Use of Slaughterhouse Sludge in the Bioremediation of an Oxyfluorfen-Polluted Soil

Vol.:(0123456789) 1 3 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 1 3 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 1 3 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 1 3 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 Con lic o in e es The au ho s decla e ha hey ha e no con lic o in e es . Open Access This a icle is licensed unde a C ea i e Commons A i- bu ion 4.0 In e na ional License, which pe mi s use, sha ing, adap a- ion, dis ibu ion and ep oduc ion in any medium o o ma , as long as you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons licence, and indica e i changes we e made. 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