PRIMARY RESEARCH PAPER

Ammonium threshold values for groundwater qualityin the EU may not protect groundwater fauna: evidencefrom an alluvial aquifer in Italy

T. Di Lorenzo • M. Cifoni • P. Lombardo •

B. Fiasca • D. M. P. Galassi

Received: 10 June 2014 / Revised: 19 August 2014 / Accepted: 21 August 2014 / Published online: 29 August 2014

� Springer International Publishing Switzerland 2014

Abstract The European Union (EU) threshold val-

ues for NHþ4 in groundwater range from a minimum of

0.084 mg l-1 to the maximum allowed of 5 mg l-1.

The aim of our study was to determine whether these

values are adequate to protect groundwater copepods

in alluvial aquifers underlying intensive agriculture.

To this end, we analyzed abiotic (including NHþ4concentration) and biological patterns (copepod

assemblages) in an alluvial aquifer in an area of

intensive agriculture. Groundwater was collected from

pre-existing farmer-owned bores. Abiotic and biolog-

ical patterns were not related to seasonal variation in

agricultural practices, and pollutant concentrations

were typically below the legal maximum threshold

values. However, both abiotic and biological variables

differed significantly between two groups of bores,

with high and low NHþ4 conditions based on separate

ecotoxicological assays that set the NHþ4 toxicity

threshold for stygobiotic copepods at 2.59 below the

current EU lowest legal threshold. In particular,

phosphorus concentration was higher, and oxygen

and copepod abundance and richness were lower in

high NHþ4 bores. Our results suggest that the present

threshold value range for NHþ4 adopted by EU

Member States may not protect stygobiotic copepod

assemblages in alluvial aquifers underlying densely

cultivated areas.

Keywords Ammonium � Aquifer � Stygobiotic �Toxicity � Groundwater � Threshold

Introduction

The use of N-fertilizers in excess of crop needs is a

widespread concern in developed and developing

countries as, generally, no more than 50% of the

applied nitrogen (N) is taken up by crops (Mosier

et al., 2004). The unused N can be lost from the soil–

plant system by volatilization as NH3, by denitrifica-

tion as reduced form NHþ4 , and by leaching as the

oxidized forms NO�3 and NO�2 (Erisman et al., 2007).

The excess of N-fertilizers (both reduced and oxidized

forms), not removed by crops, leaches into

Handling editor: Diego Fontaneto

Electronic supplementary material The online versionof this article (doi:10.1007/s10750-014-2018-y) containssupplementary material, which is available to authorized users.

T. Di Lorenzo (&) � M. Cifoni

Istituto per lo Studio degli Ecosistemi - CNR, Via

Madonna del Piano 10, Sesto Fiorentino, 50019 Florence,

Italy

e-mail: tiziana.dilorenzo@ise.cnr.it

M. Cifoni � P. Lombardo � B. Fiasca � D. M. P. Galassi

Department of Life, Health and Environmental Sciences,

University of L’Aquila, Via Vetoio, Coppito,

67100 L’Aquila, Italy

P. Lombardo

Limno Consulting, via Bedollo 303, 00124 Rome, Italy

123

Hydrobiologia (2015) 743:139–150

DOI 10.1007/s10750-014-2018-y

groundwater or reaches surface waters through runoff,

polluting water bodies. Therefore, intensive agricul-

ture is often associated with high concentrations of

nitrogen (N) compounds in groundwater (Di Lorenzo

et al., 2012; Melo et al., 2012; Morari et al., 2012;

Dahan et al., 2014), due to the use of fertilizers in the

forms of ammonium nitrate, urea, calcium nitrate,

ammonium bicarbonate, and mixtures of nitrogen,

potassium, and phosphorus (Erisman et al., 2007).

Like surface water ecosystems (e.g., Carpenter et al.,

1998; Tilman, 1999; Blann et al., 2009), groundwater

ecosystems are sensitive to changes in water quality

induced by agricultural practices (Wood et al., 2008;

Hartland et al., 2011; Korbel et al., 2013; Di Lorenzo

& Galassi, 2013; Di Lorenzo et al., 2013). Pollution

from pesticides and fertilizers and groundwater with-

drawal for irrigation purposes remain the major causes

of poor groundwater quality in several European

alluvial aquifers (Candela et al., 2008; European

Union, 2012; van Grinsven et al., 2012; Di Lorenzo &

Galassi, 2013; Caschetto et al., 2014; Poch-Massegu

et al., 2014).

Groundwater biota (i.e., stygobiotic organisms,

defined as species that are strictly confined to

groundwater and hence have evolved specific mor-

phological and physiological adaptations to cope with

the unique subterranean habitat: Gibert et al., 1994) is

essential to maintain groundwater ecosystem health

(Stein et al., 2010 and references therein). Both

groundwater microbes (such as bacteria, fungi, and

protozoa) and stygofauna (macro- and meiofaunal

invertebrates) provide important ecosystem services

which support aquifer self-purification (Kota et al.,

1999; Marshall & Hall, 2004; Boulton et al., 2008).

Stygobiotic copepods (by far the most abundant and

species-rich invertebrate group in groundwater: Ga-

lassi, 2001; Galassi et al., 2009) are highly sensitive to

NHþ4 (Di Lorenzo et al., 2014) as observed for several

other freshwater invertebrates (Jensen, 1995, 2003;

Weihrauch et al., 2004; Camargo et al., 2005;

Camargo & Alonso, 2006). However, the utility of

groundwater copepods as indicators of NHþ4 contam-

ination in groundwater is yet to be established. In the

laboratory, Di Lorenzo et al. (2014) found that the

NHþ4 chronic lethal concentration (CL) for the stygo-

biotic species Diacyclops belgicus Kiefer, 1936

(Copepoda Cyclopidae) is 0.032 mg l-1. This con-

centration is 2.59 lower than the more stringent

threshold value (TV) indicated by EU Member States

(MS) (European Commission, 2010). However, it is

unknown whether exposure to such NHþ4 concentra-

tion may affect other stygobiotic species of copepods

in their natural habitats.

The aim of our study was to determine whether

stygobiotic copepod assemblages may be affected, in

terms of species richness and/or abundance, by

exposure in natural groundwater habitats to NHþ4concentration equal to or higher than the laboratory-

determined CL concentration (0.032 mg l-1) assessed

for the stygobiotic D. belgicus by Di Lorenzo et al.

(2014). To this end, we analyzed biological data from

an alluvial aquifer in an area of intensive agriculture,

in Italy. We selected the alluvial aquifer of the River

Adige, because of its homogeneous geological and

hydromorphological features and absence of contam-

ination according to the TVs indicated in the Annexes

I and II of the Groundwater Daughter Directive 2006/

118/EC (European Commission, 2006). We tested the

hypotheses that: (1) stygobiotic copepod assemblages

are affected by environmental NHþ4 concentrations

C0.032 mg l-1; and (2) the current TVs for NHþ4 ,

indicated by EU Member States for groundwater

bodies (European Commission, 2010), are inadequate

to protect groundwater copepods in alluvial aquifers

underlying intensive agriculture.

Materials and methods

Study region

The study was carried out in the northernmost sector

(45 km2) of the alluvial plain of the River Adige in the

Trento Province, Italy (Fig. 1). The floodplain is

intensely used for agriculture, while industrial and

urban land use is less extensive. Floodplain soils are

similar across the study area, with sequences of clay,

sand, and gravel sediments (Distretto Idrografico delle

Alpi Orientali, 2010). The alluvials are 50–60 m thick

and overlie an impermeable bedrock. The distribution

of Quaternary sediments filling the plain varies

through the area with fine material (silt, clay) covered

by larger-grained sediments (gravel and sand), pro-

ducing a complex, locally multi-layered structure with

a hydraulic conductivity of 10-3–10-5 m s-1 (Dist-

retto Idrografico delle Alpi Orientali, 2010). However,

140 Hydrobiologia (2015) 743:139–150

123

the aquifer layers are hydrogeologically intercon-

nected, leading to the physico-chemical homogeniza-

tion of groundwater (Distretto Idrografico delle Alpi

Orientali, 2010). The aquifer is fed by rainfalls and

lateral discharge from the surrounding calcareous

groundwater bodies (Autorita di Bacino dell’Adige,

2008). Mean annual discharge of the aquifer is about

9 m3 s-1 (Autorita di Bacino dell’Adige, 2008). The

climate is continental, with cold winters and maxi-

mum rainfall typically in summer. The average annual

rainfall in the floodplain is about 400–500 mm year-1

(Distretto Idrografico delle Alpi Orientali, 2010).

Agriculture (predominantly orchards, especially the

renowned local apple trees) is the main activity

([85 % of the study area), with much less-extensive

industrial and urban land use, mainly within or

adjacent to agricultural areas. The aquifer is exploited

for orchard irrigation (mainly by sprinkle and drip

methods) from March through the end of September,

and to a lesser extent over the whole year for industrial

and urban uses (Distretto Idrografico delle Alpi

Orientali, 2010). Groundwater abstraction is sustain-

able (Autorita di Bacino dell’Adige, 2008). Orchard

cultivation requires nitrogen, phosphorus, and potas-

sium fertilizers, which are applied in spring and

summer, as well as herbicides and pesticides. Both

organic and synthetic fertilizers are used at a ratio of

about 10:1 (ISTAT, 2006).

Sampling regime

Groundwater biota and water samples were collected

in 33 bores, in June and again in October 2006. Study

bores were randomly selected from a pool of privately

owned bores drilled in aquifer layers of mixed gravel

and sands, and already in use for irrigation purposes.

Farmers did not use the bores during the week before

the planned sampling to allow the piezometric levels

to restore. We selected bores of various depth

(maximum depth: about 60 m), 16–20 cm in diameter,

lined with PVC casing, with a 2-m slotted screen at the

base. The range in bore depth allowed us to sample the

entire aquifer depth-wise. The distance of the study

bores from the River Adige was random, thus prox-

imity to the river was not included in our analyses as a

factor.

Sampling

Bores were equipped with immersion pumps that were

used to extract bore water, which was filtered through

a 60-lm mesh net to collect groundwater fauna. After

preliminary tests, representative samples of bore and

aquifer taxonomic richness and composition were

collected by pumping 1 m3 of groundwater (2–4 times

the bore volumes) from each bore. After fixation in the

field with 70% ethanol, specimens were sorted under a

stereomicroscope and identified to class/order level in

the laboratory, except for copepods that were identi-

fied to species. Each species, according to the degree

of adaptation to the groundwater environment (Galas-

si, 2001), was listed as stygobite (SB: species strictly

associated to groundwater habitats, where they com-

plete the life cycle) or as non-stygobite (nSB),

Fig. 1 Map of sampled bores in areas of different land use in

the River Adige alluvial aquifer, Italy (white agricultural, gray

wild vegetation, striped areas: urban areas)

Hydrobiologia (2015) 743:139–150 141

123

including stygophiles (capable of living in both

surface and groundwater habitats) and stygoxenes

(epigean species which accidentally or occasionally

enter groundwater).

After the biological sampling, specific electrical

conductivity (SEC), pH, and temperature of ground-

water were measured by a multiparametric probe

(ECM MultiTM; Dr. Lange GmbH, Dusseldorf,

Germany) in a vessel directly after pumping for three

times. During measurements, a continuous groundwa-

ter flow was ensured across the probe. Water samples

to be tested for other chemical parameters in the

laboratory were set aside after fauna had been

removed. Samples were transported to the laboratory

in a cooler at 4�C within a few hours after collection.

Groundwater quality analyses were performed by the

local environmental protection agency (APPAT).

Groundwater samples were analyzed for 30 pesti-

cides/herbicides, metals (Cu2þ and Zn2þ), calcium,

total hardness, chloride, sulfate, nitrate, nitrite, NHþ4 ,

total N, and SRP (soluble reactive phosphorus, an

operationally defined P fraction which is, to a large

extent, equivalent to ortho-phosphate).

Data analysis

We grouped bores according to the mean level of NHþ4in bore groundwater. The H (high NHþ4 concentration)

group included 16 bores with a mean NHþ4C0.032 mg l-1; the L (low NHþ4 concentration) group

included 17 bores with a mean NHþ4 \0.032 mg l-1,

where 0.032 mg l-1 is the chronic lethal concentration

of NHþ4 determined for the stygobiotic D. belgicus (Di

Lorenzo et al., 2014). Groundwater quality parame-

ters, richness, and abundance of copepods, were

analyzed using one-way analysis of variance

(ANOVA) between the two groups of bores.

As subsequent samples from the same sampling

unit (bore) were considered temporal pseudorepli-

cates according to Hurlbert (1984), in ANOVAs we

considered the mean value (data of June and

October) of each abiotic parameter and the sum of

the number of copepod individuals in the two

sampling surveys, for each sampling unit. To test if

the seasonality of the agricultural activities (irriga-

tion and fertilizer/pesticide applications) produced an

effect on groundwater quality, a one-sample t test

was performed for each groundwater quality param-

eter on the difference between June (i.e., before the

expected peak in irrigation and during fertilizer

application) and October sampling (after both the

peak in irrigation and fertilizer application), for each

bore. For the one-sample t test, we assumed a null

hypothesis that there was no difference between the

mean of each quality parameter and zero. ANOVAs

and one-sample t tests were applied only to variables

with normally distributed data (verified with the

Shapiro test). Variables with homogeneous variances

(verified with the Bartlett test) were tested with

untransformed data; data for variables with hetero-

geneous variances were log-transformed before

ANOVAs and/or one-sample t tests. Univariate

analyses were performed using R software v. 2.15.0

(R Development Core Team, 2008).

Patterns in water quality were also examined with

a multivariate approach using principal components

analysis (PCA). Patterns of copepod assemblages

were examined using non-metric multidimensional

scaling (nMDS) incorporating the Bray–Curtis sim-

ilarity coefficient. The BEST (Bio-Env ? Stepwise)

permutational procedure (Clarke et al., 2008) was

applied to examine whether the multivariate biotic

and environmental variables were correlated under

the null hypothesis of complete independence of

biotic and environmental patterns. Permutational

analyses of variance (PERMANOVA; Anderson

et al., 2008) were used to test for differences in

water quality conditions and copepod assemblages

with SEASON (levels: J = June and O = October)

and bore NHþ4 concentration (levels: H = high and

L = low) as orthogonal and fixed factors (number of

permutations = 9,999). SIMPER analysis (Clarke,

1993) was used to identify the species that contributed

mostly to the differences between groups. In nMDS,

BEST, and PERMANOVA, copepod abundance was

square-root transformed and a dummy variable of 1

was added to all samples to facilitate the inclusion of

otherwise empty (zero abundance) samples. Levene

tests were performed in order to check for homoge-

neity of variances. Water quality variables were

normalized before PCA, BEST, and PERMANOVA.

Multivariate analyses were performed using E Primer

version 6 (Clarke & Gorley, 2006). All statistical

analyses were conducted with a significance level (a)

of 0.05.

142 Hydrobiologia (2015) 743:139–150

123

Results

Abiotic variables

Mean values of groundwater quality variables

(Table 1) in H bores were not significantly different

from those in L bores, with the exception of dissolved

oxygen (DO: F = 12.81, P = 0.001), saturation oxy-

gen (%DO: F = 12.69, P = 0.001,) and nitrate

(F = 14.83, P = 0.0005), which were significantly

lower in H bores where NHþ4 concentration was

C0.032 mg l-1 (Fig. 2), and SRP (F = 8.84,

P = 0.006), which was lower in L bores. There was

no significant difference in groundwater quality due to

the seasonality of agricultural activities (one-sample

t tests: P � 0.05).

In the multivariate analyses, groundwater chemical

composition was significantly different in H and L bores

(PERMANOVA, pseudo-F = 3.15, P = 0.0173), but

SEASON (PERMANOVA, pseudo-F = 0.96, P =

0.4374) and the NHþ4 9 SEASON interaction (PER-

MANOVA, pseudo-F = 1.83, P = 0.1236) remained

non-significant. This pattern was evident also in the

PCA ordination (Fig. 3).

The fungicides Penconazole (CAS RN 66246-88-6),

Pyrimethanil (CAS RN 53112-28-0), Iprodione (CAS

36734-19-7), Metalaxil (CAS 57837-19-1), and Oxa-

dixil (CAS 77732-09-3) were the only pesticides

detected out of the 30 searched. They were found in 4

samples out of 66 (33 in June and 33 in October).

Concentrations of individual pesticides were lower than

the legal TV of B0.1 lg l-1, except for Metalaxil whose

concentration was 0.14 lg l-1 at site W32 in June.

Groundwater fauna

A total of 968 copepods, comprising 9 SB (380

individuals) and 6 nSB species (588 individuals), were

collected from bores across the aquifer (Appendix 1—

Supplementary Material). A total of eight amphipod

individuals were collected in four wells. The stygobi-

otic oligochaete Troglochaetus beranecki Delachaux

1921 was collected from bore W10 with 23 individuals

in June and 7 individuals in October.

Total (SB ? nSB) and SB copepod species rich-

ness and abundance were higher in L than in H

bores (ANOVAs: Ftot richness = 16.11 P = 0.0003;

Ftot abundance = 10.71 P = 0.0026; FSB richness = 23.11 Ta

ble

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Hydrobiologia (2015) 743:139–150 143

123

P � 0.0001, FSB abundance = 18.64 P = 0.00015;

Fig. 4), while taxonomic richness and abundance

of nSB copepods was similar in H and L bores

(FnSBrichness = 0.28 P = 0.601; FnSBabb = 0.03

P = 0.87). Species-specific copepod abundance data

were heteroscedastic, and so remained after transfor-

mation (significant two-tailed Bartlett tests), and could

not be tested by analysis of variance.

Biological variables were not significantly corre-

lated to any combination of environmental variables in

multivariate analyses (BEST: SB ? nSB q = 0.22

P = 0.681; SB q = 0.33 P = 0.102; nSB q = 0.26

P = 0.703). Yet, abundance of stygobites (PERMA-

NOVA: pseudo-FSBabb = 3.61 P = 0.0187), pre-

sence/absence of stygobiotic taxa (PERMANOVA:

pseudo-FSBrichness = 6.35 P = 0.0002), and presence/

absence of all taxa (PERMANOVA: pseudo-

Ftot richness = 4.51 P = 0.0002) were significantly differ-

ent in H and L bores. However, there was no SEASON-

based difference (PERMANOVA: pseudo-FSBabb =

1.16 P = 0.3822; pseudo-FSBrichness = 0.54

P = 0.7691; pseudo-Ftot richness = 0.60 P = 0.8093),

nor was there a significant interaction between the two

factors (PERMANOVA: pseudo-F = 0.45SBabb P =

0.7246; pseudo-FSBrichness = 0.39 P = 0.891;

pseudo-Ftot richness = 0.97 p = 0.4735). The SB spe-

cies, Diacyclops italianus and Nitocrella psammo-

phila, provided a combined 63% of the discrimination

(33% and 29% respectively; Table 2) between H and

L bores. The remaining SB species contributed for

another 32%. This pattern was evident also in the

nMDS ordination (Fig. 5).

Discussion

Abiotic variables

The study area was homogeneous in altitude, soil use,

and geological structure. Bore water levels did not

vary seasonally, contrary to what might be expected in

agriculture-intensive areas. The local water manage-

ment authority (Provincia Autonoma di Trento)

strongly supports the modernization of the irrigation

Fig. 2 Bar-plot

representation of mean

(±standard errors) for: adissolved oxygen

concentration (mg l-1); boxygen saturation (%); cnitrate concentration

(mg l-1); d soluble reactive

phosphorus concentration

(lg l-1) in high-ammonium

(H bores: NHþ4C0.032 mg l-1; n = 32) or

low-ammonium

groundwater (L bores: NHþ4\0.032 mg l-1; n = 34) in

the alluvial aquifer of River

Adige

144 Hydrobiologia (2015) 743:139–150

123

infrastructure, encouraging farmers to use highly efficient

irrigation systems to save groundwater. The absence of an

impact of irrigation on the investigated groundwater body

is likely due to this policy, keeping groundwater

withdrawals into the limits of the aquifer recharge

(Autorita di Bacino dell’Adige, 2008). Pesticides were

detected in only four bores and with concentrations near

or below the threshold of no concern for groundwater

pollution. This pattern indicates that the use of pesticides

is limited in the study area or, more probably, performed

following best agricultural management practices

(BMPs). None of the others investigated groundwater

quality parameters exhibited concentrations at or above

legal pollution levels, with the exception of NHþ4(Table 1). There were significant differences in

groundwater quality in H versus L bores. In particular,

groundwater samples from H bores (with NHþ4concentrations C0.032 mg l-1) were characterized

by lower DO and nitrate concentrations and by higher

SRP concentrations than groundwater samples from L

bores (with NHþ4 concentrations\0.032 mg l-1). The

inverse relationship between NHþ4 on one side and

NO�3 ? DO on the other side is likely a result of

abiotic and biotic nitrogen transformation. NHþ4 tends

to be oxidized to NO�3 in a two-step nitrification

process (NHþ4 ! NO�2 ! NO�3 ) by aerobic chemo-

autotrophic bacteria, primarily Nitrosomonas and

Nitrobacter (Sharma & Ahlert, 1977; McGarvey

et al., 2007). Patches of nitrification are thus well

oxygenated and characterized by NHþ4 concentration

lower than NO�3 ones, as observed in the alluvial

aquifer of River Adige (Fig. 2, Table 1). In a few

alluvial aquifers, high natural NHþ4 concentration

occurs due to proximal buried organic material in

alluvial deposits (Jiao et al., 2010, and references

therein), but this does not appear to be the case for our

study area (Distretto Idrografico delle Alpi Orientali,

2010). Considering that 85% of the floodplain of the

River Adige is densely cultivated and that 90% of

fertilization is organic, the observed groundwater

quality pattern may be reasonably ascribed to organic

NHþ4 -containing fertilizers. Application of organic

fertilizers has been long recognized as a non-point

source of both N and P to freshwater (Carpenter et al.,

1998). Considering that the municipal waste water in

the study area is managed in state-of-the-art facilities

(Distretto Idrografico delle Alpi Orientali, 2010), the

higher concentrations of SRP, coupled with those of

NHþ4 , are likely attributable to the use of fertilizers in

the study area, as observed in other studies (Raave

et al., 2014). However, a definitive, unequivocal

association of groundwater NHþ4 and agricultural

practices may come only from an analysis of nitrogen

isotopes (Di Lorenzo et al., 2012).

Groundwater fauna

The use of groundwater for agricultural purposes is

known to threaten groundwater faunal and microbial

assemblages (Rouch et al., 1993; Dumas, 2004; Di

Lorenzo & Galassi, 2013; Griebler et al., 2014).

However, in our study, the variation in SB and/or nSB

copepod assemblages was not associated with sea-

sonal irrigation practices. Similar results were

observed by Korbel et al. (2013). In contrast, Di

Lorenzo & Galassi (2013) assessed an evident impact

of groundwater withdrawal for irrigation uses on

copepod abundances in the Mediterranean alluvial

aquifer of River Vibrata, in central Italy. However, the

aquifer of River Vibrata was fed by rainfalls only,

lacking a contribution from surrounding rocky

groundwater bodies, and was thus prone to a severe

Fig. 3 Principal components analysis (PCA) of groundwater

quality parameters according to NHþ4 factor in the alluvial

aquifer of River Adige based on samples collected in June and

October 2006. Groundwater bores with NHþ4 concentration

C0.032 mg l-1 are shown with black circles and those with

NHþ4 concentration \0.032 mg l-1 are shown with gray

triangles. Cumulative variance: 47 %

Hydrobiologia (2015) 743:139–150 145

123

water shortage, especially in summer (Di Lorenzo &

Galassi, 2013).

The dominance of crustaceans, primarily copepods

(91% of total density), in the alluvial aquifer of the

River Adige, supports earlier findings for similar

ecosystems (e.g., Sket,1999; Stoch & Galassi, 2010;

Di Lorenzo & Galassi, 2013; Di Lorenzo et al. 2013;

Fiasca et al., 2014). Biotic and environmental patterns

were completely independent, with the exception of

NHþ4 (BEST analysis). Our results upheld the hypoth-

esis that N-fertilizers, mainly organic, affect the

groundwater ecosystem in the alluvial aquifer of River

Adige, inducing a potential flow-on toxic effect to

stygobiotic copepods. We detected a clear difference

in copepod assemblage structure between H and L

bores, in both univariate and multivariate analyses.

Although the occurrence of nSB species in ground-

water may be considered related to the stochastic

infiltration of surface water at the bottom of the bores,

as observed in other studies (among others: Dole-

Olivier et al., 2000; Hahn & Matzke, 2005; Schmidt

et al., 2007; Hahn & Fuchs, 2008; Galassi et al., 2009;

Stein et al., 2010; Di Lorenzo & Galassi, 2013; Di

Lorenzo et al., 2013), bore water NHþ4 concentrations

higher than 0.032 mg l-1 seemed to affect the cope-

pod assemblage as a whole, and the stygobiotic

assemblages in particular, in the study area. The

stygobiotic species, Diacyclops italianus and Nitoc-

rella psammophila, seemed to be the most affected.

Fig. 4 Bar-plot

representation of mean

(±standard errors) for:

a species richness of

stygobiotic copepods;

b abundances of stygobiotic

copepods; c total copepod

species richness; d total

copepod abundance in high-

ammonium (H bores: NHþ4C0.032 mg l-1; n = 32) or

low-ammonium

groundwater (L bores: NHþ4\0.032 mg l-1; n = 34) in

the alluvial aquifer of River

Adige

Table 2 SIMPER results between groups of samples dis-

criminated according to NHþ4 factor

Species Contrib% Cum%

Diacyclops italianus 33.09 33.09

Nitocrella psammophila 29.52 62.61

Diacyclops paolae 11.62 74.22

Graeteriella unisetigera 9.30 83.52

Speocyclops infernus 6.17 89.69

Moraria stankovitchi 4.97 94.66

Contrib% percentage of dissimilarity provided by each species,

Cum% cumulative percentage of dissimilarity

146 Hydrobiologia (2015) 743:139–150

123

The toxic effect of NHþ4 and NH3 on crustaceans is

primarily, but not exclusively, related to the alteration

of respiratory metabolism (Aarset & Aunaas, 1990).

NHþ4 binds to crustacean hemocyanins (Romano &

Zeng, 2013). Under exposure to NHþ4 , the hemolymph

pH of aquatic crustaceans changes, altering the

detoxification mechanisms (Colt & Armstrong,

1981; Chen et al., 1994; Cheng et al., 2013), and thus

increasing organism vulnerability to other toxicants.

Ammonium compounds also alter osmoregulation,

affecting ATP-K?/Na? activities in the cell membrane

(Colt & Armstrong, 1981; Chen et al., 1994; Cheng

et al., 2013). Moreover, Di Marzio et al. (2013) argued

that NHþ4 acts as an endocrine disruptor, affecting

growth in epigean and hyporheic copepods. The

negative impact on survival or growth by NHþ4 occurs

via the disruption of various physiological mecha-

nisms, including osmoregulation, immunology, acid/

base balance, gas exchanges, as well as increased

oxidative stress, and pathogenic susceptibility, and

may induce histologic damage (Romano & Zeng,

2013).

There is not a unique European threshold value

(TV) for NHþ4 in groundwater. NHþ4 is one of the

pollutants indicated in the Annex II of the Directive

2006/118/EC for which TVs are to be provided by

each Member State. Twenty-one Member States have

so far provided highly different TVs for NHþ4 , ranging

from a minimum value of 0.084 mg l-1 to the

maximum allowed TV of 5 mg l-1 (European Com-

mission, 2010). The TV for NHþ4 in Italy is 0.5 mg l-1

(Repubblica Italiana, 2009). According to the results

of our study, such TVs do not seem to adequately

protect groundwater copepod assemblages in the

alluvial aquifer of River Adige.

Conclusions

Although agricultural N loading in excess of crop

needs is a long-recognized issue for Europe’s receiv-

ing water bodies, current EU threshold values for NHþ4do not address the potential adverse impact on

groundwater biota. Our results suggest that the com-

position, richness, and abundance of groundwater

copepods can be reliable indicators of groundwater

quality and ecosystem health related to NHþ4 contam-

ination. In particular, our study showed that levels of

NHþ4 lower than the present threshold values adopted

by EU Member States may not be protective of

stygobiotic copepod assemblages in some alluvial

aquifers underlying densely cultivated areas. Despite

copepod commonness and dominance in groundwater

habitats, to our knowledge, no information currently

Fig. 5 nMDS (non-metric

Multidimensional Scaling)

ordination of stygobiotic

copepod assemblages

according to NHþ4 factor in

the alluvial aquifer of River

Adige, based on June and

October abundances per

sample. Groundwater bores

with NHþ4 concentration

C0.032 mg l-1 are shown

with black circles and those

with NHþ4 concentration

\0.032 mg l-1 are shown

with gray triangles. Vectors

defining correlations

between each species and

the ordination are also

shown. Species

abbreviations as in

Appendix 1—

Supplementary Material

Hydrobiologia (2015) 743:139–150 147

123

exists on NHþ4 effects on groundwater copepod

reproduction, egg viability, hatching rates, or lipid

metabolism. We believe that this is an area worth

investigating, in order to estimate the true extent of the

NHþ4 -induced biological damage. The time needed for

the recovery of copepod assemblage in the contami-

nated sectors of the alluvial aquifer of River Adige,

after a hypothetical reestablishment of good ground-

water quality, also is difficult to estimate. The

recovery depends on several factors, such as ground-

water flow and population sizes in the unaffected areas

from which copepods can re-colonize. However, we

think that such a process may take some time, due to

low reproductive rates, long life spans (up to

3.5 years), and long post-embryonic development

time before sexual maturity, of stygobiotic copepods.

Moreover, we focused on the effect only of NHþ4because copepods assemblage structure proved not to

be related to other chemical patterns. However, while

such a situation applies to our study aquifer, it may not

apply to other scenarios, when potential interactions

with NHþ4 and other pollutants, such as pesticides,

should be explored.

Acknowledgements The project was funded by a grant from

the Basin Authority of the River Adige (Trento, Italy). We thank

Fabio Lazzeri for the design and concept of Fig. 1, and Andrea

Marchetti and Enrico Gattone for help in field sampling.

Constructive criticism by Dr. Grant C. Hose (Macquarie

University, NSW, Australia) and an anonymous reviewer also

improved the paper.

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