Chemosphe e 291 (2022) 132758
A ailable online 1 No embe 2021
0045-6535/© 2021 The Au ho s. Published by Else ie L d. This is an open access a icle unde he CC BY-NC-ND license
(h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/).
Time-cou se e olu ion o bac e ial communi y ole ance o e acycline
an ibio ics in ag icul u al soils: A labo a o y expe imen
Vanesa San ´
as-Miguel
a
,
*
, Lau a Rod íguez-Gonz´
alez
a
, A elino Nú˜
nez-Delgado
b
,
Espe anza ´
Al a ez-Rod íguez
b
, Mon se a Díaz-Ra i˜
na
c
, Manuel A ias-Es ´
e ez
a
,
Da id Fe n´
andez-Cal i˜
no
a
a
´
A ea de Eda oloxía e Química Ag ícola, Facul ade de Ciencias, Uni e sidade de Vigo, As Lagoas 1, 32004, Ou ense, Galiza, Spain
b
Depa amen o de Eda oloxía e Química Ag ícola, Escola Poli ´
ecnica Supe io de Enxe˜
na ía, Uni e sidade de San iago de Compos ela, Campus de Lugo, Galicia, Spain
c
Depa amen o de Bioquímica Del Suelo, Ins i u o de In es igaciones Ag obiol´
ogicas de Galicia (IIAG/CSIC), San iago de Compos ela, Galicia, Spain
HIGHLIGHTS GRAPHICAL ABSTRACT
•Soil pollu ion wi h e acyclines may
inc ease bac e ial communi y ole ance.
•An ibio ic concen a ions needed o
cause hose inc eases a e highe han
500 mg/kg.
•The inc eases we e highe in soils wi h
low o ganic ca bon con en .
•The magni ude o he inc eases in bac-
e ial communi y ole ance was ime
dependen .
•Bac e ial communi y ole ance o e a-
cyclines was maximum a e 45–100
days.
ARTICLE INFO
Handling Edi o : Willie Peijnenbu g
Keywo ds:
Bac e ial g ow h
Bac e ial ole ance
Chlo e acycline
Oxy e acycline
PICT
Te acycline
ABSTRACT
The p esence o an ibio ics in soils may inc ease he selec ion p essu e on soil bac e ial communi ies and cause
ole ance o hese pollu an s. The empo al e olu ion o bac e ial communi y ole ance o di e en concen a-
ions o e acycline (TC), oxy e acycline (OTC) and chlo e acycline (CTC) was e alua ed in wo soils. The
esul s showed an inc ease o soil bac e ial communi y ole ance o TC, CTC and OTC only in samples pollu ed
wi h he highes an ibio ic concen a ions es ed (2000 mg kg
−1
). The magni ude o hose inc eases was highe in
he soil wi h he lowe o ganic ca bon con en (1.6%) han in he soil wi h an o ganic ca bon con en eaching
3.4%. In he soil wi h low o ganic ca bon con en , he ime-cou se e olu ion showed a maximum inc ease in he
ole ance o bac e ial communi ies o e acycline an ibio ics be ween 45 and 100 incuba ion days, while o
longe incuba ion imes (360 days) he ole ance dec eased. In he soil wi h high o ganic ca bon con en , a
simila beha io was ound o OTC. Howe e , o CTC and TC, sligh ly inc eases and dec eases ( espec i ely)
we e ound in he bac e ial communi y ole ance a in e media e incuba ion imes, ollowed by alues close o
ze o o TC a e 360 days o incuba ion, while o CTC hey emained highe han in he con ol. In conclusion,
soil pollu ion due o e acyclines may cause bac e ial communi y ole ance o hese an ibio ics when p esen a
high concen a ions. In addi ion, he isk is highe in soils wi h low o ganic ma e con en , and i dec eases wi h
ime.
* Co esponding au ho .
E-mail add ess: [email p o ec ed] (V. San ´
as-Miguel).
Con en s lis s a ailable a ScienceDi ec
Chemosphe e
jou nal homepage: www.else ie .com/loca e/chemosphe e
h ps://doi.o g/10.1016/j.chemosphe e.2021.132758
Recei ed 6 Sep embe 2021; Recei ed in e ised o m 29 Oc obe 2021; Accep ed 31 Oc obe 2021
Chemosphe e 291 (2022) 132758
2
1. In oduc ion
Ve e ina y an ibio ics (VAs) ha e been widely used as a ea men o
in ec ious diseases and e en as g ow h p omo e s in animals (Sapko a
e al., 2008; Li e al., 2011). The consump ion o e e ina y an ibio ics
has been inc easing in ecen decades, wi h he o e all wo ld con-
sump ion o an ibio ics es ima ed o 2030 being 105,596 ons (Van
Boeckel e al., 2015). When hese compounds a e adminis e ed o ca le,
hey a e poo ly abso bed in he animal in es ine and be ween 30 and
60% a e exc e ed as he o iginal molecule (Sa mah e al., 2006; Mass´
e
e al., 2014). Te es ial ecosys ems a e exposed o e e ina y an ibio ics
mainly h ough epea ed applica ions o manu e/slu y and sludge o
ag icul u al soils (Mon o s e al., 1999) and, once he e, an ibio ics can
a ec non- a ge o ganisms such as bac e ial communi ies (Wa man,
1980). The e ec o an ibio ics on soil mic obial communi ies has been
s udied p e iously by a ious au ho s, who obse ed how he p esence
o hese compounds in he soil can cause changes in he mic obial
communi y s uc u e (Hammes ah e al., 2008; Unge e al., 2013; U a
e al., 2019) and unc ions (Zielezny e al., 2006; To h e al., 2011; Liu
e al., 2015; Ma e al., 2016).
Te acycline an ibio ics a e one o he g oups mos widely used in he
Eu opean Union, and anking second place wo ldwide (Bansal, 2012),
mainly due o hei low cos , e ec i eness, b oad-spec um, and high
solubili y in wa e . Besides, wi hin he g oup o e acyclines he mos
consumed a e e acycline (TC), oxy e acycline (OTC), and chlo e a-
cycline (CTC) (ESVAC, 2016). In addi ion, e acyclines a e cha ac e -
ized by long hal -li es ha can las beyond 100 days in soils (Cycon
e al., 2019). The high pe sis ence o hese an ibio ics in he soil is o
pa icula conce n, as i may lead o he inc ease o an ibio ic ole ance
by soil bac e ial communi ies. In ac , he inc ease in he concen a ion
o any pollu an (o ganic o ino ganic) in soil may exe a selec ion
p essu e on he soil bac e ial communi ies, and hence cause ole ance o
ha speci ic pollu an (Blanck, 2002). This e ec is called
pollu ion-induced communi y ole ance (PICT), and i s measu emen
can be use ul in he quan i ica ion o he ha m ul e ec s o oxic sub-
s ances on soil mic oo ganisms and quan i ying he appea ance o
esis ance o soil bac e ial communi ies o oxic subs ances. I should be
no ed ha he inc ease in an ibio ic esis ance o bac e ial communi ies
in soils has become a c ucial h ea o public heal h. Exposu e o en i-
onmen al o ganisms o an imic obial agen s can c ea e ese oi s o
esis ance in soil o ganisms ha could po en ially con e esis ance o
pa hogens h ough ho izon al gene ans e and o he mechanisms
(Da ies, 1994; Knapp e al., 2010; Se weci´
nska, 2020). The esul ing
inc ease in in ec ions by pa hogens and he p oblem o bac e ia esis an
o an ibio ics ha e become a h ea o global public heal h, which needs
add essing his p oblem om wo complemen a y concep s: One Heal h
and Global Heal h (Manyi-Loh e al., 2018; He nando -Amado e al.,
2019; Se weci´
nska, 2020).
Rega ding ole ance, s udies ela ed o his opic can be ca ied ou
using DNA echniques (p esence o esis ance genes) o om a unc-
ional poin o iew ( ocusing on PICT) (Milenko ski e al., 2010). The
use o echniques such as PICT ins ead DNA inge p in ing has wo clea
ad an ages: i) he easy in e p e a ion o he da a, and ii) he de ec ion o
he p esence o ole ance in he bac e ial communi ies oge he wi h he
con i ma ion o he oxici y o he con aminan on he bac e ial com-
muni ies (Milenko ski e al., 2010). Al hough he e a e some s udies on
unc ional ole ance (PICT) o an ibio ics pe o med on bac e ial com-
muni ies (Schmi e al., 2004, 2006; Demoling and Båå h, 2008; B and
e al., 2009; Demoling e al., 2009; Liu e al., 2012), limi ed esea ch has
been ca ied ou measu ing his unc ional ole ance o e acycline
an ibio ics (Schmi , 2005; Schmi e al., 2006; Fang e al., 2014; Song
e al., 2017; Han e al., 2019). Mo eo e , none o hese wo ks ocused on
he ime-cou se e olu ion o bac e ial communi y ole ance o an ibi-
o ics. This kind o esea ch could be imp o ed aking in o accoun ha
he du a ion o he exposu e o bac e ial communi ies o oxican s mus
be long compa ed o he ime equi ed o he communi y o go h ough
i s succession o a mo e ole an s a e (Blanck, 2002). In gene al, in hose
wo ks whe e he PICT me hod has been used o an ibio ics, he imes o
exposi ion we e lowe han 9 weeks (Schmi , 2005; Schmi e al., 2006;
Fang e al., 2014; Song e al., 2017; Han e al., 2019). The e o e,
long- e m s udies dealing wi h e en ual inc eases in bac e ial commu-
ni y ole ance o e acycline an ibio ics a e needed o cla i y he po-
en ial pe sis ence o he unc ional ole ance o hese an ibio ics wi h
ime. These da a could delinea e ends in an ibio ic esis ance po en-
ially aluable o epidemiological s udies (Knapp e al., 2010).
In iew o his backg ound, he aim o he p esen wo k is o ob ain
new in o ma ion as ega ds he long- e m ime-cou se e olu ion o he
bac e ial communi y ole ance o h ee e acycline an ibio ics (TC,
OTC, and CTC) in wo soils wi h di e en o ganic ca bon con en ha
we e pollu ed wi h di e en concen a ions o hese subs ances. Spe-
ci ically, 8 di e en concen a ions o each an ibio ic we e used (2000,
500, 125, 31.3, 7.8, 2, 0.5 and 0 mg kg
−1
), and soil bac e ial communi y
ole ance o hese an ibio ics was assessed a e 45, 100, 180 and 360
days o incuba ion, using he pollu ed induced communi y ole ance
(PICT) me hodology. The esul s o he s udy could be ele an o shed
u he ligh on he a e o hese pollu an s when sp ead on en i on-
men al compa men s, and speci ically ega ding i s e ec on soil mi-
c obial communi ies o e ime.
2. Ma e ial and me hods
2.1. Chemicals
Te acycline hyd ochlo ide (TC, CAS. 64-75-5; ≥95% in pu i y),
Oxy e acycline hyd ochlo ide (OTC, CAS, 2058-46-0; ≥95% in pu i y),
and Chlo e acycline hyd ochlo ide (CTC, CAS 64-72-2; ≥97% in pu-
i y) we e supplied by Sigma–Ald ich (S einheim, Ge many). Talc (CAS
14807-96-6) was supplied by Sigma–Ald ich (S einheim, Ge many).
2.2. Soil samples
Two ag icul u al soils we e selec ed om a soil pool p e iously
cha ac e ized by Conde-Cid e al. (2018). Soils we e sampled om su-
pe icial ho izons (0–20 cm) wi h an Edelman p obe. Once in he labo-
a o y, hese samples we e ai -d ied, sie ed h ough a 2 mm mesh and
s o ed in polye hylene bo les un il analysis. The main cha ac e is ics o
he s udied soils a e shown in Table S1 (Supplemen a y Ma e ial).
B ie ly, hese soils p esen ed simila pa icle size dis ibu ion and
ex u e, being sandy clay loam o soil 1 (sand: 54.6%; clay: 23.4%; sil :
22%) and sandy loam o soil 2 (sand: 58.5%; clay: 22.5%; sil : 19.1%).
O ganic ca bon con en s we e 1.6% and 3.4% o soils 1 and 2,
espec i ely. The pH alues we e simila in bo h soils, being 4.65 o soil
1 and 4.74 o soil 2. The e ec i e ca ion exchange capaci y was 4.7
cmol
c
kg
−1
o soil 1, and 5.9 cmol
c
kg
−1
o soil 2. The concen a ions o
hea y me als de e mined in he selec ed soils we e low and simila o
hose ound in non-pollu ed soils in he s udy a ea (Macías and Cal o,
2008). In addi ion, he le els o e acycline, oxy e acycline and
chlo e acycline we e below he de ec ion limi (50 ng g
−1
) (Conde-Cid
e al., 2018).
2.3. Expe imen al design
The selec ed soil samples we e mois ened up o 60–80% o wa e
holding capaci y and incuba ed a 22 ◦C in he da k du ing 1 week, an
adequa e ime o eco e and s abilize soil bac e ial communi y g ow h
a e mois u e adjus men (Meisne e al., 2013). A e his ime, each
soil was dis ibu ed in 72 polyp opylene ubes (100 mL) (3 an ibio ics x
3 eplica es x 8 an ibio ic concen a ions), adding 20 g o soil (d y
weigh ) in each ube. The o al numbe o mic ocosms was 144 (72 pe
each soil). Then, di e en concen a ions o he h ee e acycline an-
ibio ics ( e acycline, oxy e acycline and chlo e acycline) we e
added o he soil mic ocosm indi idually (24 pe an ibio ic and soil)
V. San ´
as-Miguel e al.
Chemosphe e 291 (2022) 132758
3
using alc powde as a ca ie (Rousk e al., 2008), eaching a inal
concen a ion in he soil samples o 0, 0.5, 2, 7.8, 31.3, 125, 500 and
2000 mg an ibio ic pe kg
−1
o soil. These concen a ions we e used
p e iously in wo ks es ing di ec oxici y o e acycline an ibio ics on
he g ow h o bac e ial communi ies (San ´
as-Miguel e al., 2020a,
2020b, 2020c, 2020d). The concen a ions we e p e iously selec ed in
o de o ob ain dose- esponse cu es ha included: a) low concen a-
ions ha almos did no a ec bac e ial g ow h, and b) e y high con-
cen a ions ha caused almos comple e inhibi ion o soil bac e ial
g ow h (>90%) and hus o e es ima es o oxici y indices in a mo e
eliable way (Fox and Landis, 2016). A e pe o ming soil spiking wi h
e acycline an ibio ics, he soil mic ocosms we e incuba ed a 22 ◦C in
he da k du ing 360 days. Du ing his pe iod, bac e ial communi y
ole ance o he h ee e acycline an ibio ics was es ima ed in he mi-
c ocosms pollu ed wi h each o hem a e 45, 100, 180 and 360 days.
The soil mic ocosms we e equen ly ae a ed, while he soil mois u e
was main ained by adding wa e i necessa y.
Tole ance o bac e ial communi ies o e acycline an ibio ics was
measu ed o all mic ocosms by means o a sho - e m oxici y es ac-
co ding o Båå h (1992) and Díaz-Ra i˜
na e al. (1994) wi h ce ain
modi ica ions indica ed below, and using he leucine (Leu) inco po a-
ion me hod (Båå h, 1994; Båå h e al., 2001) o es ima e bac e ial
communi y g ow h. B ie ly, 3.5 g o soil ( esh weigh ) was mixed wi h
50 mL o dis illed wa e using a mul i o ex shake o 3 min a
maximum in ensi y, ollowed by low-speed cen i uga ion a 1000×g o
10 min, hus c ea ing a bac e ial suspension in he supe na an . An
aliquo (1.5 mL) o his suspension was ans e ed o 2 mL
mic o-cen i uga ion ubes. Then, olume aliquo s o 0.15 mL con ain-
ing di e en concen a ion o he e acycline an ibio ics we e added
be o e measu ing he leucine inco po a ion. Se en di e en concen a-
ions o he h ee an ibio ics (TC, OTC o CTC) we e used, going om 0.1
o 400 mg L
−1
. A con ol wi h dis illed wa e was also used o each
mic ocosm. Then, he bac e ial communi y g ow h was es ima ed a e a
p e-incuba ion s ep o 24 h o bac e ial suspensions wi h he di e en
an ibio ics concen a ions added be o e he leucine inco po a ion assay
(Be g e al., 2010; Fe n´
andez-Cal i˜
no e al., 2013). A e his
p e-incuba ion ime, 2
μ
L [
3
H] Leu (3.7 MBq mL
−1
and 0.574 TBq
mmol
−1
; Pe kinElme , USA) we e added wi h non-labeled Leu o each
ube, esul ing in a inal concen a ion o 275 nM Leu in he bac e ial
suspension. A e incuba ion a 22 ◦C o 3 h, he bac e ial g ow h was
s opped wi h 75
μ
L o 100% ichlo oace ic acid. Washing was pe -
o med as desc ibed by Båå h e al. (2001), and adioac i i y was
de e mined by scin illa ion liquid coun ing (T i-Ca b 2810 TR, Pe ki-
nElme , USA).
2.4. Da a analysis
The da a co esponding o es ima ed bac e ial communi y g ow h
(leucine inco po a ion) as a unc ion o he concen a ion o an ibio ics
(TC, OTC o CTC) added o he bac e ial suspension was no malized
di iding all alues by he con ol (sample wi hou an ibio ic) o each
mic ocosm, in o de o allow he subsequen compa ison among
di e en dose- esponse cu es.
The ole ance o he bac e ial communi ies o TC, OTC o CTC was
es ima ed as log IC
50
, he loga i hm o he concen a ion o an ibio ic
ha esul ed in 50% inhibi ion o bac e ial communi y g ow h (leucine
inco po a ion) in each dose- esponse cu e. Log IC
50
was es ima ed
using a logis ic model (Rousk e al., 2011; Sebaugh e al., 2011; Ra h
e al., 2016), Y =c/[1+e
b(a-x)
], whe e Y is Leu inco po a ion o each
an ibio ic concen a ion, x is he loga i hm o he concen a ion o
an ibio ic added, a is he alue o log IC
50
, b is a pa ame e ela ed wi h
he slope o inhibi ion cu es, and c is he bac e ial g ow h wi hou
an ibio ic added (con ol sample). A highe alue o log IC
50
indica es a
highe communi y ole ance, while a lowe alue indica es ha he
an ibio ics a e mo e oxic o he bac e ial communi y, i.e., i is less
ole an . The inc eases o bac e ial communi y ole ance o an ibio ics in
soils spiked wi h hem (PICT =Pollu ion Induced Communi y Tole -
ance) was calcula ed as ΔlogIC
50
, speci ically by sub ac ing logIC
50
alues es ima ed in spiked soils minus he logIC
50
alues ound in he
unpollu ed con ols o each soil.
3. Resul s and discussion
3.1. Inc eases in bac e ial communi y ole ance o e acycline an ibio ics
The inhibi ion cu es o bac e ial g ow h ob ained o each e a-
cycline an ibio ic, o each e acycline concen a ion, each soil sample,
and each ime es ed a e shown in Figu es S1-S3 (Supplemen a y Ma-
e ial). All mic ocosms showed sigmoid dose- esponse cu es, i.e., low
e acycline an ibio ics did no inhibi bac e ial g ow h, bu a high
doses, he ex en o inhibi ion inc eased wi h he dose. As a gene al
end, he e is no clea shi o he igh in he dose- esponse cu es
ob ained o e acycline an ibio ic concen a ions ≤500 mg o an i-
bio ic pe kg
−1
o soil. Howe e , a highe concen a ions o added
an ibio ic (2000 mg kg
−1
), a shi o he igh is obse ed in he dose-
esponse cu es. Howe e , his shi ing is mo e clea in he soil wi h
lowe o ganic ca bon con en (Soil 1; C =1.6%) han in he soil wi h a
highe o ganic ca bon con en (Soil 2; C =3.4%). The shi s in he dose-
esponse cu es o he igh sugges inc eases in he ole ance o bac-
e ial communi ies o he an ibio ics es ed. These inc eases in esponse
o soil pollu ion wi h his ype o subs ances may be due o physiological
o gene ic adap a ions, as was sugges ed p e iously o hea y me als
pollu ion (Díaz-Ra i˜
na and Båå h, 1996). These adap a ions may
include: 1) killing o sensi i e species due o immedia e an ibio ic
oxici y; 2) selec ion o ole an species due o di e en compe i i e
abili ies o g ow h in p esence o an ibio ics; 3) physiological o
beha io al esponses in indi idual cell popula ions; and 4) adap i e
e olu ion ia mu a ion o acquisi ion o an ibio ic esis ance ia ho i-
zon al gene ans e (B and e al., 2015). In he case o e acycline
an ibio ics, he i s mechanisms may be disca ded because molecules o
he e acycline g oup, being bac e ios a ic an ibio ics (Smilack, 1999),
may inhibi he g ow h o bac e ia bu do no kill hem.
The dose- esponse cu es we e gene ally well desc ibed by he lo-
gis ic model (Tables S2 and S3, Supplemen a y Ma e ial), wi h R
2
alues
anging om 0.899 o 0.998 o e acycline (mean R
2
=0.967), om
0.815 o 0.999 o oxy e acycline (mean R
2
=0.953), and om 0.877 o
0.988 o chlo e acycline (mean R
2
=0.980).
Fig. 1 shows he Log IC
50
alues co esponding o soil 1 o all TC,
OTC and CTC concen a ions and incuba ion imes. In gene al, he log
IC
50
alues show simila beha io s o he di e en e acycline an ibi-
o ics. O e all, ole ance o soil bac e ial communi ies o e acycline
an ibio ics was no obse ed in soil 1 a concen a ions ≤500 mg kg
−1
since no inc eases o Log IC
50
alues we e obse ed o any o he
e acycline an ibio ics es ed. Howe e , he Log IC
50
alues ob ained o
he maximum concen a ion es ed (2000 mg kg
−1
) show, in gene al, an
inc ease in ole ance o an ibio ics wi h espec o he alues ob ained
o he con ol (0 mg o an ibio ic kg
−1
o soil), o any o he incuba ion-
imes es ed. These inc eases obse ed in Log IC
50
alues anged be-
ween 0.6 and 1.9 uni s o TC, be ween 0 and 1.6 uni s o OTC, and
anged be ween 0.9 and 3.0 uni s o CTC. The e o e, soil bac e ial
communi ies showed inc eases in ole ance o e acycline an ibio ics in
soils o he an ibio ics concen a ion o 2000 mg kg
−1
. The esul s ob-
ained in his s udy ag ee wi h hose epo ed by o he au ho s using he
PICT me hod (Hund-Rinke e al., 2004; Schmi e al., 2006). These
au ho s did no obse e inc eases in soil bac e ial communi y ole ance
o e acycline an ibio ics a low an ibio ics concen a ions (Hund-Rinke
e al., 2004; Song e al., 2017), howe e , o highe concen a ions
(1000 mg o an ibio ic kg
−1
o soil) inc eases in bac e ial communi y
ole ance ha e been ound a e one week o incuba ion (Schmi e al.,
2006). Schmi e al. (2006) also obse ed ha inc eases in ole ance o
e acycline an ibio ics can occu a lowe concen a ions (100 mg
kg
−1
), bu his was no obse ed in ou s udy.
V. San ´
as-Miguel e al.
Chemosphe e 291 (2022) 132758
4
Fig. 1. Time-cou se a ia ion o bac e ial communi y ole ance o e acycline (TC; A, D, G and J), oxy e acycline (OTC; B, E, H and K) and chlo e acycline (CTC;
C, F, I and L) (exp esed as Log IC
50
), as a unc ion o an ibio ic concen a ion added o soil 1, a e 45, 100, 180 and 360 days o incuba ion. *The concen a ion
inhibi ing he 50% o popula ion was no es ima ed because he bac e ial communi ies showed o al eco e y, he e o e he Log IC
50
asigned was 2.6, co esponding
o he maximum e acycline concen a ion es ed in he PICT es s.
V. San ´
as-Miguel e al.
Chemosphe e 291 (2022) 132758
5
The log IC
50
alues ob ained o soil 2, ega ding TC, OTC and CTC,
and all incuba ion imes a e shown in Fig. 2. The inc eases obse ed in
Log IC
50
alues we e ≤0.1 uni s o TC, and ≤0.5 uni s o CTC, o all
an ibio ic concen a ions and incuba ion imes. Howe e , in he case o
OTC he e was a small inc ease in bac e ial communi y ole ance o he
an ibio ic a e soil spiking wi h 2000 mg kg
−1
and a lowe incuba ion
imes (45 and 100 days), bu being ≤0.9 uni s in bo h cases. The e o e,
mos o he inc eases in he ole ance o bac e ial communi ies o
Fig. 2. Time-cou se a ia ion o bac e ial communi y ole ance o e acycline (TC; A, D, G and J), oxy e acycline (OTC; B, E, H and K) and chlo e acycline (CTC;
C, F, I and L) (exp esed as Log IC
50
), as a unc ion o an ibio ic concen a ion added o soil 2, a e 45, 100, 180 and 360 days o incuba ion.
V. San ´
as-Miguel e al.

Chemosphe e 291 (2022) 132758
6
e acycline an ibio ics occu a 2000 mg kg
−1
. These esul s ag ee wi h
he alues obse ed o soil 1 and wi h da a epo ed by o he au ho s
(Hund-Rinke e al., 2004; Schmi e al., 2006; Song e al., 2017).
Howe e , in he cu en wo k he magni ude o hese inc eases is
di e en o each o he soils s udied. Speci ically, he inc ease in he
magni ude o he ole ance o e acycline an ibio ics is lowe o soil 2
(wi h high ca bon con en ) han o soil 1 (wi h low ca bon con en ),
sugges ing ha hese inc eases in he ole ance o he bac e ial com-
muni ies o an ibio ics may be in luenced by he o ganic ma e con en
o he soil. This e ec may be due o he key ole played by o ganic
ma e in he adso p ion o e acycline an ibio ics in soils (Tolls, 2001;
Gu and Ka hikeyan, 2008; Ling-Ling e al., 2010; Conde-Cid e al.,
2019). The so p ion o e acycline an ibio ics on o soils educes he
bioa ailabili y o hese molecules o bac e ial communi ies and, he e-
o e, i could make lowe he selec i e p essu e on bac e ia and educe
he p omo ion o an ibio ic esis ance o he mic oo ganisms.
3.2. Time-cou se e olu ion o bac e ial communi y ole ance o
e acycline an ibio ics
The bac e ial communi ies showed inc eases in ole ance o an i-
bio ic concen a ions o 2000 mg kg
−1
. The e o e, he ime-cou se
e olu ion (45, 100, 180 and 360 days) o Δlog IC
50
alues was e alu-
a ed o he maximum concen a ion es ed (2000 mg kg
−1
) (Fig. 3). In
soil 1 (soil wi h low ca bon con en , C =1.6%), he ole ance o bac e ial
communi ies o an ibio ics inc eased wi h ime up o a maximum Δlog
IC
50
alue eached a 45 days o CTC (Fig. 3C), while i was achie ed a
incuba ion imes be ween 45 and 100 days o OTC (Fig. 3B), and a e
100 days o incuba ion o TC (Fig. 3A). A e hese incuba ion imes, he
ole ance o he bac e ial communi ies o e acycline an ibio ics g ad-
ually dec eased, eaching a e 360 days Δlog IC
50
alues close o ze o
(−0.2) o OTC, bu emaining close o 0.9 and 1.6 o TC and CTC,
espec i ely. Howe e , in soil 2 (wi h high ca bon con en , C =3.4%)
soil bac e ial communi ies ole ance o CTC (Δlog IC
50
) sligh ly
inc eased wi h ime, eaching he maximum alue (0.5 uni s) a e 360
days (Fig. 3F). The bac e ial communi y ole ance o TC sligh ly de-
c eases o in e media e incuba ion imes (up o −0.5), eaching Δlog
IC
50
alues close o ze o (−0.1) a e 360 days (Fig. 3D). Howe e , he
bac e ial communi y ole ance o OTC showed a ime-cou se e olu ion
simila o he end ound in soil 1 o all h ee an ibio ics, wi h a
maximum inc ease o ole ance o OTC be ween 45 and 100 days
(Fig. 3E), bu showing alues lowe han hose ound o soil 1 (0.9, 0.8,
ins ead o 1.5, 1.6). Fo longe incuba ion imes bac e ial communi y
ole ance o OTC g adually dec eased, eaching a Δlog IC
50
alue close
o ze o (0.2) a e 360 days o incuba ion.
The e is a lack o esea ch dealing wi h he s udy o ime-cou se
e olu ion o soil bac e ial communi y ole ance o an ibio ics in soils
pollu ed wi h his ype o eme ging pollu an s. The p esen wo k is one
o hose ocusing on his ield. Some o he in e es ing and ecen s udies
a e hose by San ´
as-Miguel e al. (2020e), o he pape by Zhong e al.
(2021), whe e he au ho s in es iga ed he po en ial e ec o Cu and
o he hea y me als on he bac e ial ole ance o esis ance o an ibio ics.
P e iously, a simila esea ch was pe o med, bu s udying he
ime-cou se e olu ion o bac e ial communi y ole ance o di e en
hea y me als (Diaz-Ra i˜
na and Båå h, 1996). Resul s om ha p e ious
s udy showed ha he bac e ial communi y ole ance o Cu, Zn and Cd in
soils pollu ed wi h hese hea y me als inc eased wi h ime, eaching
maximum alues a he end o he expe imen (14 mon hs o incuba-
ion). In he case o soils pollu ed wi h Zn, Diaz-Ra i˜
na and Båå h (1996)
also s udied he bac e ial communi y ole ance o Zn up o 1024 days,
inding he highe alues a his p olonged incuba ion ime. In he cu -
en wo k, he esul s pa ially i wi h hose ound o hea y me als, i.e.
ini ial bac e ial communi y ole ance inc eases wi h ime. Howe e , o
longe incuba ion imes he bac e ial communi y ole ance o an ibio ics
dec eased, eaching in some cases Δlog IC
50
alues close o ze o. In
addi ion, ou esul s also i , a leas pa ially, wi h s udies measu ing
he abundance o an ibio ics esis ance genes (ARGs) in ag icul u al soils
ea ed wi h an ibio ic-pollu ed manu es, which in some cases showed a
simila ime-cou se e olu ion as ha we ound o bac e ial communi y
ole ance o an ibio ics (Hu e al., 2016; Zhang e al., 2017). Hu e al.
(2016) epo ed inc eases o β-lac am ARGs up o 63 incuba ion days
ollowed by dec eases a 140 days, while, o e acycline and o he
an ibio ics, ARGs inc eases we e de ec ed a 63 days and main ained
un il he end o he incuba ion (140 days). Zhang e al. (2017) assessed
he abundance o ARGs in ag icul u al soils a e he addi ion o di e en
ypes o manu es spiked and no-spiked wi h ylosin, showing ha he
gene al abundance o ARGs dec eased wi h ime.
The inc ease o ole ance wi h ime may be due o he slow a e o
Fig. 3. Time-cou se e olu ion o inc eases in bac e ial communi y ole ance (ΔLog IC
50
) o e acycline an ibio ics: Te acycline (TC), Oxy e acycline (OTC) and
Chlo e acycline (CTC). Soil 1: TC (A), OTC (B) and CTC (C). Soil 2 TC (D), OTC (E) and CTC (F). ΔLog IC
50
=Log IC
50
o samples pollu ed wi h 2000 mg kg
−1
o
e acycline an ibio ic (TC, OTC o CTC) minus Log IC
50
o 0 mg o an ibio ic kg
−1
(con ol soil).
V. San ´
as-Miguel e al.
Chemosphe e 291 (2022) 132758
7
ole ance de elopmen mechanisms, i.e. he p ocess o inc ease o bac-
e ial communi y ole ance o any oxican needs enough ime (Día-
z-Ra i˜
na and Båå h, 1996). Fu he mo e, he dec ease in bac e ial
communi y ole ance o di e en e acycline an ibio ics ound o long
incuba ion pe iods may be due o di e en causes:
1) e acycline an ibio ics show a s ong adso p ion by soil componen s
such o ganic ma e and clays (Tolls, 2001; Figue oa e al., 2004; Pils
and Lai d, 2007; Gu and Ka hikeyan, 2008; Teixid´
o e al., 2012;
Conde-Cid e al., 2019). This high so p ion may inc ease o e ime
ia ageing p ocesses (Loibne e al., 2006; He e al., 2019), causing
ha e acycline an ibio ics became less bioa ailable o mic oo -
ganisms, and he e o e less oxic. To no e ha less oxici y also
means less capaci y o inc ease he bac e ial communi y ole ance o
an ibio ics.
2) deg ada ion o e acycline an ibio ics in soils wi h ime (Maki e al.,
2006; Wang and Ya es, 2008; Pan and Chu, 2017). Al hough he
deg ada ion o e acycline an ibio ics in he soil is a slow p ocess
depending on he ini ial concen a ion o he an ibio ic (Bansal,
2012; Cyco´
n e al., 2019), in 360 days hey may be highly deg aded
because he hal -li e alues a e a ound 100 days when he concen-
a ions o e acycline an ibio ics a e high (Cyco´
n e al., 2019). Less
concen a ion o e acycline an ibio ics wi h ime lead o less
oxici y p essu e, and he e o e o educ ions in bac e ial communi y
ole ance o hese subs ances.
3.3. En i onmen al implica ions
The concen a ions o e acycline an ibio ics de ec ed in ag icul-
u al soils a e e y a iable among soil ype and di e en egions (Ka cı
and Balcıo˘
glu, 2009; Hu e al., 2010; Conde-Cid e al., 2018), wi h
maximum alues anging be ween 2.9 ×10
−3
and 1 mg kg
−1
o TC,
be ween 7 ×10
−4
and 2.7 mg kg
−1
o OTC, and be ween 9 ×10
−4
and
11 mg kg
−1
o CTC (Conde-Cid e al., 2020). These alues a e much
lowe han alues which caused inc eases in bac e ial communi y
ole ance o TC, OTC o CTC in he p esen wo k (>500 mg kg
−1
).
The e o e, o ci cums ances simila o hose es ed in he cu en
esea ch, e acycline an ibio ics concen a ions usually ound in ag i-
cul u al soils would p esen low isk o inc easing he bac e ial com-
muni y ole ance o hese subs ances. This inding could ha e an
impo an implica ion, in he sense ha al hough many s udies showed
ha he p esence o low concen a ions o an ibio ics in he soil may
inc ease he p esence o an ibio ics esis ance genes, including e acy-
cline ARGs (Tang e al., 2015; Xie e al., 2018; Liu e al., 2020), ou s udy
shows ha om a unc ional poin o iew he isk would be lowe . In
addi ion, i some ype o an ibio ic solu ions spills happen in he a ms,
high concen a ions o e acycline an ibio ics may be eached in limi ed
soil su aces as an ibio ics ho spo s (Kaesebe g e al., 2018), and
he e o e inc eases in bac e ial communi y ole ance o his ype o
an ibio ics could be possible. Iden i ying hese ho spo s is cu en ly a
challenge, dese ing special a en ion because he p esence o unc ional
ole ance o soil bac e ial communi ies o an ibio ics may p esen
impo an isk implica ions o human heal h, especially due o ole -
ance ansmission o human pa hogens (Fo sbe g e al., 2012). The e-
o e, he iden i ica ion o en i onmen s whe e he e a e high densi ies o
an ibio ic- ole an bac e ia is impo an o ounding he basis ega ding
he design o new o complemen a y mi iga ion s a egies ha will allow
achie ing p og ess in he con ol o an ibio ic esis ance (Be endonk
e al., 2015).
4. Conclusions
The esul s om he p esen s udy showed ha soil pollu ion due o
he e acycline an ibio ics e acycline, oxy e acycline and chlo e -
acycline may induce inc eases in bac e ial communi y ole ance o
hese subs ances. Howe e , he an ibio ic concen a ions needed a e
>500 mg kg
−1
, alues no usually ound in ag icul u al soils. The e was
a di e ence in he magni ude o hose inc eases as a unc ion o soil ype,
being highe in he soil wi h lowe o ganic ca bon con en (1.6%),
compa ed wi h he soil wi h highe o ganic ma e con en (3.4%).
Rega ding he ime-cou se e olu ion o bac e ial communi y ole ance
o e acycline an ibio ics, as gene al end, i inc eased wi h ime up o
45–100 incuba ion days, hen dec easing o longe incuba ion pe iods.
This e ec was mo e e iden in he soil wi h lowe o ganic ca bon
con en . These esul s could be o ele ance o shed ligh on he en i-
onmen al a e o his kind o eme ging pollu an s, and on he e olu ion
o hei e ec s on soil mic obial communi ies o e ime.
Au ho s a emen
Vanesa San ´
as-Miguel: Me hodology, W i ing – o iginal d a .
Lau a Rod íguez-Gonz´
alez: Me hodology A elino Nú˜
nez-Delgado:
Concep ualiza ion, W i ing- Re iewing and Edi ing. Espe anza
´
Al a ez-Rod íguez: Me hodology, In es iga ion. Mon se a Díaz-
Ra i˜
na : Concep ualiza ion, W i ing – o iginal d a . Manuel A ias-
Es ´
e ez: Da a cu a ion, In es iga ion, Supe ision. Da id Fe n´
andez-
Cal i˜
no: Concep ualiza ion, In es iga ion, W i ing- Re iewing and
Edi ing
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 .
Acknowledgmen
This s udy has been unded by he Spanish Minis y o Economy and
Compe i i eness h ough he p ojec s CGL 2015-67333-C2-1-R and -2-R
(FEDER Funds) and by Xun a de Galicia ia BV1 esea ch g oup
(ED431C 2017/62-GRC). Da id Fe n´
andez Cal i˜
no holds a Ram´
on y
Cajal con ac (RYC-2016-20411) inanced by he Spanish Minis y o
Economy, Indus y and Compe i i eness. Vanesa San ´
as-Miguel and
Lau a Rod íguez Gonz´
alez holds a p e-doc o al ellowship (ED481A-
2020/089 and ED481A-2021/309, espec i ely) inanced by Xun a de
Galicia.
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.chemosphe e.2021.132758.
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