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Sensi i i y o mac oin e eb a e indica o axa o me al g adien s in mining a eas in 1
No he n Spain 2
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N. COSTAS1, I. PARDO1,*, L. MÉNDEZ-FERNÁNDEZ2, M. MARTÍNEZ-MADRID3 and P. RODRÍGUEZ2
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1Dp . Ecology and Animal Biology. Uni e si y o Vigo. 36310 Vigo, Spain. 10
2Dp . Zoology and Animal Cell Biology. Uni e si y o he Basque Coun y, Apdo. 644. 48080 11
Bilbao, Spain. 12
3Dp . Gene ics, Physical An h opology and Animal Physiology. Uni e si y o he Basque Coun y 13
Apdo. 644. 48080 Bilbao, Spain 14
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*Co esponding au ho : Tel.: +34 986812585; ax: +34 986812556; e-mail:20
[email p o ec ed]
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Decla a ions o in e es : none 24
This is he accep ed manusc ip o he a icle ha appea ed in inal o m in Ecological Indica o s 93 : 207-218 (2018), which has
been published in inal o m a h ps://doi.o g/10.1016/j.ecolind.2018.04.059. © 2018 Else ie unde CC BY-NC-ND license
(h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/)
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Abs ac 26
The s udy o mac oin e eb a e communi ies using a Re e ence Condi ion App oach 27
(RCA) is a powe ul ool o de ec ing he impac s o mining ac i i ies due o hea y me al 28
con amina ion in i e s. Howe e , s udies on his opic based on an RCA ully sa is ying he 29
Wa e F amewo k Di ec i e (WFD) c i e ia o e e ence si e selec ion a e s ill sca ce. Following 30
a WFD-complian RCA design, a o al o 14 e e ence and 15 es si es we e in ensi ely sampled 31
du ing July 2015 in he Nalón Ri e basin (As u ias, No he n Spain). Mac oin e eb a e samples, 32
wa e physicochemis y, me al concen a ions in sedimen s and habi a quali y we e e alua ed 33
a each selec ed si e. To de e mine whe he inc easing hea y me al le els in sedimen s ha e 34
signi ican ecological impac s in he s uc u e o mac oin e eb a e communi ies in he s udy 35
a ea, we applied a combina ion o non-pa ame ic mul i a ia e and uni a ia e p ocedu es, as 36
well as g adien analysis and Th eshold Indica o Taxa ANalysis (TITAN) o e he bio ic and 37
en i onmen al da ase s. We ound signi ican al e a ions in he mac oin e eb a e communi y 38
s uc u e wi h espec o he e e ence condi ion (REF g oup) o hose es si es wi hin me cu y 39
(HG g oup) and gold (AU g oup) mining a eas, wi h Epheme op e a, Plecop e a and T ichop e a 40
(EPT) ichness and abundance being he communi y desc ip o s showing he s onges 41
al e a ions in ela ion o mining p essu es in he s udied a ea. Me al concen a ions in 42
sedimen s we e signi ican ly co ela ed o he me ics o communi y s uc u e ela ed o 43
abundance, ichness and biodi e si y, wi h As and Hg being he wo me als showing highe 44
biological e ec s on mac oin e eb a e assemblages. TITAN also allowed us o es ablish a se o 45
mac oin e eb a e amilies speci ically sensi i e o me al concen a ions in sedimen s, mos o 46
which co esponded o EPT amilies. Ou esul s p o e ha de e mined sensi i e axa could be 47
conside ed as eliable indica o s o me al pollu ion in he mining a eas o No he n Spain. F om 48
a WFD pe spec i e, ou s udy clea ly indica es ha he esponsi e communi y desc ip o s ound 49
in his s udy a e ac ually key pa ame e s in he e alua ion o he ecological s a us in he i e s 50
o No he n Spain. These da a a e highly ele an o he u u e de elopmen o en i onmen al 51
quali y s anda ds o i e sedimen s in Spain. 52
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Key-Wo ds: Hea y me als; ben hic mac oin e eb a es; me ics; communi y s uc u e; mines 58
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1. In oduc ion 61
F eshwa e ecosys ems a e one o he mos endange ed ecosys ems wo ldwide, mainly 62
due o human dependence on hem (Sala e al. 2000; Dudgeon e al. 2006). Inc easing an h opic 63
p essu e on unning wa e s has led o se e e le els o en i onmen al deg ada ion in hese 64
ecosys ems. Indeed, indus ialisa ion, u banisa ion, land-use changes and wa e cou se 65
al e a ions a e he main d i e s h ea ening unning wa e s (Malmq is & Rundle 2002). Among 66
he exis ing p essu es, mining ac i i ies may well be one o he mos pe sis en impac s on i e s 67
and s eams (Ma qués e al. 2001a), and me al pollu ion has become a majo conce n ega ding 68
he p ese a ion o aqua ic sys ems in and downs eam o mining a eas (Luoma e al. 2010). In 69
gene al, oge he wi h suspended pa icula e ma e ial, sedimen s ep esen he main s o ing 70
si e o hea y me als in i e s (mainly in he ine ac ion, < 63 µm) (ERMITE-Conso ium 2004), 71
and bo h sedimen deposi ion and esuspension may ep esen po en ial sou ces o pollu ion a 72
di e en spa ial scales (Ma in 2000). 73
Assessing he isk o he ecological impac s om human ac i i ies is he co e o an 74
e ec i e managemen o eshwa e ecosys ems, and in his ega d, he e alua ion o he 75
ela ionships be ween biological esponses and en i onmen al p essu es is c ucial. The Wa e 76
F amewo k Di ec i e (WFD, EC 2000) app oach is ocused on he de elopmen o ecological 77
s a us classi ica ion sys ems o wa e bodies based on assessmen s o he deg ee o de ia ion 78
in biological condi ion a pa icula s udy si es om he so-called Re e ence Condi ion (EC 2000). 79
In Eu ope, his concep is de ined by he use o minimally dis u bed si es (sensu S odda d e al. 80
2006) cha ac e ised by ype-speci ic biological condi ions and he accompanying 81
physicochemis y and hyd omo phology, in which he absence o signi ican human 82
dis u bances has been assessed (Pa do e al. 2012). Wi hin his con ex , he use o di e en 83
biological quali y elemen s (i.e., mac ophy es, phy oben hos, phy oplank on, ben hic 84
mac oin e eb a es and ish) is p omo ed by he WFD as an e ec i e assessmen ool o 85
de ec ing impac s de i ed om an h opogenic p essu es, such as eu ophica ion o o ganic 86
pollu ion (Sánchez-Mon oya e al. 2010; Pa do e al. 2014). Among hem, me ics based on 87
mac oin e eb a e communi y s uc u e a e p obably he mos widely used indica o s o wa e 88
quali y (Me cal e 1989; Reynoldson & Me cal e-Smi h 1992; Bi k e al. 2012; Ga cía e al. 2014). 89
In ac , wo ldwide mining-de i ed wa e and sedimen con amina ion by me als was 90
demons a ed o ha e a di ec e ec on di e en ben hic communi y s uc u al componen s, 91
such as abundance, di e si y, ichness, o me ics ela ed o sensi i e species (Hi s e al. 2002; 92
Solà e al. 2004; Doi e al. 2007; Qu e al. 2010; Poul on e al. 2010; Ruiz-Ga cía e al. 2012; By ne 93
e al. 2013). Fo all ha , s udies we e e e ence condi ions a e designa ed acco ding o WFD 94
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p ecep s a e desi able owa ds c ea ing an adequa e backg ound o allow being able o 95
e icien ly use ben hic mac oin e eb a es as indica o s o me al pollu ion. 96
The Nalón Ri e basin was one o he main mining-exploi ed a eas in No he n Spain, 97
whe e he Texeo coppe mines (loca ed in he Riosa dis ic ) and he me cu y mines (Mie es, 98
Pola de Lena and Somiedo dis ic s) we e s ill ac i e un il he ea ly 1970s (Méndez-Fe nández e 99
al. 2015). Spoil heaps de i ed om pas mining ac i i ies can s ill ep esen an impo an sou ce 100
o local con amina ion o i e sedimen s (Lo edo e al 2006, 2010). P e ious s udies ha e 101
epo ed sedimen pollu ion in i e s a ec ed by mine spoil heaps, as well as me al 102
bioaccumula ion and oxici y in he Nalón Ri e basin (O doñez e al. 2013; Méndez-Fe nández 103
e al. 2015). The en i onmen al quali y s anda ds (EQS) di ec i e (EC 2013) ecognised he 104
impo ance o bo h sedimen s and bio a ma ices o wa e quali y policies in Eu ope. Howe e , 105
bo h sedimen and bio a EQSs o me als and me alloids (hence o h, me als) need o be 106
de eloped by membe s a es. Di e en p oposals ha e a emp ed o ela e me al issue 107
concen a ions o me al-induced ecological impac s on di e en mac oin e eb a e me ics 108
(Luoma e al. 2010; Schmid e al. 2011; Rainbow e al. 2012; De Jonge e al. 2013; Be oe s e 109
al. 2016). Howe e , he in eg a ion o me al le els in he sedimen due o mining ac i i ies and 110
he ecological impac s on i e mac oin e eb a e communi ies should be he objec o deepe 111
esea ch, especially in egions like he Nalón Ri e basin wi h a long his o y o mining ac i i ies. 112
Thus, he e alua ion o me al concen a ions in sedimen s wi hin a Re e ence Condi ion 113
App oach (RCA) may be ele an no only in he de elopmen o use ul ools o assessing he 114
isk o mining ac i i ies de i ed om me al pollu ion, bu also in he de elopmen o EQSs in 115
Spain o a Eu opean le el. 116
A key issue in biomoni o ing is he le el o axonomic esolu ion used. Al hough ecen 117
s udies poin ed ou he bene i s o using ine axonomic le els in s eam assessmen (Gue old 118
2000; Lena & Resh 2001; S i ock e al. 2014), he amily le el con inues o be p e e ed as i 119
was demons a ed o p o ide aluable ecological in o ma ion (Lena & Ba bou 1994) and is able 120
o espond signi ican ly o di e en p essu es ( o example, see Pa do e al. 2014). Mo eo e , 121
di e en s udies demons a ed simila esponse pa e ns o di e en s esso s a bo h he 122
genus and amily le els (i.e., Bowman & Bailey 1997; Pond e al. 2008). Conside ing ha amily 123
cons i u es he le el o esolu ion used in ecological quali y assessmen in he con ex o he 124
WFD in No he n Spain (Pa do e al. 2014; METI 2015), e alua ing he abili y o his axonomic 125
le el o de ec signi ican me al con amina ion is o high ele ance. 126
The gene al aim o his s udy is o de ec and es ima e he possible e ec s o sedimen 127
me al pollu ion on he mac oin e eb a e communi ies inhabi ing he i e s loca ed in mining 128
a eas o he Nalón Ri e basin in o de o imp o e assessmen , managemen and conse a ion 129
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e o s. We hypo hesise ha me al pollu ion plays a undamen al ole in s uc u ing 130
mac oin e eb a e communi ies in hese a eas. The speci ic objec i es include 1) o desc ibe he 131
communi y composi ion o mac oin e eb a es a bo h e e ence and po en ially impac ed 132
si es, 2) o assess he esponse o a ben hic communi y s uc u e o a g adien o me al 133
concen a ion in he sedimen s based on he me al le els measu ed a each si e as a a io wi h 134
a p e-selec ed e e ence condi ion in he s udy a ea, 3) o de ec ben hic axa ha a e sensi i e 135
o me al pollu ion using a wide exposu e ange o sedimen concen a ions, and 4) o de e mine 136
whe he he a ia ion in me ics a he amily le el can be de ec ed by al e a ions due o 137
sedimen me al concen a ions. O e all, ou esea ch on he mac oin e eb a e communi ies 138
wi hin he mining a eas o he Can ab ian egion using a e e ence condi ion app oach may 139
acili a e he iden i ica ion o a ge -sensi i e amilies o he de elopmen o adequa e EQSs, 140
bo h a egional and Eu opean scales. 141
2. Ma e ials and me hods 142
2.1. S udy a ea 143
This s udy was conduc ed in he Nalón Ri e basin (As u ias, No he n Spain), including 144
bo h he Nalón and Na cea i e s and some o hei ibu a ies (Fig. 1). This basin is he la ges 145
one wi hin he Can ab ian wa e dis ic , wi h a o al leng h o 140.8 km co e ing a o al a ea o 146
4907 km2. The dominan clima e is oceanic, cha ac e ised by mild win e s and summe s, and 147
abundan ain all h oughou he yea (a mean annual p ecipi a ion o 1296 mm). As a 148
consequence o i s li hological cha ac e is ics, he s udy egion also wi nessed his o ical mining 149
ac i i y, wi h mo e han 800 si es ac i e be ween 1950 and 1975 (Águeda-Villa & Sal ado -150
González 2008; O dóñez e al. 2008). 151
A o al o s eam 29 s eam si es we e sampled once du ing July 2015 (Fig. 1). The 152
sampling design conside ed wo main g oups o si es: e e ence si es (n = 14) and es si es (n = 153
15), and co e ed ou o he mac oin e eb a e communi y-based i e ypologies (Types 1, 2, 154
4 and 5) o he Can ab ian egion (Pa do e al. 2014). Re e ence si es (REF g oup) we e selec ed 155
acco ding o WFD c i e ia (EC 2000) and alida ed ollowing he p ocedu e and c i e ia o he 156
absence o p essu es desc ibed in Pa do e al. (2012). Eigh o he ou een e e ence si es 157
ac ually belong o he spa ial ne wo k used by he Can ab ian Hyd og aphical Con ede a ion 158
(CHC) he Nalón Ri e Wa e Managemen Agency (unpublished da a). Tes si es we e selec ed 159
co e ing i e s nea his o ic (me cu y and coppe mines) and ac i e (gold mine) mining 160
indus ies, and hus subjec o known inpu s o hea y me als. Th ee o hem we e loca ed in a 161
coppe mine a ea (CU g oup), 8 in me cu y mine a eas (HG g oup) and 4 in a gold mine a ea (AU 162
g oup) (Fig. 1). 163
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Fig. 1. Loca ion o he Nalón Ri e basin (No he n Spain) and he 29 sampling si es (wi h hei 164
co esponding iden i ica ion codes) included in his s udy. ● = Re e ence si es (REF), ○ = si es wi hin 165
me cu y-mining a eas (HG), = si es wi hin gold-mining a eas (AU), = si es wi hin coppe -mining a eas 166
(CU). 167
168
169
2.2. Da a collec ion and labo a o y p ocessing
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2.2.1. Biological da a 171
Collec ion and p ocessing o mac oin e eb a e samples was done acco ding o he 172
Spanish o icial p o ocol ML-R -I-2013 (see Pa do e al. 2014). The sampling p o ocol ollowed a 173
mul ihabi a p ocedu e adap ed om Ba bou e al. (1999). A each s udy si e, a o al o wen y 174
‘sample uni s’ o 0.125 m2 each (dis ibu ed p opo iona ely in he main habi a s exis ing along 175
a 100 m each) we e collec ed using a kick-ne (500 µm mesh size), and combined in o a single 176
mixed sample (a o al sampled a ea o 2.5 m2). A e collec ion, he samples we e p ese ed 177
wi h 70% e hanol and s o ed. All we e washed and sepa a ed in o h ee ac ions using a se ies 178
o sie es o di e en mesh sizes (5, 1 and 0.5 mm). The in e eb a es we e so ed in each 179
ac ion and subsamples aken in he 1 and 0.5 mm ac ions only when necessa y (W ona e al. 180
1982). Taxon ichness and abundance we e e alua ed a he amily le el (excep o aqua ic 181
oligochae es and Hyd aca ina) unde 57x magni ica ion (Olympus SZX9). 182
2.2.2. En i onmen al da a 183
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Wa e samples om each si e we e collec ed in 100 ml polyp opylene bo les and kep 184
ozen un il hei analysis a CACTI (Cen o de Apoio Cién i ico e Tecnolóxico á In es igación, 185
Uni e si y o Vigo, Spain). Nine a iables we e measu ed: ni i es (N-NO2−), ni a es (N-NO3−), 186
phospha es (P-PO4-3), ammonium (N-NH4+) and chlo ides (Cl−) by means o a con inuous- low 187
analyse (Au o-Analyse AA3, B an+Luebbe, Ge many); ca ions o calcium (Ca+2), magnesium 188
(Mg+2), po assium (K+) and sodium (Na+) we e analysed by ICP-OES (Op ima 4300 DV, Pe kin-189
Elme ). Se e al physicochemical a iables we e measu ed in si u using po able de ices: wa e 190
empe a u e, pH and dissol ed oxygen (O ion 5-s a , The moScien i ic), and elec ic conduc i i y 191
(O ion 3-S a , The moScien i ic). 192
Sedimen samples om he uppe 5-10 cm laye o ine sedimen s we e collec ed along 193
a 25 m each o he i e bank a each s udy si e using a s ainless-s eel spade and s o ed a 4°C 194
in he da k. Pa icle-size dis ibu ions (exp essed as a pe cen age o he d y weigh ), o al o ganic 195
ca bon pe cen ages (%TOC: B yan e al. 1985; USEPA 1990) and me al concen a ions (Al, As, 196
Cd, C , Cu, Fe, Hg, Mn, Ni, Pb, Se, and Zn, epo ed as d y weigh , µg g-1 dw) we e measu ed in 197
he sedimen samples acco ding o me hods desc ibed in Méndez-Fe nández e al. (2017); o 198
Al, Fe and Mn, hei co esponding limi s o quan i ica ion (LOQ) we e 0.05 µg l-1, 0.03 µg l-1 and 199
0.1 µg l-1, espec i ely. Reco e y a es we e wi hin ce i ied alues (Al has no ce i ied alues) in 200
he s anda d sedimen s (Bu alo Ri e sedimen : RM8704, USA and Sewage Sludge-3: CRM 031-201
040, UK), used as quali y con ols. 202
The Flu ial Habi a Index IHF (Pa do e al. 2002) was calcula ed a each si e. This index 203
is a measu emen composed o se e al aspec s o habi a he e ogenei y di ided in o se en 204
blocks: subs a e inclusion in i les and sedimen a ion in pools, equency o i les, subs a e 205
di e si y, low eloci y and dep h egime, shade co e , he e ogenei y in bo h physical habi a 206
di e si y and ood esou ces, and aqua ic ege a ion co e age and composi ion. 207
2.3. S a is ical analysis 208
2.3.1. Mul i a ia e analysis o mac oin e eb a e communi ies 209
The en i e biological da ase consis ed o a log(x+1)- ans o med ‘ axon x si e’ ma ix o 210
abundances. The e ec s o me al le els in he sedimen s and o he chemical and physical 211
en i onmen al ac o s on he ben hic communi y s uc u e we e examined using a 212
pe mu a ional mul i a ia e analysis o a iance (PERMANOVA) (Ande son 2001). One-way 213
models ( ixed ac o s = ‘Mining ac i i y’), using 9999 maximum un es ic ed pe mu a ions, 214
ollowed by pai wise compa isons (a 999 pe mu a ions) we e pe o med a a signi icance le el 215
o α < 0.05. The B ay-Cu is index was chosen as a sui able mul i a ia e dis ance measu e o 216
axon composi ion and s uc u e (B ay & Cu is 1957). Due o he lowe numbe o possible 217
pe mu a ions o he pai wise es s, a Mon e-Ca lo sampling p ocedu e was applied o ob ain 218
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mo e eliable P alues (Ande son e al. 2008). To help cla i y he na u e o he PERMANOVA 219
esul s, a pe mu a ional analysis o mul i a ia e dispe sions (PERMDISP) (a α < 0.05) was also 220
applied o es o homogenei y o dispe sions be ween he mining ac i i y g oups o si es, in 221
o de o de e mine i any signi ican PERMANOVA esul may be in ac due he e ec o he 222
conside ed ac o o i a dispe sion e ec is p esen and u he da a explo a ion is desi able. 223
Simila i y pe cen age analyses (SIMPER) (Cla ke & Go ley 2006) we e applied o de e mine 224
which axa con ibu ed mos o he wi hin-g oup simila i ies and dissimila i ies be ween g oups. 225
All analyses we e conduc ed using PRIMER .6 so wa e (Cla ke & Go ley 2006). 226
2.3.2. Uni a ia e desc ip o s o mac oin e eb a e communi y s uc u e 227
In he sea ch o speci ic desc ip o s o al e a ions in communi y s uc u e, 228
mac oin e eb a e da a we e used o calcula e a se o abundance and di e si y me ics, mainly 229
selec ed on he basis o p e ious s udies assessing mining impac s (G ay & Delaney 2008; Qu e 230
al. 2010; Poul on e al. 2010; By ne e al. 2013): o al abundance, abundance o EPT 231
(Epheme op e a, Plecop e a and T ichop e a) axa, % o 3 dominan axa, % o oligochae es, 232
axon ichness, ichness o EPT axa, and he Ma gale ichness and Shannon-Wiene di e si y 233
indices. K uskal-Wallis non-pa ame ic es s ollowed by mul iple compa isons wi h he Dunn-234
Bon e oni pos -hoc es , we e used o assess o signi ican di e ences in he selec ed indices 235
o he p ede ined mining ac i i y ea men s (REF, HG, CU and AU si e g oups). Compa isons 236
we e conduc ed using he IBM® SPSS S a is ics 22 so wa e. 237
2.3.3. En i onmen al g adien s 238
The habi a ma ix consis ed o all measu ed en i onmen al da a ha migh po en ially 239
a ec he dis ibu ion o mac oin e eb a e assemblages along he s udy a ea (29 samples x 30 240
a iables). Mul i a ia e g adien analyses we e employed o explo e he a iabili y in he habi a 241
cha ac e is ics and isualise majo ends wi hin he da ase . Spa ial o dina ion o 242
en i onmen al a iables (p e iously log- ans o med and s anda dised) was examined h ough 243
P incipal Coo dina e Analysis (PCoA) in PRIMER .6 so wa e (Cla ke & Go ley 2006), and he 244
con ibu ion o each a iable o he o dina ion was calcula ed by Pea son co ela ion analysis o 245
iden i y he s esso g adien s ha be e cha ac e ised he s udy a ea. P io o PCoA, edundan 246
a iables we e elimina ed by means o a Spea man’s co ela ion ma ix (a α < 0.05), es ablishing 247
= 0.7 as he cu -o le el. 248
To assess me al le els in he sedimen s, we i s es ima ed backg ound me al le els as 249
he concen a ion ange in sedimen s measu ed in p e-es ablished e e ence si es, i.e., in he 250
absence o iden i ied an h opogenic p essu es (al hough di use pollu ion could no be 251
discoun ed). Th eshold le els we e de ined as he high pe cen ile o he backg ound da a 252
dis ibu ion in e e ence si es o he Nalón Ri e basin, and in e p e ed as no-e ec 253
9
concen a ions. Me al h eshold le els we e calcula ed as he 90 h pe cen ile (P90) (wi h hei 254
95% con idence in e als) om he dis ibu ion o he log- ans o med da a a he e e ence 255
si es, calcula ed a e boo s apping (wi h 1000 es a s) using IBM® SPSS S a is ics 22 so wa e, 256
and hen an i-logged o ans o m hem back. 257
2.3.4. P essu e- esponse ela ionships 258
To asce ain whe he inc easing le els o me als in he sedimen s we e in ac associa ed 259
wi h he possible de-s uc u ing e ec s de ec ed in he mac oin e eb a e communi ies h ough 260
mul i a ia e and uni a ia e compa isons, wo app oxima ions we e conside ed: a) Pea son 261
co ela ion coe icien s be ween he log- ans o med (when necessa y) alues o each 262
uni a ia e me ic and he concen a ions o hea y me als and SedPoll sco es, calcula ed wi h 263
IBM® SPSS S a is ics 22 so wa e; b) indi idual in e eb a e esponses o s ess g adien s 264
e alua ed by means o Th eshold Indica o Taxa ANalysis (TITAN) (Bake & King 2010) ( un wi h 265
he TITAN 2.0 package in R.3.3.2.). The TITAN me hod uses indica o alue (IndVal) sco es 266
ob ained om indica o species analysis (Du êne & Legend e 1997) and s anda dised o z-267
sco es o de ec bo h he loca ion o axon-speci ic change poin s and he esponse di ec ion 268
along an en i onmen al g adien (Bake & King 2010). Depending on he axa associa ed wi h 269
ei he side o he change poin , hey a e designa ed as declining (z-) o inc easing (z+), and a e 270
used o ace cumula i e esponses: sum(z-) and sum(z+) (Bake & King 2010). La ge alues o 271
sum(z) sco es imply ha many axa show s ong esponses a a simila alue o he 272
en i onmen al g adien . Synch onous signi ican changes among axa a e conside ed as 273
e idence o a communi y-le el h eshold (Bake & King 2010). By means o boo s apping, TITAN 274
iden i ies eliable and pu e indica o axa as well as he unce ain y a ound ob ained change 275
poin s (bo h a he axon and communi y le els). We pe o med TITAN on he log(x+1)-276
ans o med abundances o hose mac oin e eb a e axa occu ing in a leas 3 samples (Bake 277
& King 2013). 278
Taxon esponses we e e alua ed along he gene al g adien s ob ained om he PCoA 279
and along a me al pollu ion g adien in he sedimen de ined by he deg ee o di e ence o he 280
es si es o he e e ence condi ion. A each si e, e e y single me al concen a ion in he 281
sedimen was di ided by he co esponding P90 alue calcula ed in he e e ence si es o ge a 282
me al quo ien (P90-Q). The me al concen a ion in he sedimen was hen classi ied in o i e 283
po en ial ca ego ies on he basis o he me al quo ien s as ollows: 1) Simila - o- e e ence si es 284
whe e P90-Q ≤ 1.0, 2) Low me al concen a ion whe e P90-Q = 1.1-2.0, 3) Medium me al 285
concen a ion whe e P90-Q = 2.1-10.0, 4) High me al concen a ion whe e P90-Q = 10.1-50.0, 286
and 5) Ve y high me al concen a ion whe e P90-Q ≥ 50.1. Nex , a each si e he ele an me als 287
we e selec ed (see he Resul s sec ion) and P90-Q alues summed and s anda dised by he 288
16
3.3. P essu e- esponse ela ionships 426
The signi ican di e ences in communi y s uc u e p e iously shown in sec ion 3.1, 427
appea ed o be clea ly associa ed wi h me al le els in he sedimen acco ding o he p essu e-428
esponse analyses. 429
Co ela ion analyses be ween me al concen a ions in he sedimen s and he me ic 430
desc ip o s o communi y s uc u e showed a signi ican co ela ion (Table 5). As and Hg we e 431
he wo me als showing highe e ec s on he mac oin e eb a e communi y abundance, 432
ichness and biodi e si y, wi h 6 and 5 me ics a ec ed, espec i ely (Table 5); o Cd, Cu and Se 433
only 1 o 3 me ics we e al e ed, and Pb was no signi ican ly co ela ed wi h any o he 434
e alua ed me ics. The s onges obse ed associa ions we e EPT abundance wi h As ( = -0.800, 435
P < 0.001), and EPT ichness wi h As and Hg ( = -0.646 and = -0.659 espec i ely, P < 0.01) 436
(Table 5). Indeed, abundance me ics we e mo e equen ly a ec ed by se e al me als (4), 437
ollowed by o al ichness (3 me als), sugges ing ha hese me ics migh be use ul in de ec ing 438
me al mix u e p essu es in ield si ua ions. Resul s ha a e ein o ced by he signi ican 439
co ela ions obse ed also be ween me ics and he SedPoll sco es (Table 5). 440
441
Table 5. Pea son co ela ion coe icien s o he me al concen a ions in sedimen s (used as ele an o 442
TITAN) and he 9 selec ed desc ip o s o ben hic communi y s uc u e. Signi ican co ela ions a e 443
indica ed by * P < 0.05; ** P < 0.01 and *** P < 0.001. 444
445 On he whole, 20 amilies we e iden i ied as indica o axa o he s ess g adien s 446
de ined by he i s wo axes o he en i onmen al PCoA (PCo1 and PCo2) (Fig. 4). Fou een o 447
hese axa showed synch onous declines in esponse o he inc easing le els o As, Cd, Cu, Se, 448
Cl-and % o he ine sedimen ac ion (< 63 µm) de ined by PCo1 (see Fig. 3 and sec ion 3.2). 449
Among hese 14 axa, 9 we e EPT axa, 3 we e Coleop e a and 2 Dip e a (Fig. 4). Rega ding he 450
g adien de ined by PCo2, ou amilies (Ancylidae, B achycen idae, Glossossoma idae and 451
Se icos oma idae) declined wi h inc easing le els o Hg, P-PO4-3 and Cl-, while 2 amilies 452
(Hyd ome idae and Hyd aca ina) seemed o be ole an (z+) o hese inc eases (Fig. 4). 453
Howe e , conside ing ha he men ioned PCo2 g adien is de ined bo h by nega i e (Se and Cd) 454
and posi i e (Hg) co ela ions wi h me als, in e p e ing o wha ex en hey a e ole an o Hg 455
17
o sensi i e o Se and Cd is di icul . Howe e , i was no possible o es ablish change h esholds 456
a he communi y le el because acco ding o he TITAN p emises, a eliable communi y 457
h eshold may be based on synch onous changes in he abundance o many indica o axa wi hin 458
a na ow ange o he conside ed p edic o g adien (Bake & King 2010). Howe e , in ou da a, 459
he e was a limi ed numbe o eliable indica o axa, and hey exhibi ed b oad con idence 460
in e als. By conside ing he 95 h quan ile in e al (QI) as indica i e o he maximum esis ance 461
o each sensi i e indica o axon o he mining g adien s de ined by he PCoA axes, we ound 462
ha mos amilies showed di e en ial sensi i i ies o he de ined s esso s bo h be ween o de s 463
and wi hin he same o de (95 h QI ange: -0.15 o 4.33) (Table 6). In gene al, he dip e an amily 464
Tipulidae (95 h QI = -0.15) and he caddis lies Philopo amidae (95 h QI = 0.44) and 465
Se icos oma idae (95 h QI = 0.80) we e he mos sensi i e axa, while he i le bee le amily 466
Elmidae (95 h QI = 4.33) showed he highes esis ances among he ob ained indica o axa 467
(Table 6). 468
Using he sedimen pollu ion sco es (SedPoll) g adien , a o al o 9 axa (see Fig. 4) 469
showed synch onous dec eases wi h he inc easing me al pollu ion g adien . When conside ing 470
sum (z-) he ob ained change poin was 3.7, wi h a con idence in e al anging be ween 0.55 471
and 12.25. Among he indica o axa, Bae idae appea ed as he mos esis an o SedPoll (95 h 472
QI = 26.25), while Se icos oma idae (95 h QI = 3.70), ollowed by Sci idae (95 h QI = 5.48) and 473
Hep ageniidae (95 h QI = 9.25), we e less esis an (Table 6). 474
475
Fig. 4. Change poin s o signi ican mac oin e eb a e indica o axa (pu i y ≥ 0.95, eliabili y ≥ 476
0.95, P < 0.05) iden i ied in Th eshold Indica o Taxa ANalysis (TITAN), ac oss he g adien s 477
de ined by he PCo1 and PCo2 axes o he en i onmen al P incipal Coo dina e Analysis (PCoA) 478
and Sedimen Pollu ion g adien (SedPoll) ac oss he 29 sampling si es. Solid and do ed lines 479
ep esen he 5 h-95 h quan ile in e als o each change poin , illed symbols (●) co espond o 480
sensi i e axa (z-) and emp y symbols (○) o ole an axa (z+) (see ex in he Resul s sec ion o 481
mo e de ails on his las case). 482
483
18
Table 6. TITAN indica o axa o he en i onmen al g adien s ob ained om he sedimen pollu ion 484
g adien (SedPoll) and PCoA ( wo main axes: PCo1 and PCo2). The indica o alue (IndVal), en i onmen al 485
change poin based on z maximum (zen .cp) and quan ile in e als (QI) o 5% and 95% a e gi en o each 486
axon. Response di ec ions (z- o z+) assignmen s a e also indica ed. 487
488
4. Discussion 489
Mining impac s on eshwa e ecosys ems ha e been he objec o in ense esea ch 490
wo ldwide in ecen decades, and ad anced knowledge has been ob ained a se e al 491
physicochemical and biological le els (Kelly 1988; ERMITE-Conso ium 2004). In his sense, 492
mul iple s udies ha e demons a ed he nega i e ecological impac s o such ac i i ies on ben hic 493
communi ies h ough mul iple lines o e idence: bioaccumula ion, oxici y, o communi y 494
s uc u e changes, bo h a an in e na ional scale (Clemen s e al. 2002; Ma e e al. 2003; 495
Smolde s e al. 2003; G ay & Delaney 2008; Rainbow e al. 2012; S i ok e al. 2014) and a 496
egional scales, in pa icula , in No he n Spain (Ga cía-C iado e al. 1999; Ga cía-C iado & Aláez 497
2001; Ma qués e al. 2001b, 2003). Howe e , assessmen s o me al impac s on 498
mac oin e eb a e communi ies whe e he conside ed e e ence condi ion sa is y he igo ous 499
WFD selec ion c i e ia a e s ill sca ce. The p esen s udy p o ides new in o ma ion on he le el 500
o me al pollu ion impai men due o mining ac i i ies in he Nalón Ri e basin, and on he 501
e ec s ha he inc easing hea y me al le els in sedimen s ha e on he s uc u e o 502
mac oin e eb a e communi ies. This in o ma ion cons i u es a c ucial da abase o he u u e 503
de elopmen o en i onmen al quali y s anda ds (EQSs) o i e sedimen s, since EU 504
en i onmen al legisla ion on wa e policy highligh s he ele ance o he bio ic componen s o 505
de ining en i onmen ally ele an sedimen EQSs. 506
19
The applica ion o a WFD-complian ‘ e e ence o es a ios’, ha is an RCA design, o 507
he e alua ion o sedimen me al concen a ions, allowed us o es ablish a g oup o me als (As, 508
Cd, Cu, Pb, Se and Hg) ha signi ican ly di e om he e e ence condi ion in he di e en 509
mining impac ed g oups o si es in he Nalón Ri e basin. Mo eo e , PCoA analyses iden i ied 5 510
(As, Cd, Cu, Se and Hg) o he men ioned me als as he main con ibu o s o he si es o dina ion 511
acco ding o en i onmen al s esso s. Al hough impac ed si es wi h me al pollu ion also seem 512
o be a ec ed by a sligh habi a dis u bance assessed by IHF acco ding o PCoA, he ac is ha 513
his esul is only a e lec ion o he g ea conse a ion s a us o he selec ed e e ence si es. 514
Howe e , i doesn´ imply ha he le el o habi a al e a ion in he es si es each such a high 515
deg ee o al e also ben hic communi ies o a g ea ex en . In gene al, acco ding o Pa do e al. 516
(2002) IHF alues o e 50 can p ope ly sus ain a di e se mac oin e eb a e communi y in 517
Medi e anean i e s. Besides, al hough he e is no o icial index assessmen o he Can ab ian 518
a ea, i e s loca ed in o he No he n Spain a eas wi h same i e ypologies as hose o he 519
p esen s udy, es ablished an IHF alue o 66 as he e e ence condi ion and 60 as he limi 520
be ween high/good s a us (RD 1/2016). Only 6 o ou s udy si es showed alues unde 60, om 521
which only 2 we e unde 50. Thus, indica ing ha hyd omo phological al e a ions may no be a 522
key s esso o ben hic communi ies and ha me al pollu ion ep esen s he main 523
en i onmen al s esso wi hin ou s udy a ea. 524
Resul s also p o ed he exis ence o signi ican esponses o mac oin e eb a e 525
assemblages o he speci ic mining condi ions exis ing wi hin he Nalón Ri e basin whe e, as 526
said, one kind o s esso domina es, i.e., me al pollu ion. In ou s udy, uni a ia e es s we e no 527
o ally e icien in de ec ing mining impac s o e mac oin e eb a e communi ies, when 528
analyses we e pe o med a he g oup le el (i.e., REF, CU, HG and AU mining ac i i y). Mos 529
gene ic me ics (i.e., gene al ichness, di e si y indices, o al abundance) seemed no o be 530
sensi i e o communi y changes ha occu unde me al s ess, when conside ing his app oach. 531
These esul s a e in acco dance wi h p e ious s udies indica ing ailu e in he applica ion o 532
uni a ia e es s o some adi ional communi y indices o de ec ing mining ecological impac s 533
(see o example By ne e al. 2013). Howe e , in ou case i should be conside ed ha his 534
absence o esponse could be a ibu ed o he unbalanced si es design (REF n= 14, HG n=8, AU 535
n=4, CU n=3) p e en ing he achie emen o su icien s a is ical powe o de ec he 536
di e ences, o pe haps due o he exis ence o some uncon amina ed si es in he HG and CU 537
g oups masking he e ec s. In ac , when g adien analyses we e applied, as in o he s udies (Qu
538
e al. 2010), signi ican associa ions we e ound be ween he men ioned communi y desc ip o s 539
and he hea y me al le els in sedimen s (bo h conside ing he me als a indi idual le el and he 540
addi i e e ec h ough SedPoll). Simila ly, mul i a ia e modelling in combina ion wi h TITAN 541
20
was e icien in de ec ing sedimen me al pollu ion as a ele an s esso o bio ic communi ies 542
in he s udy a ea, e en in he p esence o o he en i onmen al s esso s (Bu on & Johns on 543
2010). In gene al, signi ican esponses bo h a a global le el o change in he mac oin e eb a e 544
communi y s uc u e (gene al composi ion, abundance and di e si y), al e a ions in di e en 545
sensi i e axa me ics (i.e., EPT ichness and abundance) and ega ding he indi idual 546
sensi i i ies o speci ic axa (mainly EPT amilies) we e ound. These esul s indica e ha 547
sedimen me al pollu ion due mining ac i i ies clea ly impac mac oin e eb a e communi ies, 548
al hough his ac is highly masked h ough g oup analyses. This ac highligh s he need o using 549
mul iple lines o e idence o iden i y eliable changes in mac oin e eb a e communi y 550
s uc u es wi hin mining a eas. 551
Iden i ying eliable indica o axa and hei esponses o mining p essu e is o majo 552
conce n o he de elopmen o managemen ools in wa e quali y policy. In his s udy, he 553
s a is ical ea men based on indica o species (IndVal) pe o med h ough TITAN was 554
sa is ac o y o de ec ing he sensi i i y le els o indica o axa o he conside ed me al 555
g adien s. Al hough i was no possible o es ablish change h esholds a he communi y le el, 556
TITAN allowed us o de e mine a g oup o mac oin e eb a e amilies speci ically sensi i e o 557
me al concen a ions in he sedimen s measu ed as SedPoll sco es. Nine axa (se en EPT plus 558
wo Coleop e a) ollowed a sensi i i y g adien , om he Se icos oma idae wi h maximum 559
sensi i i y o he Bae idae wi h he lowes . In gene al, hese esul s we e in acco d wi h many 560
o he s udies showing mos o he men ioned axa as sensi i e o me al pollu ion (Hi s e al. 561
2002; Solà e al. 2004; Pond e al. 2008), excep o wo cases ha we e epo ed as ole an in 562
he p io li e a u e, such as he Elmidae (Clemen s e al. 2000; Qu e al. 2010) and 563
Hyd opsychidae (Clemen s e al. 2000; Luoma e al. 2010). On he o he hand, he men ioned 564
TITAN esul s o he sedimen pollu ion sco es (SedPoll) we e highly coinciden wi h hose 565
epo ed o he PCoA g adien s in e ms o he sensi i e indica o s de ec ed (Fig. 4), sugges ing 566
ha he d i e s o he e ec s de ec ed by TITAN o he PCoA g adien s we e p obably due o 567
he me al concen a ions in he sedimen s, a he han he o he en i onmen al a iables 568
loading bo h PCo1 and PCo2. These esul s suppo he hypo hesis ha me al pollu ion plays a 569
undamen al ole in s uc u ing mac oin e eb a e communi ies in he Nalón mining a eas and 570
p opose a se o indica o axa sui able o u u e s udies on ield oxici y and bioaccumula ion. 571
The p esence o esis an axa should be u he explo ed as a possible ac o p e en ing he 572
obse a ion o g ea e impac s wi hin he mac oin e eb a e communi ies in his a ea, since he 573
apid acquisi ion o esis ance o me al con amina ion in sedimen s has been documen ed (e.g., 574
o aqua ic oligochae es, by Kle ks & Le in on 1989). Al hough indica o axa esul s do no allow 575
clea conclusions on his sense, i should be no ed ha 2 o he 3 axa conside ed posi i ely 576
21
associa ed wi h me al s ess in TITAN analyses, a e su ace dwelling Hemip e a ha may no be 577
exposed o me al pollu ion o he same ex en han o he axa in close con ac wi h he 578
con amina ed sedimen s. In ac , su ace-dwelling Hemip e a usually ended o be classi ied as 579
me al- ole an (Ge ha d i e al. 2004). On he o he hand, he use o indi idual axa change 580
poin s and hei QI in elabo a ing p e en i e h esholds o me als wi h he po en ial o impac 581
s uc u al axa a di e en le els (see Pa do & Ga cía 2016) can be he subjec o deepe 582
esea ch o imp o e conse a ion policies. 583
F om an applied pe spec i e ou indings a e aluable as hey ep esen a i s s ep in 584
he de elopmen o speci ic ools o assess he impac o me al s ess in he a ea, no only by 585
gi ing a se o indica o axa o sedimen me al pollu ion, bu also by p o iding a eliable 586
measu e o me al s ess in he a ea h ough a WFD complian RCA e alua ion (i.e., SedPoll). In 587
o he hand om a WFD pe spec i e, ou esul s a e consis en wi h he cu en ecological s a us 588
classi ica ion sys ems used in i e s o No he n Spain, he mul ime ic index METI (METI 2015) 589
and he p edic i e model NORTI (Pa do e al., 2014). As he main communi y desc ip o s 590
a ec ed by me al pollu ion in ou s udy (i.e, composi ion, abundance and EPT me ics) ep esen 591
ac ually key pa ame e s wi hin hese classi ica ion sys ems. This ac , may be indica i e ha he 592
men ioned classi ica ion sys ems could be al eady sensi i e o communi y al e a ions due o 593
me al s ess. Indeed, he e alua ion o his aspec ep esen s he nex s ep o ou esea ch. In 594
addi ion, he selec ed axonomic esolu ion le el (i.e., he amily le el in mos ins ances) used 595
in ou s udy was e y use ul in de ec ing ecological impai men s ela ed o mining p essu e 596
wi hin he Nalón Ri e basin, in acco dance wi h he esul s o o he s udies (Clemen s e al. 597
2000; Ma qués e al. 2003; Pond e al. 2008; W igh & Ryan 2016). Aspec ha is also ele an , 598
as he men ioned classi ica ion sys ems o No he n Spain wo k wi h his le el o esolu ion. 599
Gene al conclusions 600
In ou s udy, we p o e ha pa o he mac oin e eb a e communi ies (sensi i e axa) 601
could be conside ed as eliable indica o s o me al pollu ion in Cu, Hg and Au-mining a eas in 602
No he n Spain. As he communi y as a whole did no show a clea h eshold esponse o mining 603
p essu e, speci ic me ics based on sensi i e axa we e success ully esponsi e o he obse ed 604
mining g adien s. The iden i ica ion o hose speci ic axa p o iding a eliable signal o he o e all 605
me al s ess measu ed h ough SedPoll sco es (calcula ed wi hin he amewo k o a WFD-606
complian ‘ e e ence- es si es’ design), eme ges as a i s aluable s ep in o de o de elop 607
speci ic ools o e alua ing impac o me al s ess in he con ex o he WFD di ec i e. Thus, he 608
applied me hodology and esul s a e ele an o de e mining he impo ance o hea y me al 609
con amina ed sedimen s as s esso s o bio ic communi ies in he s udied a ea. 610
22
The discussed ela ionships be ween he communi ies’ s uc u e, sensi i e me ics and 611
indi idual axa wi h exis ing me al g adien s in he s udy a ea can be used by wa e au ho i ies 612
o assess he le el o me al impac s wi hin hei moni o ing schedule. Simila ly, i can help o 613
es ablish ei he he le els o deg ada ion o he eco e y le els o be achie ed, helping o 614
suppo he decisions o be aken and he p og amme o measu es o be implemen ed in he 615
wa e managemen o mining a eas. 616
Acknowledgemen s 617
This in es iga ion was suppo ed by he esea ch p ojec CGL2013-44655-R, sponso ed by he 618
Spanish Go e nmen , Minis y o Economy and Compe i i eness (MINECO). D . Méndez-619
Fe nández was inanced by a pos doc o al posi ion a he Uni e si y o he Basque Coun y 620
(UPV/EHU, Spain). We g ea ly app ecia e he suppo p o ided by Amanda Mi anda and he s a 621
o he Can ab ian Hyd og aphical Con ede a ion, who assis ed in he selec ion and sampling o 622
he si es in he Nalón Ri e basin. The au ho s hank Claudio Padilla, Odei Ba edo and Iñigo 623
Mo eno o he in ense days o help du ing he ield su eys. Liliana Ga cía is also acknowledged 624
o s a is ical ad ice. 625
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