En i onmen al Pollu ion 315 (2022) 120472
A ailable online 19 Oc obe 2022
0269-7491/© 2022 The Au ho s. Published by Else ie L d. This is an open access a icle unde he CC BY-NC license (h p://c ea i ecommons.o g/licenses/by-
nc/4.0/).
Compa a i e oxicological assessmen o h ee soils pollu ed wi h di e en
le els o hyd oca bons and hea y me als using in i o and in
i o app oaches
Sand a de la Pa a
a
, Ve ´
onica Gonz´
alez
b
, Pa icia Sol´
o zano Vi es
b
, Sand a Cu iel-Aleg e
a
,
c
,
Blanca Velasco-A oyo
a
, Ca los Rad
c
, Rocío Ba os
a
, Juan An onio Tamayo-Ramos
a
,
Ca los Rumbo
a
,
*
a
In e na ional Resea ch Cen e in C i ical Raw Ma e ials o Ad anced Indus ial Technologies-ICCRAM, Uni e sidad de Bu gos, Plaza Misael Ba˜
nuelos s/n, 09001,
Bu gos, Spain
b
LEITAT Technological Cen e , c/Palla s 179-185, 08005, Ba celona, Spain
c
Resea ch G oup in Compos ing (UBUCOMP). Uni e sidad de Bu gos, Facul y o Sciences. Plaza Misael Ba˜
nuelos s/n. 09001, Bu gos, Spain
ARTICLE INFO
Keywo ds:
HepG2 cells
Saccha omyces ce e isiae
Pseudomonas pu ida
Enchy aeus c yp icus
Soil con amina ion
ABSTRACT
The biological e ec s induced by he pollu an s p esen in soils, oge he wi h he chemical and physical cha -
ac e iza ions, a e good indica o s o p o ide a gene al o e iew o hei quali y. Howe e , he exis ence o
s udies whe e he oxici y associa ed o soils con amina ed wi h mix u es o pollu an s applying bo h in i o and
in i o models a e sca ce. In his wo k, h ee soils (namely, Soil 001, Soil 002 and Soil 013) pollu ed wi h
di e en concen a ions o hyd oca bons and hea y me als we e e alua ed using di e en o ganisms ep esen-
a i e o human (HepG2 human cell line) and en i onmen al exposu e ( he yeas Saccha omyces ce e isiae, he
G am-nega i e bac e ium Pseudomonas pu ida and, o he in i o e alua ion, he annelid Enchy aeus c yp icus). In
i o assays showed ha he soluble ac ion o he Soil 001, which p esen ed he highes le els o hea y me als,
ep esen ed a g ea impac in he iabili y o he HepG2 cells and S. ce e isiae, while o ganic ex ac s om Soils
002 and 013 caused a sligh dec ease in he iabili y o HepG2 cells. In addi ion, in i o expe imen s showed ha
Soils 001 and 013 a ec ed he su i al and he ep oduc ion o E. c yp icus. Al oge he , hese esul s p o ide a
gene al o e iew o he po en ial haza ds associa ed o h ee speci ic con amina ed si es in a a ie y o o gan-
isms, showing how di e en concen a ions o simila pollu an s a ec hem, and highligh s he ele ance o
es ing bo h o ganic and soluble ex ac s when in i o sa e y assays o soils a e pe o med.
1. In oduc ion
Soils a e essen ial o he global ecosys ems unc ioning since hey
a e in ol ed in se e al c ucial p ocesses including clima e con ol,
nu ien dynamics o es ablishing he habi a o a wide a ie y o o -
ganisms (Vogel e al., 2019). Thus, and closely connec ed o hese im-
plica ions, hese sys ems ha e had a de e mining ole in he
es ablishmen and de elopmen o ci iliza ions, cons i u ing a highly
aluable na u al esou ce o human beings p o iding ood and o he
ele an p oduc s such as ib e (Muelle e al., 2010).
Soil quali y, also e e ed o as soil heal h, is de ined as “ he capaci y
o a li ing soil o unc ion, wi hin na u al o managed ecosys em
bounda ies, o sus ain plan and animal p oduc i i y, main ain o
enhance wa e and ai quali y, and p omo e plan and animal heal h”
(Do an, 2002; Do an e al., 1996). Toge he wi h ai and wa e quali ies,
hese h ee elemen s de ine he en i onmen al quali y (Bünemann e al.,
2018). Among o he s, he pollu ion le el is one o he main ac o s ha
c i ically a ec s soil quali y, ep esen ing a di ec impac o heal h and
he en i onmen (A ellano e al., 2015; Guo e al., 2012; Oke ea o e al.,
2020; Poggio e al., 2009). Fo his eason, and conside ing ha soils a e
a non- enewable sou ce, hei p o ec ion has become one o he main
p io i ies o he in e na ional communi y and se e al legal app oaches
we e app o ed by he Eu opean Union in his ega d (P´
e ez and Euge-
nio, 2018).
Di e en chemicals wi h po en ial oxic e ec s o he o ganisms and
he ecosys em can be de ec ed in pollu ed soils. Speci ically, o ganic
* Co esponding au ho .
E-mail add ess: [email p o ec ed] (C. Rumbo).
Con en s lis s a ailable a ScienceDi ec
En i onmen al Pollu ion
jou nal homepage: www.else ie .com/loca e/en pol
h ps://doi.o g/10.1016/j.en pol.2022.120472
Recei ed 5 Augus 2022; Recei ed in e ised o m 30 Sep embe 2022; Accep ed 15 Oc obe 2022
En i onmen al Pollu ion 315 (2022) 120472
2
con aminan s such as mine al oil o polycyclic a oma ic hyd oca bons
(PAHs), and hea y me als a e o pa icula in e es o he en i on-
men al ield and ela ed a eas, since acco ding o a e iew p epa ed by
Panagos e al., bo h a e he main pollu an s in soils and g oundwa e o
he Eu opean Union (Panagos e al., 2013). These subs ances can each
soils h ough na u al and an h opogenic means, being an h opogenic
ac i i ies, including acciden al and delibe a e p ocesses, he p ima y
cause o con amina ion o he soils. Thus, sec o s such as mining and
pe ochemical indus ies, as well as he imp ope use o was ewa e s and
o ganic was es in ag icul u al ac i i ies, a e impo an sou ces o hea y
me al pollu ion in soils (Wuana and Okieimen, 2011), while PAHs
showed o ha e a py ogenic o igin in u ban soils, sugges ing he
impo an ole o ehicle emissions (Mo illo e al., 2007).
In addi ion o chemical and physical analysis, he use o biological
assays is a aluable ool o e alua e he quali y o a soil. These analyses
a e based on he applica ion o ep esen a i e in i o and in i o models
ha p o ide in o ma ion abou he po en ial haza ds ha a con ami-
na ed si e may ep esen o he human heal h and o he en i onmen .
The e o e, he oxici y associa ed o di e en eal pollu ed soils was
epo ed in p e ious wo ks using se e al model o ganisms such as
ea hwo ms (Li e al., 2020; Oli ei a Resende e al., 2018), plan s
(Lou ei o e al., 2006; Massa e al., 2018), o cell lines, being he la e
exposed o con aminan ex ac s di ec ly ob ained om he soils
(Bade na e al., 2014, 2013; Husejno ic e al., 2018). Besides in
con amina ed soils, hese assays also showed o be an app op ia e
app oach o s udy he oxici y o land ill soils (Alimba e al., 2016; Swa i
e al., 2017), as well as in o he pollu ed subs a es such as sedimen s
(Pin o e al., 2014a, 2014b).
The aim o he p esen s udy was o e alua e and compa e he quali y
o h ee con amina ed soils p esen ing di e en le els o hyd oca bons
and hea y me als om a oxicological pe spec i e. The i s soil unde
s udy (named as Soil 001 in his wo k) was aken om an indus ial a ea
loca ed in Toledo, Spain, which is 3 km om an u ban nucleus. This
emplacemen has been used o decades o machine y epai ing and
main enance ac i i ies, and he soil sample e alua ed has been selec ed
om exca a ion and emo al o soil a ec ed by acciden al spills, so i is
pollu ed wi h he p esence o di e en ypes o hyd oca bons, mine al
oils, and hea y me als om uel and engine oil leaks. The second soil
analysed (named as Soil 002 in his wo k) was aken om a ood
manu ac u ing si e loca ed in he ou ski s o Ca low. This ac o y was
ope a i e om 1920 o 2006. Since i s closu e, his si e has been un-
de going a Closu e, Res o a ion and Residues Managemen plan. Du ing
he si e in es iga ion a numbe o small pocke s o hyd oca bon
con amina ed soil we e iden i ied, o igina ing om a leaking hea y uel
oil ank used o un he gene a o s on si e du ing i s ope a ion. Finally,
he hi d soil selec ed (named as Soil 013 in his wo k) was ob ained
om one o he la ges oil ields o China: Shengli Oil Field. Speci ically,
his soil was aken om a si e loca ed in Gudao, and i is pollu ed wi h
c ude oil. The oxici y associa ed o hese h ee soils was analysed
h ough he applica ion o a ba e y o assays combining in i o and in
i o models. To pe o m he in i o assays, soluble and o ganic ex ac s
we e ob ained om he soils, and hei e ec on he iabili y o di e en
o ganisms was s udied sepa a ely. The HepG2 cell line, a model o
human li e , oge he wi h he yeas Saccha omyces ce e isiae and he
bac e ia Pseudomonas pu ida, bo h ep esen a i e o en i onmen al o -
ganisms, we e applied in hese assays. Addi ionally, he e es ial
annelid Enchy aeus c yp icus was used o ca y ou he in i o assays,
s udying he e ec s on he su i al and he ep oduc ion o his o gan-
ism a e being di ec ly exposed o di e en concen a ions o he
pollu ed soils. The ob ained esul s p o ide in o ma ion abou he bio-
logical quali y o he h ee soils unde s udy, showing how he di e en
concen a ions o he con aminan s in luence his pa ame e .
2. Ma e ials and me hods
2.1. Selec ed si es and soil samples p epa a ion
Th ee soils pollu ed wi h di e en le els o hyd oca bons and hea y
me als, and in ended o being ea ed wi h di e se bio emedia ion
echnologies, we e selec ed o be oxicologically analysed in his s udy.
The loca ion and con amina ion sou ce o hese si es a e summa ised in
supplemen a y ma e ial (Table S1). Soils 001 and 013 we e exca a ed
and homogenized, and app op ia e amoun s om hem we e p epa ed
o bo h chemical and oxicological analyses. In he case o Soil 002, he
samples analysed we e aken om an ecopile cons uc ed wi h an
exca a ed soil ha was ea ed wi h N:P e ilise and inocula ed wi h 1
L m
−3
o a To al Pe oleum Hyd oca bons (TPH) deg ading inoculum. As
wi h he o he soils s udied in his wo k, app op ia e amoun s o i we e
p epa ed o chemical and oxicological analysis.
Be o e hei p epa a ion o he di e en analysis, all he soil samples
we e ai d ied and sie ed a 2 mm.
2.2. Chemical cha ac e iza ion
2.2.1. TPH quan i ica ion
Fo he TPH ex ac ion, 1 g o each soil p e iously d ied a 30 ◦C was
weigh ed, and 20 mL o an ace one:hexane mix u e (1:1 / ) was added
in a mic owa e-assis ed ex ac ion equipmen (E hos X, Miles one,
So isole, I aly) o 20 min a 150 ◦C. A e his ime, samples we e cooled
down and cen i uged a 2500×g o 30 min. The supe na an ob ained
was il e ed (po e size 0.22
μ
m) and e apo a ed o a olume o 1 mL in a
speed- ac e apo a o (SAVANT SPD111V, The mo).
F ac iona ion o Ex ac ed Pe oleum Hyd oca bons (EPHs) be ween
linea and a oma ic hyd oca bons was ca ied ou using he solid phase
ex ac ion (SPE) me hod acco ding wi h Jim´
enez e al. (2014). Fo his
pu pose, Isolu e EPH ac iona ion ca idges (25 mL/5 g, Bio age,
Uppsala, Sweden) we e used in a SPE-24G column p ocesso (JT Bake ).
The ca idges we e i s ac i a ed by adding 30 mL o hexane and,
be o e i d ied, he loading s ep was s a ed, dissol ing he samples in 1
mL o hexane. Then, he elu ion s age was ca ied ou wi hou applying
p essu e a a speed o 2–3 mL min
−1
adding 12 mL o hexane o collec
he alipha ic ac ion, and 20 mL o dichlo ome hane o collec he a -
oma ic ac ion. Bo h ac ions we e concen a ed in a speed- ac e ap-
o a o o 1 mL. Quan i ica ion o EPHs and PAHs was pe o med in a
Va ian 3900 gas ch oma og aphy ins umen equipped wi h a FID de-
ice and a Va ian CP8907 capilla y column (25 m, 0.25 mm inne
diame e , nominal ilm hickness 0.25 mm).
2.2.2. T ace elemen s quan i ica ion
To pe o m he ex ac ion, 0.2 g o each soil we e weighed, and 10
mL o ni ic acid we e added o Te lon ubes. Samples we e diges ed a
180 ◦C du ing 20 min in a mic owa e-assis ed diges ion sys em (E hos
One, Miles one, So isole, I aly). The diges ed samples we e hen il e ed
using a double il e pape in o 50 mL olume ic lasks and made up
wi h ul apu e wa e . All he ma e ials used we e p e iously cleaned in
dilu ed HNO
3
.
Those me als ha we e expec ed o be p esen a high concen a ions
(Al and Fe) we e analysed by induc i ely coupled plasma - op ical
emission spec ome y (ICP-OES) using a SPECTRO GENESIS spec-
ome e . In he case o hose elemen s ha we e expec ed o be p esen
a e y low concen a ions (As, Cd, C , Co, Cu, Mn, Mo, Ni, Pb, Ti and
Zn), he quan i ica ion was pe o med by induc i ely coupled plasma
mass spec ome y (ICP-MS) using an Agilen 8900 ICP-QQQ ins umen
in he Uni e si y o Bu gos. A 21-mul ielemen s anda d solu ion (100
mg L
−1
, VWR Chemicals, Leu en, Belgium) was used o he calib a ion-
e i ica ion p ocedu e.
S. de la Pa a e al.
En i onmen al Pollu ion 315 (2022) 120472
3
2.3. In i o assays
2.3.1. Sample p epa a ion
2.3.1.1. O ganic ac ion ex ac ion. The pollu an ex ac ion was pe -
o med ollowing a p o ocol adap ed om Pin o e al. (2014a). Fi s ly,
samples we e d ied a 40 ◦C in he da k, pul e ized and homogenized
wi h a mo a be o e being ex ac ed wi h a mix u e o dichlo ome hane
(DCM):me hanol (2:1 / ). To ca y ou his s ep, 3.3 mL o he sol en
mix u e we e added o e e y 2 g o d y pul e ized soil sample, and he
ex ac ion was done mechanically by ecip ocal shaking du ing 15 min
(60 pm). Samples we e hen cen i uged (2000×g, 20 min) and he
supe na an was eco e ed and e apo a ed using a o a y e apo a o ,
adding he supe na an li le by li le. To p epa e he o ganic ex ac
s ocks 2 mL o dime hyl sul oxide (DMSO) we e added o each ube, and
he samples we e submi ed o gen le shaken (300 pm) combined wi h
se e al 1-h sonica ion s eps in an ul asonic ba h du ing 1 week wi h he
aim o sol e as much as possible he ob ained p ecipi a e. The inal soil:
ex ac p opo ion was 1 g soil d y weigh pe mL o ex ac . Finally,
samples we e il e ed wi h a 0.22
μ
m nylon il e . Th ee independen
ex ac ions pe each soil we e pe o med. A blank sample was c ea ed
ollowing he same ex ac ion p ocess wi hou soil.
2.3.1.2. Soluble ac ion ex ac ion. P io o he ex ac ion, soil samples
we e comple ely ehyd a ed. The ex ac ion was pe o med adding
wa e a oom empe a u e in in a a io 1:4 (w/ ) (4 mL pe g am o
d ied weigh soil). Then, samples we e o exed and sonica ed in an
ul asonic ba h wi h ice cold wa e o 1 h. A e his ime, samples we e
o exed again, cen i uged a 4000×g o 10 min, and he supe na an s
we e collec ed and e apo a ed un il a concen a ion equi alen o 1 g
soil d y weigh pe mL o wa e . Finally, samples we e il e ed wi h a
0.22
μ
m polye he sul one il e .
2.3.2. O ganisms and cul u e condi ions
HepG2 cell line was cul u ed in comme cial Eagle’s Minimum
Essen ial Medium (EMEM) supplemen ed wi h 10% ( / ) e al bo ine
se um (FBS), 1% non-essen ial amino acids, 1% sodium py u a e and
100 U mL
−1
penicillin and 100 mg L
−1
s ep omycin. This cell line was
incuba ed unde s anda d condi ions in 37 ◦C humidi ied 5% CO
2
a mosphe e.
The BY4741 s ain o S. ce e isiae was main ained in s anda d Yeas
ex ac Pep one-Dex ose (YPD) medium (1% yeas ex ac , 1% yeas
bac o-pep one, 2% glucose). Liquid cell cul u es we e done on a o a y
shake a 185 pm a 30 ◦C.
P. pu ida CECT 4064 (DSMZ 548) was main ained a 30 ◦C in Muelle -
Hin on b o h o aga .
2.3.3. Viabili y analysis o HepG2 cells
The neu al ed up ake assay was ca ied ou o de e mine he
iabili y o HepG2 cells a e being exposed o he di e en con aminan
ac ions. B ie ly, cells we e seeded in 96 well pla es (4 ×10
4
cells pe
well) and, 24 h a e seeding, cells we e incuba ed o 24 h in p esence o
he con aminan s dilu ed in esh cul u e medium. Fo he o ganic
ac ion, which was esuspended in DMSO, he concen a ion es ed was
10 mg o soil equi alen pe mL (s.e. mL
−1
). Fo he expe imen s ana-
lysing he soluble ac ion, cul u e medium 10 ×was p e iously p e-
pa ed and di ec ly dilu ed o 1 ×in he samples, being he concen a ion
es ed 900 mg s.e. mL
−1
. F om his poin on, he assay was pe o med
ollowing he p o ocol p e iously explained in o he wo ks de eloped by
ou g oup (Rumbo e al., 2021, 2020).
2.3.4. Viabili y analysis o S. ce e isiae
S. ce e isiae cells we e g own on YPD medium in an o bi al shake
(185 pm) a 30 ◦C un il each an O.D.
600 nm
=1. Cells we e hen
exposed o 2 and 24 h o 10 mg s.e. mL
−1
o he o ganic ac ion and 900
mg s.e. mL
−1
o he soluble ac ion in 24-well pla es ( inal olume o 1
mL). Yeas colony o ming uni s we e de e mined inocula ing cells on
solid YPD medium (6% aga ) and incuba ed a 30 ◦C. Resul s we e
exp essed as pe cen age o con ol (CFUs in absence o con aminan s).
2.3.5. Expe imen s using P. pu ida: g ow h cu es analysis
The in luence o bo h con aminan ac ions in mic obial g ow h was
moni o ed using a Syne gy HT mic opla e eade (BioTek Ins umen s,
Inc.). Cul u es o P. pu ida we e i s g own in MHB o e nigh a 30 ◦C
wi h shaking. Cul u es we e hen dilu ed 1:100 in medium alone (as
con ol) and in medium supplemen ed wi h 10 and 900 mg s.e. mL
−1
o
he o ganic ex ac s and he soluble ac ions espec i ely. The g ow h
a e was moni o ed e e y hou eco ding he op ical densi y a 600 nm
in 24-well pla es o 20 h.
2.4. In i o assays using Enchy aeus c yp icus
Toxici y es wi h E. c yp icus was ca ied ou acco ding o OECD 220
(OECD, 2004). Loamy sand soil (LUFA-Speye 2.2, Sp 2121, Ge many,
2009) wi h a pH-CaCl
2
o 5.5, a o al o ganic ca bon con en o 2.09%, a
ca ion exchange capaci y (CEC) o 10 meq/100 g and a wa e -holding
capaci y (WHC) o 46.5% was used. A e mix u es (con amina ed soil
+LUFA 2.2) p epa a ion, addi ional Milli-Q was added o achie e a soil
mois u e con en o 50% o he maximum WHC.
The o ganisms we e cul u ed in dishes con aining Bac o aga , kep a
20 ±1 ◦C wi h a 12/12 h pho ope iod, and ed wi h boiled oa meal.
Animals wi h clea ly isible cli ellum (sexually ma u e) and he same
size we e used in he expe imen . Selec ed o ganisms we e ans e ed
om he cul u e o a Pe i dish wi h wa e and hen in oduced in he
es con aine s illed wi h 30 g o mois soil. Fo each concen a ion, ou
eplica e es con aine s we e used. Tes con aine s we e closed wi h
pe o a ed aluminium oil, and 3 mg c ushed oa meal was added as ood.
Then, con aine s we e placed in a clima e oom a 22 ◦C, wi h 75% o
ela i e humidi y and 12/12 h ligh /da k cycle. Twice a week, con-
aine s we e checked o wa e loss and compensa ed o by weighing,
and addi ional ood was added i needed. A e 21 days, enchy aeids
we e ixed adding a solu ion o 10 mL e hanol o each es con aine , and
ho oughly s i ing he con aine . A e 1 min, he suspension was
ans e ed o a plas ic ja and 100 mL o dis illed wa e was added. The
enchy aeids we e s ained by adding 300
μ
L o a 1% Bengal ose solu-
ion. The samples we e shaken again igo ously and incuba ed o 24 h
in he e ige a o a app ox. 4 ◦C o achie e an op imal dying e ec .
Then he b igh pink colou ed enchy aeids we e isola ed by sie ing
o e 160
μ
m and coun ed in 80 ×50 pho o ays using a magni ying
glass. The numbe o su i ing adul s and ju eniles p oduced we e
de e mined in each es con aine .
2.5. S a is ical analysis
Da a a e p esen ed as means ±SD. S a is ical analysis was pe o med
by he one-way analysis o a iance (ANOVA), ollowed by Tukey pos
hoc es o mul iple compa isons. S a is ical es s we e ca ied ou using
P ism 8.0.2 (G aphPad P ism, G aphPad So wa e, Inc.), conside ing he
di e ences signi ican a P ≤0.05.
3. Resul s
3.1. Chemical cha ac e iza ion o he soils
The h ee soils unde s udy we e analysed o cha ac e ize hei main
physico-chemical pa ame e s (Supplemen a y Ma e ial, Table S2) and
hei con aminan le els (Table 1). In hese soils, he To al Pe oleum
Hyd oca bons (TPHs; TPHs =EPHs +VPH) ac ion was equi alen o
ex ac able ac ion (EPHs), since he ola ile ac ion (VPH) was almos
comple ely deple ed, he e o e only emaining he medium and high
molecula weigh , in bo h linea alipha ic and a oma ic hyd oca bons.
S. de la Pa a e al.
En i onmen al Pollu ion 315 (2022) 120472
4
All he soils displayed me allic and o ganic con aminan s, being he
concen a ions o he EPHs and some pa icula ace elemen s abo e
hei co esponden h eshold le els, acco ding o legisla ion. Clea
di e ences in he le els o he di e en con aminan s be ween he soils
we e obse ed. Thus, he le els o EPHs in Soil 013 we e ≈3- and 9- old
highe han in Soils 001 and 002 espec i ely. Linea alipha ic hyd o-
ca bons (LAHs) we e he mos abundan hyd oca bons in his soil,
speci ically hose o >C22 – C35 chains. Rega ding he a oma ics ac-
ion, hose o >EC17 - EC35 n-alkane equi alen C chains we e p e-
dominan in Soil 013. Soil 001 ep esen ed he in e media e sample in
e ms o EPHs pollu ion. LAHs >C22 – C35 co esponded o he majo
ac ion, as well as in he a oma ics ac ion, whe e his equi alen C-
chain ange was also p edominan . Finally, Soil 002 showed he lowe
EPHs con en , being simila he le els o LAHs and a oma ics and, as
obse ed in Soil 001, >C22 – C35 n-alkane chain hyd oca bons we e he
mos ep esen a i e in bo h ac ions.
Rega ding me als and me alloids con en , Al and Fe we e he mos
abundan elemen s in hese h ee soils. In Soil 001, he le els o Mn we e
pa icula ly high, being he concen a ion o his elemen ≈8- and 14-
old highe han in Soils 002 and 013 espec i ely. In addi ion, he
concen a ions o As, Cd, Pb and Zn s and ou o e he le els ound in he
o he soils. In Soil 002, Mn, oge he wi h Al and Fe, cons i u ed he mos
abundan me als, while in Soil 013, Ti anged he 3 d posi ion, jus
abo e he Mn.
3.2. E ec o pollu ed soil ex ac s in human HepG2 cell line
The e ec o bo h o ganic and soluble ex ac s ob ained om he
h ee con amina ed soils in he iabili y o he human hepa oma cell line
HepG2 was e alua ed using he neu al ed up ake assay. Fig. 1 shows
he esul s ob ained a e exposing HepG2 cells o 10 mg s.e. mL
−1
o he
di e en o ganic ex ac s o 24 h. Due o he di icul y o edissol e he
o ganic condensa e ob ained a e he e apo a ion, and he impossibili y
o achie e i s comple e dissolu ion, h ee independen ex ac ions om
each soil we e pe o med, and he edissol ed ex ac s we e es ed
sepa a ely o ensu e ha he p ocedu e was homogeneous ac oss he
p ocess, and o con i m ha he esul s ob ained we e simila . Ex ac s
om Soil 001 showed a sligh dec ease in he pe cen age o iable cells
(5–10%) (Fig. 1A). In he case o he ex ac s om Soils 002 and 013,
bo h caused a dec ease o ≈10% in he iabili y o HepG2 cells, being
hese alues s a is ically signi ican when compa ed o con ol in he
h ee samples ob ained om Soil 002.
Mo eo e , cells we e exposed o a concen a ion o 900 mg s.e. mL
−1
o he soluble ex ac s om he h ee soils o 24 h. Resul s showed ha
he ex ac s om Soil 001 p esen ed a c i ical impac in he iabili y o
HepG2 cells, whe e he pe cen age o dead cells was almos 100%
(Fig. 1B). On he o he hand, soluble ex ac s om Soil 002 caused a
sligh dec ease in he iabili y o hese cells (≈10%), while hose
exposed o soluble ex ac s om Soil 013 p esen ed a pe cen age o
iable cells simila o ha o he con ol (Fig. 1B).
3.3. E ec o pollu ed soil ex ac s in S. ce e isiae
To de e mine he oxicological po en ial o bo h o ganic and soluble
soil ex ac s, he ungal model S. ce e isiae was included in he assays.
Thus, he iabili y o his o ganism was e alua ed a wo exposu e imes
(2 and 24 h). As displayed in Fig. 2A, any o he o ganic ex ac s caused a
nega i e e ec on he iabili y o S. ce e isiae when exposed o a
Table 1
Con amina ion le els (pe con aminan class) o he h ee soils.
Soil Sample
Soil 001 Soil 002 Soil 013
Con aminan
O ganic (mg kg
−1
)
a
EPHs 3723 1355.2 13,162
LAHs >C10 – C12 2 0.2 26
>C13 – C16 117 6 897
>C17 – C21 112 41 1750
>C22 – C35 2661 462 5857
>C35 208 134 1143
∑LAHs 3100 643.2 9673
A oma ics >EC10 - EC12 15 3 37
>EC13 - EC16 11 2 99
>EC17 - EC21 111 152 1690
>EC22 - EC35 340 400 1639
>EC35 146 155 24
∑A oma ics 623 712 3489
Ino ganic (mg kg
−1
)
b
Al 11,000 8200 17,000
Fe 32,000 15,000 24,000
Mn 4200 530 295
As 77.28 3.20 4.26
Cd 7.78 3.26 0.06
C 14.9 14.2 16.7
Co 5.8 4.4 5.3
Cu 8.5 21.4 15.2
Mo 1.71 0.71 <d.l.
Ni 9.9 15.1 14.6
Pb 339 41 10
Ti 188 85 398
Zn 681 70 44.5
<d.l., below de ec ion limi .
a
Values ob ained conside ing he pe cen age o he measu emen unce ain y
associa ed o he analy ical me hod (5%).
b
Values ob ained conside ing he pe cen age o he measu emen unce ain y
associa ed o he analy ical me hod (10% o Al and Fe; 4% o Mn, As, Cd, C ,
Co, Cu, Mo, Ni, Pb, Ti, Zn).
Fig. 1. HepG2 cells iabili y a e being exposed o 10 mg s.e. mL
−1
o he o ganic ex ac s (A) and o 900 mg s.e. mL
−1
o he soluble ex ac s (B) o 24 h. Resul s a e
exp essed as % o con ol (un ea ed cells). Da a ep esen he mean o a leas 5 biological eplica es (±s anda d de ia ion, SD) ob ained in wo independen
expe imen s (A) o he mean o a leas 6 biological eplica es (±s anda d de ia ion, SD) ob ained in h ee independen expe imen s (B). Di e ences we e es ablished
using a One-way ANOVA ollowed by mul iple compa isons es (Tukey es ) and conside ed signi ican a P ≤0.05. Di e en le e s indica e s a is ically signi ican
di e ences be ween ea men s.
S. de la Pa a e al.
En i onmen al Pollu ion 315 (2022) 120472
5
concen a ion o 10 mg s.e. mL
−1
. On he o he hand, and as i was
obse ed in HepG2 cells, soluble ex ac s om Soil 001 a 900 mg s.e.
mL
−1
showed o ha e a c i ical impac on his o ganism. Fig. 2B shows
ha e en a 2 h o incuba ion, he iabili y o hese cells was dec eased
≈95%, being his pe cen age simila a 24 h. In e es ingly, soluble ex-
ac s om Soil 002 d as ically educed he iabili y o S. ce e isiae a 2 h
o incuba ion bu p esen ing his o ganism a simila pe cen age o iable
cells han in he con ol a e a longe exposu e ime (24 h). Finally, he
soluble ex ac s om Soil 013 did no cause any nega i e e ec on he
iabili y o S. ce e isiae a any o he ime poin s selec ed, being e en
signi ican ly highe han con ol a 24 h.
Fig. 2. Colony o ming uni s (CFUs) o S. ce e isiae
cells exposed o 10 mg s.e. mL
−1
o he o ganic ex-
ac s (A) and o 900 mg s.e. mL
−1
o he soluble
ex ac s (B), a wo exposu e imes (2 and 24 h). Re-
sul s a e exp essed as he pe cen age (%) o CFUs
de e mined o each exposu e condi ion using as
e e ence alue he non-exposed cells condi ion,
which was assigned a alue o 100%. Da a ep esen
he mean o a leas 5 biological eplica es
(±s anda d de ia ion, SD) ob ained in wo indepen-
den expe imen s (A) o 6 biological eplica es
(±s anda d de ia ion, SD) ob ained in h ee inde-
penden expe imen s (B). Di e ences we e es ab-
lished using a One-way ANOVA ollowed by mul iple
compa isons es (Tukey es ) and conside ed signi i-
can a P ≤0.05. Di e en le e s indica e s a is ically
signi ican di e ences be ween ea men s.
S. de la Pa a e al.
En i onmen al Pollu ion 315 (2022) 120472
6
3.4. E ec o pollu ed soil ex ac s in P. pu ida
Pseudomonas pu ida, a ep esen a i e bac e ium ound in wa e and
soil, was used o e alua e he oxici y o he di e en soil ex ac s. The
oxic e ec o bo h o ganic and soluble ex ac s on bac e ial g ow h was
e alua ed i s pe o ming MICs, showing ha any o he concen a ions
es ed inhibi ed he g ow h o his bac e ium (Supplemen a y Ma e ial;
S1, Table S3). Mo eo e , o analyse i he highes concen a ions o he
soil ex ac s could ep esen any pe u ba ion in bac e ial g ow h in
spi e o no inhibi ing i , g ow h cu es we e ca ied ou . Fig. 3 shows
ha nei he o ganic no soluble ex ac s caused any nega i e e ec on
bac e ial g ow h, p esen ing all condi ions simila cu es han he
con ol condi ion.
3.5. E ec o pollu ed soil in E. c yp icus su i al and ep oduc ion
Con ol pe o mance o he es animals was e alua ed using he
quali y c i e ia desc ibed in es guidelines. Fo he enchy aeid,
E. c yp icus su i al should be ≥80% wi h ≥25 ju eniles pe es
con aine and a coe icien o a ia ion ≤50% (OECD, 2004). In ou
expe imen , he enchy aeids p oduced la ge numbe s o ju eniles
(a e age =321 ju eniles) and he coe icien o a ia ion was less han
he 50% limi . Soils 001 and 013 showed a s a is ically signi ican oxic
e ec on enchy aeid su i al and ep oduc ion. Howe e , no oxic e -
ec was obse ed when o ganisms we e exposed o Soil 002 (Fig. 4).
Mo eo e , o con amina ed Soils 001 and 013, he EC
50
alues o
su i al and ep oduc ion we e also e alua ed exposing he o ganisms
o mix u es o he con amina ed soil wi h con ol soil LUFA 2.2 a
di e en concen a ions. Dose esponse cu es we e pe o med showing
ha , o Soil 001, he EC
50
alues we e 80% [67–109] o su i al, and
57% [36–81] o ep oduc ion, while in Soil 013, hese alues we e 57%
[53–77] and 46% [36–49] o su i al and ep oduc ion espec i ely,
being hus highligh ed he g ea e oxici y o he la e (Supplemen a y
Ma e ial, Fig. S1).
4. Discussion
Hyd oca bons and hea y me als a e among he main pollu an s in
he soils o he Eu opean Union, ep esen ing a ound 60% o soil
con amina ion acco ding o es ima ions published in p e ious wo ks
(Panagos e al., 2013). In addi ion, in he speci ic case o As, Cd, C , Cu,
Pb, Zn, Sb, Co and Ni, i was desc ibed ha in he 28.3% o he o al
su ace a ea o he Eu opean Union, one o mo e o hese me als and
me alloids exceed he applied h eshold concen a ion (T´
o h e al.,
2016). Conside ing all o his, oge he wi h he pe nicious e ec s ha
bo h hyd oca bons and hea y me als pose o human heal h and he
en i onmen , he a ailabili y o da a conce ning he po en ial oxici y o
soils p esen ing hese pollu an s is c i ically impo an o de e mine
hei associa ed haza ds.
Physicochemical cha ac e iza ion me hods a e adi ionally applied
o analyse he quali y o a soil. Howe e , his me hodology does no
en i ely e lec he global e ec s ha xenobio ic mix u es may induce in
li ing o ganisms. Fo his eason, he employmen o oxicological as-
says using di e en model o ganisms a e essen ial ools o elucida e he
po en ial impac o a soil. In he p esen s udy, h ee pollu ed soils
p esen ing di e en concen a ions o hyd oca bons and hea y me als
ha e been e alua ed om a biological pe spec i e.
The chemical cha ac e iza ion analysis e ealed ha EPH le els in
he h ee soils we e abo e o 1000 mg kg
−1
, he eby sugges ing no able
hyd oca bon pollu ion. Speci ically, Soil 013, which co esponds o an
oil ex ac ion ield, showed o ha bou by a he highes concen a ion
o hyd oca bons, ollowed by Soil 001 and Soil 002. The p esence o
ace elemen s was also s udied. In compa ison wi h he o he s, espe-
cially signi ican we e he amoun s o Mn in Soil 001, as well as hose o
As, Cd, Pb and Zn, being he appea ance o he la e wo elemen s
compa ible wi h he con amina ion by uel and engine oil leaks since
bo h me als a e p esen in used engine oils (Kashi e al., 2018; S ou
e al., 2018).
The use o o ganisms belonging o di e en biology kingdoms p o-
ides a mo e accu a e o e iew abou he eal implica ions ha a
pollu ed soil may ep esen o he whole en i onmen since, due o
hei pa icula i ies, hey will espond di e en ly o s ess and, he e-
o e, hey will no be equally a ec ed when exposed o a speci ic
pollu an a pa icula concen a ions. Bea ing his in mind, a b oad
po olio o model o ganisms including human cells, ungus, bac e ia
and wo ms we e applied, combining in i o and in i o me hodologies o
e alua e he po en ial haza dous e ec s associa ed o hese h ee eal
pollu ed soils. In addi ion, he ac ha all he soils we e pollu ed wi h a
mix u e o hea y me als and hyd oca bons, p o ided aluable in o -
ma ion ex ac ed om eal si es abou he implica ions o di e en le els
o simila con aminan s and mix u es in he iabili y o di e en
o ganisms.
To ca y ou he in i o assays, he HepG2 cell line, he yeas
S. ce e isiae and he G am-nega i e bac e ium P. pu ida we e used in he
expe imen s. HepG2 cell line is conside ed a sui able o ganism o be
applied in oxicological s udies since he li e is one o he main a ge s
o xenobio ics and, in addi ion, he egula ion o xenobio ic genes in his
cell line is simila han in p ima y hepa ocy es (Bade na e al., 2013).
The e o e, HepG2 cells ha e been applied in p e ious wo ks o analyse
he cy o oxici y o ex ac s ob ained om en i onmen al con amina ed
samples (Bade na e al., 2014; Pin o e al., 2014b). Rega ding
S. ce e isiae, his mic oo ganism was included in his wo k since yeas s
a e p esen in soils, in ol ed in se e al p ocesses such as nu ien
ans o ma ions o he main enance o i s s uc u e (Yu ko , 2018). In
addi ion, his unicellula ungus is among he mos widely applied
model o ganisms in s udies ocused on a b oad ange o biological pa-
ame e s such as in egula ion o gene exp ession o me abolic analyses
(Ka a hia e al., 2011). Mo eo e , i was also p oposed as a sui able
Fig. 3. G ow h cu es in he p esence o 10 mg s.e. mL
−1
o he o ganic ex ac s (A) and 900 mg s.e. mL
−1
o he soluble ex ac s (B). Da a ep esen he mean o 3
biological eplica es (±s anda d de ia ion, SD).
S. de la Pa a e al.
En i onmen al Pollu ion 315 (2022) 120472
7
model o s udy he oxici y o soils con amina ed wi h pes icides (Gil
e al., 2015). Likewise, he G am-nega i e bac e ium P. pu ida can be
ound in soils, pa icula ly in hizosphe e, whe e i in e ac s wi h plan s
s abilising commensal ela ionships, and being in ol ed in se e al
impo an unc ions such as plan g ow h p omo ion (Molina e al.,
2020). This mic oo ganism was also used in p e ious wo ks o s udy he
oxici y o con amina ed and bio emedia ed soil ex ac s (Ah iainen
e al., 2002; Hund and T aunspu ge , 1994). To pe o m in i o assays,
he annelid E. cyp icus was employed as model o ganism. Soil-dwelling
annelids o he genus Enchy aeus a e ecologically ele an species o
eco oxicological es ing since hese o ganisms play an impo an ole in
o ganic ma e decomposi ion and soil bio u ba ion. E. c yp icus has a
la ge ole ance ange o soil p ope ies such as pH (4.4–8.2), clay
(1–29%) and o ganic ma e con en (1.2–42%) (Kupe man e al., 2006;
Van Ges el e al., 2011), making i a use ul ool o assess he eco oxici y
o soils wi h di e en p ope ies.
To pe o m he in i o oxicological e alua ion, soluble and o ganic
ex ac s we e ob ained om he soils. The o ganic ex ac s we e ob-
ained h ough a DCM:me hanol ex ac ion. This me hodology was
selec ed since, acco ding o a p e ious wo k whe e he oxici y o sed-
imen s con aining hea y me als, PAHs, pes icides and polychlo ina ed
biphenyls was e alua ed, i has he abili y o ap a complex mix u e o
oxican s, e lec ing he o iginal con amina ion pa e n o he samples
(Pin o e al., 2014b). Ou esul s showed ha a concen a ion o 10 mg s.
e. mL
−1
caused a sligh dec ease in he iabili y o HepG2 cells, mainly
in he case o hose om Soils 002 and 013, bo h p esen ing he highe
le els o TPHs. On he o he hand, no e ec was obse ed in S. ce e isiae
and P. pu ida. The e alua ion o ex ac s om soils o analyse hei
biological impac was p e iously applied in o he wo ks. Bade na e al.
e alua ed he isk o soils wi h low le els o pollu an s loca ed in wo
semi- u al a eas in he No h o I aly using soil o ganic ex ac s in hei
analysis (Bade na e al., 2014, 2013). These au ho s ound ha only high
doses o pollu ed soil equi alen s esul ed in cy o oxic e ec s. Mo eo e ,
Pin o e al. also used his cell line o s udy he po en ial geno oxici y and
mu agenici y o di e en ex ac s o impac ed es ua ine sedimen s
(Pin o e al., 2014a, 2014b), obse ing ha sedimen s ob ained om
indus ial a eas we e signi ican ly mo e geno oxic and mu agenic han
hose om u al a eas. In ou case, despi e he la ge a ia ions obse ed
in he le els o pollu an s be ween he h ee soils, he expe imen s
analysing he o ganic ex ac s did no show no able di e ences a he
used concen a ion (10 mg s.e. mL
−1
) o any o he h ee model o -
ganisms. This alue was selec ed as he highes allowable concen a ion
ha does no exceed he 1% o sol en (DMSO), which could nega i ely
a ec he o ganisms pe se.
The ex ac ion o con aminan s in me al pollu ed soils applying
wa e was p e iously applied in o he wo ks (Husejno ic e al., 2018;
Vidic e al., 2009), ollowing simila me hodologies han ha desc ibed
he e. In ou s udy, he mos accessible con aminan s om he soils we e
ex ac ed applying sonica ion and, gi en ha he used sol en is wa e , a
highe concen a ion o soil equi alen (900 mg s.e. mL
−1
) could be
es ed in he di e en o ganisms o s ablish compa isons. In his case,
ema kable di e ences we e obse ed be ween he h ee soils in e ms
o hei e ec s as well as in hei me als and me alloids con en (Sup-
plemen a y Ma e ial, Table S4). Thus, he Soil 001 ex ac s c i ically
a ec ed he iabili y o HepG2 cells and S. ce e isiae. In he case o he
ex ac s om Soil 002, hey showed o in e e e in he iabili y o
S. ce e isiae only a low incuba ion imes, while ex ac s om Soil 013
did no show any nega i e e ec . Li ing o ganisms a e exposed in he
en i onmen o di e en me als ha , in combina ion, may pose a isk
and cause ad e se e ec s e en when hei indi idual concen a ions a e
below oxic le els (Wu e al., 2016). Ou esul s show ha , in gene al,
he speci ic mix u es o me als and me alloids ound in he ex ac s om
Soils 002 and 013 a e sa e in e ms o iabili y e ec s, while hose om
Soil 001 esul in a c i ical impac in HepG2 cells and S. ce e isiae.
Se e al ac o s a e in ol ed in he oxici y o he hea y me als, including
dose, ou e and ime o exposu e. In he case o indi idual me als, hei
acu e and ch onic e ec s ha e been desc ibed o some o hem (Bala-
li-Mood e al., 2021; Jaishanka e al., 2014). Howe e , ega ding he
impac o mix u es o me als and me alloids, he numbe o exis en
wo ks in he cu en li e a u e is sca ce, since mos o hem a e ocused
on hei indi idual e ec . Fo his eason, li le is known abou he
combined oxici y o hea y me als, which can p esen addi i e, an ag-
onis ic o syne gis ic e ec s. The huge di e ences in hei whole con en
be ween Soil 001 ex ac s and he o he s a e he mos likely cause o he
ad e se biological impac o his soil. A P incipal Componen Analysis
(PCA) o he chemical p ope ies o he soluble ex ac s and hei asso-
cia ed oxici y on HepG2 cells and S. ce e isiae was also pe o med o
iden i y hose elemen s ha a e mo e likely o be associa ed wi h he
oxic e ec s obse ed (Supplemen a y Ma e ial, S2). This analysis
showed ha he high concen a ion o some ace elemen s such as Zn,
Mn, As, C o Co could be ela ed wi h he oxici y agains bo h human
cells and he yeas (Supplemen a y Ma e ial, Tables S5–S6; Fig. S2).
Pa icula ly, he high le els o Mn de ec ed in his sample p obably
ep esen a de e minan ac o in he obse ed haza d e ec s. Excessi e
le els o Mn ha e been epo ed o induce cell dea h h ough apop osis
(Alaimo e al., 2014; Hi a a, 2002) o neu odegene a i e damage in
mammals (Pe es e al., 2016). In hepa oma cell lines, including HepG2
cells, i has been epo ed ha hei iabili y can be a ec ed when
exposed o MnCl
2
a concen a ions in he millimola and mic omola
ange (Chen e al., 2022; Tillman, 2018). Mo eo e , in S. ce e isiae, 0.5
mM o highe concen a ions o Mn showed o inhibi he g ow h o his
o ganism (Blackwell e al., 1998). The e o e, he le els o Mn, ac ing
syne gis ically wi h he o he elemen s, could cause he c i ical dec ease
obse ed in he iabili y o bo h HepG2 cells and S. ce e isiae. In he case
o P. pu ida, and as i was obse ed in he e alua ion o he o ganic
ex ac s, his mic oo ganism was no a ec ed by any o he soil samples.
In gene al e ms, he membe s o his species ha e he abili y o g ow
and de elop in ad e se en i onmen s, including pollu ed si es (Ramos
e al., 2015). Thus, ou esul s show ha he le els o con aminan s
p esen in hese soils a e unde he ole ance limi s o P. pu ida.
Fig. 4. E ec o con amina ed soils (Soil 001, Soil 002 and Soil 013) on su i al (A) and ep oduc ion (B) o E. c yp icus a e 28 days o exposu e. LUFA 2.2 was used
as con ol soil. Da a ep esen he mean o 4 eplica es (±s anda d de ia ion, SD). Di e ences we e es ablished using a One-way ANOVA ollowed by mul iple
compa isons es (Tukey es ) and conside ed signi ican a P ≤0.05. Di e en le e s indica e s a is ically signi ican di e ences be ween ea men s.
S. de la Pa a e al.
En i onmen al Pollu ion 315 (2022) 120472
8
Rega ding he in i o oxicological e alua ion o he h ee soils, he
di e ences in EPH le els in Soil 013 compa ed o he o he s could
explain he high e ec o his soil on su i al and ep oduc ion o
E. c yp icus. E ec s o c ude oil con amina ion on enchy aeids su i al
we e obse ed a concen a ions o 10,000 mg TPH kg
−1
and 0.4 mg
PAH kg
−1
. In addi ion, su i al has been epo ed as an index o hea y
c ude oil con amina ion o soil because co ela ed linea ly wi h TPH and
PAH (Filimono a and Poka zhe skii, 2000). The e ec o Soil 001 could
be pa ly explained by he high concen a ions o As and Zn, which we e
closed o and up o bibliog aphic alues o LC50 and EC50, su i al and
ep oduc ion, espec i ely (LC50: 72.8 mg As kg
−1
(Li e al., 2021);
EC50: 212 mg Zn kg
−1
(Wel je e al., 1995); and 229 mg As kg
−1
(Lock
and Janssen, 2001)). A PCA pe o med o p ospec o he possible in-
luence o soil physical and chemical p ope ies on he obse ed oxici y
in wo ms (Supplemen a y Ma e ial, Tables S7–S8; Fig. S3) showed ha
he load o o ganic soil pollu an s and some ace me als such as Al, Ti
and C , as well as As, Mn, Pb and Zn, could be associa ed wi h ha m ul
e ec s o e he iabili y and he ep oduc ion o E. c yp icus. Mo eo e ,
o he p ope ies such as he elec ical conduc i i y and he pe cen age o
sand seems o a ec bo h pa ame e s.
5. Conclusions
The biological impac o h ee soils pollu ed wi h di e en le els o
hyd oca bons and hea y me als was e alua ed in his wo k. The selec ed
analysis included in i o assays using o ganic and soluble ex ac s om
he soils, and in i o assays di ec ly using he soil samples. Di e en
model o ganisms ep esen a i e o human (cell line HepG2) and en i-
onmen al (S. ce e isiae, P. pu ida and E. c yp icus) exposu es we e
employed in he oxicological e alua ion.
The ob ained esul s showed ha he soluble con aminan s o he Soil
001, which p esen ed he highes le els o me als and me alloids, p o-
oked a c i ical dec ease in he iabili y o he HepG2 cells and
S. ce e isiae. Mo eo e , in i o expe imen s exhibi ed ha his soil,
oge he wi h Soil 013, which p esen ed he highes le els o hyd o-
ca bons, display a signi ican e ec o e he iabili y and he ep o-
duc ion o E. c yp icus.
In summa y, hese esul s p o ide a gene al o e iew o he impac
o h ee eal pollu ed soils in he human heal h and he en i onmen . In
addi ion, his wo k b ough ou he impo ance o e alua ing di e en
ex ac s o he soils when pe o ming in i o assays, and he ele ance o
using di e en ep esen a i e o ganisms combining in i o and in i o
assays o ob ain accu a e in o ma ion abou he po en ial oxici y ha a
pollu ed si e may ep esen .
C edi au ho s a emen
Sand a de la Pa a: Fo mal analysis, In es iga ion, W i ing - Re iew
& Edi ing. Ve ´
onica Gonz´
alez: Fo mal analysis, In es iga ion, W i ing -
Re iew & Edi ing. Pa icia Sol´
o zano Vi es: Fo mal analysis, In es i-
ga ion, W i ing - Re iew & Edi ing. Sand a Cu iel-Aleg e: Fo mal
analysis, In es iga ion, W i ing - Re iew & Edi ing. Blanca Velasco
A oyo: Fo mal analysis, W i ing - Re iew & Edi ing. Ca los Rad:
Fo mal analysis, W i ing - Re iew & Edi ing. Rocío Ba os: Fo mal
analysis, Supe ision, W i ing - Re iew & Edi ing, Funding acquisi ion
Juan An onio Tamayo-Ramos: Concep ualiza ion, Me hodology,
W i ing - Re iew & Edi ing, Supe ision. Ca los Rumbo: Concep uali-
za ion, Me hodology, In es iga ion, Fo mal analysis, W i ing - O iginal
D a , W i ing - Re iew & Edi ing, Supe ision.
Decla a ion o compe ing in e es
The au ho s decla e ha hey ha e no known compe ing inancial
in e es s o pe sonal ela ionships ha could ha e appea ed o in luence
he wo k epo ed in his pape .
Da a a ailabili y
Da a will be made a ailable on eques .
Acknowledgmen s
This wo k ecei ed unding om he GREENER p ojec o he Eu-
opean Union’s Ho izon 2020 esea ch and inno a ion p og am (G an
Ag eemen No. 826312). S. Cu iel-Aleg e was g an ed wi h a p e-
doc o al con ac by Jun a de Cas illa y Le´
on and he Eu opean Social
Fund (ORDEN EDU/1508/2020, de 15 de diciemb e). We hank Mi eya
Ped e o and And ea Ma ínez o hei echnical suppo . We also hank
Ins i u e o Technology Ca low and Shandong Academy o Sciences o
kindly p o iding us wi h soil samples.
Appendix A. Supplemen a y da a
Supplemen a y da a o his a icle can be ound online a h ps://doi.
o g/10.1016/j.en pol.2022.120472.
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