Chemosphere 112 (2014) 9–17

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Chemosphere

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Deformities of chironomid larvae and heavy metal pollution:From laboratory to field studies

http://dx.doi.org/10.1016/j.chemosphere.2014.03.0530045-6535/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author. Tel./fax: +39 075 5855733.E-mail addresses: enzo.goretti@unipg.it, benthos@unipg.it (E. Goretti).

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

Article history:Received 18 July 2013Received in revised form 12 March 2014Accepted 16 March 2014Available online 21 April 2014

Handling Editor: S. Jobling

Keywords:Mentum deformitiesChironomus ripariusSediment toxicityHeavy metalsLivestock breeding

a b s t r a c t

Mouthpart deformities of Chironomus riparius larvae (Diptera) have been investigated to evaluate thetoxic effects of contamination by heavy metals in the Genna Stream (Central Italy), situated in an areasubjected to intensive swine farms (40000 heads). The livestock farming (fertirrigation) contributes tometal pollution of the Genna Stream with an increase of copper, zinc, cadmium, chromium and nickelin the sediments of the downstream stations. The incidence of mentum deformities was very high atall sampling stations, about 56%. The highest values of deformities were found in the intermediate riverreach (St. 3: 65%) and in March (66%), mainly due to an increase in severe deformities. The high incidenceof severe deformities (30%) is attributed to the high pollution level by heavy metals in the sediments, inparticular to copper and zinc, which showed the highest average value at St. 3 and in March. This fieldstudy reflected the relationships between sediment metal concentrations and chironomid mouthpartdeformities, previously observed in laboratory tests, and highlighted these deformities as toxicityendpoints. This feature paves the way for their use as an effective tool in freshwater bioassessmentmonitoring programs to evaluate the toxic effects of metal contamination in freshwater ecosystems.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Rivers are prone to pollution since they serve as conveyors fordissolved and particulate matter, and sediments in particular playan important role in the accumulation and transport of nutrients,metals, and other contaminants through river systems (Orkunet al., 2011).

Stream sediments can act as metal reservoirs, with the primaryexchange modes being adsorption or precipitation, and thesediment-associated metals can be released into the water columnand/or accumulate in plants and animals, thus entering the foodweb (Gibbs, 1977; Jain and Sharma, 2001; Filgueiras et al., 2002;Orkun et al., 2011). Moreover, in sediments, heavy metals can bepresent in a number of chemical forms, exhibiting different

interactions, mobility, biological availability and potential toxicity(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 andon 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 maintenanceof various physiological processes in animals, are used in highdoses because of their generally very low assimilation efficiency.

Due to improper disposal of farm effluents, a substantial part ofthe metals contained in animal food may end up in the receivingwater bodies (Mantovi and Bonazzi, 2004). Aquatic organisms

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

can bioaccumulate, biomagnificate or biotransfer these metalsreaching 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 freshwaterbiomonitoring programs (Hare and Tessier, 1998; Nahmani andRossi, 2003; Santoro et al., 2009). Moreover, many insect taxa arestrongly related to the sediment, making them useful bioindicatorsof sediment quality, representing local environmental conditions.

Most species of the large insect family Chironomidae live infreshwater environments. Among these, the species Chironomusriparius Meigen, 1804 is a common and widely distributed organ-ism, present in high densities also under eutrophic conditions. Itis often used as a key organism in biotic indexes, in environmentalassessing programs and in toxicity tests (Ristola et al., 1999; DeHaas et al., 2004; OECD, 2004; León Paumen et al., 2008; Di Veroliet 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 interestingendpoint for freshwater ecosystems assessments. Chironomidsdeformation analysis is applicable under both field and laboratoryconditions, so deformities screening offers an attractive biologicalinstrument 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 andheavy metal concentrations in sediments (Di Veroli et al., 2012b)in laboratory tests with C. riparius larvae. Specifically, we focusedon 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 anddeformities.

Also, we demonstrated significant correlations between metalsconcentration in sediments and deformities, only when larvae

Fig. 1. Map of the study area and lo

were exposed to a known matrix of sediment artificially contami-nated with respect to natural sediments, by including a complexunknown toxic mixture. In fact, in the natural sediments toxicantsmay 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 approachto the field, by evaluating the incidence of mouthpart deformitiesof C. riparius larvae as a measure of the toxicity of environmentalpollutants, in particular heavy metals, in a water course drainingan 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 atributary of the Tiber River (Central Italy). The hydraulic regimeof 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) to199 m a.s.l. (St. 4). The last station is located about 0.7 km beforethe confluence with the Nestore River and about 2 km downstreamfrom the wastewater treatment plant (biodigester) from the swinefarms (40000 heads, at full capacity) (Fig. 1). Since summer 2009, alarge number of swine farms in the catchment area of the GennaStream no longer employed the sewage treatment plant whichwas shutdown, because of management decisions. Currentlyfarmers store the wastes in lagoons next to the farms, to then dis-pose them through agricultural fertirrigation on soils surroundingthe catchment area of the Genna Stream without treatment. This

cation of the sampling stations.

A. Di Veroli et al. / Chemosphere 112 (2014) 9–17 11

situation is increasing the pollution load carried to the receivingstream.

2.2. Sampling campaign

A preliminary survey was carried out (May 2010 – February2011) at Station 4 to analyze the presence and consistency of larvaldeformities of C. riparius. The actual sampling campaign coveredthe entire stream and consisted of three seasonal samplings foreach 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 samplingsite/season). Water samples were collected in 500 ml polyethylenebottles (5 sub-samples of water, 100 ml each), and then stored in arefrigerator at 5 �C until analysis. Chironomid larvae samples werecollected with a dragnet equipped with a 335-lm mesh. The sam-ples were transported to the laboratory for in vivo sorting, and thenfixed in 70% alcohol. Sediment samples were also taken for heavymetals determination. Samplings were carried out with a handdredge 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 wasperformed by colorimetric method. The concentrations of anionand cation species were determined in the laboratory bySuppressed Ion Chromatography after filtration of the sampleswith cellulose filters (0.2 lm) (Selvaggi et al., 2010).

2.4. Mouthpart deformities of chironomid larvae

Chironomus riparius (Diptera, Chironomidae) is non-bitingmidge 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 sedimentsand develop in four instars. In this study we investigatedmorphological deformities of the mentum of chironomid larvaebelonging to the last instar (IV instar). Each specimen was classi-fied according to a morphological response protocol (modifiedfrom Janssens de Bisthoven et al., 1998; Di Veroli et al., 2010): class1 (CL. 1 – specimens without morphological deformity); class 2 (CL.2 – specimens with weak deformity); class 3 (CL. 3 – specimenswith strong deformity).

2.5. Heavy metals analysis

Concentrations of heavy metals (Cr, Ni, Cu, Zn, Cd and Pb) insediments, larval tissues and water samples were determined byInductively 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 (CETACTechnologies, U-5000AT). Commercially produced (ICP multi-element standard solution IV CertiPUR�, VWR Merck Chemicalsand Reagents) standard solutions (1000 mg L�1) in nitric acid wereused to prepare appropriate elemental calibration standards.

The water samples were acidified with ultrapure nitric acid(Fluka, TraceSELECT�, for trace analysis P69%); while an aciddigestion was performed on the sediment (MATT and APAT,2005) and on the larval tissue samples (Türkmen and Ciminli,

2007). Each measure of the metals in sediments, in larval tissuesand in water was based on two replicates.

The sediment samples were air-dried, disaggregated using amortar and pestle to pass through a 2 mm mesh sieve, dried at105 �C for 24 h and digested as following: 15 mL concentratedultrapure nitric acid (Fluka, TraceSELECT�, for trace analysisP69%) were added to the sample (2.0 g) and heated to 160 �C for1 h, using an aluminum block heating digester (VELP Scientific,Inc.); then, the vessel was cooled to room temperature and10 mL ultrapure concentrated hydrochloric acid (Fluka,TraceSELECT�, for trace analysis P37%) were added and the flaskwas heated to 160 �C for 1 h. The mixture was cooled, filtered(Whatman Grade No. 42, particle retention 2.5 lm) and dilutedwith ultrapure water to 50 mL.

Chironomus riparius larvae were washed with double-distilledwater and frozen at �18 �C in glass containers.

Then, about 300/500 larvae per station were taken and thawedfor removing the head capsule to analyze the mouthpart deformi-ties. The remaining parts of the larval bodies were pooled andplaced in an oven at 105 �C until reaching a constant weight. Diges-tion of larval tissues was carried out using acid digestion withultrapure concentrated nitric acid: the samples (0.5 g) were heatedto 160 �C with HNO3 (2.5 ml) for 1 h, using an aluminum blockheating digester (VELP Scientific, Inc.); then vessels were cooledto 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 ultrapurewater was added to reach the desired volume (25 ml).

Therefore, metal concentrations were determined in pooledchironomid larvae samples, while deformities were checked persingle individual.

2.6. Statistical analysis

The observed differences of the deformities incidences wereexplored with Chi-square test (v2).

Principal component analysis (PCA), as ordination technique,was used to establish associations among the sampling sites andthe 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 PCextracted 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 atStations 1–4, and were not performed for the 2010 exploratorycampaign, because of the reduced set of data (monitoring of heavymetals only in the sediments and deformities at Station 4).

The statistical treatment was performed with the R softwarepackage (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 conditionof strong alteration of the environment, particularly for the chem-ical and physical–chemical parameters of the water, indicative of astrong organic pollution, and of the high concentrations of heavymetals in the sediments and the high incidence of malformationsfor C. riparius larvae (IV instar). The actual sampling campaign(Stations 1–4: year 2012) confirmed this state of degradation forthe entire stream (Tables 1–3). The results of the survey arediscussed below.

The chemical and physical–chemical parameters of the watersamples showed that the environmental condition of the GennaStream was affected by strong pollution, as evidenced by high

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Table 2Incidence of morphological deformity classes of C. riparius larvae from the stations (St.1–4; year 2012) of the Genna Stream (CL. 1 – specimens without any morphologicaldeformity, CL. 2 – specimens with weak deformity, CL. 3 – specimens with strongdeformity) and from St. 4 (years 2010–2011), during the explorative sampling; ‘‘–’’,dry stream.

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

4 June 2010 245 42 23 310 20.97 7.424 August 2010 162 60 71 293 44.71 24.234 October 2010 88 67 153 308 71.43 49.684 February 2011 113 69 128 310 63.55 41.29

Tot. 608 238 375 1221 50.20 30.71

1 March 2012 – – – – – –1 June 2012 146 93 64 303 51.82 21.121 October 2012 – – – – – –2 March 2012 195 148 155 498 60.84 31.122 June 2012 174 94 26 294 40.82 8.842 October 2012 147 105 54 306 51.96 17.65

Tot. 516 347 235 1098 53.01 21.40

3 March 2012 110 112 271 493 77.69 54.973 June 2012 175 88 45 308 43.18 14.613 October 2012 106 71 128 305 65.25 41.97

Tot. 391 271 444 1106 64.65 40.14

4 March 2012 203 155 140 498 59.24 28.114 June 2012 133 100 77 310 57.10 24.844 October 2012 127 90 92 309 58.90 29.77

Tot. 463 345 309 1117 58.55 27.66

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

values of conductivity, COD, nitrogen (particularly ammonium)and low values of D.O. In March and October 2012, St. 1 was dry(Table 1).

In total 4875 larvae of C. riparius (IV instar) (Table 2) were sam-pled during the whole investigation. The incidence of mentumdeformities (CL. 2 + 3) was generally high, about 56.2% (29.5% inCL. 3) with higher values at Station 3 (64.7%), mainly because ofthe higher incidence of strong deformities (40.1% in CL. 3). In year2012, weak deformities (CL. 2) showed, with the exception of Sta-tion 3 (24.5%), constant values around 31%.

June presented the lowest incidence of deformities (CL. 2 + 3) atall stations, while March the highest one, with average valuesrespectively of 48.2% (17.4% in CL. 3) and of 65.9% (38.1% in CL.3). The Chi-square test (year 2012: stations 1–4) showed that theobserved differences were statistically significant (v2 = 327.56,p < 0.01) among normal (CL. 1), weakly (CL. 2) and strongly (CL.3) deformed specimens.

During the year 2012, the mean concentrations of Cd, Cr and Niin the sediments were higher at St. 4, Zn and Cu at St. 3, and Pb atSt. 2. In larval tissues the highest average concentrations were de-tected at St. 4 for all metals except for Ni (St. 3). In the water theaverage highest metal concentrations were found at St. 2 exceptfor Zn (St. 4).

In sediments the mean concentrations of Cd, Zn and Cu werehigher in March, Cr and Ni in October and Pb in June. In larval tis-sues the highest average concentrations were found in June for allmetals except for Zn (October). In water the average highest metalconcentrations were detected in June.

TEC (Threshold Effect Concentration) and PEC (Probable EffectConcentration) reference values were used to predict sedimenttoxicity by heavy metals, accordingly to the SQGs (SedimentQuality Guidelines) (Mac Donald et al., 2000).

The concentrations of Cd registered in the sediments of theGenna Stream, were always higher than the TEC reference value,except in June 2010 (explorative sampling) at St. 4. Cd exceededthe PEC value only in October 2010 (explorative sampling) at St.4. Pb exceeded the TEC value in August and October 2010 at St. 4and in June 2012 at St. 1 and 2. Ni concentrations exceeded theTEC value except in March and June 2012 at St. 2. The values of

Table 3aHeavy 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).

a) Date Sediments (mg kg�1) Larval tissues (mg kg�1) Water (lg L�1)

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 14.704 August 2010 1.30 49.38 79.31 16.47 33.19 27.184 October 2010 5.66 36.18 109.60 26.57 38.03 27.144 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 0.141 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 5.492 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 31.242 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 3.493 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 2.043 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 6.014 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 0.144 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 31.6PEC 4.98 128 459 111 48.6 149

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13

Table 3bBSAFs (Biota to Sediment Accumulation Factor: ratio between metal in tissues and inthe sediments) in C. riparius larvae; ‘‘–’’, dry stream.

b) BSAF Cd Pb Zn Cr Ni Cu

1 March 2012 – – – – – –1 June 2012 0.38 0.1 3.1 0.5 1.2 0.431 October 2012 – – – – – –

2 March 2012 0.2 0.1 4.0 0.6 0.3 0.82 June 2012 0.4 0.0 3.0 0.3 1.2 0.62 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

3 March 2012 0.1 0.0 2.4 0.1 0.1 0.43 June 2012 0.7 0.2 2.4 0.5 1.5 0.83 October 2012 0.5 0.3 3.0 0.4 0.4 1.6

Mean 0.44 0.18 2.60 0.33 0.67 0.93

4 March 2012 0.09 0.08 2.19 0.10 0.09 0.574 June 2012 0.52 0.41 5.80 0.46 0.47 1.754 October 2012 0.37 0.17 4.91 0.23 0.26 1.11

Mean 0.33 0.22 4.30 0.26 0.27 1.14

Table 4aCorrelation between the degree of contamination by heavy metals, based on the PCextracted from the PCA analysis, and the incidence of deformities (CL.) (arcsintransformed percent values); Dim 1 and Dim 2, first two axes of PCA; r, Pearson’scorrelation coefficient; p, probability value.

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

Cu exceeded the TEC in June 2012 at St. 1 and in March and June2012 at St. 3. Zn and Cr never exceed the TEC values (Table 3a).

Eventually, in year 2012, the mean concentration of heavy met-als in the sediments for each station never exceeded the PECthreshold, while the TEC threshold was exceeded for Cd at all thestations, for Ni at St. 3 and 4, and for Cu at St. 3.

The BSAFs (Biota to Sediment Accumulation Factor: ratio be-tween metal concentration in tissues and sediments) in C. ripariuslarvae were >1 for Zn (all samples), for Ni (June 2012, St. 1–3) andfor Cu (October 2012, St. 3 and 4; June 2012, St. 4). The BSAF forGenna Stream was metal-specific, with Zn and Cu always showingthe highest accumulation factors and Pb always at the lower limitof the series. Specifically, as far as the stations were concerned,

-4 -2 0 2 4

-20

Dim1 (33.80%)

Dim

2 (2

7.80

%)

2Mar3Mar

4Mar

1Jun

2Jun

3Jun

4Jun

2Oct 3Oct

4Oct

-0.5 0.0 0.5

-0.5

0.5

0.0

Dim1 (33.80%)

Dim

2 (2

7.80

%)

Cd(s)

Pb(s)

Zn(s)

Cr(s) Ni(s)

Cu(s)

Cd(t)

Pb(t)Zn(t)

Cr(t)

Ni(t)

Cu(t)

Cd(w)

Pb(w)

Zn(w)

Cr(w)

Ni(w)

Cu(w)

(A)

(B)

2-4

Fig. 2. PCA (first two axes) carried out on heavy metals (Cr, Ni, Cu, Zn, Cd and Pb) indifferent compartments: sediments (s), water (w), and larval tissues (t), at thesampling stations of Genna Stream over the year 2012; (A), sampling sites; (B),heavy metals in different compartments.

BSAF decreased, as follows: St. 2: Zn > Cu > Ni > Cr > Cd > Pb; St.3: Zn > Cu > Ni > Cd > Cr > Pb; St. 4: Zn > Cu > Cd > Ni > Cr > Pb; asfar as the seasons were concerned, BSAF decreased, as follows:June: Zn > Cu, Ni > Cd > Cr > Pb; March: Zn > Cu > Cr > Ni > Cd > Pb;October: Zn > Cu > Cd > Ni > Cr > Pb (Table 3b).

PCA carried out on metals in sediments, water and larval tissuesallowed the level of contamination due to heavy metals to be ana-lyzed at the stations of the Genna Stream over the year 2012.

Dim 1 (33.80%) showed a seasonal gradient, where March andJune were placed on the left and right side of the axis respectively.In March, the stations were mainly associated to heavy metals inthe sediments, in particular Zn and Cu, whereas in June the stationswere associated to metals in water and larval tissues. In October,the stations were in an intermediate position.

PCA in Dim 2 (27.80%) showed a gradient partially reflecting thelongitudinal profile of the Genna Stream over each seasons, wheredownstream station (St. 4) was placed on the upside of Dim 2 andmainly associated to Cd, Ni and Cr in the sediments and to Zn, Pband Cu in larval tissues. The remaining stations (in particular atJune) were placed on the downside of Dim 2 and mainly affectedby metals Cu, Cr, Pb, Ni in water, and by Pb in the sediments andby Ni in larval tissues (Fig. 2a–b).

No significant correlations between PC (Dim 1 and 2), extractedfrom PCA analysis, and the morphological deformity classes ofC. riparius (angular transformation was applied to percent values)nor between single heavy metals in different compartments

Heavy metals: all toxicity

CPA Dim Deformity r p

Dim 1 CL. 2 0.368 0.295Dim 1 CL. 3 �0.542 0.105Dim 1 CL. 2 + 3 �0.570 0.086Dim 2 CL. 2 �0.055 0.880Dim 2 CL. 3 0.504 0.138Dim 2 CL. 2 + 3 0.522 0.122

Table 4bCorrelation between the degree of contamination by single heavy metals in differentcompartments (sediments, larval tissues and water) and the incidence of deformities(CL.) (arcsin transformed percent values); r, Pearson’s correlation coefficient; p,probability value.

Heavy metals: Sediments Larval tissues Water

Deformity r p r p r p

Cd CL. 2 0.208 0.564 0.234 0.515 0.436 0.208Cd CL. 3 0.293 0.411 �0.346 0.327 0.570 0.030Cd CL. 2 + 3 0.387 0.270 �0.390 0.265 �0.701 0.024Pb CL. 2 0.401 0.251 �0.001 0.998 0.298 0.403Pb CL. 3 �0.658 0.039 �0.026 0.944 �0.735 0.015Pb CL. 2 + 3 �0.657 0.039 �0.082 0.822 �0.756 0.011Zn CL. 2 �0.231 0.521 0.323 0.363 0.380 0.279Zn CL. 3 0.191 0.597 0.036 0.922 �0.263 0.463Zn CL. 2 + 3 0.176 0.626 0.080 0.826 �0.204 0.086Cr CL. 2 0.329 0.353 0.290 0.416 0.258 0.472Cr CL. 3 0.041 0.911 �0.315 0.375 �0.643 0.045Cr CL. 2 + 3 0.116 0.749 �0.359 0.308 �0.639 0.086Ni CL. 2 0.153 0.673 0.222 0.538 0.277 0.438Ni CL. 3 0.072 0.842 �0.638 0.047 �0.553 0.086Ni CL. 2 + 3 0.080 0.826 �0.712 0.021 �0.564 0.090Cu CL. 2 �0.284 0.426 �0.271 0.448 0.215 0.551Cu CL. 3 0.163 0.653 0.150 0.680 �0.477 0.163Cu CL. 2 + 3 0.151 0.677 0.048 0.895 �0.439 0.204

A. Di Veroli et al. / Chemosphere 112 (2014) 9–17 15

(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 mouthpartdeformities of C. riparius larvae are the endpoint of toxicity, afterinvestigating the relationships between such deformities andmetal contamination in natural conditions.

The contamination of the stream appeared to be related to theto discharges of the South-west zone of the city of Perugia (about150000 inhabitants). In addition, towards the valley part of thecatchment of the Genna Stream is subjected to intensive swinefarms (about 80 farms), which contribute (via agricultural fertirri-gation) to metal pollution (except lead) from St. 2 to the confluencewith the Nestore River (St. 4), thus explaining the increase of zincand copper at the St. 3, and of cadmium, chromium, nickel and zincat St. 4.

Mouthpart deformities of C. riparius larvae observed in the pres-ent work, resulted an efficient endpoint of toxicity assessment inthe Genna Stream, because more than the half (56%) of the exam-ined C. riparius larvae (about 5000) were deformed (CL. 2 + 3). Thisresult reflects the high level of heavy metals contamination in thestream (sediments, in particular), and confirms previous laboratorystudies, in which mouthpart deformities were demonstrated to beactual 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; Maddenet 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 onlya few chemical substances, in particular heavy metals (Kosawaltand Knight, 1987; Vermeulen et al., 2000). In experimentalconditions, 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 (DiVeroli et al., 2012b).

In the present study, among heavy metals examined in naturalconditions in different compartments (sediments, larval tissuesand water), the metals in the sediments, particularly copper andzinc, 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 meanconcentrations 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%) werenot 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 watercontamination.

These results of the effects of heavy metals examined in differ-ent compartments (sediments, larval tissues and water) are inagreement with previous investigations carried out undercontrolled conditions, where significant correlations betweendeformities and metal concentrations have been observed onlyfor sediments artificially contaminated, but not for larval tissues(Di Veroli et al., 2012b). In particular, laboratory tests indicated a

direct dependence of the severity of deformities with the heavymetal concentrations in the sediments.

The concentrations of metals in larval tissues of C. riparius arenot correlated with those in sediments and with deformities, mostlikely because the physiological and metabolic functions and theenvironmental conditions influenced the bio-availability of themetals, 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 BSAFfor the six heavy metals in different stations and months. In partic-ular, zinc and copper (essential micro-elements, toxic only in casethey exceed a particular threshold) were always the metals withthe highest rate of bioaccumulation, while lead was the lowest.

Notwithstanding the high incidence of deformities detected inthe Genna Stream (in particular the severe ones, CL. 3: about30%), the correlation between heavy metals in sediments anddeformities 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 responsiblefor the incidence of the larval deformities of C. riparius in the GennaStream (Di Veroli et al., 2012b).

Our results suggest that the major cause of morphologicaldeformities could be due to the synergistic effect of a mixture oftoxicants, more than of a single contaminant, in agreement withprevious 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 pivotalrole on the morphological deformities, manly on the severe ones,observed in chironomid larvae.

These data are in agreement with previous experimental testsand highlight that chironomid mouthpart deformities representan effective endpoint for freshwater bioassessment monitoringprograms to evaluate the toxic effects of contaminants, such asheavy 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 wastewaterof the livestock farming.

The assessment of heavy metals, among the most commonenvironmental pollutants, in river sediments can be of greatimportance for human and environmental health (Graney andEriksen, 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 evaluatethe toxic effects of contamination in freshwater ecosystems bycarrying on, in parallel, investigations with the help of laboratorybioassays, 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-tions. A special thanks goes to Prof. E. Gaino for her valuable sug-gestions. Finally, we express our gratitude to the FoundationCassa di Risparmio di Perugia for the financial support, and tothe anonymous referees for their helpful suggestions, which mark-edly improved the content of the text.

References

Allen, H.E., Hansen, D.J., 1996. The importance of trace metal speciation to waterquality criteria. Water Environ. Res. 68, 42–54.

Arnason, J.G., Fletcher, B.A., 2003. A 40-year record of Cd, Hg, Pb and U deposition insediments of Patroon Reservoir, Albany County, NY, USA. Environ. Pollut. 123,383–391.

ARPA (Agenzia Regionale per la Protezione Ambientale) Umbria, 2010. Bacinoidrografico del Fiume Nestore monitoraggio chimico e microbiologico di acque escarichi. Relazione Tecnica, Perugia.

Bervoets, L., Blust, R., De Wit, M., Verheyen, R., 1997. Relations of river sedimentcharacteristics to trace metal concentrations in oligochaetes and chironomids.Environ. Pollut. 95, 345–356.

Besser, J.M., Brumbaugh, W.G., Ivey, C.D., Ingersoll, C.G., Moran, P.W., 2008.Biological and chemical characterization of metal bioavailability in sedimentsfrom Lake Roosevelt, Columbia River, Washington, USA. Arch. Environ. Contam.Toxicol. 54, 557–570.

Bird, G.A., Schwartz, W.J., Joseph, D.L., 1995. The effect of 210Pb and stable lead onthe induction of menta deformities in Chironomus tentans larvae and on theirgrowth and survival. Environ. Toxicol. Chem. 14, 2125–2130.

Bird, G.A., 1994. Use of chironomid deformities to assess environmentaldegradation in the Yamaska River, Quebec. Environ. Monit. Assess. 30, 163–175.

Canfield, T.J., Kemble, N.E., Brumbaugh, W.G., Dwyer, F.J., Ingersoll, C.G., Fairchild,J.F., 1994. Use of benthic invertebrate community structure and the sedimentquality triad to evaluate metal-contaminated sediment in the upper Clark ForkRiver, Montana. Environ. Toxicol. Chem. 13, 1999–2012.

Cottrill, B.R., 2003. The scope for reducing trace element supplementation oflivestock diet. Atti del workshop ‘‘Assessment and reduction of heavy metalinputs into agro-ecosystems’’, concerted action Aromis. <http://www.ktbl.de/english/projects/aromis/program.htm>.

De Haas, E.M., Léon Paumen, M., Koelmans, A.A., Kraak, M.H.S., 2004. Combinedeffects of copper and food on the midge Chironomus riparius in whole sedimentbioassays. Environ. Pollut. 127, 99–107.

De Souza, C.M.M., Pestana, M.H.D., Drude de Lacerda, L., 1986. Geochemicalpartitioning of heavy metals in sediments of three estuaries along the coastal ofRio de Janeiro (Brazil). Sci. Total Environ. 58, 63–72.

Desrosiers, M., Gagnon, C., Masson, S., Martel, L., Babut, M.P., 2008. Relationshipsamong total recoverable and reactive metals and metalloid in St. LawrenceRiver sediment: bioaccumulation by chironomids and implications forecological risk assessment. Sci. Total Environ. 389, 101–114.

Di Veroli, A., Selvaggi, R., Pellegrino, R.M., Goretti, E., 2010. Sediment toxicity anddeformities of chironomid larvae in Lake Piediluco (Central Italy). Chemosphere79, 33–39.

Di Veroli, A., Selvaggi, R., Goretti, E., 2012a. Chironomid mouthpart deformities asindicator of environmental quality: a case study in Lake Trasimeno (Italy). J.Environ. Monit. 14, 1473–1478.

Di Veroli, A., Goretti, E., Leo9n Paumen, M., Kraak, M.H.S., Admiraal, W., 2012b.Mouthpart deformities in Chironomus riparius larvae exposed to toxicants.Environ. Pollut. 166, 212–217.

Eckel, H., Mantovi, P., Roth, U., 2003. Options for heavy metal input reduction. Attidel workshop ‘‘Assessment and reduction of heavy metal inputs into agro-ecosystems’’, concerted action Aromis. <http://www.ktbl.de/english/projects/aromis/program.htm>.

Faria, M.S., Lopes, R.J., Malcato, J., Nogueira, A.J.A., Soares, A.M.V.M., 2008. In situbioassays with Chironomus riparius larvae to biomonitor metal pollution inrivers and to evaluate the efficiency of restoration measures in mine areas.Environ. Pollut. 151, 213–221.

Filgueiras, A.V., Lavialla, I., Bendicho, C., 2002. Chemical sequential extraction formetal partitioning in environmental solid samples. J. Environ. Monit. 4, 823–857.

Gibbs, R.J., 1977. Transport phases of transition metals in the Amazon and Yukonrivers. Geol. Soc. Am. Bull. 88, 829–843.

Graney, J.R., Eriksen, T.M., 2004. Metals in pond sediments as archives ofanthropogenic activities: a study in response to health concerns. Appl.Geochem. 19, 1177–1188.

Hamilton, A.L., Saether, O.A., 1971. The occurrence of characteristic deformities inthe chironomid larvae of several Canadian lakes. Can. Entomol. 103, 363–368.

Hare, L., Tessier, A., 1998. The aquatic insect Chaoborus as a biomonitor of tracemetals in lakes. Limnol. Oceanogr. 43, 1850–1859.

Jain, C.K., Sharma, C.K., 2001. Distribution of trace metals in the Hindon riversystem, India. Water Res. 38, 81–90.

Janssens de Bisthoven, L.G., Nuyts, P., Goddeeris, B., Ollevier, F., 1998. Sublethalparameters in morphologically deformed Chironomus larvae: clues tounderstand their bioindicator value. Freshwater Biol. 39, 179–191.

Janssens De Bisthoven, L., Postma, J., Vermeulen, A., Goemans, G., Ollevier, F., 2001.Morphological deformities in Chironomus riparius Meigen larvae after exposureto cadmium over several generations. Water, Air, Soil Pollut. 129, 167–179.

Kosawalt, P., Knight, A.W., 1987. Chronic toxicity of copper to a partial life cycle ofthe midge, Chironomus decorus. Arch. Environ. Contam. Toxicol. 16, 283–290.

Lacerda, L.D., Fernandez, M.A., Calazans, C.F., Tanizaki, K.F., 1992. Bioavailability ofheavy metals in sediments of two coastal lagoons in Rio de Janeiro, Brazil.Hydrobiologia 228, 65–70.

León Paumen, M., Borgman, E., Kraak, M.H.S., Van Gestel, C.A.M., Admiraal, W., 2008.Life cycle responses of the midge Chironomus riparius to polycyclic aromaticcompound exposure. Environ. Pollut. 152, 225–232.

Liu, J., Li, Y., Zhang, B., Cao, J., Cao, Z., Domagalski, J., 2009. Ecological risk of heavymetals in sediments of the Luan River source water. Ecotoxicology 18, 748–758.

Luoma, S.N., 1983. Bioavailability of trace metals to aquatic organisms—a review.Sci. Total Environ. 28, 1–22.

Mac Donald, D.D., Ingersoll, C.G., Berger, T.A., 2000. Development and evaluation ofconsensus-based sediment quality guidelines for freshwater ecosystems. Arch.Environ. Contam. Toxicol. 39, 20–31.

Madden, C.P., Suter, P.J., Nicholson, B.C., Austin, A.D., 1992. Deformities inchironomid larvae as indicators of pollution (pesticide) stress. Neth. J. Aquat.Ecol. 26, 551–557.

Madden, C.P., Austin, A.D., Sutter, P.J., 1995. Pollution monitoring using chironomidlarvae: what is a deformity? In: Craston, P. (Ed.), Chironomids from Genes toEcosystems. CSRIO Publication, East Melbourne, pp. 89–94.

Mantovi, P., Bonazzi, G., 2004. Riduzione del tenore di rame e zinco nei mangimi.Inf. Agr. 4, 61–64.

Marking, L.L., 1985. Toxicity of chemical mixtures. In: Rand, G.M., Petrocelli, S.R.(Eds.), Fundamentals of Aquatic Toxicology. Hemisphere PublishingCorporation, New York, pp. 164–176.

Martinez, E.A., Moore, B.C., Schaumloffel, J., Dasgupta, N., 2002. The potentialassociation between menta deformities and trace elements in Chironomidae(Diptera) taken from a heavy metal contaminated river. Arch. Environ. Contam.Toxicol. 42, 286–291.

Martinez, E.A., Moore, B.C., Schaumloffel, J., Dasgupta, N., 2003. Morphologicalabnormalities in Chironomus tentans exposed to cadmium and copper-spikedsediments. Ecotoxicol. Environ. Saf. 55, 204–212.

MATT (Ministero dell’Ambiente e della Tutela del Territorio), APAT (Agenzia per laProtezione dell’Ambiente e per i Servizi Tecnici), 2005. Progetto nazionale dimonitoraggio delle acque superficiali (IRSA, CNR). Gli ecosistemi e i sedimenti:caratterizzazione dei sedimenti. Linee guida, protocolli operativi, attivitàsperimentali.

Michailova, P., Belcheva, R., 1990. Different effect of lead on external morphologyand polytene chromosomes of Glyptotendipes barbipes (Staeger) (Diptera,Chironomidae). Folia Biol. (Krakow) 38, 83–88.

Nahmani, J., Rossi, J.P., 2003. Soil macroinvertebrates as indicators of pollution byheavy metals. C. R. Biol. 326, 295–303.

Naimo, T.J., 1995. A review of the effects of heavy-metals on freshwater mussels.Ecotoxicology 4, 341–362.

OECD, 2004. Guideline 21: Sediment–water Toxicity Test Using Spiked Sediment.Organization for Economic Co-operation and Development, Paris, France.

Orkun, D.I., Seckin, G., Ersu, C.B., Yilmaz, T., Sari, B., 2011. Heavy metal content anddistribution in surface sediments of the Seyhan River, Turkey. J. Environ.Manage. 92, 2250–2259.

Péry, A.R.R., Geffard, A., Conrad, A., Mons, R., Garric, J., 2008. Assessing the risk ofmetal mixtures in contaminated sediments on Chironomus riparius based oncytosolic accumulation. Ecotoxicol. Environ. Saf. 71, 869–873.

R Development Core Team, 2008. R: A Language and Environment for StatisticalComputing. R Foundation for Statistical Computing, Vienna, Austria. <http://www.R-project.org>.

Ristola, T., Kukkonen, J.V.K., Pellinen, J., 1999. Body residues and responses ofthe midge Chironomus riparius to sediment-associated 2,4,5-trichlorophenolin subchronic and chronic exposures. Arch. Environ. Contam. Toxicol. 37,42–49.

Santoro, A., Blo, G., Mastrolitti, S., Fagioli, F., 2009. Bioaccumulation of heavy metalsby aquatic macroinvertebrates along the Basento River in the south of Italy.Water, Air, Soil Pollut. 201, 19–31.

Selvaggi, R., Colonna, N., Lupia, F., Murgia, M.S., Poletti, A., 2010. Water quality andsoil natural salinity in the Southern Imera Basin (Sicily, Italy). Ital. J. Agron./Riv.Agron. 3, 81–89.

Singh, K.P., Mohan, D., Singh, V.K., Malik, A., 2005. Studies on distribution andfractionation of heavy metals in Gomti river sediments – a tributary of theGanges, India. J. Hydrol. 312, 14–27.

Türkmen, M., Ciminli, C., 2007. Determination of metals in fish and mussel speciesby inductively coupled plasma-atomic emission spectrometry. Food Chem. 103,670–675.

Van De Guchte, C., Van Urk, G., 1989. Discrepancies in the effects of field andartificially heavy metal contaminated aquatic sediments upon midge larvae. In:Heavy Metals in the Environment, 7th International Conference, 12–15September 1989, Geneva.

Vermeulen, A.C., Liberloo, G., Dumont, P., Ollevier, F., Goddeeris, B.R., 2000.Exposure of Chironomus riparius larvae (Diptera) to lead, mercury, and b-sitosterol: effects on mouthpart deformation and moulting. Chemosphere 41,1581–1591.

Vermeulen, A.C., 1995. Elaboration of chironomid deformities as bioindicators oftoxic sediment stress: the potential application of mixture toxicity concepts.Ann. Zool. Fennici 32, 265–285.

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

Warwick, W.F., 1985. Morphological abnormalities in Chironomidae (Diptera)larvae as measures of toxic stress in freshwater ecosystems: indexing antennaldeformities in Chironomus Meigen. Can. J. Fish. Aquat. Sci. 42, 1881–1914.

Warwick, W.F., 1988. Morphological deformities in Chironomidae (Diptera) larvaeas biological indicators of toxic stress. In: Evans, M.S. (Ed.), ToxicContaminants and Ecosystem Health. A Great Lakes focus. John Wiley andSons, New York, pp. 281–320.

Watts, M.M., Pascoe, D., 1996. Use of the freshwater macroinvertebrate Chironomusriparius (Diptera: Chironomidae) in the assessment of sediment toxicity. WaterSci. Technol. 34, 101–107.

Wiederholm, T., 1984. Incidence of deformed chironomid larvae (DipteraChironomidae) in Swedish Lakes. Hydrobiologia 109, 243–249.