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In e na ional Jou nal o En i onmen al Resea ch (2021) 15:723–731
h ps://doi.o g/10.1007/s41742-021-00351-z
RESEARCH PAPER
Use o Slaugh e house Sludge in heBio emedia ion
o anOxy luo en‑Pollu ed Soil
PalomaÁ ila‑Pozo1· JuanPa ado2· PabloCaballe o2· Ma aDíaz‑López3· FelipeBas ida3· ManuelTejada1
Recei ed: 5 Janua y 2021 / Re ised: 10 May 2021 / Accep ed: 17 June 2021 / Published online: 25 June 2021
© The Au ho (s) 2021, co ec ed publica ion 2021
Abs ac
The use o o ganic ma e is a highly accep ed en i onmen al p ac ice among scien is s o he bio emedia ion o pollu ed
soils. In his manusc ip we s udy unde labo a o y condi ions he bio emedia ion capaci y o a new bios imulan ob ained
om slaugh e house sludge in a soil pollu ed by he oxy luo en a a a e o 4l ha−1 (manu ac u e ’s a e ecommended)
o e a 90-day pe iod. We de e mined i s e ec s on dehyd ogenase, u ease, β-glucosidase and phospha ase ac i i ies, he soil
mic obial communi y s uc u e and he e olu ion o he he bicide in soil. Possibly due o he high con en o low molecula
weigh p o eins in he bios imulan , he enzyma ic ac i i ies we e s imula ed mainly a he beginning o he expe imen . Soil
biological pa ame e s we e inhibi ed in oxy luo en-pollu ed soil. A he end o he expe imen and compa ed wi h he con ol
soil, dehyd ogenase, u ease, β-glucosidase, and phospha ase ac i i ies signi ican ly dec eased by 47.8%, 50.5%, 36.4%, and
45.5% in he oxy luo en-pollu ed soil. A 5days in o he expe imen , he use o he bios imulan in oxy luo en-pollu ed soils
dec eased soil enzyma ic ac i i ies and mic obial communi y inhibi ion. A he end o he incuba ion pe iod he oxy luo en
concen a ion had dec eased by 60% in he pollu ed soil and amended wi h bios imulan s. These esul s sugges ed ha he
use o his bios imulan wi h highe amoun s o low molecula weigh p o eins and pep ides had a posi i e e ec on he
emedia ing oxy luo en-pollu ed soils. The e o e, his s udy p o ides he use o a new bios imulan ob ained om slaugh-
e house sludge by enzyma ic hyd olysis p ocesses used in he bio emedia ion o a soil pollu ed by he oxy luo en he bicide.
A icle Highligh s
• Oxy uo en he bicide caused a nega i e e ec on soil biological p ope ies
• The applica ion o bios imulan s ob ained by enzyma ic hyd olysis om slaugh e house sludge dec eased he oxic
ac ion o oxy luo en
• The low molecula weigh p o ein o bios imulan s inc eased he deg ada ion o he bicide
The o iginal online e sion o his a icle was e ised due o add
unding no e.
* Manuel Tejada
[email p o ec ed]
1 G upo de In es igación Eda ología Ambien al,
Depa amen o de C is alog a ía, Mine alogía y Química
Ag ícola, E.T.S.I.A. Uni e sidad de Se illa, C a de U e a
km. 1, 41013Se ille, Spain
2 Depa amen o de Bioquímica y Biología Molecula ,
Facul ad de Fa macia, Uni e sidad de Se illa, C/P o . Ga cía
González 2, 41012Se ille, Spain
3 CEBAS-CSIC, Campus Uni e si a io de Espina do,
30100Mu cia, Spain
724 In e na ional Jou nal o En i onmen al Resea ch (2021) 15:723–731
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G aphic abs ac
Keywo ds Slaugh e house sludge· Bios imulan · Soil oxy luo en pollu ion· Soil biological p ope ies
In oduc ion
In he las decades he e has been a no able inc ease in he
amoun o sludge esul ing om slaugh e house was ewa e
ea men , a consequence o he inc ease in mea p oduc ion.
Bouwman e al. (2013) sugges ha by 2050 mea p oduc-
ion will ha e doubled. Consequen ly, i is e y likely ha
he inc ease in he numbe o slaugh e house acili ies o
lead o an inc ease in he olume o slaugh e house sludge
(Bus illo-Lecomp e and Meh a 2015).
Slaugh e house was ewa e has been classi ied by he
Uni ed S a es En i onmen al P o ec ion Agency as was e-
wa e ha m ul o he en i onmen (Al onso-Muniozgu en
e al. 2021) mainly due o i s high con en o o ganic ma e ,
suspended solids, oil and a s and nu ien s (Aziz e al. 2019;
Menegassi e al. 2020).
725In e na ional Jou nal o En i onmen al Resea ch (2021) 15:723–731
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Cu en ly, one o he mos widely used bio emedia ion
echniques among he scien i ic communi y is he applica-
ion o o ganic ma e o pollu ed soil, because i is a e y
cheap, e ec i e echnique ha can be pe o med insi u
(Da in e al. 2018). In hese cases, o ganic ma e educes
he concen a ion o con aminan in he soil ia wo di e -
en pa hways. Fi s ly, his o ganic ma e has he capaci y o
adso b he oxins, dec easing hei concen a ion in he soil
solu ion and consequen ly educing hei oxici y. Second,
he addi ion o his o ganic ma e s imula es he g ow h o
hose mic oo ganisms ha a e ole an o he oxins p esen s
in he soil. In doing so, he mic oo ganisms inc ease he
a e o pollu an deg ada ion and so dec ease hei soil con-
cen a ion (Gómez e al. 2014; Lipczynska-Kochany 2018;
O iz-Bo ella e al. 2021). Howe e , o his o ganic ma e
o be able o s imula e hese soil mic oo ganisms, he e is
a delay, while i is mine alized in o simple o ms ha a e
mo e easily a ailable o he said mic oo ganisms (O iz-
Bo ella e al. 2021).
Using his slaugh e house sludge o manu ac u e new
edaphic bios imulan (BS) ob ained by enzyma ic hyd olysis
p ocesses would, he e o e, ha e impo an socioeconomic
and en i onmen al impac s. On he one hand, he e would be
a solu ion o he accumula ion o his o ganic was e and, on
he o he , he sludges could be o g ea use o bio emedia -
ing pes icide-pollu ed soils.
This s udy was p omp ed o ob ain a new BS ob ained
om slaugh e house sludge o bio emedia e a soil con ami-
na ed wi h he oxy luo en he bicide, which exe s a pe sis-
en oxic e ec on soil biological p ope ies (Gómez e al.
2014; Rod íguez-Mo gado e al. 2014; F anco-And eu e al.
2016; Campos e al. 2019). Ou hypo hesis is suppo ed by
he exis ence o o he BS ob ained by enzyma ic hyd olysis
p ocess om di e en o ganic was es such as sewage sludge,
chicken ea he s, oka a, ice b an, e c. used in he bio e-
media ion o soils pollu ed by a ious pes icides (Gómez
e al. 2014; Tejada e al. 2010, 2011a, b, 2014; Rod íguez-
Mo gado e al. 2014, 2015a; O s e al. 2017). These BS a e
cha ac e ized by p esen ing a high numbe o low molecula
weigh pep ides, amino acids, e c. ha a e easily abso bed
by soil mic oo ganisms, hus accele a ing he deg ada ion o
he pollu an in he con amina ed soil.
The e is an abundan bibliog aphy ha sugges s unde -
s anding he beha iou o any xenobio ic in soil equi es he
s udy o biological pa ame e s (Campos e al. 2019; Wołejko
e al. 2020). This is because hese biological pa ame e s
eac much as e han physical and chemical ones (Kadian
e al. 2012; F anco-And eu e al. 2016; O s e al. 2017).
The e o e, he s udy o soil enzyma ic ac i i ies could be
e y use ul o unde s and he e ec o slaugh e house sludge
on he bio emedia ion o oxy luo en-pollu ed soils.
Wi hin he ame wo k o his hypo hesis, he aims we e:
(1) o ob ain a new bios imulan c ea ed om slaugh e house
sludge ia enzyma ic hyd olysis p ocesses, and (2) o s udy
he e ec i eness o slaugh e house sludge as a BS in he
bio emedia ion o a soil pollu ed by oxy luo en, p incipally
s udying i s epe cussion on he a ge soil’s biochemical
p ope ies.
Ma e ial andMe hods
Cha ac e is ics o O ganic Was es, Soil andHe bicide
The slaugh e house sludge was supplied by he “Ma ade o
del Su ” company, loca ed in Sal e as (Se ille, Spain). This
sludge was subjec ed o wo di e en ea men s o ob ain
wo di e en o ganic p oduc s.
The i s o he ea men s consis ed o concen a ing he
slaugh e house sludge un il eaching a d y ma e alue o
15%, since a his alue, an easily manageable p oduc is
ob ained. This concen a ion was ca ied ou a 75°C wi h
a o a y e apo a o . This i s o ganic compound is called
concen a ed slaugh e house sludge (SS).
The second o ganic compound was ob ained by subjec -
ing he slaugh e house sludge o an enzyma ic hyd olysis
p ocess. The hyd olysis p ocess was pe o med in a bio e-
ac o acco ding o he pH–s a me hodology (Adle -Nissen
1977). Figu e1 shows he condi ions unde which his bio-
chemical p ocess was pe o med. Once he soluble p oduc
was ob ained, i was also concen a ed o 15% a 75°C wi h
a o a y e apo a o . This second o ganic compound is called
BS ob ained om slaugh e house sludge.
Table1 shows he chemical composi ion o bo h o ganic
compounds. The me hodology measu ing each chemical
pa ame e is de ailed in Rod íguez-Mo gado e al. (2015b).
The expe imen al soil used was a Calca ic Regosol (FAO
1989). The soil cha ac e is ics a e shown in Table1. The
analy ical me hods used in de e mining hese soil pa ame e s
a e de ailed in Tejada e al. (2014).
Oxy luo en was used as expe imen al he bicide a a e
o 4l ha−1 ( ecommended applica ion a e). The comme cial
o mula ion Fen en (24% p −1, 240g l−1) was pu chased
om Lainco, S.A (Spain).
Expe imen al Design
Th ee hund ed g ams o d ied soil was mixed wi h oxy luo -
en and ea ed wi h SS a a andom a e o 3% (9g o p od-
uc ) and BS a a a e o 3.9% (11.7g o p oduc ) o apply he
same amoun o o ganic ma e o he soil (5.84g o ganic
ma e pe kg o soil). A non-amended and non-pollu ed soil
was used as con ol. The incuba ion ea men s we e de ailed
in Table2.
Bo h o ganic compounds we e liquid and we e solubi-
lized in dis illed wa e be o e applying.
726 In e na ional Jou nal o En i onmen al Resea ch (2021) 15:723–731
1 3
T iplica e ea men s we e kep in mic ocosms a
25 ± 1°C o 90days. Dis illed wa e was added o each
soil o each 30–40% o i s wa e -holding capaci y and he
mois u e losses we e compensa ed by adding dis illed wa e .
Soil Analysis
Dehyd ogenase, u ease, β-glucosidase and phospha ase
ac i i ies o each ea men we e de e mined in iplica e a
days 5, 10, 20, 35, 55 and 90 using he me hods desc ibed
by Ga cía e al. (1994), Kandele and Ge be (1988), Ei azi
and Taba abai (1988) and Taba abai and B emne (1969).
Fo each expe imen al ea men , mic obial a y acids
we e de e mined in iplica e a days 5 and 90 o he expe i-
men . Es e -linked mic obial a y acids we e ex ac ed
and quan i ied acco ding o he me hodology desc ibed
by Mon es de Oca-Vásques e al. (2020). The a y acids
i15:0, a15:0, i16:0, and i17:0 we e ep esen a i e o he
G am + (G +) bac e ial biomass, whe eas he a y acids
18:1ω c, 18:1ω9 , cy17:0, and cy19:0 we e ep esen a i e
o he G am- (G-) bac e ial biomass (Ba dge e al. 1996;
Dungai e al. 2010). The a y acid 18:2ω6 we e ep esen a-
i e o he ungal biomass (Zelles e al. 1992; Båå h 2003).
A days 5, 10, 20, 35, 55 and 90, he soil oxy luo en
con en was de e mined. Soil oxy luo en was ex ac ed
acco ding o he Anas assiades e al. (2003) me hod. The
me hodology measu ing he he bicide is desc ibed in
Rod íguez-Mo gado e al. (2014). The limi o de ec ion
(LOD) was 0.006mg kg−1 and he limi o quan i ica ion
(LOQ) 0.01mg kg−1.
Fig. 1 Enzyma ic hyd olysis p ocess used o ob aining bios imulan
om slaugh e house sludge
Table 1 Chemical cha ac e is ics and p o ein molecula weigh dis-
ibu ion (mean ± s anda d e o , n = 3) o soil, slaugh e house sludge
and bios imulan ob ained by slaugh e house sludge by enzyma ic
hyd olysis p ocess
Files ollowed by he same le e (s) a e no signi ican ly di e en
acco ding o he Tukey es (p < 0.05)
SS slaugh e house sludge, BS bios imulan ob ained by slaugh e -
house sludge
Pa ame e s Soil SS BS
pH (H2O) 7.6 ± 0.2
Sand (g kg−1)554 ± 28
Sil (g kg−1)134 ± 21
Clay (g kg−1)312 ± 31
D y ma e (%) 15.2a ± 2.1 15.8a ± 1.7
O ganic ma e (g kg−1)18.3b ± 1.8 789a ± 37 649a ± 22
N (g kg−1)0.9b ± 0.2 3.5a ± 1.4 3.0a ± 1.1
P (g kg−1)5.6a ± 2.1 5.4a ± 1.8
K (g kg−1)9.1a ± 2.6 8.9a ± 2.7
S (g kg−1)12.8a ± 3.3 11.7a ± 2.1
Ca (g kg−1)29.4b ± 5.8 15.3a ± 1.6
Mg (g kg−1)1.6a ± 0.4 1.5a ± 0.7
Fe (g kg−1)8.4b ± 1.7 3.0a ± 0.9
Cu (mg kg−1)139a ± 28 85.7a ± 14.3
Mn (mg kg−1)148b ± 14 44.3a ± 13.5
Zn (mg kg−1)510b ± 21 333a ± 18
Pb (mg kg−1)10.6b ± 2.3 5.7a ± 2.0
Ni (g kg−1)10.4a ± 1.7 7.8a ± 1.1
P o ein molecula weigh dis ibu ion (Da)
 > 10,000 77.4b ± 3.4 50.0a ± 2.7
10,000–5000 3.4a ± 1.6 4.8a ± 1.7
5000–3000 2.1a ± 0.5 2.9a ± 1.1
3000–1000 3.8a ± 1.2 5.9a ± 1.3
1000–300 3.3a ± 1.7 6.8a ± 1.6
 < 300 10.0a ± 1.9 29.6b ± 2.4
Table 2 Scheme o he incuba ion ea men s pe o med
(1) C, con ol soil, non-amended and wi hou he bicide
(2) C + SS, soil amended wi h SS and wi hou he bicide
(3) C + BS, soil amended wi h BS and wi hou he bicide
(4) C + Ox, soil non-o ganically amended and wi h he bicide
(5) C + Ox + SS, soil amended wi h SS and wi h he bicide
(6) C + Ox + BS, soil amended wi h BS and wi h he bicide
727In e na ional Jou nal o En i onmen al Resea ch (2021) 15:723–731
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S a is ical Analysis
To de e mine he di e ences be ween he esul s ob ained
o each pa ame e analysed, he da a we e submi ed o
wo-way analysis o a iance (ANOVA) using wo ac o s
( ea men s and sampling ime). This was hen ollowed by
he Tukey pos -hoc es (HSD, p < 0.05). The ANOVA was
pe o med using he S a g aphics Plus 2.1 so wa e package.
Fo he s a is ical analysis, iplica e da a we e used o each
ea men and each day o incuba ion.
Resul s andDiscussion
A e he enzyma ic hyd olysis p ocess and wi h espec o
he SS, he BS showed a signi ican (p < 0.05) dec ease in
he Ca and mic onu ien s concen a ion (Table1). I was
also obse ed ha signi ican changes occu ed in he p o ein
molecula weigh dis ibu ions. In his espec , and compa ed
wi h he SS, he p o eins wi h he highes molecula weigh
(> 1000 KDa) in he new BS dec eased by 35.4%. Also,
and wi h espec o he SS, he 1000–300Da and < 300Da
molecula weigh p o eins in he new BS inc eased signi i-
can ly by 51.5% and 66.2%.
The applica ion o SS and BS signi ican ly (p < 0.05)
s imula ed he soil enzyma ic ac i i ies s udied (Tables3 and
4). Compa ed wi h he C ea men , he applica ion o SS o
he soil p og essi ely inc eased he dehyd ogenase, u ease,
β-glucosidase and phospha ase ac i i ies by 65.7%, 70.3%,
78.8% and 68%, espec i ely. Howe e , he beha io o he
enzyma ic ac i i ies s udied a e applying he BS o he
soils was e y di e en . Dehyd ogenase, β-glucosidase and
phospha ase ac i i ies eached maximum s imula ion 5days
a e he s a o he expe imen , hen g adually dec easing
un il 35days a e he expe imen . F om his da e, he dehy-
d ogenase, β-glucosidase and phospha ase ac i i y began
o inc ease again un il he end o he incuba ion pe iod,
eaching a highe alue han ha ob ained in he con ol
ea men . In his sense, he s imula ion o dehyd ogenase,
β-glucosidase and phospha ase ac i i ies was 54%, 68.6%
and 59.8.
Compa ed wi h he C + SS ea men , a e he appli-
ca ion o he BS o he soil, he beha io o he u ease
ac i i y was e y simila h oughou he incuba ion pe iod
(Table3).
We hink ha he cause o he di e en beha iou in he
wo expe imen al o ganic was es can be due o he di e -
ence in he molecula p o ein weigh dis ibu ion pe cen -
age. Ou esul s highligh ha in bo h o ganic was es he
p o ein size dis ibu ion di e s mainly in sizes o > 1000Da
and < 300Da. The SS has a highe pe cen age o high molec-
ula weigh p o eins, while BS showed a highe pe cen age
o low molecula weigh p o eins. Since he mic oo gan-
isms in he soil canno di ec ly abso b he high molecula
weigh p o eins, o ob ain ene gy hese mic oo ganisms need
o deg ade hem. This mine alisa ion p ocess is usually slow,
and he e o e, mic obial s imula ion in he SS-amended soil
inc eased p og essi ely h oughou he expe imen al pe iod.
Acco ding o Rod íguez-Mo gado e al. (2015b), when
applying BS o he soil, mic oo ganisms di ec ly abso b
hese low molecula weigh p o eins, a ou ing hei s imu-
la ion. The subsequen dec ease in hei enzyma ic ac i i ies
o alues simila o he con ol a day 35 sugges ha by
hen soil mic oo ganisms had al eady consumed hese low
Table 3 E olu ion o
dehyd ogenase and u ease
ac i i ies (mean ± s anda d
e o , n = 3) in soils amended
wi h slaugh e house sludge
(SS) and hyd olysa e
slaugh e house sludge (BS) and
wi h oxy luo en du ing he
expe imen al pe iod
Columns ollowed by he same le e (s) a e no signi ican ly di e en (p > 0.05)
INTF 2-p-iodo-3-ni ophenyl o mazan
Incuba ion days
5 10 20 35 55 90
Dehyd ogenase ac i i y (µg INTF g−1 h−1)
C 2.7b ± 0.6 2.8b ± 0.4 2.5b ± 0.3 2.3b ± 0.2 2.4b ± 0.3 2.3b ± 0.3
C + SS 4.1c ± 0.9 4.9c ± 1.0 5.1c ± 1.2 5.4c ± 1.1 6.1c ± 1.4 6.7c ± 1.5
C + BS 15.2d ± 2.6 6.8c ± 1.7 5.7c ± 0.8 4.2c ± 0.5 4.5c ± 0.8 5.0c ± 1.1
C + Ox 1.3a ± 0.2 1.4a ± 0.3 1.1a ± 0.1 1.4a ± 0.3 1.5a ± 0.2 1.2a ± 0.3
C + Ox + SS 2.7b ± 0.5 3.3b ± 0.8 4.4c ± 1.0 4.7c ± 0.8 4.9c ± 1.1 5.4c ± 0.8
C + Ox + BS 10.3d ± 1.9 5.5c ± 0.7 4.0c ± 0.7 3.7bc ± 0.6 3.7bc ± 0.7 4.1c ± 1.0
U ease ac i i y (µg NH4+ g−1 h−1)
C 1.7b ± 0.2 1.7b ± 0.2 1.8b ± 0.1 1.7b ± 0.2 1.8b ± 0.3 1.9b ± 0.3
C + SS 2.7b ± 0.9 3.2bc ± 0.6 3.7c ± 0.8 4.8c ± 1.0 5.2c ± 0.9 6.4c ± 1.7
C + BS 2.9b ± 1.1 3.4bc ± 0.8 4.0c ± 1.0 4.7c ± 0.8 5.8c ± 0.6 6.9c ± 0.8
C + Ox 0.94a ± 0.13 0.98a ± 0.12 0.91a ± 0.13 0.83a ± 0.17 0.90a ± 0.09 0.94a ± 0.11
C + Ox + SS 1.7b ± 0.6 2.2b ± 0.6 2.9b ± 0.7 3.0b ± 0.8 3.8c ± 0.6 4.5c ± 1.0
C + Ox + BS 2.2b ± 0.4 2.5b ± 0.4 2.7b ± 0.5 2.7b ± 0.6 3.1bc ± 0.8 3.4c ± 0.7

728 In e na ional Jou nal o En i onmen al Resea ch (2021) 15:723–731
1 3
molecula weigh p o eins. The p og essi e inc ease om
day 35 on he soil biochemical ac i i y is a consequence
o he ac ha he BS s ill showed a high numbe o high
molecula weigh p o eins, implying ha , as o his day,
soil mic oo ganisms began o exc e e ex acellula enzymes
o ob ain ene gy. This means ha he e was a p og essi e
inc ease in he soil biochemical ac i i y un il he end o he
incuba ion pe iod.
The soil beha iou o his new BS ob ained om slaugh-
e house sludge di e s g ea ly om ha ob ained om o he
BSs also by enzyma ic hyd olysis using he same enzyme
om sewage sludge, chicken ea he s, ice b ain, condensed
dis illa es o soluble whea , and oka a, which, a e applica-
ion, caused an inc ease in he soil biological ac i i y du -
ing he i s days o incuba ion. Subsequen ly his ac i i y
dec eased and showed a biochemical ac i i y simila o ha
o a non-pollu ed soil (Tejada e al. 2010, 2011b, 2014;
Rod íguez-Mo gado e al. 2015b; O s e al. 2017). This is a
consequence o he ac ha in his ype o BS, he amoun
o low molecula weigh p o eins (< 300Da) was g ea e
han ha ob ained wi h he BS o slaugh e house sludge.
The e o e, when he BS ob ained om he slaugh e house
sludge was applied o he soil, he mic oo ganisms quickly
assimila ed he low molecula weigh pep ides. Once hese
had been abso bed, he mic oo ganisms had o ob ain hei
ene gy om he high molecula weigh p o eins, exc e ing
ex acellula enzymes o deg ade said p o eins.
Tejada and Bení ez (2020) also obse ed ha he bio-
chemical ac i i y was highe in soils amended wi h o ganic
ma e wi h a highe pe cen age o low molecula weigh
pep ides. These au ho s also concluded ha he highe soil
mic obial s imula ion was possibly a consequence o a
highe abso p ion o low molecula weigh pep ides by he
mic oo ganisms.
Applying oxy luo en o non-amended soil caused a sig-
ni ican (p < 0.05) inhibi ion in he soil enzyma ic ac i i ies
du ing he expe imen (Tables2 and 3). A he end o he
expe imen and compa ed wi h he C ea men , dehyd oge-
nase, u ease, β-glucosidase, and phospha ase ac i i ies sig-
ni ican ly dec eased by 47.8%, 50.5%, 36.4%, and 45.5%,
espec i ely (Tables3 and 4).
Applying oxy luo en o non-pollu ed soil caused an
inhibi o y e ec on he soil biochemical ac i i y and mic o-
bial popula ion. These esul s a e in acco dance wi h Nadi-
je e al. (2013), Gómez e al. (2014), F anco-And eu e al.
(2016), Campos e al. (2019) and Wołejko e al. (2020), who
highligh ed he oxic e ec o oxy luo en on soil biochemi-
cal ac i i y.
The applica ion bo h o ganic compounds dec eased he
soil oxy luo en concen a ion (Fig.2). A he end o he
expe imen , he soil oxy luo en had dec eased 44.4% com-
pa ed o he concen a ion a day 5. Applying bo h o ganic
compounds o he pollu ed soil caused a signi ican dec ease
(p < 0.05) in he soil oxy luo en concen a ion. Compa ed
wi h he C + Ox ea men , he oxy luo en concen a ion had
dec eased by 33.3% in he C + Ox + SS ea men and by
60% in he C + Ox + BS ea men .
Gómez e al. (2014) and Rod íguez-Mo gado e al.
(2014) obse ed a signi ican dec ease in he concen a-
ion o oxy luo en a e he applica ion o a ious o ganic
compounds o con amina ed soil. These au ho s obse ed
ha his dec ease was g ea e in soils amended wi h o ganic
ma e wi h a high con en o low molecula weigh p o-
eins, possibly due o he ac ha oxy luo en- ole an soil
Table 4 E olu ion o
β-glucosidase and phospha ase
ac i i ies (mean ± s anda d
e o , n = 3) in soils amended
wi h slaugh e house sludge
(SS) and hyd olyza e
slaugh e house sludge (BS) and
wi h oxy luo en du ing he
expe imen al pe iod
Columns ollowed by he same le e (s) a e no signi ican ly di e en (p > 0.05)
PNP p-ni ophenol
Incuba ion days
5 10 20 35 55 90
β-glucosidase ac i i y (mmol PNP g−1 h−1)
C 1.4b ± 0.2 1.3b ± 0.2 1.3b ± 0.1 1.3b ± 0.2 1.2b ± 0.3 1.1b ± 0.1
C + SS 2.7c ± 0.8 2.9c ± 0.9 3.2c ± 1.2 3.8c ± 0.8 4.4cd ± 1.0 5.2d ± 1.2
C + BS 8.4d ± 1.8 5.9c ± 1.1 3.1c ± 0.6 2.4c ± 0.4 3.0c ± 0.5 3.5c ± 0.8
C + Ox 0.72a ± 0.09 0.79a ± 0.11 0.75a ± 0.08 0.76a ± 0.08 0.74a ± 0.12 0.70a ± 0.14
C + Ox + SS 2.3c ± 0.8 2.2c ± 0.4 2.5c ± 0.2 2.6c ± 0.7 2.9c ± 0.3 3.1c ± 0.8
C + Ox + BS 5.8c ± 0.4 4.1c ± 1.3 2.7c ± 0.3 1.7b ± 0.3 1.9bc ± 0.7 2.1c ± 0.5
Phospha ase ac i i y (µmol PNP g−1 h−1)
C 3.0b ± 1.0 3.3b ± 0.9 3.4b ± 1.2 3.4b ± 1.2 3.2b ± 1.1 3.3b ± 0.9
C + SS 4.9b ± 1.4 5.4b ± 1.7 6.2c ± 1.0 7.5c ± 1.8 8.6c ± 1.4 10.3d ± 2.1
C + BS 20.4d ± 3.1 11.5d ± 2.2 12.7d ± 1.4 7.2c ± 1.8 7.8c ± 1.1 8.2c ± 1.5
C + Ox 1.7a ± 0.4 1.9a ± 0.3 1.8a ± 0.4 1.9a ± 0.2 1.8a ± 0.3 1.8a ± 0.3
C + Ox + SS 4.4b ± 1.1 4.7b ± 1.5 5.2b ± 1.3 6.1c ± 1.0 6.8c ± 1.2 7.2c ± 1.8
C + Ox + BS 15.8d ± 2.0 10.4d ± 2.4 6.4c ± 1.8 4.2b ± 1.4 4.2b ± 1.2 4.6b ± 1.0
729In e na ional Jou nal o En i onmen al Resea ch (2021) 15:723–731
1 3
mic oo ganisms abso b hese pep ides mo e easily, a o ing
hei p oli e a ion in he soil and inc easing he deg ada ion
o said he bicide in soil.
This dec ease in he soil oxy luo en concen a ion caused
a dec ease in he enzyma ic ac i i ies inhibi ion shown in he
C + Ox ea men (Tables3 and 4). Also, he chemical com-
posi ion o bo h o ganic was es in luenced his ac ion di e -
en ly. Fo example, a he end o he expe imen al pe iod and
compa ed wi h he C + Ox ea men , dehyd ogenase ac i i y
had inc eased by 87.7% and 70.7% in he C + Ox + SS and
C + Ox + BS ea men s. U ease ac i i y inc eased by 79.1%
and 72.3% in he C + Ox + SS and C + Ox + BS ea men s.
β-glucosidase ac i i y had inc eased by 77.4% and 66.7% in
he C + Ox + SS and C + Ox + BS ea men s, espec i ely.
Phospha ase ac i i y inc eased by 75% and 60.9% in he
C + Ox + SS and C + Ox + BS ea men s.
The oxy luo en deg ada ion was highe in he BS-
amended soil han wi h SS. The highe pe cen age o low
molecula weigh p o eins (< 300Da) in he BS enabled
he he bicide- ole an mic oo ganisms o he said pep ides
mo e quickly. This s imula ed he mic obial popula ion and
consequen ly led o a g ea e deg ada ion o he he bicide.
The high pe cen age o high molecula weigh p o ein in
SS esul s in a lowe s imula ion in he he bicide- ole an
mic obial popula ion. Consequen ly, o e ime oxy luo en
deg ada ion is slowe han when SS is applied.
Applying he he bicide o non-o ganic amended soil
did no change he mic oo ganisms popula ion du ing he
expe imen al pe iod (Table5). Simila esul s we e ound
by Rod íguez-Mo gado e al. (2014) and Gómez e al.
(2014) in a soil wi h simila physicochemical cha ac e is-
ics and con amina ed wi h he same dose o oxy luo en
du ing a pe iod o 120days.
The applica ion o o ganic ma e o he soil caused an
inc ease in o al bac e ial PLFA and o al ungal PLFA
popula ion. Tian e al. (2017) and Zheng e al. (2021) also
Fig. 2 E olu ion o oxy luo en
(mean ± s anda d e o , n = 3)
in soils du ing he expe imen al
pe iod. Columns ollowed by
he same le e (s) a e no signi i-
can ly di e en (p > 0.05)
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
51020355
59
0
gkgm(ne oul yxO -1)
C+Ox C+Ox+SSC+Ox+BS
aa
aa
a
a
a
a
aa
ab b
ab
ab bbb
c
Table 5 E olu ion o bac e ial
G am+, bac e ial G am−,
o al bac e ial and ungal
PLFAs (nmol g−1), du ing he
expe imen al pe iod
Da a a e he means o h ee samples. Columns (mean ± S.E.) ollowed by he same le e (s) a e no signi i-
can ly di e en (p > 0.05)
T ea men Incuba ion days bac e ial G am+bac e ial G am−To al bac e ial PLFA Fungal PLFA
C 5days 28.1a ± 2.4 18.6a ± 1.8 47.7a ± 3.9 3.1a ± 1.1
90days 28.9a ± 2.0 19.5a ± 2.2 48.4a ± 4.4 2.8a ± 0.9
C + SS 5days 31.4a ± 2.5 24.4a ± 2.0 55.8a ± 4.3 3.8a ± 1.1
90days 49.9b ± 3.1 50.2b ± 2.7 100.1b ± 5.5 5.2b ± 1.2
C + BS 5days 50.2b ± 2.9 30.6ab ± 3.1 80.8b ± 6.3 3.7a ± 1.2
90days 50.0b ± 3.6 38.9b ± 2.2 88.9b ± 5.6 4.0a ± 1.7
C + Ox 5days 29.9a ± 1.3 23.5a ± 1.6 53.4a ± 2.7 3.6a ± 1.1
90days 27.3a ± 2.6 22.9a ± 1.2 50.2a ± 3.6 3.2a ± 0.8
C + Ox + SS 5days 29.8a ± 2.9 25.7a ± 2.6 55.5a ± 5.6 4.0a ± 1.7
90days 47.0b ± 1.6 49.1bc ± 3.3 96.4b ± 4.7 4.8ab ± 1.5
C + Ox + BS 5days 49.3b ± 2.7 30.4ab ± 2.4 79.7ab ± 4.8 4.0a ± 1.0
90days 46.7b ± 2.9 33.8b ± 1.7 80.5b ± 4.5 3.8a ± 1.2
730 In e na ional Jou nal o En i onmen al Resea ch (2021) 15:723–731
1 3
ound an inc ease in mic obial biodi e si y in soils wi h
a high con en o o ganic ma e . These au ho s sugges
ha he applica ion o o ganic ma e o he soil p o ides
subs a es ha can inc ease he o al abundance o PLFA.
When oxy luo en was applied o soils amended wi h
bo h o ganic compounds, he bac e ia and ungi popula-
ion was no di e en om ha obse ed o C + SS and
C + BS ea men s. This ac is possibly due o he ac ha
oxy luo en did no change he mic obial biodi e si y o
he soil, as p e iously men ioned.
Conclusions
Acco ding o he au ho s' knowledge, he main no el y o his
manusc ip is he i s s udy in which bio emedia ion es s ha e
been ca ied ou on soils pollu ed by he oxy luo en he bicide
using slaugh e house sludge, bo h concen a ed and in he o m
o bios imulan s a e subjec ing said sludge concen a ed o
an enzyma ic hyd olysis p ocess. The esul s ob ained in his
expe imen indica ed ha slaugh e house sludge dec eased
he inhibi o y e ec o he oxy luo en on he soil’s biological
p ope ies. Consequen ly, slaugh e house sludge could be con-
side ed as being e y use ul in bio emedying oxy luo en-pol-
lu ed soils. Howe e , he g ea es bio emedia ion e ec s we e
ob ained when he slaugh e house sludge was ans o med in o
a bios imulan by enzyma ic p ocesses ich in low molecula
weigh p o eins. These p o eins a e easily assimila ed by oxic-
ole an soil mic oo ganisms which accele a ed he deg ada-
ion o he he bicide in soil.
Howe e , he bio emedia ion e ec o slaugh e house
sludge should be s udied u he . The ype o con aminan
in he soil, as well he soil p ope ies, a e cha ac e is ics
o be conside ed o u u e s udies o be e unde s and he
e ec o hese o ganic was es on he bio emedia ion o pol-
lu ed soils.
Acknowledgemen s This wo k was suppo ed by he Minis e io de
Ciencia, Inno ación y Uni e sidades (Spain), Plan Nacional I+D+I
wi h e e ence RTI2018-097425-B-100, Jun a de Andalucia (Conse-
je ía de Economía y Educación), P oyec os I+D+i FEDER Andalucía
2014-2020, wi h e e ence US-1263885 and Comisión Eu opea, Ho i-
zon e 2020 wi h e e ence 3052/0048.
Funding Open Access unding p o ided hanks o he CRUE-CSIC
ag eemen wi h Sp inge Na u e.
Decla a ions
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