Chemical composi ion o wild i e ash p oduced in
con as ing ecosys ems and i s oxici y o Daphnia magna
Ashleigh R. Ha pe
A
,
E
,C is ina San in
A
,
B
,S e an H. Doe
A
,
Cyn hia A. F oyd
B
,Dania Albini
B
,Xose Luis O e o
C
,Lucia Vin
˜as
D
and
Begon
˜aPe
´ ez-Fe na
´ndez
D
A
Depa men o Geog aphy, Swansea Uni e si y, Single on Pa k, Swansea, SA2 8PP, UK.
B
Depa men o Biosciences, Swansea Uni e si y, Single on Pa k, Swansea, SA2 8PP, UK.
C
Depa men o Edaphology and Ag icul u al Chemis y, Facul y o Biology, Uni e si y o San iago
de Compos ela, 15782 San iago de Compos ela, Spain.
D
Ins i u o Espan
˜ol de Oceanog a ı´a (IEO), Cen o Oceanog a
´ ico de Vigo, Subida a Radio Fa o 50,
36390 Vigo, Spain.
E
Co esponding au ho . Email: [email p o ec ed]
Abs ac . I is well es ablished in he wo ld’s i e-p one egions ha wild i es can conside ably change he hyd ological
dynamics o eshwa e ca chmen s. Limi ed esea ch, howe e , has ocused on he po en ial impac s o wild i e ash
oxici y on aqua ic bio a. He e, we assess he chemical composi ion and oxici y o ash gene a ed om wild i es in six
con as ing ege a ion ypes dis ibu ed globally (UK g assland, Spanish pine o es , Spanish hea hland, USA chapa al,
Aus alian eucalyp o es and Canadian sp uce o es ). Acu e (48 h) immobilisa ion es s we e conduc ed on he
ex ensi ely s udied aqua ic mac oin e eb a e Daphnia magna, a sensi i e indica o o aqua ic con aminan s. We ound
signi ican di e ences be ween he chemical composi ion and oxici y o hese ash ypes. The UK and Spanish ash had no
de ec able oxici y o Daphnia magna, whe eas he Aus alian eucalyp , USA chapa al and Canadian sp uce ash all caused
signi ican oxici y (immobilisa ion). The p incipal cha ac e is ics o he la e ash ypes we e hei high pH, and NO
3
,
Cl
and conduc i i y le els. Ele a ed wa e -soluble and o al concen a ions o me als (e.g. Mn, Fe, Zn, Pb, Cu and As) and
o al polycyclic a oma ic hyd oca bons (PAHs) we e no linked o oxici y.
Addi ional keywo ds: bioassays, eco oxicology, polycyclic a oma ic hyd oca bons (PAH), wild i e impac s.
Recei ed 15 No embe 2018, accep ed 20 June 2019, published online 6 Augus 2019
In oduc ion
Fi es a e a na u al p ocess in many habi a ypes wo ldwide
(Bixby e al. 2015), bu hey can be a social and en i onmen al
conce n, po en ially impac ing public heal h, sa e y, in a-
s uc u e, biodi e si y, land-use, wa e and ai pollu ion (Bladon
e al. 2014;B i o e al. 2017). Fi e ac i i y is p ojec ed o
inc ease in many loca ions and eco ypes as a esul o clima e
and socie al changes, making he ull unde s anding o hei
impac s c ucial (Scholze e al. 2006;Chen e al. 2018).
Du ing wildland i es, combus ion o uels eleases a wide
ange o o ganic and ino ganic componen s in o he a mosphe e
bu also concen a es some o hem in o wild i e ash le on he
g ound (Bodı´ e al. 2014). F esh wild i e ash ypically consis s
o mine al ma e ials and cha ed o ganic componen s, is non-
cohesi e, has a low densi y, and is no a ached o he soil, which
acili a es i s mobilisa ion and anspo a ion by pos - i e wa e
and wind e osion (Bodı´ e al. 2014;Ab aham e al. 2017). The
elease o soluble elemen s and pa icula e ma e om e oded
ash and unde lying soil in o aqua ic sys ems ollowing i es can
cause inc eases in wa e u bidi y, pH, o ganic ma e , sus-
pended sedimen and conduc i i y and deple ion o dissol ed
oxygen, among o he e ec s (Smi h e al. 2011;Tsai e al. 2017).
Ash is howe e no usually examined as a dis inc pa o he
pos - i e sedimen and ew labo a o y s udies ha e cha ac e ised
he composi ion o wild i e ash (Bodı´ e al. 2014).
The majo i y o he gene al s udies in o he e ec s o wild i e
on wa e quali y ha e ocused on uno amoun s and nu ien
le els and only mo e ecen ly has inc eased esea ch a en ion
been gi en o py oly ic subs ances, chemical elemen s and
biological eac i i y (Shakesby and Doe 2006;Campos e al.
2012;Sil a e al. 2015). Key a eas ecei ing pa icula a en ion
as a esul o conce n abou hei en i onmen al e ec s a e he
p oduc ion and mobilisa ion o polycyclic a oma ic hyd oca -
bons (PAHs) and hea y me als (e.g. Vila-Escale´ e al. 2007;
Campos e al. 2012;Oli ei a-Filho e al. 2018). Bo h p esen
majo biological conce n owing o hei ca cinogenic po en ial,
pe sis ence wi hin ecosys ems and endency o bio-accumula e
(Smi h e al. 2011;Chen e al. 2018). These con aminan s a e
CSIRO PUBLISHING
In e na ional Jou nal o Wildland Fi e 2019,28, 726–737
h ps://doi.o g/10.1071/WF18200
Jou nal Compila ion ÓIAWF 2019 Open Access CC BY-NC-ND www.publish.csi o.au/jou nals/ijw
SPECIAL ISSUESPECIAL ISSUE
hough o ha e complex impac s on wa e quali y and he
biological e ec s o hese in aqua ic sys ems ha e been
obse ed o pe sis ac oss long spa ial and empo al scales
(Ea l and Blinn 2003;Cos a e al. 2014).
Ash has also begun o ecei e inc easing ecogni ion as a
sou ce o di use con amina ion in eshwa e sys ems and
de imen al impac s on bo h lake and s eam bio a, including
ish (e.g. Nunes e al. 2017;Oli ei a-Filho e al. 2018;Gonino
e al. 2019a), amphibians (Pilliod e al. 2003), mac oin e e-
b a es (B i o e al. 2017) and algae (Campos e al. 2012) ha e all
been obse ed. Highly a iable impac s o ash con amina ion on
eshwa e bio a ha e been epo ed be ween di e en ecosys-
ems, ypes o ash, i es and species (Smi h e al. 2011;Sil a
e al. 2015;Oli ei a-Filho e al. 2018). Campos e al. (2012) and
Sil a e al. (2015), o example, ound no signi ican impac o
eucalyp ash on he plank onic c us acean Daphnia magna
ep oduc ion o immobilisa ion a es o e a ch onic (21 day)
and acu e (48 h) exposu es espec i ely. Toxici y was, howe e ,
obse ed on se e al lowe ophic le el species in hese s udies,
he bac e ia Vib io ische i, algae Pseudoki chne iella subcapi-
a a and he mac ophy e Lemna mino . A simila s udy by B i o
e al. (2017) es ed oxici y o e acu e exposu es (48 h) o h ee
ypes o ash om he B azilian Ce ado eco egion on he
plank onic c us acean Ce iodaphnia dubia, he ish Danio e io
and he mollusc Biomphala ia glab a a and ound ha all ash
ypes caused oxici y o C. dubia, none a ec ed B. glab a a and
only one ype was oxic o D. e io. A highe ophic le els,
nega i e impac s o B azilian suga cane ash ha e also been
obse ed on se e al na i e ish species (As yanax lacus is,
Moenkhausia boni a and M. o es ii) o e 24-h acu e exposu es
bu no o wo non-na i e ish species (O eoch omis nilo icus
and Poecilia e icula a)(Gonino e al. 2019b). These s udies
demons a e he a iabili y and complexi y o in luencing ac-
o s in ela ion o he impac s o ash con amina ion on aqua ic
bio a, highligh ing he limi ed b ead h o a ailable esea ch in
his a ea (Hallema e al. 2018).
To enhance ou unde s anding o he impac s o ash con am-
ina ion on aqua ic bio a, he p esen s udy aimed o (1) de e -
mine he chemical composi ion o wild i e ash p oduced in six
con as ing ecosys ems; (2) examine he eco oxicological e ec
o hese ash ypes on he eshwa e indica o species Daphnia
magna; and (3) e alua e he ela ionship be ween chemical
composi ion and obse ed oxici y and i s implica ions o he
ela i e wa e con amina ion po en ial o ash p oduced in hese
di e ing ecosys ems. To he bes o ou knowledge, his con-
s i u es he i s eco oxicology assessmen allowing he di ec
compa ison o he composi ion and oxici y o ash om se e al
globally dis ibu ed con as ing ecosys ems.
Ma e ials and me hods
Ash samples
Six composi e ash samples we e collec ed a e wildland i es,
be o e any ain all, in each o he selec ed ecosys ems ypes
(Table 1): Aus alian eucalyp o es (AUS), USA chapa al
(USA), Canadian sp uce o es (CAN), Spanish hea hland
Table 1. Fi e and ege a ion cha ac e is ics o he six ash ypes used in he p esen s udy
Sample name Loca ion Vege a ion co e Bu n desc ip ion
Aus alian eucalyp
(AUS)
Wes o Sydney (3385201400S;
15083600100E)
Open, d y scle ophyll o es wi h a dense sh ubby
unde g ow h. Key species: i onba k (Eucalyp us
ib osa), s ingyba ks (Eucalyp us eugenioides,
Eucalyp us oblonga), Banksia sp., Lep ospe num
sp., Acacia sp. and Pe ophile sp.
Mode a e- o high-se e i y p esc ibed i e in 2014.
Fi e did no a ec ee canopy bu comple e
combus ion o unde s o ey uels and mos ly ine
ash, ligh in colou , was gene a ed (San ı´n e al.
2018)
USA chapa al
(USA)
Sou h-wes e n Cali o nia
(3482501400N; 11983003900W)
Mixed chapa al wi h dominan species including
coas li e oak (Que cus ag i olia), oyon
(He e omeles a bu i olia), coyo e b ush
(Baccha is pilula is), holly-lea che y (P unus
ilici olia)
La ge-scale, ex emely high-se e i y wild i e
(Thomas i e) in 2018, achie ing almos com-
ple e combus ion o abo e-su ace uel
Canadian sp uce
(CAN)
No h-wes Te i o ies
(6183405500N; 11781105500W)
Ve y dense ee canopy comp ising mos ly black
sp uce (Picea ma iana) and jack pines (Pinus
banksiana) wi h e y li le unde s o ey ege a-
ion wi h he excep ion o young sp uce and moss
species
Ve y-high-in ensi y expe imen al c own i e in
2015, all ine uels abo e g ound we e consumed.
The o es loo was only sligh ly a ec ed
(,1 cm dep h o bu n)
Spanish hea hland
(URIA)
No h-wes e n Spain
(438601700N; 685005200W)
Dominan species including hea he (Calluna
ulga is), wes e n go se (Ulex gallii) and a
a ie y o E ica sp. (e.g. E ica e alix)
Ex eme ho and d y condi ions p oduced a se e e
expe imen al wild i e in 2017. Combus ion
comple eness e y high (mos ine uel
consumed)
Spanish pine o es
(SPA)
Eas e n Spain (4081803600N;
180105900W)
Fo es ed a ea domina ed by Aleppo pine (Pinus
halepensis)
High-se e i y wild i e p oduced a e y high le el
o combus ion comple eness. All su ace uel
consumed
UK g assland (UK) Sou h Wales (5185001100N;
382504400W and 5184100200N;
383803700W)
Upland g aminoid-dominan a eas. Dominan
ege a ion in hese species-poo a eas consis ing
o pu ple moo -g ass (Molinia cae ulea), so
ush (Juncus e uses), ma -g ass (Na dus s ic a)
and hea h plai -moss (Hypnum ju landicum)
Mode a e-se e i y wild i es in 2018. Consumed
mos abo e-su ace uel and li e , bu did no
pene a e soil su ace. Composi e om wo i es
c ea ed in weigh a io 2 : 1 (g) o ash om si e
one and si e wo espec i ely
Eco oxicology o wild i e-gene a ed ash In . J. Wildland Fi e 727
(URIA), Spanish pine o es (SPA), UK g assland (UK). Fi e
and ege a ion cha ac e is ics a e summa ised in Table 1. Each
composi e ash sample was sie ed h ough a 1-mm mesh be o e
chemical cha ac e isa ion o use in he bioassays.
Chemical cha ac e isa ion
Chemical cha ac e isa ion o he six ash ypes collec ed
was unde aken o de e mine he o al and wa e -soluble con-
cen a ions o majo (Ca, Cl
,Mg,Na,Si,SO
42
,NO
3
)and
ace elemen s and compounds (Al, B, Cu, F
,Fe,Ni,NH
4
þ
,
As, Cd, Hg, Pb and PO
43
), in addi ion o pH, dissol ed o ganic
ca bon (DOC) and elec ic conduc i i y. This cha ac e isa ion
was unde aken using es ablished me hods (Plumlee e al.
2007;San ı´n e al. 2015,2018; see supplemen a y ma e ial o
ull de ails).
The concen a ions o 35 PAHs we e also de e mined
acco ding o Pe´ ez-Fe na´ndez e al. (2015) and Vin
˜as e al.
(2009) wi h gas ch oma og aphy-mass spec ome y (GC/MS)
(The mo mod DSQ II, The mo Elec on Co po a ion). The i s
s ep consis ed o a 10-h Soxhle ex ac ion wi h a 1 : 3 ace one :
hexane mix u e. The ex ac was hen ea ed o e nigh wi h
ac i a ed coppe o elemen al sul u emo al and hen cleaned
using column ch oma og aphy wi h deac i a ed alumina (see
supplemen a y ma e ial o ull de ails).
Daphnia oxici y es ing
Eco oxicological assays consis ing o acu e ash exposu es (48 h)
we e conduc ed using he plank onic c us acean Daphnia
magna. This species is ex ensi ely used in ecological and ox-
icological s udies as a sensi i e indica o o he e ec s o
con aminan s on aqua ic bio a (OECD 2004; USEPA 2016).
Daphnia spp. a e also pa icula ly ele an o eshwa e len ic
ecosys ems (lakes, ese oi s and ponds) and ideal o in es i-
ga ing con amina ion po en ial in downs eam wa e bodies
(Robinson and Tho n 2005;Nikinmaa 2014).
A monoclonal s a e cul u e o D. magna was ob ained om a
long- e m (2-yea ) ea ing p og am. The new cul u e was ea ed
and main ained acco ding o ecommended guidelines (OECD
2004; USEPA 2016), unde con olled empe a u e (20 28C)
and ligh condi ions (uni o m illumina ion o cool-whi e ype,
app oxima ely 5000 lx; pho ope iod 16 h ligh : 8 h da k) and ed
e e y 2 days wi h a dis illed suspension o Pseudoki chne iella
subcapi a a a ,0.1–0.2 mL pe Daphnid pe day.
To p oduce he es solu ions, each ash sample was combined
wi h a cul u e medium (syn he ic ha dwa e medium – (ASTM
1996)) a he a io 1 : 10 (mass : olume) (e.g. 100 g o ash in 1 L
o medium). The samples we e hen homogenised in an o bi al
shake o 4 h and s o ed a 48C (maximum 24 h) be o e using in
he eco oxicological assays.
The acu e oxici y es s we e conduc ed acco ding o he
OECD 202 (OECD 2004) guidelines, wi h he excep ion o ull
pH adjus men . pH was no adjus ed o con ol le els (pH
7.2 0.2) in he bioassays o ep oduce as close o na u al
condi ions as possible, gi en pH is one o he mos impo an
ac o s a ec ing he oxici y and bioa ailabili y o elemen s o
eshwa e species (F anklin e al. 2000). OECD 202 guidelines
acknowledge ha es s should be ca ied ou wi hou he adjus -
men o pH whe e alues a e wi hin pH 6–9 a he highes es
concen a ion (OECD 2004). I is c ucially impo an ha pH
adjus men does no cause signi ican changes o he es sub-
s ances and owing o he complex and a ying composi ions and
eac i i y o wild i e ash, po en ial in e ac ions a e unclea .
Li le is known o da e on wild i e ash concen a ions in wa e
bodies; he e o e, a wide ange o ash concen a ions was es ed,
ying o ep esen he po en ial a iabili y o di e en na u al
scena ios. Six di e en concen a ions o he ash–medium
solu ions we e used du ing es ing (3.12, 6.25, 12.5, 25.0,
50.0, 75.0 g L
1
), plus ou con ols pe concen a ion.
Tes s we e ini ia ed using new-bo ns less han 24 h old,
o igina ing om he hi d o i h b ood o he cul u e. Fo each
ash ype, 150 daphnids we e used. This sample size was di ided
in o i e indi iduals pe es essel o each concen a ion wi h
ou eplica es and one con ol pe concen a ion. The es was
conduc ed o 48 h and he immobilisa ion o neona es was
documen ed a 24 and 48 h. Immobilisa ion o neona es is de ined
he e as indi idualsno able o swimwi hin 15 s o gen le agi a ion
o he es essel. Du ing his pe iod, he same empe a u e
(20 28C) and pho ope iod (pho ope iod 16 h ligh : 8 h da k)
condi ions as du ing ea ing we e main ained. D. magna we e no
ed du ing he acu e exposu e (USEPA 2016).
S a is ical analysis
The wa e -soluble (leacha es) chemical composi ion esul s
we e subjec ed o p incipal componen analysis (PCA) (RS udio
e sion 5.4.1) o iden i y cons i uen s mos s ongly co ela ed
wi h he di e en ash ypes. This app oach o assessing he
cha ac e is ic componen s in a gi en sample is widely used in
en i onmen al esea ch when dealing wi h complex da ase s
(B i o e al. 2017). The leacha es da a we e chosen o his
analysis, as opposed o he o al elemen s da a, because his is
likely he mos bioa ailable ac ion and, he e o e, he mos
likely o ha e a ec ed he daphnia o e an acu e exposu e.
To iden i y h esholds in he D. magna oxici y esul s and in
ag eemen wi h s anda d p ocedu es (Musse 2006), he da a
we e subjec ed o single- ac o analysis o a iance es s
(RS udio e sion 5.4.1). Whe e signi ican esul s we e iden i-
ied, pos -hoc Dunne ’s analysis was used o es i he esponse
a each concen a ion was signi ican ly di e en o he con ol
g oups and, he e o e, iden i y c i ical h esholds (le hal
concen a ions) in he esponse ela ionships. This enables he
e ec concen a ions (EC
10
¼concen a ion a which 10% o
indi iduals a e immobilised and EC
50
¼concen a ion a which
50% o indi iduals a e immobilised) o each ash o be in e po-
la ed, along wi h he lowes obse ed e ec concen a ion
(LOEC) (Musse 2006). A signi icance le el o 5% (0.05) was
used in all s a is ical es s.
Resul s
Ash chemis y
The o al elemen al composi ion o he six ash ypes o e all
con ained se e al po en ial con aminan s, bu in highly a iable
concen a ions (Supplemen a y ma e ial Table S1). The mos
abundan elemen in all samples was Ca ( ange 11 800–
177 000 mg kg
1
) wi h Al ( ange 1320–22 600 mg kg
1
) and Fe
( ange 979–30 600 mg kg
1
) bo h p esen in high concen a ions
h oughou . The elemen s ound in he lowes o al
728 In . J. Wildland Fi e A. R. Ha pe e al.
concen a ions we e: As ( ange 0.46–9.67 mg kg
1
), Cd ( ange
0.17–1.13 mg kg
1
) and Hg ( ange 0–0.05 mg kg
1
)
(supplemen a y ma e ial Table S1).
pH and elec ical conduc i i y (EC) measu ed in he leacha es
no ably a ied ac oss ash samples, wi h pH le els anging om
mode a ely alkaline in he UK ash (7.9), o s ongly alkaline in
he USA ash (11.2). Equally, EC le els a ied g ea ly om
233 mScm
1
in he SPA ash o 3880 mScm
1
in he AUS ash.
High pH and EC alues we e bo h cha ac e is ic ea u es o he
ash ypes p oducing immobilisa ion o Daphnia magna es ed
(see Acu e oxici y es sec ion). pH wi hin he bioassays hem-
sel es, howe e , was no ably less a iable (7.31–9.08), likely due
o di e ences in dilu ion be ween leacha es and he bioassay
es ing, and he addi ion o he cul u e medium in he la e .
The wa e -soluble (leacha e) composi ion o he ash ypes
was also highly a iable (Table 2), wi h he mos abundan
componen s being SO
42
( ange 1203–10 180 mg kg
1
), Cl
( ange 228–1509 mg kg
1
) and Na ( ange 17–3893 mg kg
1
).
The mino me al and me alloids elemen s we e simila ly he
componen s ound in he lowes concen a ion in he leacha es:
Cd ( ange 0–7 mgkg
1
), Ni ( ange 60–844 mgkg
1
), Zn ( ange
0–140 mgkg
) and Hg ( ange 1–2 mgkg
1
)(Table 2).
Some soluble elemen s occu ed in pa icula ly high le els,
highligh ing he a ia ion in elemen con en wi hin he ash
(Table 2). Fo example, in he UK sample, PO
43
(620 mg kg
1
)
and me als such as Fe (4378 mgkg
1
) and Mn (9292 mgkg
1
)
we e no ably high in compa ison wi h he o he ash ypes. The e
we e also no ably high le els o , o example, Ca
(5864 mg kg
1
) and SO
42
(32 289 mg kg
1
) in he CAN
sample; B (85 mg kg
1
) and Na (3893 mg kg
1
) in he AUS
sample; and Cu (5158 mgkg
1
) and As (329 mgkg
1
) in he
URIA sample (Table 2).
The wa e -soluble concen a ions o each elemen we e ela-
i ely low when compa ed wi h he o al d y concen a ion wi hin
each ash ype (Table 2). On a e age, he p opo ions o wa e -
soluble Al, Pb, Mn, Fe, Zn we e ,1% o al d y weigh ; As, Si,
Ca, P,Ni, Cu, Cd we e ,5% and Mg was ,10%. The le els o Na
(2–77%) and Hg (5–57%) solubili y we e highly a iable and
hey a e clea ly he mos soluble o he componen s analysed.
PCA iden i ied h ee p ima y componen s explaining 79% o
he o al leacha es da ase a iance (PC1 ¼41%; PC2 ¼23%;
PC3 ¼15%) (Table 3). PC1 is mos s ongly posi i ely co e-
la ed wi h Mn, Fe, Zn, As, Pb and PO
43
le els and mos
s ongly nega i ely co ela ed wi h pH, EC, NO
3
,Cl
,Hg
and SO
42
(Table 3 and Fig. 1). A biplo o he s anda dised PC1
and PC2 alues (Fig. 1) shows which componen s bes cha -
ac e ised each ash ype and pH, EC,NO
3
,Cl
, Hg and SO
42
we e mos closely co ela ed wi h he h ee ash ypes p oducing
signi ican immobilisa ion o D. magna, whe eas Al, Cu, Ni,
NH
4
þ
, As, Fe, Mn, PO
43
, Pb, Cd we e mo e closely co ela ed
wi h he h ee non- oxic ash ypes (Fig. 1).
Thi y- i e PAHs we e analysed ac oss he ash ypes includ-
ing he 16 USEPA p io i y PAHs, which p o ide he ocus o he
ollowing discussion (Table 4). The o al concen a ion o hese
Table 2. Wa e -soluble chemical composi ion o he six ash ypes ob ained by leaching es s
Solubili y o elemen s p o ided in b acke s as a pe cen age (%) o he o al ash composi ion. Elec ical conduc i i y (EC) gi en in mScm
1
. The symbol (–) is
used o deno e alues no able o be calcula ed owing o he d y weigh o he componen no being es ed o he alue being 0
Ash ypes
AUS USA CAN URIA SPA UK
(mg kg
1
) pH 11.1 – 11.2 – 10.3 – 10.3 – 9.1 – 7.9 –
EC 3880 – 2570 – 2500 – 1505 – 233 – 293 –
Al 0 – 4 (0.02) 0 – 20 (0.2) 0 – 0 –
Si 45 (2.17) 182 (8.81) 27 (1.54) 133 (5.59) 25 (1.13) 27 (1.86)
Ca 55 (0.03) 136 (0.06) 5864 (3.60) 580 (1.97) 1101 (0.83) 114 (0.97)
PO
43–
10 (0.65) 10 (0.06) 1 (0.01) 27 (0.35) 10 (0.17) 620 (7.50)
NH
4
þ
8 – 9 – 0 – 33 – 4 – 20 –
DOC 496 – 130 – 1331 – 1272 – 93 – 198 –
Cl
1509 – 1494 – 1139 – 955 – 230 – 228 –
NO
3
207 – 232 – 206 – 104 – 24 – 26 –
SO
42
4065 – 10 180 – 32 289 – 5600 – 3370 – 1203 –
B 85–17– 6 –12–4–1–
Na 3893 (77.2) 831 (18.1) 860 (27.6) 1766 (49.6) 17 (1.49) 148 (22.3)
Mg 377 (3.81) 26 (0.12) 3067 (25.6) 328 (5.13) 232 (4.23) 172 (6.36)
(mgkg
1
)F
340 – 3260 – 460 – 5080 – 9300 – 800 –
Mn 0 – 0 – 68 (0.01) 656 (0.07) 136 (0.04) 9292 (0.65)
Fe 205 – 643 – 553 (0.06) 2172 (0.03) 406 – 4378 (0.06)
Ni 0 – 0 – 0 – 844 (3.84) 0 – 59 (0.37)
Cu 423 (2.01) 280 (0.54) 198 (0.68) 5158 (12.9) 147 (0.49) 340 (0.68)
Zn 0 – 0 – 0 – 0 – 0 – 140 (0.08)
As 18 (1.17) 26 (1.12) 6 (1.21) 329 (7.40) 102 (1.05) 259 (5.96)
Cd 1 (0.42) 0 – 1 (0.59) 0 – 7 (2.83) 2 (0.18)
Hg 1 (44.9) 2 (57.4) 2 (10) 1 (5.96) 1 (17.9) 1 (4.91)
Pb 16 (0.05) 7 (0.02) 3 (0.01) 5 (0.01) 7 (0.01) 64 (0.06)
Eco oxicology o wild i e-gene a ed ash In . J. Wildland Fi e 729
p io i y con aminan s anged om 1155 o 14 078 ng g
1
ash,
he highes o al being ound in he UK ash o igina ing om an
upland g assland ecosys em in sou h Wales (P16 EPA PAHs:
12 336 ng g
1
ash) (Table 4). No ably high PAHs concen a ions
we e also ound in he CAN (P16 EPA PAHs: 7486 ng g
1
) and
he SPA ash (P16 EPA PAHs: 4393 ng g
1
ash) (Table 4).
The p opo ion o he me hyla ed and non-me hyla ed PAHs
was e y simila in all he samples wi h app oxima ely h ee
imes mo e non-me hyla ed PAHs in each ash ype, excep he
USA ash, which con ained o e 15 imes he amoun o non-
me hyla ed PAHs (Table 4). The e was also a p edominance o
wo- ing PAHs in all he samples. Gene ally, he quan i y o
each ing ype dec eases sequen ially wi h he numbe o ings,
2.3.4.5 and 6 wi h he excep ion o he USA sample,
which had a ela i ely simila quan i y o h ee-, ou -, i e-
and six- ing PAHs. The p edominan wo- ing PAH in all
samples was naph halene. Phenan h ene was he mos common
h ee- ing PAH, excep in he UK sample whe e i was ace-
naph hylene. All h ee o hese abundan PAHs (naph halene,
phenan h ene and acenaph hylene) a e classi ied as EPA p io -
i y con aminan s (Table 4).
Acu e oxici y es
High le els o D. magna immobilisa ion we e eco ded a bo h
24- and 48-h exposu e o h ee o he six ash ypes es ed: AUS,
USA and CAN (P,0.001 o all h ee ash ypes) (Fig. 2;Tables 5
and 6). The esponse ela ionships iden i y he AUS ash as he
mos oxic, wi h a 100% immobilisa ion o D. magna indi iduals
a less han 25 g ash L
1
wi hin he i s 24 h o exposu e (Table 5;
Fig. 2). The immobilisa ion e ec o bo h he No h Ame ican ash
samples (USA and CAN) we e ela i ely simila , wi h 48-h EC
50
being achie ed a 20 and 26 g ash L
1
espec i ely, despi e he
no ably di e en sou ce ege a ion (Table 6;Fig. 2). In con as ,
no signi ican immobilisa ion occu ed in esponse o he
emaining h ee ash ypes (URIA, SPA and UK) (Tables 5 and 6).
The UK ash did no p oduce any obse able immobilisa ion
ac oss any o he es concen a ions a e 48 h o exposu e. The
Spanish samples (URIA, SPA) only p oduced low a es o
immobilisa ion a he highes concen a ions (Tables 5 and 6).
Discussion
O e all ash chemical p ope ies
The o al concen a ion o each elemen wi hin he six ash ypes
showed a wide a iabili y (Supplemen a y ma e ial Table S1).
These a ia ions may be explained by he accumula i e capaci y
o he di e en ege a ion ypes, aking up di e en le els o
elemen s om he soil and su ounding en i onmen (Pe al a-
Videa e al. 2009;B i o e al. 2017). Fi e dynamics (e.g. bu n
empe a u e) and soil p ope ies a e also impo an ea u es in
he composi ion o elemen s wi hin ash (Pi man 2006;Bodı´ e al.
2014;Chen e al. 2018). In gene al, oxides and hyd oxides o Ca,
Mg, Si and P pa icula ly end o be abundan in wild i e ash
(Pe ei a and U
´beda 2010;Sil a e al. 2015) as ound in he ash
es ed he e (Supplemen a y ma e ial Table S1).
O e all, he wa e solubili y o he s udied elemen s in all ash
ypes was low (,20% excep o Na and Hg). This ag ees wi h
p e ious indings (Khanna e al. 1994;San ı´n e al. 2015;Sil a
e al. 2015;B i o e al. 2017). The mos abundan ions in all
Table 3. Rela i e con ibu ion o he 24 wa e -soluble ash cons i uen s
and pa ame e s o ou o he signi ican p incipal componen s (PCs) o
he six ash ypes de i ed om p incipal componen s analysis
Cumula i e p opo ion (%) o he a iance explained by each p inciple
componen also p o ided. EC, elec ical conduc i i y; DOC, dissol ed
o ganic ca bon
PC1 PC2
pH 0.29 0.11
EC 0.26 0.02
Al 0.00 0.40
Si 0.10 0.26
Ca 0.13 0.18
PO
43
0.27 0.08
NH
4
þ
0.15 0.36
DOC 0.12 0.16
Cl
0.27 0.08
NO
3
0.27 0.01
SO
42
0.18 0.15
B0.14 0.03
Na 0.16 0.14
Mg 0.14 0.17
F
0.05 0.10
Mn 0.28 0.07
Fe 0.27 0.07
Ni 0.04 0.39
Cu 0.02 0.40
Zn 0.27 0.10
As 0.23 0.27
Cd 0.11 0.18
Hg 0.19 0.14
Pb 0.26 0.10
Cumula i e p opo ion (%) 0.41 0.64
–0.5
–0.5
–2
–1
0
1
2
0
0.5
0
–1–2 0 1 2
0.5
PC1
PC2
Fig. 1. Rep esen a ion o he o dina ion in he i s wo axes (PC1 and PC2)
p oduced by p incipal componen analysis (PCA) o he wa e -soluble chemi-
cal composi ion o he six ash ypes s udied. EC, elec ical conduc i i y.
730 In . J. Wildland Fi e A. R. Ha pe e al.
leacha es we e SO
42
,Cl
and Na
þ
(Table 2), likely owing o
hem o ming e y soluble sal s (i.e. sul a es o chlo ides). These
componen s a e hus commonly ound in high concen a ions in
he dissol ed esidue o ash (F ei as and Rocha 2011;San ı´n
e al. 2015)(Table 2). In con as , hea y me als such as Cd, Ni
and Zn showed he lowes concen a ion in he leacha es owing
o being ela i ely insoluble in alkaline (pH 8–10) condi ions,
p ecipi a ing mainly as hyd oxides (Weine 2012). These esul s
a e simila o hose ound in o he s udies assessing pos - i e
uno and ash leacha es in a ange o ecosys em ypes (Jung
e al. 2009;Pe ei a e al. 2011) and in ag eemen wi h he
gene al concen a ion end o alkali (Na, K) .alkaline
(Ca, Mg) .. hea y me als (Pb, Cd and Hg) ound by San ı´n
e al. (2015) in eucalyp o es ash.
Ash ypes and elemen solubili y
Despi e he o e all simila i ies in ash solubili y in he ash lea-
cha es, he e a e also subs an ial a ia ions among he ash ypes,
making hei chemical p o iles no ably di e en . B i o e al.
(2017), assessing B azilian Ce ado ash ypes, also ound he e
we e li le quali a i e di e ences in he o e all composi ion o
he di e en ash es ed, bu la ge a ia ions in he concen a ion
o he chemical elemen s be ween sampling a eas.
The PCA analysis allowed de ec ion o key di e ences in
he composi ion o he ash ypes s udied he e. The UK ash
leacha e has a dis inc ly soluble p o ile in compa ison wi h
he o he s. PCA analysis shows se e al hea y me als (Mn,
Fe, Zn and Pb) and PO
43
o be cha ac e is ic elemen s o
he UK ash leacha e (Fig. 1). This leacha e shows high
Table 4. Concen a ion and composi ion o polycyclic a oma ic hyd oca bons (PAHs) ound in each ash ype
PAHs wi h he no a ion † a e US En i onmen al P o ec ion Agency p io i y PAHs (Kei h 2015)
Ash ype
PAH (ng g
1
) AUS USA CAN URIA SPA UK
Naph halene† 744.9 1148.6 4540.3 2861.4 1147.4 8010.9
Biphenyl 293.5 654.3 1851.1 1953.3 1019.1 1677.6
Acenaph hylene† 75.2 9.7 377.3 323.7 28.1 3337.2
Acenaph hene† 13.2 1.9 84.5 44.3 9.6 198.2
Fluo ene† 18.1 3.4 99.7 104.0 26.7 380.4
Dibenzo hiophene 4.7 3.5 45.6 11.9 7.7 29.2
Phenan h ene† 140.5 121.2 1049.5 487.4 170.5 1131.8
An h acene† 19.1 10.4 126.3 76.1 15.6 193.2
Fluo an hene† 36.1 27.5 285.6 128.1 26.7 262.2
Py ene† 37.3 16.9 215.8 112.9 20.7 257.8
Benzo(c)phenan h ene 2.9 3.0 12.7 9.7 2.9 12.7
Benz(a)an h acene† 11.9 8.4 32.3 23.9 4.4 35.3
T iphenylene 7.7 53.7 44.6 16.0 7.1 14.7
Ch ysene† 14.3 26.5 38.9 27.4 7.3 36.3
Benzo(b) luo an hene† 16.3 29.2 335.0 83.1 7.9 95.4
Benzo(k) luo an hene† 4.2 8.9 92.8 25.0 3.0 32.3
Benzo(e)py ene 11.3 44.1 291.8 90.9 8.8 57.3
Benzo(a)py ene† 7.2 6.1 74.9 31.7 2.4 34.2
Pe ylene 3.4 1.3 20.1 14.8 1.9 13.2
Indeno (1,2,3-c,d)py ene† 5.2 5.5 30.2 15.9 1.5 27.0
Dibenzo(a,h)an h acene† 1.9 2.6 10.5 14.8 0.9 5.2
Benzo(g,h,i)pe ylene† 9.6 18.2 92.5 33.0 2.8 40.7
2-Me hylnaph alene 225.7 66.9 1702.3 751.4 274.3 2118.2
1-Me hylnaph halene 168.2 34.3 1204.2 683.7 318.7 1663.5
2,3-Dime hylnaph halene 43.2 7.1 388.0 138.0 166.8 235.0
2,3,6-T ime hylnaph alene 19.4 3.4 140.5 50.0 20.9 80.0
4-Me hyldibenzo hiophene 7.9 1.0 32.5 49.5 32.8 35.0
2-Me hylphenan h ene 21.2 6.3 154.3 87.7 44.7 153.6
2,8-Dime hyldibenzo hiophene 3.0 0.7 14.1 12.0 5.2 29.4
1,6-Dime hylphenan h ene 31.9 5.5 147.2 109.0 51.5 120.0
2,4,7-T ime hyldibenzo hiophene 0.3 0.1 0.8 1.0 0.5 3.3
1,2,8-T ime hylphenan h ene 8.4 2.1 67.2 47.8 43.4 35.5
1-Me hylpy ene 9.8 1.4 24.5 31.3 11.3 32.9
2-Me hylch ysene 3.8 1.7 4.5 5.9 1.9 6.4
7,12-Dime hylbenz(a)an h acene 2.9 0.5 1728.4 64.2 90.9 12.5
S16 PAHS 1155 1445 7486 4393 1476 14 078
S35 PAHS 2024 2336 15 360 8521 3586 20 408
SMe hyla ed 546 131 5608 2031 1063 45 25
SNon-me hyla ed 1479 2205 9752 6489 2523 158 83
% Me hyla ed 27 6 37 24 30 22
Eco oxicology o wild i e-gene a ed ash In . J. Wildland Fi e 731
0
Con ol
NOEC
AUS 24-h concen a ion– esponse ela ionship AUS 48-h concen a ion– esponse ela ionship
LOEC
EC10 EC50
3.12 6.25 12.5 25 50 75
10
20
30
40
50
60
70
80
90
100
0
Con ol
NOEC
LOEC
EC10 EC50
3.12 6.25 12.5 25 50 75
10
20
30
40
50
60
70
80
90
100
0
Con ol
NOEC
USA 24-h concen a ion– esponse ela ionship
Su i al a e (%)
USA 48-h concen a ion– esponse ela ionship
LOEC
EC10 EC50
3.12 6.25 12.5 25 50 75
10
20
30
40
50
60
70
80
90
100
0
Con ol
NOEC LOEC
EC10 EC50
3.12 6.25 12.5 25 50 75
10
20
30
40
50
60
70
80
90
100
0
Con ol
NOEC
CAN 24-h concen a ion– esponse ela ionship CAN 48-h concen a ion– esponse ela ionship
LOEC
Concen a ion (
g
L–1)
EC10
EC50
3.12 6.25 12.5 25 50 75
10
20
30
40
50
60
70
80
90
100
0
Con ol
NOEC
LOEC
EC10 EC50
3.12 6.25 12.5 25 50 75
10
20
30
40
50
60
70
80
90
100
Fig. 2. Concen a ion esponse ela ionship a e 24 and 48 h o exposu e. NOEC, no obse ed e ec concen a ion; LOEC, lowes obse ed e ec
concen a ion; EC
10
, e ec concen a ion a which 10% o daphnids a e immobilised; EC
50
, e ec concen a ion a which 50% o daphnids a e
immobilised.
Table 5. Immobilisa ion pe cen age o Daphnia magna a 24 h
Es ima es o Lowes Obse ed E ec Concen a ion (LOEC) (Dunne ’s es ; P,0.05), EC
10
and EC
50
(g L
1
). One-way analysis o a iance P alues also
p o ided, es ing i obse ed immobilisa ion o each ash ype was signi ican ly di e en o he con ol. E ec concen a ion (EC
x
) e e s o he concen a ion o
subs ance equi ed o p oduce x% (10 o 50) o he es indi iduals immobilised. The symbol (–) is used o deno e alues no able o be calcula ed
Concen a ion (g L
1
) pH LOEC EC
10
EC
50
P alue
Con ol 3.12 6.25 12.5 25 50 75 (g L
1
)(gL
1
)(gL
1
)
AUS 0 5 10 75 100 100 100 8.81 6.25 6.25 11 ,0.001
USA 0 0 0 5 35 100 100 8.78 25 14 30 ,0.001
CAN 0 0 0 5 10 100 100 8.23 50 25 37 ,0.001
URIA 0 0 0 0 0 0 5 8.17 – – – 0.451
SPA 0 0 0 0 0 5 0 7.88 – – – 0.451
UK 0 0 0 0 0 0 0 7.56 – – – –
732 In . J. Wildland Fi e A. R. Ha pe e al.
concen a ions o soluble Fe, Mn and PO
43
in compa ison
wi h he o he ash ypes (Table 2). The pH (7.9) o he UK
leacha e was 1 o 3 uni s lowe han he ex ac s om he
o he samples (Table 2). These less alkaline condi ions
a ou he solubili y o me als and P compa ed wi h he
o he samples whe e he me als end o p ecipi a e as hyd o-
xides o pH alues abo e 8–9 and he phospha e as
hyd oxyapa i e o pH alues .8.5 ( o example see: Diaz
e al. 1994;S umm and Mo gan 1995).
A cha ac e is ic componen o he CAN sample (iden i ied by
PCA, Fig. 1) was he high le els o soluble Ca, despi e he o al
concen a ion in d y ash being ela i ely simila o ha o he
AUS, SPA and USA ash (Table 2). I is unclea why he solubili y
o Ca is no ably highe in he CAN ash in compa ison wi h he
o he ash ypes (Jung e al. 2009;B i o e al. 2017), bu i may be
esponsible o he educed PO
43
le els (1.2 mg kg
1
) in he
CAN leacha e as P has a endency o p ecipi a e in he p esence o
Ca (Diaz e al. 1994). This P–Ca in e ac ion may in luence algal
and cyanobac e ial g ow h (and hus, eu ophica ion) by egula -
ing P le els in eshwa e sys ems (Bladon e al. 2008;Blake
e al. 2009). In he b oade con ex , Ca is no no mally conside ed
haza dous, bu can signi ican ly in luence he o e all oxici y o
ash elua es (e.g. i s s ong ela ionship wi h SO
42
leaching)
(Moun e al. 1997;Tian e al. 2018). S ie ns o¨m e al. (2013)
e en p opose ha Ca migh be one o he key elemen s esponsi-
ble o he eco oxici y o ash elua es on he c us acean Ni oc a
spinipes, despi e Ca no being classi ied as indi idually eco oxic.
The CAN ash es ed he e p oduced signi ican immobilisa ion o
Daphnia magna o e he 48-h exposu e also po en ially in lu-
enced by i s high Ca concen a ion.
Fo he AUS ash sample, he le els o soluble B and Na a e
highe han in he o he ash ypes (Fig. 1,Table 2). These
elemen s a e o en ound in high concen a ions in ash leacha es
(Jung e al. 2009;Pe ei a e al. 2011), pa icula ly B in o he
eucalyp us ash es ed (F ei as and Rocha 2011). High Na
þ
le els
in eshwa e sys ems can p esen an issue o wa e pu i ica ion
p ocesses as hey canno be emo ed using con en ional me h-
ods (Smi h e al. 2011). Unlike epo ed by Sil a e al. (2015),
whe e he p incipal po en ial oxic componen s o hei eucalyp
ash we e Mn and Zn, nei he o hese elemen s we e ound in he
eucalyp (AUS) ash analysed he e. This u he highligh s he
di e ences in ash composi ion compa ing indi idual i e e en s
and ecosys em ypes (Bodı´ e al. 2014).
In he URIA ash, he mos de ining componen s we e Cu,
Al, Ni, NH
4
þ
and As (Fig. 1). This ash con ained compa a-
i ely high concen a ions o soluble Cu (5158 mgkg
1
) and
he ca cinogen As (329 mgkg
1
). Simila ele a ed soluble
le els o Cu ha e, howe e , been ound in mixed eucalyp us
ash (Cu 5100–6200 mgkg
1
)bySan ı´n e al. (2015). The
eason o he signi ican ly highe solubili y a e o Cu in his
hea hland ash (URIA 12.9%, ange excluding URIA 0.49–
2.01%) is wo h u he conside a ion o iden i y a eas o
componen s likely o inc ease he isk o Cu con amina ion.
The concen a ion o As, al hough ele a ed in he URIA (and
UK) sample he e, has been epo ed as highe in se e al o he
wild i e ash samples (e.g. 4000–7300 mgkg
1
in San ı´n e al.
(2015); 42 000 mgkg
1
in Sil a e al. (2015)) and despi e being
abo e he 0.01-mg L
1
Wo ld Heal h O ganiza ions d inking-
wa e guideline (Wo ld Heal h O ganiza ion 2011), i did no
appea o cause signi ican immobilisa ion o D. magna in he
URIA o UK ash.
The SPA ash has a ela i ely insoluble o e all p o ile wi h
no ably high concen a ions o he me als Al, Fe, Zn, Cb, Pb and
he me alloid As in he d y ash (Supplemen a y ma e ial S1) bu
limi ed o no leaching o Al, Fe, Zn and Pb in o he wa e -soluble
ex ac (Table 2). Despi e his, Cd p esen ed as a dis inc
p incipal componen o he SPA ash wi h a compa a i ely high
soluble concen a ion (7 mgkg
1
) and as he only sample o
egis e a solubili y pe cen age g ea e han 1% (2.85%). Simila
d y quan i ies o Cd we e eco ded by B i o e al. (2017)
assessing B azilian Ce ado ash ypes (0.1–0.3 mg kg
1
) bu
Cd solubili y was lowe in hese ash ypes (,0.01%).
PAHs composi ion
The o ganic ac ion o ash may also con ain o ganic con-
aminan s o biological conce n (Vila-Escale´ e al. 2007;Chen
e al. 2018). The da a a ailable on PAHs elease ollowing a i e,
howe e , a e qui e limi ed (Vila-Escale´ e al. 2007;Kim e al.
2011;Campos e al. 2012;Rey-Salguei o e al. 2018).
The concen a ions o PAHs ound in he ash analysed he e
a e also widely a iable, wi h a ange o 1155 ng g
1
in he
AUS ash o 14 078 ng g
1
in he UK ash (16 USEPA p io i y
PAHs) (Table 4). The alues con ained wi hin he ash es ed
he e a e subs an ially highe han hose p esen ed by Oli ella
e al. (2006) es ing wild i e ash om pine and oak o es s
(P12PAHs:1–19ngg
1
ash). The lowes concen a ion,
Table 6. Immobilisa ion pe cen age o Daphnia magna a 48 h
Es ima es o Lowes Obse ed E ec Concen a ion (LOEC) (Dunne ’s es ; P,0.05), EC
10
and EC
50
(g L
1
). One-way analysis o a iance P alues also
p o ided, es ing i obse ed immobilisa ion o each ash ype was signi ican ly di e en o he con ol. E ec concen a ion (EC
x
) e e s o he concen a ion o
subs ance equi ed o p oduce x% (10 o 50) o he es indi iduals immobilised. The symbol (–) is used o deno e alues no able o be calcula ed
Concen a ion (g L
1
) pH LOEC EC
10
EC
50
P alue
Con ol 3.12 6.25 12.5 25 50 75 (g L
1
)(gL
1
)(gL
1
)
AUS 0 10 15 85 100 100 100 8.93 6.25 5.5 9.5 ,0.001
USA 0 0 5 5 65 100 100 9.08 6.25 14 20 ,0.001
CAN 0 0 0 5 40 100 100 7.81 25 14 26 ,0.001
URIA 0 0 0 0 0 0 10 8.03 – – – 0.451
SPA 0 0 0 0 0 5 0 7.58 – – – 0.451
UK 0 0 0 0 0 0 0 7.31 – – – –
Eco oxicology o wild i e-gene a ed ash In . J. Wildland Fi e 733
oundin heAUSash ype(
P16 EPA PAHs: 1155 ng g
1
ash), was o a compa able le el wi h hose ound by
Sil a e al. (2015) assessing d y wild i e ash in a p edomi-
nan ly eucalyp ecosys em in Po ugal (P16 EPA PAHs:
1100 ng g
1
ash). The ull ange o PAH concen a ions
ound he e a e wi hin he ange 1000–50 000 ng g
1
(P16 EPA
PAHs) ound by San ı´n e al. (2017) analysing PAHs in pine
o es loo and wood unde wild i e cha ing and slow
py olysis.
The UK ash shows a much highe PAHs concen a ion han
he o he ypes (Table 4). I is unclea why his is he case as
no o he esea ch has been conduc ed on he PAHs composi-
ion o wild i e ash o igina ing om compa able g assland
ecosys ems. The ype o uel and a ia ions in combus ion
empe a u es and oxygen a ailabili y a e hough o s ongly
a ec he concen a ion and ype o PAHs in ash (Enell e al.
2008;Rey-Salguei o e al. 2018). Chen e al. (2018) ound
ha PAHs concen a ions we e signi ican ly highe in black
wild i e ash (mode a e bu n se e i y) in compa ison wi h
whi e wild i e ash (high bu n se e i y). This was also ue
o he ash ypes es ed he e wi h he da ke (da k g ey-black)
ash samples (UK, URIA, CAN) con aining a much highe
concen a ion o PAHs han he ligh e (ligh g ey-whi e)
samples (AUS, USA, SPA) (Table 4). Al hough a ia ions
in combus ion comple eness could be ela ed o PAHs con en
he e, he p opo ion o me hyla ed PAHs in he UK ash is
simila o ha o he o he samples es ed (Table 4). The
p opo ion o me hyla ed/ o al PAHs is usually conside ed an
indica o o combus ion comple eness as du ing combus ion,
he me hyla ed componen o he compound is los i s
(Keiluwei e al. 2012)(Table 4).
The high p esence o low-molecula -weigh and he e o e,
g ea e - ola ili y PAHs (i.e. Naph halene (Naph) and Phenan-
h ene (Phe)) in he ash es ed he e may seem con adic o y as i
can be expec ed ha hese compounds would be los du ing a
i e. I is, howe e , likely ha hese PAHs p e e en ially econ-
dense in he ash laye and a e e ained in mic opo ous s uc u es
o py ogenic ma e ial (San ı´n e al. 2017). O he s udies suppo
his idea, epo ing high concen a ions o Naph and Phe (Kim
e al. 2011) o Naph, Ch ysene (Ch y), Benz(a)an h acene and
Acenaph hylene (Campos e al. 2012) om wood bu ning. Ash
s udies o beech and simila species (Bund e al. 2001) we e
domina ed by Naph and by Benzo(ghi)pe ylen, Benzo(b)-
luo an hene, Benzo(k) luo an hene, Ch y, T iphenylene and
Phe bu a lowe concen a ions.
Cau ion is equi ed when making compa isons be ween he
PAHs alues ac oss s udies as he e a e impo an a ia ions in
he me hodologies employed. Some s udies examine PAHs in
ash (Enell e al. 2008;Sil a e al. 2015) o sedimen (Oli ella
e al. 2006;Kim e al. 2011) and o he s in s eam wa e (Oli ella
e al. 2006), pond wa e (Vila-Escale´ e al. 2007), uno wa e
(Campos e al. 2012) o aqueous ex ac s (Enell e al. 2008;Sil a
e al. 2015), meaning concen a ion and composi ional di e -
ences a e o be expec ed. I is likely he high o e y high PAHs
concen a ions eco ded in he ash s udied he e would be
d ama ically educed i he leachable ac ion o he samples
we e es ed, as opposed o o al concen a ions, he e o e mak-
ing he po ion mo e accessible o in e ac wi h aqua ic auna
lowe (F isˇ a´k e al. 2019).
Implica ions o oxicology
The wild i e ash analysis conduc ed he e no only demons a es
he high a iabili y in he concen a ion o chemical componen s
o ash p oduced in con as ing ecosys ems (Table 2), bu also he
di e ences in hei po en ial oxic e ec s in aqua ic sys ems
(Table 5 and 6). Signi ican oxici y was obse ed o D. magna
o e he acu e exposu es o h ee o he six ash ypes es ed:
AUS, USA and CAN (Fig. 2;Table 5 and 6). Ash ype and
composi ion, he e o e, seem c ucial o he le el o oxici y on
cladoce an species, as also demons a ed p e iously (Campos
e al. 2012;Sil a e al. 2015;B i o e al. 2017).
The combina ion o he chemical da a wi h he D. magna
immobilisa ion esul s highligh s se e al possible ela ionships
(Fig. 1). The PCA iden i ied pH and EC as wo o he pa ame e s
s ongly cha ac e is ic o he h ee ash ypes causing signi ican
D. magna immobilisa ion (AUS, USA, CAN) (Table 3,Fig. 1).
I is well es ablished ha ex eme alues o pH and EC ha e a
de imen al impac on zooplank on species (Moun e al. 1997;
F anklin e al. 2000;Sil a e al. 2015). The pH alues in he
bioassays hemsel es, howe e , we e no ably lowe and less
a iable han in he leacha e esul s used du ing he PCA
analysis and wi hin a ange hough accep able o he su i al
o D. magna and simila cladoce an species (OECD 2004)
(Table 5 and 6). C ucially, howe e , he ela ionship be ween
pH and immobilisa ion is e y simila be ween he leacha es and
bioassays pH esul s, wi h highe pH alues cha ac e is ic o he
ash ypes p oducing immobilisa ion in D. magna. This pe haps
sugges s ha pH has an indi ec e ec on D. magna immobilisa-
ion as pH can also in luence he dissolu ion o elemen s om
ash in o wa e and he e o e he ela i e oxic po en ial o o he
ash componen s (Fedje e al. 2010). Low pH alues, o exam-
ple, encou age he leaching o oxyanion- o ming (As, B, C , Sb
and V) and ca ion- o ming elemen s (Ca), and neu al pH g ea ly
educes he leaching o ampho e ic elemen s (e.g. Al, Cd, Cu, Pb
and Zn) (Fedje e al. 2010). The mo e neu al pH o he UK
sample, howe e , does no seem o ha e educed he leaching o
Al, Cd, Cu and Pb. pH has an inconsis en ela ionship wi h
oxici y, and, o en, esul s a e di icul o in e p e (Wilde e al.
2006;Sil a e al. 2015).
The in luence o key nu ien s on D. magna immobilisa ion is
pe haps less well es ablished (Smi h e al. 2011)(Fig. 1), as ions
such as Cl
and NO
3
a e equi ed a minimum le els o suppo
aqua ic li e. Howe e , he PCA also iden i ied high concen a-
ions o Cl
and NO
3
as being key cha ac e is ic componen s
o he h ee oxic ash ypes, pa icula ly he mo e oxic AUS and
USA ash (Table 3 and Fig. 1). Many an h opogenic (e.g. oil and
gas p oduc ion, i iga ion me hods and indus ial and ag icul-
u al p ocesses) and na u al (e.g. sedimen po e wa e s and
bu ning) condi ions ha e been shown o inc ease nu ien con-
cen a ions o oxic le els (e.g. Hoke e al. 1993;Fe ei a e al.
2005;Mas and Clow 2008). Sco and C unkil on (2000)
demons a ed NO
3
p oduces immobilisa ion o D. magna a
462 mg L
1
wi h no obse able e ec concen a ion a
358 mg L
1
. Simila ly, Moun e al. (1997) es ima ed a
concen a ion o 1000–2000 mg L
1
as he concen a ion o
Cl
equi ed o p oduce EC
50
in Ce iodaphnia dubia. This
sugges s despi e he co ela ions be ween NO
3
and Cl
wi h
he oxic ash ypes ound he e, he ela i ely low quan i ies o
734 In . J. Wildland Fi e A. R. Ha pe e al.
