic toxicity of copper to a partial life cycle of
Cassa di Risparmio di Perugia for the financial support, and to the midge, Chironomus decorus. Arch. Environ. Contam. Toxicol. 16, 283–290.
the anonymous referees for their helpful suggestions, which mark- Lacerda, L.D., Fernandez, M.A., Calazans, C.F., Tanizaki, K.F., 1992. Bioavailability of
heavy metals in sediments of two coastal lagoons in Rio de Janeiro, Brazil.
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