1
Con as ing en i onmen al impac s o nano-biocha and
1
con en ional biocha on a ious o ganisms
2
Monika Raczkiewicz1, Aleksand a Bogusz2, Bo Pan3, Baoshan Xing4, Pa yk Oleszczuk1*
3
4
1Depa men o Radiochemis y and En i onmen al Chemis y, Facul y o Chemis y, 3 Ma ia
5
Cu ie-Skłodowska Squa e, 20-031 Lublin, Poland
6
2Depa men o Eco oxicology, Ins i u e o En i onmen al P o ec ion - Na ional Resea ch
7
Ins i u e, Ks. T oszynskiego S . 9, Wa saw 01-693, Poland
8
3Facul y o En i onmen al Science and Enginee ing, Kunming Uni e si y o Science and
9
Technology, Kunming, 650500, People's Republic o China
10
4 S ockb idge School o Ag icul u e, Uni e si y o Massachuse s, Amhe s , MA 01003, Uni ed
11
S a es
12
13
Co esponding au ho : Pa yk Oleszczuk, [email p o ec ed]
14
2
Abs ac
15
16
The en i onmen al haza ds o nanobiocha (n-BC) equi e a en ion due o limi ed
17
knowledge. This s udy is he i s o explo e he e ec s o biocha size educ ion ac oss a ious
18
o ganisms, including bac e ia (Alli ib io ische i), plan s (Lemna mino , Lepidium sa i um),
19
and in e eb a es (Daphnia magna, Folsomia candida). Bulk biocha (b-BC) and n-BC we e
20
applied in bo h liquid and solid-phase es s o assess i s eco oxici y. The esul ing leacha es
21
we e es ed a concen a ions o 2, 10, and 100 mg/L o o ganisms like Daphnia magna and
22
Lemna mino . In he solid-phase es s, b-BC and n-BC we e added o s anda d soil a
23
concen a ions o 1% and 5% o e alua e oxici y in Folsomia candida and a concen a ions o
24
1% o e alua e oxici y in Lepidium sa i um.We ound n-BC o be signi ican ly mo e oxic (by
25
18% o 2886%) o A. ische i han bulk biocha (b-BC), wi h oxici y inc easing o e ime. Low
26
doses (1%) o bo h b-BC and n-BC did no cause mo ali y o inhibi ep oduc ion in F. candida,
27
hough b-BC enhanced ep oduc ion (by 30% o 56%) compa ed o n-BC. A a 5% dose, bo h
28
b-BC and n-BC inhibi ed ep oduc ion F. candida, wi h n-BC being 0.5 o 1.8 imes mo e oxic.
29
Nei he b-BC no n-BC immobilized D. magna, bu bo h inhibi ed ep oduc ion (by 28% o
30
35%). The nanoscale dimensions o n-BC acili a ed bioaccumula ion in D. magna, leading o
31
adhesion on he o ganism’s body. The n-BC had a g ea e impac on plan s, bo h b-BC and n-
32
BC we e non- oxic o L. mino , bu all n-BC inhibi ed oo g ow h in L. sa i um. These indings
33
highligh he impo ance o conside ing biocha size, eeds ock, and py olysis condi ions when
34
e alua ing en i onmen al isks, ensu ing sa e use in sus ainable ag icul u e. Fu u e di ec ions
35
o his s udy should ocus on explo ing he long- e m en i onmen al impac s o n-BC,
36
including i s pe sis ence and ans o ma ion in a ious ecosys ems. Fu he esea ch is needed
37
o elucida e he mechanisms d i ing i s inc eased oxici y and bioaccumula ion ac oss di e en
38
o ganisms. Addi ionally, s udies should e alua e he e ec s o a ying eeds ocks, py olysis
39
3
condi ions, and unc ionaliza ion s a egies o op imize n-BC's en i onmen al sa e y and
40
e icacy in sus ainable ag icul u e.
41
Keywo ds: oxici y; leacha es; sewage sludge; ag icul u e esidues; biomass; pa icle size;
42
nanoscale
43
4
1. INTRODUCTION
44
As global en i onmen al challenges con inue o g ow, nano echnology eme ges as a
45
pi o al ool o de eloping inno a i e solu ions o pollu ion in wa e and soil. Among hese
46
ad ancemen s, nanobiocha s ands ou as a p omising ma e ial due o i s ema kable abili y o
47
adso b bo h o ganic and ino ganic con aminan s. Nanobiocha (n-BC) ep esen s an inno a i e
48
o m o well-known ca bonaceous ma e ial called biocha (BC) ob ained h ough py olysis. The
49
n-BC, de ined as BC wi h pa icle sizes anging om 1 o 100 nm (Rashid e al., 2023) is
50
cu en ly a he o e on o esea ch. The cu en app oaches o n-BC p oduc ion and u iliza ion
51
include en i onmen ally iendly syn hesis me hods and highligh ing ene gy-e icien
52
echniques acco ding o he di ec ions o g een chemis y in nano echnology (Lyu e al., 2018).
53
Among a ious syn hesis n-BCs me hods, such as sonica ion (Oleszczuk e al., 2016) o
54
con en ional g inding ollowed by sie ing he aqueous suspension o n-BC (Li e al., 2017),
55
ecen ly ball milling s ands ou as he mos commonly employed echnique (Naghdi e al.,
56
2017).
57
The inc eased in e es in n-BC can be a ibu ed o n-BC’s unique p ope ies de e mined
58
by pa icle size like highe speci ic su ace a ea (Huang e al., 2020), educed hyd odynamic
59
adius, mo e nega i e ze a po en ial (Song e al., 2019), inc eased p esence o oxygen (O)-
60
con aining unc ional g oups, and he occu ence o ca bon de ec s compa ed o p is ine
61
( egula ) BC(Webe and Quicke , 2018). These dis inc i e p ope ies po en ially a ec n-BC
62
adso p ion capaci y o ace me als (TMs) and o ganic con aminan s (e.g., polycyclic a oma ic
63
hyd oca bons - PAHs) (Ramanayaka e al., 2020), which can be used o was ewa e ea men
64
and immobiliza ion o con aminan s. Howe e , by changing he men ioned p ope ies du ing
65
ball milling, he s eng h o in e ac ion be ween hyd ophobic o ganic con aminan s and BC may
66
dec ease, making p e iously inaccessible PAHs, mo e a ailable and mobile (Wang e al., 2017).
67
Releasing hese oxic subs ances in o he en i onmen and inducing ha m ul e ec s on he
68
5
ecosys em. A he same ime, mo e unc ional g oups c ea ed du ing ball milling may inc ease
69
he a ini y (Liu e al., 2022) o n-BC o TMs and simul aneously dec ease he TM’s mobili y.
70
Mo eo e , n-BC exhibi s a highe c i ical coagula ion concen a ion (CCC) han o he
71
ca bonaceous nanoma e ials (e.g. ca bon nano ubes o g aphene oxides) (Song e al., 2019),
72
which induces i g ea e agg ega ion o accumula ion and colloidal s abili y (Sun e al., 2018).
73
This be e agg ega ion and s abili y o n-BC han o he ca bon ma e ials may in luence he
74
oxicological cha ac e o nanopa icles. Agg ega ed o ms o nanopa icles can ha e dis inc
75
biological e ec s compa ed o indi idual nanopa icles (Sani e al., 2023). Fo example, he
76
educed bioa ailabili y o agg ega ed nanopa icles may in luence hei in e ac ions wi h
77
biological sys ems (Zhang, 2014).
78
Conside ing ha bulk biocha (b-BC) and n-BC a e p ima ily used in ag icul u e and
79
soil emedia ion (Pa hak e al., 2024; P adhan e al., 2024; Sa ma e al., 2024; Vadakkan e al.,
80
2024), i is c ucial o assess hei impac on plan s and di e en g oups o o ganisms. The
81
physicochemical cha ac e iza ion and he con aminan con en a e insu icien o e alua ing
82
he en i onmen al isks associa ed wi h n-BC (Ndoung e al., 2021; Osman e al., 2024). This
83
is due o he complex in e ac ions be ween n-BC and a ious en i onmen al ma ices, as well
84
as he dynamic na u e o con aminan elease. Fu he mo e, he po en ial o bioaccumula ion
85
in ecosys ems unde sco es he need o comp ehensi e eco oxicological s udies and long- e m
86
moni o ing o ully unde s and hei impac s. While chemical analyses s ill emain essen ial and
87
gi e an o e all look a possible beha io and indi ec and po en ial isk o n-BC,
88
eco oxicological es s can complemen in o ma ion abou he di ec isk and o e aluable
89
insigh s in o he di ec e ec s on li ing o ganisms, such as hei g ow h, beha io , ep oduc ion,
90
and o e all physiological heal h o e he long e m (Domene e al., 2015; Oleszczuk e al.,
91
2013). In his con ex , he biological assays should no be seen as a eplacemen o chemical
92
analyses bu a he as a supplemen a y ool, which ex ends ou knowledge abou po en ial
93
6
h ea s and addi ionally allowing he explana ion o in e ac ions be ween a ious con aminan s,
94
p o iding c ucial e idence in he assessmen p ocess.
95
Nume ous s udies ha e examined he e ec s o b-BC on o ganisms and plan s
96
(Godlewska e al., 2021; Kim e al., 2020; Ma cińczyk e al., 2024; Wei e al., 2024). These
97
s udies ha e e ealed ha b-BC can ha e a ying impac s on mic oo ganisms, plan s, and soil
98
in e eb a es (Kim e al., 2020; Ma cińczyk e al., 2024; Wei e al., 2024) and he e ec e y
99
o en depends on he soil ypes. Cu en ly, s udies speci ically ocused on he eco oxici y o n-
100
BCs a e ex emely limi ed, highligh ing he no el y and signi icance o ou esea ch. The e is
101
a lack o in o ma ion ega ding he in luence o n-BC depending on eeds ocks and py olysis
102
condi ions on di e en ecosys ems. I is e y c ucial o explain he e ec o he ini ial
103
pa ame e s o b-BC in he con ex o he oxicological p ope ies o n-BC. A c i ical knowledge
104
gap exis s ega ding whe he size- ela ed changes in n-BC p ope ies in luence po en ial
105
oxici y. Add essing his gap is necessa y o p edic ing n-BC's en i onmen al impac s based
106
on p is ine BC p ope ies and o he e en ual widesp ead use o n-BC.
107
The aim o his s udy was o assess he impac o n-BC on a ious g oups o o ganisms
108
like bac e ia (Ali ib io ische ii), plan s (Lemna mino , Lepidium sa i um), and in e eb a es
109
(Daphnia magna, Folsomia candida), speci ically examining hei e ec s on iabili y,
110
ep oduc ion, and g ow h. E alua ing o ganisms om a ious ophic g oups can p o ide
111
comp ehensi e in o ma ion on he po en ial isks o in oducing n-BC in o he en i onmen .
112
Unde s anding in e ac ions among o ganisms a di e en ophic le els is c ucial o p edic ing
113
n-BC's en i onmen al impac s. Analyzing hese in e ac ions may e eal changes in ecosys em
114
dynamics and po en ial cascading e ec s.
115
7
2. MATERIALS AND METHODS
116
2.1. Bulk and nano-biocha s
117
De ailed in o ma ion abou p oduc ion condi ions and physicochemical p ope ies o bulk-
118
(b-BC) and nano-biocha (n-BC) was p esen ed in ou p e ious wo k (Raczkiewicz e al.,
119
2024b). The b-BCs used in his expe imen we e ob ained om willow (WL), ice husk (RH),
120
oilseed ape (OSR), and a ious sewage sludges 1-3 (SSL 1-3) collec ed om h ee was ewa e
121
ea men plan s. The b-BCs we e p oduced a 550°C unde a ni ogen a mosphe e. The WL-
122
de i ed b-BC we e p oduced a addi ional empe a u es o 450°C, 650°C, and 750°C o
123
examine he empe a u e-dependen e ec s on b-BC p ope ies (Tomczyk e al., 2020), which
124
may in luence n-BC p ope ies and eco oxici y. The SSL-de i ed b-BC was also p oduced in a
125
ca bon dioxide a mosphe e, which may al e b-BC p ope ies and oxici y (Kończak e al., 2020)
126
A e wa d, he esul ing b-BCs we e ozen (-80°C ) and g ound using a ball milling machine
127
(PM100, Re sch Co po a ion, Ge man) o achie e BC pa icle sizes smalle han 100 nm
128
acco ding o he me hod desc ibed p e iously (Raczkiewicz e al., 2024b). The con aminan s
129
con en (PAHs and TMs,) and hei bioa ailabili y a e p esen ed in ou p e ious wo k
130
(Raczkiewicz e al., 2024a).
131
2.2. Eco oxicological es s
132
The e ec o he b-BC and n-BC on es ed o ganisms was e alua ed in liquid- and solid-
133
phase es s. The “liquid-phase es s” in es iga e he impac o leacha es ob ained om BC on
134
o ganisms (mainly wa e o ganisms) and can be called indi ec es because he pa o chemical
135
(leached om solid) is e alua ed, whe eas solid-phase es s examine he di ec e ec o he
136
whole ma e ial on he es ed o ganism. Fi e eco oxicological es s we e employed o assess and
137
compa e he eco oxici y o b-BC and n-BC. The liquid phase (leacha e) was p epa ed acco ding
138
o he EN 12457-2 p o ocol (2003). The b-BC and n-BC we e mixed wi h deionized wa e a a
139
8
p opo ion o 1:10 (solid:liqiud, w/ ) and we e subsequen ly shaken on a ho izon al shake o
140
24 hou s (Model 358A, ELPINSC+, Poland). The esul ing ex ac was il e ed h ough a 0.45
141
μm sy inge il e .
142
2.3. Liquid phase es
143
The e alua ion o he leacha e oxici y o Ali ib io ische ii was pe o med based on
144
Mic o ox® es s using a Mic o ox M500 analyze (Mic o ox M500, Mode n Wa e , UK)
145
acco ding o he es p o ocol (SDI, 1992). Be o e he es , he pH was adjus ed o he ange o
146
6-8 using 0.1M HCl/NaOH o ensu e es accu acy. Luminescence inhibi ion was measu ed
147
a e 5 and 15 minu es o exposu e o A. ische i o he leacha es. The Mic o ox Omni so wa e
148
was employed o he analysis o he esul s.
149
The oxici y assessmen o b-BC and n-BC o Daphnia magna was conduc ed ollowing he
150
s anda d p ocedu es acco ding o OECD 202 (OECD, 2004) and OECD 211 guidelines (OECD,
151
1998). The immobiliza ion and ep oduc ion o D. magna a e he endpoin s o his es . To
152
assess he impac o b-BC and n-BC on immobiliza ion, h ee concen a ions (2, 10, and 100
153
mg/L) o each b-BC o n-BC we e p epa ed by adding speci ic amoun s in deionized wa e . In
154
exposu e con aine s, i e young daphnids we e placed in 100 mL o he es solu ion, wi h h ee
155
eplica es o each concen a ion o b-BC o n-BC. The con aine s wi hou BCs we e used as a
156
con ol. Tes condi ions, including ligh and empe a u e, we e consis en wi h he cul u e
157
condi ions, and he exposu e medium was no ae a ed. Following he immobiliza ion es , he
158
impac o b-BC and n-BC on ep oduc ion was u he assessed o selec ed b-BC/n-BC (whe e
159
he e ec o he adhesion induced by n-BC was obse ed unde a mic oscope). Two
160
concen a ions o b-BC o n-BC (2 and 10 mg/L) we e selec ed. A young emale D. magna was
161
exposed o each concen a ion o 21 days, and a he end o he es , he o al numbe o li ing
162
o sp ing p oduced by he exposed D. magna was assessed.
163
9
In o de o assess he oxici y o b-BC and n-BC solu ions o aqua ic plan s, he duckweed
164
Lemna mino was used acco ding o he p ocedu e p oposed by OECD 221 guidelines (OECD,
165
2006). The p ima y objec i e o he es was o quan i y subs ance- ela ed e ec s on ege a i e
166
g ow h o e a es pe iod, based on assessmen s o selec ed measu emen a iables, including
167
ond numbe and o al ond a ea. Th ee concen a ions o b-BC o n-BC (2, 10, and 100 mg/L)
168
we e applied. Fo each concen a ion, 3 colonies wi h 3-4 isible onds each we e placed in o
169
measu emen essels wi h a o al olume o 100 mL. The o ganisms we e exposed in g ow h
170
medium o 3 weeks p io o e alua ing he pa icula endpoin s. The cul u es we e incuba ed
171
in a empe a u e-con olled en i onmen (a 24 ± 2°C) unde con inuous illumina ion wi h
172
luo escen lamps). The ond coun ing was manually pe o med a he beginning o he es (0
173
days) and a e 2, 5, and 7 days using he ImageJ 1.54 so wa e (ImageJ 1.54, Ja a, USA). The
174
a e age speci ic g ow h a e (μi) was calcula ed o each eplica e a e 7 days, o assess he
175
heal h s a us o plan s ollowing equa ion:
176
μ𝑖=ln(𝑁𝑗) − ln(𝑁𝑖)
177
Whe e µi-a e age speci ic g ow h a e om ime i o j, Ni- measu emen a iable in he es
178
o con ol essel a he ime I, Nj-measu emen a iable in he es o con ol essel a he ime
179
j, - ime pe iod om i o j.
180
2.4. Solid phase es
181
An eco oxicological es wi h Collembola (Folsomia candida) was pe o med ollowing he
182
s anda d p ocedu e based on OECD 232 guidelines (OECD, 2016). The es examines he
183
mo ali y and ep oduc ion o F. candida. A de ailed desc ip ion o he es is p esen ed in ou
184
p e ious publica ions (Koł owski e al., 2017; Oleszczuk e al., 2019; Tomczyk e al., 2021).
185
B ie ly, he adul indi iduals (aged 10-12 days) we e placed in a pe i dish wi h s anda d soil
186
(OECD) mixed wi h 1% and 5% o b-BC o n-BC. The s anda d soil (OECD) was used as a
187
16
nu ien s such as po assium, phospho us, and calcium (Hossain e al., 2020), which a e
336
bene icial o plan g ow h. Consequen ly, a highe ash con en in n-BC may esul in a g ea e
337
a ailabili y o nu ien s o L. mino (Bisane e al., 2023).
338
3.3. Toxici y o Daphnia magna
339
The Daphnia magna immobiliza ion es e ealed ha none o he es ed concen a ions
340
caused s a is ically signi ican immobiliza ion o hese o ganisms o b-BCs and n-BCs (Table
341
S3). Howe e , de ailed mic oscopic obse a ions o he o ganisms e ealed a signi ican
342
adhe ence o BC’s pa icles o he bodies o D. magna (Fig. 4). This adhe ence may pose long-
343
e m isks, po en ially leading o ch onic oxici y. The adhe ence e ec was no obse ed in
344
he sys em wi h b-BC. Following his obse a ion, n-BC and b-BC ep esen ing di e en
345
eeds ocks we e selec ed o a ep oduc ion es o assess he long- e m e ec s o he pa icles.
346
Bo h n-BC and b-BC a ec ed he ep oduc ion o D. magna (Table 1), bu a highe educ ion
347
compa ed o con ol was obse ed o n-BC han b-BC, especially in he sys em wi h a highe
348
concen a ion o pa icles. The ha m ul e ec was obse ed o n-BC o wo applied
349
concen a ions, while o b-BC he e ec was only no ed o he highes concen a ion o
350
pa icles. Rep oduc ion inhibi ion clea ly depended on he eeds ock used o BC p oduc ion.
351
Excep o b-BC-WL-750 and n-BC-WL-750, highe ep oduc ion inhibi ion was obse ed o
352
n-BC han b-BC (Table 1). Rega ding he age o i s ep oduc ion, i was gene ally no ed ha
353
he alues we e e y close o hose o he con ol g oup. Howe e , o n-BC-OSR-550and b-
354
BC-Wl-750, he age o i s ep oduc ion was signi ican ly longe (>11 days) compa ed o he
355
con ol (9 days). This delay may be explained by he po en ial p esence o inhibi o y
356
compounds in he speci ic eeds ocks o by he la ge impac o hese pa icula BC on he
357
o ganisms' o e all ene gy budge , possibly due o he leaching o speci ic TMs o o ganic
358
compounds ha in e e e wi h ep oduc i e iming.
359
17
3.4. Toxici y o Lepidium sa i um
360
No no able impac o end was de ec ed ega ding he inhibi ion o Lepidium sa i um seed
361
ge mina ion (Table S6). O he s udies epo ha unde s ess condi ions, he addi ion o n-BC
362
can inc ease he ge mina ion a e o plan s. Xin e al. (2024) obse ed maximum inc eases in
363
ge mina ion a es o 24.8% and 20.8% o BC’s nanopa icles and BC, espec i ely, compa ed
364
o he con ol g oup. In con as , Zhang e al.(2020) did no obse e any e ec o BC’s
365
nanopa icles a di e en concen a ions (0.5, 5, and 50 mg/L) on he seed ge mina ion a es o
366
ice and oma o.
367
Ne e heless, a s a is ically signi ican e ec (p≤ 0.05) o BCs was obse ed on oo g ow h
368
inhibi ion (Fig. 5). When compa ing di e en eeds ocks, only he SSL-de i ed b-BC (b-BC-
369
SSL1-550, b-BC-SSL2-500, and b-BC-SSL3-500) among he es ed b-BC exhibi ed oxici y
370
owa ds L. sa i um, causing oo g ow h inhibi ion anging om 18% o 23%. In con as , he
371
ag icul u al esidues-de i ed b-BC (b-BC-RH-550 and b-BC-OSR-550) and WL-de i ed b-BC
372
(b-BC-WL-550) p oduced a he same empe a u e we e ound o be non- oxic and e en
373
s imula ed he oo g ow h by 6% o 15%. SSL-de i ed b-BC p oduced a lowe empe a u es
374
(500-550°C) ends o ha e highe concen a ions o ola ile o ganic compounds (VOCs), which
375
can dis up oo de elopmen (Goldan e al., 2022). Addi ionally, SSL-de i ed b-BC has lowe
376
ca bon con en and highe le els o ash (Raczkiewicz e al., 2024b). Highe ash con en is
377
associa ed wi h an inc eased p esence o ino ganic con aminan s, such as hea y me als, which
378
can be oxic o plan s (Goyal e al., 2020) and in BC can also cause luc ua ions in soil pH and
379
elec ical conduc i i y (EC), c ea ing an un a o able en i onmen o plan g ow h (Ma a e
380
al., 2018). Inc easing he py olysis empe a u e also enhanced he posi i e e ec o b-BC on L.
381
sa i um, which co ela ed wi h a educ ion in he con en o he con aminan s in hese BC (Fig.
382
7b) (Table S4). S a is ical analysis e ealed a s ong nega i e co ela ion be ween he C o al
383
PAHs and he oo g ow h o L. sa i um. I was p e iously sugges ed ha PAHs con ained in
384
18
BCs could be esponsible o hei nega i e impac on plan s (Godlewska e al., 2022; Jajoo,
385
2017; S e aniuk and Oleszczuk, 2016). Addi ionally, a posi i e co ela ion was obse ed
386
be ween he ca bon con en (C) and oo g ow h, while a nega i e co ela ion was ound
387
be ween he ni ogen con en (N) in b-BC and oo g ow h. These co ela ions could be
388
a ibu ed o he di e ing physicochemical p ope ies o he BC associa ed wi h i s chemical
389
composi ion. Highe C con en may enhance soil s uc u e, wa e e en ion, and nu ien
390
a ailabili y, he eby p omo ing plan g ow h (Mu aza e al., 2024). On he o he hand, he N
391
con en in BC may in luence mic obial ac i i y in he soil, which could also a ec plan g ow h
392
(Liu e al., 2018). Insu icien N le els may lead o ad e se e ec s such as inhibi ed plan
393
g ow h, explaining he obse ed nega i e co ela ion. Fu he mo e, eplacing he ca ie gas
394
om N2 o CO2 showed no signi ican impac (p ≤ 0.05) on he inhibi ion o L. sa i um oo
395
g ow h. These indings align wi h simila ends obse ed in p e ious s udies ega ding he
396
in luence o py olysis condi ions on BC’s oxici y (Kończak e al., 2020; Sun e al., 2023).
397
The educ ion o BC’s size o he nanoscale esul ed in a shi om a s imula o y e ec o
398
oxici y (BC-WL, BC-RH, BC-OSR) o a signi ican inc ease in he inhibi ion o L. sa i um
399
oo g ow h (Fig. 5). The Tukey es esul s in he Figu e 5 indica e ha n-BC gene ally causes
400
highe oo g ow h inhibi ion in L. sa i um compa ed o bulk biocha b-BC, as indica ed by he
401
highe pe cen age inhibi ion alues o n-BC ba s. S a is ically signi ican di e ences in oo
402
g ow h e ec s a e deno ed by di e en le e s. Zhang e al. (2020) indica ed ha BC’s
403
nanopa icles could pose a po en ial h ea o bio a, p ima ily due o phenolic compounds
404
deposi ed on he la ge BC’sspeci icsu ace a ea, which ha e been shown o nega i ely impac
405
plan s. The e o e, he isk posed by n-BC o bio a likely o igina es om hese phenolic
406
compounds on i s inc eased su ace. All n-BCs displayed a consis en end o changes simila
407
o hose obse ed wi h b-BC conce ning he eeds ock used and py olysis condi ions. S a is ical
408
analysis indica ed ha he physicochemical ac o s de e mining oxici y a e he same in bo h b-
409
19
BC and n-BC (Table S4). Howe e , due o he educ ion in size, ha m ul subs ances may be
410
eleased mo e easily. These ends be ween b-BC and n-BC we e also obse ed o F. candida
411
(Fig. 6). Fu he mo e, n-BCs demons a ed signi ican ly highe EC alues compa ed o b-BCs
412
(Table S2) (Raczkiewicz e al., 2024b). These ele a ed EC alues may exceed he ole ance
413
le els o plan s o sal p esence, especially as BC concen a ion inc eases. Nume ous s udies
414
ha e epo ed ha high EC alue esul ing om soil amendmen s wi h BC is a key ac o
415
con ibu ing o he de imen al e ec s o BC on L. sa i um (Gell e al., 2011; Muhammad and
416
Hussain, 2010; Nhan e al., 2019).
417
3.5. Toxici y o Folsomia candida
418
Bo h b-BC and n-BC, when added o soil a wo di e en doses did no a ec he mo ali y
419
o F. candida (Table S7), ne e heless, hey did in luence hei ep oduc ion (Fig. 6). A 1%
420
dose, b-BC s imula ed he ep oduc ion o F. candida in he ange om 9% o 90%. Conce ning
421
he eeds ock used, he mos isible e ec was obse ed o SSL-de i ed b-BCs, while he
422
lowes alues we e no ed o b-BC-OSR-550. An inc ease in py olysis empe a u e esul ed in
423
a g adual inc ease in ep oduc ion wi h he highes empe a u e yielding a ep oduc ion a e
424
25% highe han ha obse ed a he lowes empe a u e (Fig. 6). Changing he ca ie gas om
425
N2 (b-BC-SSL3-500) o CO2 (b-BC-SSL3CO2-500) did no a ec ep oduc ion o F. candida
426
(Fig. 6).
427
The addi ion o 1% o n-BC had a less a o able impac on he s imula ion o F. candida
428
ep oduc ion o hei espec i e bulk coun e pa s (Fig. 6a). The ep oduc ion s imula ion o n-
429
BC was om 38% o 45% lowe o WL-de i ed n-BCs, om 30% o 44% o SSL-de i ed n-
430
BCs, and om 46% o 56% o ag icul u al esidues-de i ed n-BC (n-BC-RH-550 and n-BC-
431
OSR-550) compa ed o co esponding b-BC. The obse ed end in esponse o he applied
432
eeds ock and py olysis pa ame e s o n-BC exhibi ed a s ong co ela ion wi h he end no ed
433
o b-BC.
434
20
An inc ease o he b-BC dose o 5% signi ican ly diminished he s imula o y e ec obse ed
435
a lowe doses (Fig. 6), esul ing in he inhibi ion o F. candida ep oduc ion o WL- ( om 4%
436
o 16%) and o ag icul u al esidues-de i ed b-BC ( om 35% o 60%) (Fig. 6b). Only SSL-
437
de i ed b-BC did no nega i ely a ec he ep oduc ion o F. candida, showing no signi ican
438
di e ence om he con ol. An inc ease py olysis empe a u e, which educed oxici y and
439
eplacing N2 wi h CO2, which showed no signi ican e ec , exhibi ed a consis en end as
440
obse ed a lowe b-BC dose.
441
A highe dose o n-BC, a simila end was obse ed as o b-BC ega ding o he applied
442
eeds ock and py olysis condi ions (Fig. 6b). The n-BCs exhibi ed a g ea e oxic e ec on F.
443
candida han co esponding b-BCs. In gene al, he ep oduc ion inhibi ion was om 62% o
444
81% highe o WL-de i ed n-BC and om 15% o 51% o ag icul u al was e-de i ed n-BCs
445
han b-BCs.
446
The obse ed oxic o s imula o y e ec s we e signi ican ly co ela ed (p ≤ 0.05) wi h he
447
le els o con aminan s in he BC, including PAHs and TMs (Fig. 7). This obse a ion aligns
448
wi h indings om p e ious esea che s (Eom e al., 2007; S e d up e al., 2002), who
449
demons a ed he sensi i i y o sp ing ails (Folsomia candida and Folsomia ime a ia) o PAHs,
450
pa icula ly ega ding ep oduc ion ju enile. In he case o he 5% dose o b-BC, ano he
451
po en ial explana ion o he de imen al impac on ep oduc ion could be he pH le els. P io
452
s udies (Luo e al., 2022; S y isha e e al., 2010) ha e shown ha he alkaline pH may
453
nega i ely a ec F. candida, especially in e ms o ep oduc ion. The op imal pH o he
454
g ow h and ep oduc ion o F. candida alls wi hin he ange o 5 o 6. The pH alues o he
455
es ed b-BC anged om 8.2 o 11. Wi h he addi ion o 1% b-BC, he pH impac was minimal
456
and did no cause a oxic e ec , bu a he a s imula ing one. This could be due o he ela i ely
457
low concen a ion o b-BC, which migh no signi ican ly al e he o e all pH o c ea e
458
condi ions ha a e ha m ul o he o ganisms. Ins ead, he small amoun o b-BC migh p o ide
459
21
essen ial nu ien s o c ea e a mildly a o able en i onmen ha enhances ep oduc ion. In he
460
case o n-BC, he pH a e size educ ion dec eased o he ange o 7.4 o 10. Fo n-BC, besides
461
he abili y o adso b nu ien s, i may be easie o he pa icles o pene a e o ganisms o
462
accumula e, inc easing he chance o in oducing con aminan s ha could ha e a di ec oxic
463
e ec . Addi ionally, nano-sized pa icles may mo e easily deposi in issues/cells han mac o-
464
sized pa icles, which pass h ough he o ganism and a e exc e ed o no e en abso bed.
465
Nanopa icles, due o hei small size, a e mo e p one o accumula ion, po en ially causing
466
long- e m ad e se e ec s on o ganisms (Rajpu e al., 2020). Due o i s la ge speci ic su ace
467
a ea and size e ec , n-BC exhibi s g ea e adso p ion p ope ies han b-BC. When added o soil,
468
n-BC can adso b essen ial na i e nu ien s om he soil solu ion, leading o hei deple ion. This
469
p ocess can induce he ansloca ion o nu ien s in o deepe soil laye s due o he enhanced
470
mig a o y capabili ies o n-BC, which a e associa ed wi h hei g ea e su ace cha ge in soil
471
p o iles, he eby diminishing nu ien e en ion wi hin he su ace ho izons (Li e al., 2024).
472
Consequen ly, he educed physical a ailabili y o hese elemen s in he su ace laye s o F.
473
candida may exace ba e oxicological e ec s. Ou s udy showed ha low doses o n-BC (1%
474
dose) do no lead o signi ican soil nu ien deple ion su icien o inhibi ep oduc ion o cause
475
mo ali y in adul indi iduals, bu i is e iden ha ep oduc ion is educed compa ed o b-BC
476
(Fig. 6a). The obse ed de imen al e ec a ele a ed doses o n-BC (5% dose) may be
477
a ibu able o he adso p ion capaci y o n-BC su passing he na u al eplenishmen a e o soil
478
nu ien s a his concen a ion. Fu he mo e, a highe dose o n-BC is associa ed wi h inc eased
479
nu ien adso p ion, which hen mig a es deepe in o he soil p o ile, he eby educing hei
480
a ailabili y in he su ace ho izons. The p onounced opposing e ec o BC, a ibu able o he
481
applied dose (s imula ion s. de imen al e ec ) may sugges he exis ence o a h eshold
482
concen a ion o n-BC/b-BC beyond which i s p esence in he soil becomes oxic o sp ing ails.
483
While lowe doses o n-BC may s ill con e bene i s wi hou eaching his h eshold, he
484
22
cumula i e e ec o highe doses could p o e de imen al. Bielská e al. (2018) obse ed ha
485
wi h inc easing doses o BC, he oxici y owa ds F. candida also inc eased. In hei s udy,
486
ep oduc ion a es in soil wi h he addi ion o 1% wood and ice husk BC we e compa able o
487
hose o con ol soil. Howe e , when he BC dose was inc eased o 10%, ep oduc ion a es
488
dec eased by 38% and 27% o wood BC and ice husk BC, espec i ely, compa ed o con ol
489
soil. Mo eo e , he ele a ed con en o dissol ed o ganic ca bon (DOC) in n-BC may in luence
490
i s eco oxici y by enhancing he bioa ailabili y and mobili y o oxic subs ances (like PAHs o
491
TMs) associa ed wi h DOC (Ma e al., 2021). This is con i med by he s a is ical analysis o
492
co ela ions be ween DOC and ep oduc ion o F. candida (Fig. 8c). Despi e he educed
493
o ganic-sol en ex ac able (C o al) and eely dissol ed (C ee) PAHs con en in n-BC han in b-
494
BC, he inc eased speci ic su ace a ea and DOC con en in n-BC acili a e enhanced
495
in e ac ions be ween PAHs in he soil-n-BC sys em.
496
4. CONCLUSION
497
This s udy p o ides a sys ema ic assessmen o he eco oxicological e ec s o nanobiocha
498
(n-BC) ac oss mul iple biological sys ems, elucida ing he en i onmen al implica ions o i s
499
applica ion. The indings highligh he pi o al ole o eeds ock p ope ies, py olysis condi ions,
500
and pa icle size in in luencing BC’s in e ac ions wi h o ganisms. The key ou comes a e as
501
ollows:
502
⎯ Bac e ia: n-BC exhibi ed signi ican ly highe oxici y o A. ische i compa ed o bulk
503
biocha (b-BC), likely due o enhanced su ace ac i i y, leaching o oxic subs ances,
504
and lowe pH.
505
⎯ Aqua ic and e es ial plan s: While n-BC was non- oxic o L.mino , p omo ing
506
ond g ow h po en ially due o he elease o bioa ailable nu ien s, i inhibi ed oo
507
g ow h in L. sa i um, possibly as a esul o ele a ed elec ical conduc i i y (EC)
508
su passing he plan ’s sal ole ance.
509
23
⎯ Soil in e eb a es: Highe concen a ions o n-BC induced ep oduc i e oxici y o
510
F.candida, which may be a ibu ed o inc eased dissol ed o ganic ca bon (DOC) and
511
deepe soil mig a ion o n-BC pa icles.
512
⎯ Aqua ic in e eb a es: Exposu e o ele a ed doses o n-BC inhibi ed D. magna’s
513
ep oduc ion and esul ed in no able pa icle adhesion o he o ganisms’ su aces,
514
indica ing po en ial bioaccumula ion and long- e m oxic e ec s.
515
Ou esea ch e eals he pi o al ole o eeds ock and pa icle size in de e mining he
516
en i onmen al isks o biocha . By me hodically analyzing bo h bulk- and nano-sized
517
biocha ac oss a ious ophic le els, we unlock new pe spec i es on he ecological impac s
518
o biocha usage. These insigh s can d i e u u e inno a ions in he de elopmen and
519
esponsible applica ion o biocha echnologies wi hin en i onmen al managemen .
520
Ul ima ely, ou s udy unde sco es he impe a i e o in eg a ing mul iple ac o s, such as
521
eeds ock, py olysis condi ions, and pa icle size, when e alua ing he oxicological p o iles
522
o biocha . Addi ionally, i highligh s he u gency o con inued esea ch o ully g asp he
523
en i onmen al ami ica ions and o de ise s a egies o he sa e, sus ainable deploymen o
524
biocha ma e ials. Fu he in es iga ions should ocus on long- e m ecological moni o ing,
525
mechanis ic s udies o biocha -o ganism in e ac ions, and he de elopmen o guidelines o
526
he sa e and sus ainable deploymen o biocha in en i onmen al managemen .
527
528
529
Acknowledgmen s
530
The p ojec was unded by he Na ional Science Cen e g an ed based on he decision numbe
531
DEC- 2021/42/A/ST10/00161.
532
Compe ing in e es s
533
The au ho s ha e no ele an inancial o non- inancial in e es s o disclose.
534
24
Au ho Con ibu ions
535
Concep ualiza ion was pe o med by M.R and P.O.; Ma e ial p epa a ion, da a collec ion and
536
analysis we e pe o med by M.R. and A.B.; The i s d a o he manusc ip was w i en by
537
M.R. and all au ho s commen ed on p e ious e sions o he manusc ip . All au ho s ead and
538
app o ed he inal manusc ip .
539
5. REFERENCES
540
Bala, S., Ga g, D., Thi umalesh, B.V., Sha ma, M., S idha , K., Inba aj, B.S., T ipa hi, M., 2022. Recen
541
S a egies o Bio emedia ion o Eme ging Pollu an s: A Re iew o a G een and Sus ainable
542
En i onmen . Toxics 10, 484. h ps://doi.o g/10.3390/ oxics10080484
543
Bielská, L., Škulco á, L., Neuwi ho á, N., Co nelissen, G., Hale, S.E., 2018. So p ion, bioa ailabili y
544
and eco oxic e ec s o hyd ophobic o ganic compounds in biocha amended soils. Sci. To al
545
En i on. 624, 78–86. h ps://doi.o g/10.1016/j.sci o en .2017.12.098
546
Bisane, M., Chak a a y, I., Mehe e, S., Mukhe jee, P., Manda gane, S.A., 2023. Biomass ash as a
547
sou ce o nu ien ; a case s udy on co on (Gossypium), in: F ui and Vege able Was e
548
U iliza ion and Sus ainabili y. Else ie , pp. 301–308. h ps://doi.o g/10.1016/B978-0-323-
549
91743-8.00005-8
550
Council, E. U. Council Decision 2003/33/EC o 19 Decembe 2002 es ablishing c i e ia and p ocedu es
551
o he accep ance o was e a land ills pe suan o A icle 16 o and Annex II o Di ec i e
552
1999/31/EC. O icial Jou nal o he Eu opean Communi ies, 16(2003), L11., 2003.
553
Doe, K., Jackman, P., Sc oggins, R., McLeay, D., Wohlgescha en, G., 2005. Solid-Phase Tes o
554
Sedimen Toxici y Using he Luminescen Bac e ium, Vib io Fische i, in: Blaise, C., Fé a d, J.-F.
555
(Eds.), Small-Scale F eshwa e Toxici y In es iga ions. Sp inge -Ve lag, Be lin/Heidelbe g, pp.
556
107–136. h ps://doi.o g/10.1007/1-4020-3120-3_3
557
Domene, X., Ende s, A., Hanley, K., Lehmann, J., 2015. Eco oxicological cha ac e iza ion o biocha s:
558
Role o eeds ock and py olysis empe a u e. Sci. To al En i on. 512–513, 552–561.
559
h ps://doi.o g/10.1016/j.sci o en .2014.12.035
560
Eom, I.C., Ras , C., Vebe , A.M., Vasseu , P., 2007. Eco oxici y o a polycyclic a oma ic hyd oca bon
561
(PAH)-con amina ed soil. Eco oxicol. En i on. Sa . 67, 190–205.
562
h ps://doi.o g/10.1016/j.ecoen .2006.12.020
563
Gell, K., Van G oenigen, J., Cayuela, M.L., 2011. Residues o bioene gy p oduc ion chains as soil
564
amendmen s: Immedia e and empo al phy o oxici y. J. Haza d. Ma e . 186, 2017–2025.
565
h ps://doi.o g/10.1016/j.jhazma .2010.12.105
566
Godlewska, P., Kończak, M., Oleszczuk, P., 2022. E ec o ca ie gas change du ing sewage sludge o
567
sewage sludge and willow py olysis on eco oxici y o biocha -amended soil. Eco oxicol.
568
En i on. Sa . 247, 114224. h ps://doi.o g/10.1016/j.ecoen .2022.114224
569
Godlewska, P., Ok, Y.S., Oleszczuk, P., 2021. THE DARK SIDE OF BLACK GOLD: Eco oxicological aspec s
570
o biocha and biocha -amended soils. J. Haza d. Ma e . 403, 123833.
571
h ps://doi.o g/10.1016/j.jhazma .2020.123833
572
Goldan, E., Nede , V., Ba san, N., Culea, M., Tomozei, C., Panain e-Lehadus, M., Mosnegu u, E.,
573
2022. E alua ion o he Use o Sewage Sludge Biocha as a Soil Amendmen —A Re iew.
574
Sus ainabili y 14, 5309. h ps://doi.o g/10.3390/su14095309
575
Gondek, K., Mie zwa-He sz ek, M., Sm eczak, B., Ba an, A., Kopeć, M., M óz, T., Janowski, P., Bajda,
576
T., Tomczyk, A., 2016. Con en o PAHs, ac i i ies o γ- adionuclides and eco oxicological
577
25
assessmen in biocha s. Pol. J. Chem. Technol. 18, 27–35. h ps://doi.o g/10.1515/pjc -2016-
578
0067
579
Goyal, D., Yada , A., P asad, M., Singh, T.B., Sh i as a , P., Ali, A., Dan u, P.K., Mish a, S., 2020. E ec
580
o Hea y Me als on Plan G ow h: An O e iew, in: Naeem, M., Ansa i, A.A., Gill, S.S. (Eds.),
581
Con aminan s in Ag icul u e. Sp inge In e na ional Publishing, Cham, pp. 79–101.
582
h ps://doi.o g/10.1007/978-3-030-41552-5_4
583
Hossain, M.Z., Baha , M.M., Sa ka , B., Donne, S.W., Ok, Y.S., Palansoo iya, K.N., Ki kham, M.B.,
584
Chowdhu y, S., Bolan, N., 2020. Biocha and i s impo ance on nu ien dynamics in soil and
585
plan . Biocha 2, 379–420. h ps://doi.o g/10.1007/s42773-020-00065-z
586
Huang, J., Zimme man, A.R., Chen, H., Gao, B., 2020. Ball milled biocha e ec i ely emo es
587
sul ame hoxazole and sul apy idine an ibio ics om wa e and was ewa e . En i on. Pollu .
588
258, 113809. h ps://doi.o g/10.1016/j.en pol.2019.113809
589
Jagodzinski, L.S., O’Donoghue, M.T., He e nan, L.B., Van Pel , F.N.A.M., O’Hallo an, J., Jansen,
590
M.A.K., 2018. Wood ash esidue causes a mix u e o g ow h p omo ion and oxici y in Lemna
591
mino . Sci. To al En i on. 625, 667–676. h ps://doi.o g/10.1016/j.sci o en .2017.12.233
592
Jajoo, A., 2017. E ec s o En i onmen al Pollu an s Polycyclic A oma ic Hyd oca bons (PAH) on
593
Pho osyn he ic P ocesses, in: Hou, H.J.M., Naja pou , M.M., Moo e, G.F., Allakh e die , S.I.
594
(Eds.), Pho osyn hesis: S uc u es, Mechanisms, and Applica ions. Sp inge In e na ional
595
Publishing, Cham, pp. 249–259. h ps://doi.o g/10.1007/978-3-319-48873-8_11
596
Kim, J.-Y., Oh, S., Pa k, Y.-K., 2020. O e iew o biocha p oduc ion om p ese a i e- ea ed wood
597
wi h de ailed analysis o biocha cha ac e is ics, hea y me als beha io s, and hei
598
eco oxici y. J. Haza d. Ma e . 384, 121356. h ps://doi.o g/10.1016/j.jhazma .2019.121356
599
Koł owski, M., Cha mas, B., Skubiszewska-Zięba, J., Oleszczuk, P., 2017. E ec o biocha ac i a ion by
600
di e en me hods on oxici y o soil con amina ed by indus ial ac i i y. Eco oxicol. En i on.
601
Sa . 136, 119–125. h ps://doi.o g/10.1016/j.ecoen .2016.10.033
602
Kończak, M., Pan, B., Ok, Y.S., Oleszczuk, P., 2020. Ca bon dioxide as a ca ie gas and mixed
603
eeds ock py olysis dec eased oxici y o sewage sludge biocha . Sci. To al En i on. 723,
604
137796. h ps://doi.o g/10.1016/j.sci o en .2020.137796
605
Li, L., Zhang, K., Chen, L., Huang, Z., Liu, G., Li, M., Wen, Y., 2017. Mass p epa a ion o mic o/nano-
606
powde s o biocha wi h wa e -dispe sibili y and hei po en ial applica ion. New J. Chem.
607
41, 9649–9657. h ps://doi.o g/10.1039/C7NJ00742F
608
Li, P., Yan, M., Li, M., Zhou, T., Li, H., Si, B., 2024. Mig a ion Rules and Mechanisms o Nano-Biocha in
609
Soil Columns unde Va ious T anspo Condi ions. Nanoma e ials 14, 1035.
610
h ps://doi.o g/10.3390/nano14121035
611
Liu, Q., Zhang, Y., Liu, B., Amone e, J.E., Lin, Z., Liu, G., Ambus, P., Xie, Z., 2018. How does biocha
612
in luence soil N cycle? A me a-analysis. Plan Soil 426, 211–225.
613
h ps://doi.o g/10.1007/s11104-018-3619-4
614
Liu, Z., Xu, Z., Xu, L., Buyong, F., Chay, T.C., Li, Z., Cai, Y., Hu, B., Zhu, Y., Wang, X., 2022. Modi ied
615
biocha : syn hesis and mechanism o emo al o en i onmen al hea y me als. Ca bon Res.
616
1, 8. h ps://doi.o g/10.1007/s44246-022-00007-3
617
Luo, X., Zhu, L., Xu, G., Zhang, J., Xu, J., Yu, S., Chen, X., 2022. E ec s o acid deposi ion on he
618
a oidance beha io o Folsomia candida (Collembola, Iso omidae). Soil Ecol. Le . 4, 164–170.
619
h ps://doi.o g/10.1007/s42832-021-0099-x
620
Lyu, H., Gao, B., He, F., Zimme man, A.R., Ding, C., Tang, J., C i enden, J.C., 2018. Expe imen al and
621
modeling in es iga ions o ball-milled biocha o he emo al o aqueous me hylene blue.
622
Chem. Eng. J. 335, 110–119. h ps://doi.o g/10.1016/j.cej.2017.10.130
623
Lyu, H., He, Y., Tang, J., Hecke , M., Liu, Q., Jones, P.D., Codling, G., Giesy, J.P., 2016. E ec o
624
py olysis empe a u e on po en ial oxici y o biocha i applied o he en i onmen . En i on.
625
Pollu . 218, 1–7. h ps://doi.o g/10.1016/j.en pol.2016.08.014
626
Ma, P., Yang, C., Zhu, M., Fan, L., Chen, W., 2021. Leaching o o ganic ca bon enhances mobili y o
627
biocha nanopa icles in sa u a ed po ous media. En i on. Sci. Nano 8, 2584–2594.
628
h ps://doi.o g/10.1039/D1EN00409C
629
