1
VARCLIREFOR
FORWARDS p ojec
G an Ag eemen G-04-2023-3
The P ojec unde his Ag eemen is unded unde G an Ag eemen 101084481 — FORWARDS
— HORIZON-CL6-2022-CLIMATE-01
Deli e able 1.2: VARCLIREFOR Final echnical epo
René Ve bu g, Ma ijke an Kuijk, Robin Aans oo (U ech Uni e si y)
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P e ace
This Deli e able 1.2 desc ibes he me hods applied in he VARCLIREFOR p ojec , unded wi hin he
FORWARDS conso ium.
The VARCLIREFOR p ojec was conduc ed be ween May 2024 (M1) and Augus 2025 (M16) in he
U ech se Heu el ug a ea, which is a ca. 20,000 ha la ge o es a ea ound in he cen al pa o he
Ne he lands. The o es s on he U ech se Heu el ug a e domina ed by P. syl es is, bu local co-
dominance o o he species occu like P. menziesii, B. pendula, Q. obu and F. syl a ica. Mos o es
is loca ed on o me sandy (glacial) mo aines, and soils a e highly a ec ed by con inuous ni ogen
deposi ion om a ic, indus y and ag icul u e, soil acidi ica ion and inc easing d ough pe iods due
o clima e change. In mos a eas, only deep g ound wa e le els a e ound, indica ing mos o he
o es is solely ain ed and ph ea ic wa e s o ed in he uppe soil laye s a e he mos impo an
wa e sou ces o ege a ion g ow h.
The VARCLIREFOR p ojec s udied su i al and g ow h o na u ally egene a ed and plan ed ee
saplings in new ield ials in one expe imen al si e on he U ech se Heu el ug a ea. Fu he mo e,
pa e ns in soil ac o s in exis ing o es plo s wi hin he la ge o es a ea whe e s udied.
New ials (new o es and sapling plo s) whe e loca ed in one o he Fo es Managemen Uni s
(FMU) o he Du ch S a e Fo es y Se ice (FMU uni ‘AUS’), while he exis ing ials ca. 781 o es
plo s) whe e loca ed in h ee sepa a e FMUs (‘AUS’, ‘AML’ and ‘VUU’).
Da a collec ed du ing he execu ion o VARCLIREFOR a e s o ed in da a iles and uploaded o he
Fo es Obse a o y o FORWARDS.
The au ho s
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Table o con en s
P e ace ........................................................................................................................................ 2
1. In oduc ion ............................................................................................................................. 4
2.Plo se up and sapling plan ing in WP2........................................................................................ 6
2.1 Exis ing plo s wi hin he WP2 wo king a ea ........................................................................... 6
2.2 Addi ional semi-pe manen plo s ......................................................................................... 6
2.2.1 Addi ional plo le el da a .................................................................................................. 7
2.2.2 T ee le el da a o ali e and s anding dead ees ................................................................. 9
2.2.3 Lying dead wood ............................................................................................................ 10
2.2.4 Na u al egene a ion ...................................................................................................... 11
2.3 Sapling plan ing ................................................................................................................. 11
2.3.1 Sapling ea men ........................................................................................................... 13
2.3.2 Sapling measu emen s .................................................................................................... 14
2.4 Na u al ec ui men measu emen s ................................................................................... 15
3. Soil sampling and ield measu emen s ..................................................................................... 16
3.1 Soil sampling in WP2 ......................................................................................................... 16
3.2 Soil sampling in WP3 ......................................................................................................... 16
3.3 Li e collec ion and p ocessing in WP3............................................................................... 16
3.4 Ligh a ailabili y ................................................................................................................ 16
3.5 Decomposi ion measu emen s ........................................................................................... 17
3.5.1 Tea Bag P epa a ion, deploymen , and p ocessing ........................................................ 17
3.5.2 Decomposi ion ime se ies .......................................................................................... 18
3.5.3 Tea Bag Index pa ame e calcula ions .......................................................................... 20
3.6 Soil auna eeding ac i i y .................................................................................................. 20
4. Soil and nu ien p ocessing..................................................................................................... 23
4.1 Soil Analyses ..................................................................................................................... 23
4.1.1 Soil pH........................................................................................................................ 23
4.1.2 Ano ganic ni ogen: NOx, NO3, NO2, NH4, PO4................................................................ 24
4.1.3 Ca ion concen a ions ................................................................................................. 24
4.1.4 Soil o ganic ca bon and C:N a io ................................................................................. 24
4.1.5 Soil O ganic Ma e Con en ........................................................................................ 25
5. WP 3 Fo es plo da a, p ocessing and calcula ions ................................................................... 26
5.1. Plo layou exis ing ield ials (WP3).................................................................................. 26
5.2 T ee g ow h measu emen s ............................................................................................... 26
5.3 Allome ic ela ions ........................................................................................................... 27
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1. In oduc ion
The VARCLIREFOR p ojec consis s o wo wo k packages, each wi h i s unique aim and
me hodologies. This deli e able desc ibes he applied me hods ( ield and labo a o y echniques)
ca ied ou in he wo k packages 2 and 3. All da a we e collec ed in he Na ional Pa k U ech se
Heu el ug in h ee o es managemen uni s: Vuu sche (VUU), Aus e li z (AUS) and Ame ongen-
Lee sum (AML).
WP2
Wo k package 2 ocused on newly es ablished ield ials in he o es managemen uni o
Aus e li z, designed o e alua e ee species ha may be be e adap ed o he inc easingly d y
condi ions o he U ech se Heu el ug. In hese expe imen al plo s, saplings o a ious species we e
plan ed in a con olled se up. Among hese a e so-called “ ich li e species” — species known o
p oduce nu ien - ich li e ha may con ibu e o he de elopmen o a mo e a o able o ganic
laye in he soil. This laye plays a key ole in suppo ing soil biodi e si y and imp o ing nu ien
cycling, po en ially enhancing soil quali y and ecosys em esilience unde changing clima ic
condi ions. The expe imen al se up consis ed o he addi ion o a combina ion o nu ien s and
myco hiza in he plan ing holes o plan ed saplings o suppo he g owing condi ions.
To be e unde s and he g owing condi ions o he plan ed saplings, a ange o soil and
en i onmen al pa ame e s we e measu ed wi hin he plo s. These include canopy co e , soil o ganic
ma e con en , pH, soil o ganic ca bon and CN a io, NH4-N, NO3-N, NO2-N and NOx-N and se e al
ca ions. Addi ionally, soil decomposi ion ac i i y was assessed using he Tea Bag Index (TBI), which
p o ides insigh in o mic obial ac i i y and o ganic ma e b eakdown in he opsoil.
Alongside he plan ed saplings, he na u al egene a ion o ee species was moni o ed o e a pe iod
o ou mon hs, wi h a ocus on heigh g ow h and su i al.
The wo king a ea o WP2 also con ained a ange o exis ing plo s ha o e his o ical da a on ee
diame e a b eas heigh (DBH) and his o ical g ow h. A subse o hese plo s also con ain da a on
dead ees, lying dead wood and eju ena ion.
WP3
Wo k Package 3 ocuses on ee g ow h-, soil- and en i onmen al da a om h ee o es
managemen uni s on he U ech se Heu el ug: Vuu sche, Aus e li z, and Ame ongen-Lee sum. As in
WP2, a comp ehensi e se o pa ame e s was collec ed o cha ac e ize si e condi ions, in a andom
se o 150 plo s. Sampling in hese plo s included li e biomass, canopy co e , soil o ganic ma e
con en (SOM), pH, soil o ganic ca bon and C/N a io, concen a ions o NH4-N, NO3-N, NO2-N and
NOx-N and in a sub-se o hese selec ed plo s, a ious ca ion concen a ions. In WP3, bai lamina
s ips we e used in addi ion o he Tea Bag Index (TBI) me hod o measu e decomposi ion a es,
p o iding in o ma ion on he eeding ac i i y o soil in e eb a es. These measu emen s p o ide a
baseline o unde s anding how local abio ic ac o s in luence o es dynamics and soil p ocesses
ac oss di e en o es s ands.
The ield measu emen s a e complemen ed by long- e m plo da a on ee g ow h measu emen s
de i ed om ca 800 plo s in hese h ee o es managemen uni s. These da a included diame e a
b eas heigh (DBH) measu emen s eco ded in h ee di e en yea s ha co e ed a ime span o
oughly 30 yea s, and ee heigh measu emen s o a selec ion o ees in hese plo s. This da ase
allows o he analysis o g ow h ends o e ime in ela ion o en i onmen al a iables. In addi ion,
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his o ical me eo ological da a we e in eg a ed o help in e p e how long- e m clima ic condi ions
may ha e in luenced o es de elopmen and si e p oduc i i y.
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2.Plo se up and sapling plan ing in WP2
2.1 Exis ing plo s wi hin he WP2 wo king a ea
The WP2 wo king a ea includes 11 plo s ha all wi hin he same g id o plo s as he WP3 plo s as
desc ibed in sec ion 5.1. Da a in hese plo s is collec ed using he same me hodology as in he plo s
o WP3 in h ee di e en pe iods (1994, 2006, 2019). In 2023, using he same me hodology, 23 new
plo s we e es ablished and moni o ed o ee DBH o ca. 20 ees. The o iginal plo s ha e he o ma
Loca ion_Le e Numbe (AUS_G18), he new 2023 plo s ha e he o ma Loca ion_numbe (AUS_1).
No e ha in hese plo s, da a on he exac loca ion o indi idual ees was no collec ed.
In 2024, 38 addi ional semi-pe manen plo s we e ins alled in he WP2 wo king a ea, using a sligh ly
di e en moni o ing me hodology ha allows o indi idual ee moni o ing, as desc ibed in sec ion
2.2.
Table 2.1: O e iew o plo ypes and used moni o ing me hodology in he WP2 wo king a ea.
Plo ype
Me hodology
Indi idual ee loca ion eco ded
Exis ing plo s (1994, 2006, 2019)
See sec ion 5.1.
No
2023 exis ing plo s
See sec ion 5.1.
No
Addi ional semi-pe manen plo s
See sec ion 2.2.
Yes
2.2 Addi ional semi-pe manen plo s
In No embe and Decembe 2024 (M7-M8 o he p ojec ), 38 new semi-pe manen ci cula plo s
we e ins alled in he o es managemen uni o Aus e li z. The plo s a e loca ed in 11
‘demons a ion s ands’, i.e. in 7 s ands domina ed by Pinus syl es is (Sco s pine) (28 plo s o al) and
4 s ands domina ed by Que cus ub a (Ame ican oak) (10 plo s o al) (see Figu e 2.1). Measu emen s
ook place a plo le el and a ee le el, as desc ibed below.
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Figu e 2.1: Loca ion o 38 semi-pe manen ci cula plo s in he FMU o Aus e li z, es ablished in No -
Dec 2024.
2.2.1 Addi ional plo le el da a
In Aus e li z, in each demons a ion s and a 1 ha a ea was de ined in which 2 o 4 ci cula plo s we e
ins alled. Ci cula plo s consis ed o 15m adius ci cles and nes ed 8m adius ci cles (see Figu e 2.2).
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Figu e 2.2: The semi-pe manen ci cula plo s consis o a ci cle wi h 15m adius in which ees
≥10cm DBH we e measu ed and a smalle ci cle wi h 8m adius in which ees ≥5cm DBH we e
measu ed.
Pe plo he ollowing was de e mined:
• coo dina es o he cen e
• yea o o igin
• dominan ee species
• a e age heigh o 2-4 indi iduals o dominan species
• de elopmen phase o he ege a ion, i.e. successional s age
• canopy co e
Coo dina es o he cen e o each plo we e eco ded wi h a GPS. The yea o o igin/es ablishmen
was es ima ed based on da a o he s a e o es se ice. The dominan ee species was de e mined
and de ined as he ee species ha is mos abundan . Heigh o 2-4 indi iduals o he dominan
species was de e mined by using lase equipmen and a e aged. The de elopmen phase o he
o es consis s o se e al classes o successional s ages:
Table 2.2: classi ica ion o successional s ages
Class
Successional s age
1
open phase (no ees)
2
es ablishmen phase (young ees p esen )
3
hicke phase
4
pole s age (closed o es )
5
ee/climax phase
6
de e io a ion phase
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We de e mined canopy co e om he cen e poin o h ee indi idual ees ha we e measu ed o
ex a ai s in May 2025 (see 2.2.2). Canopy co e was measu ed wi h he CanopyApp a ailable a
he AppleS o e, a a heigh o 1.8m.
Addi ionally, in he 8m adius plo s he ollowing was measu ed:
• ee co e
• sh ub co e
The ee co e (abo e 6m) and sh ub co e (0.5-6m) consis o he ollowing pe cen age classes:
Table 2.3: pe cen age classes o ee and sh ub co e
Code
Co e in %
1
0-0.1
2
0.1-1
3
1-5
4
5-10
5
10-25
6
25-50
7
50-75
8
75-90
9
90-100
2.2.2 T ee le el da a o ali e and s anding dead ees
Wi hin he 15m adius ci cle plo s o ees wi h a dbh ≥10cm and wi hin he nes ed 8m adius ci cle
plo s o ees wi h a dbh ≥5cm, he ollowing a iables we e measu ed:
• species
• DBH
• angle and dis ance om cen e
Each ee was iden i ied o species le el and DBH was measu ed wi h a calipe . The loca ion o each
ee was de e mined using he pola coo dina e sys em. The dis ance om he cen e o he plo o
he ee (cen e o he unk base) was measu ed wi h a measu ing ape . The de ia ion (angle) o
each ee is de e mined clockwise ( igh ) in deg ees ela i e o no h using a compass, whe e no h
is a 0°.
S anding dead ees we e also measu ed in he 15m adius ci cle plo s. S anding dead wood was
measu ed in he same way as he ali e s anding ees bu a e eco ded on a sepa a e ab in he
da a ile.
In Ma ch 2025 (M13) we selec ed 3 andom ees in/nea each plo o addi ional measu emen s
including:
• c own dimensions
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3. Soil sampling and ield measu emen s
3.1 Soil sampling in WP2
In he WP2 expe imen al loca ion and he new ield ial sec ion a selec ion o he 153 plan ed sapling
g oups was made so ha 64 g oups we e selec ed o soil analyses. Fi e plan ing g oups pe species
we e andomly selec ed o sampling. In o al, 65 soil samples we e collec ed du ing he ini ial
measu emen pe iod.
Soil was ex ac ed a ou di e en poin s wi hin he plan ing g oup using a soil auge , collec ing he
op 20 cm o soil. These ou subsamples we e combined o o m one composi e sample pe g oup.
To ensu e ou measu emen s we e no in luenced by he ea men s gi en o he saplings, he samples
we e aken a leas 0.5m away om he plan ed saplings.
Upon a i al a he labo a o y, all soil samples we e s o ed a 4°C in a cold oom o p ese e hei
physiochemical p ope ies o subsequen analyses.
3.2 Soil sampling in WP3
In WP3, a o al o ca. 800 semi-pe manen plo s exis in he h ee o es managemen uni s, and a
s a i ied andom selec ion o 150 plo s we e made in such way ha o each FMU ca 50 plo s we e
sampled o soil p ope ies. En i onmen al da a was collec ed o assess abio ic ac o s in luencing
decomposi ion dynamics. Soil samples and ligh a ailabili y measu emen s we e aken di ec ly in he
ield, while addi ional da a on o es s uc u e and composi ion o he es ablished plo s we e ea lie
p o ided by he S a e Fo es y Se ice.
In each o he 150 selec ed plo s h ee soil samples we e collec ed using a cylind ical auge . Fo each
o he h ee soil sub-samples, he o ganic ma e laye hickness was measu ed wi h a ape and
eco ded in he ield be o e ex ac ing he op 20 cm o soil. The sub-samples we e hen combined
in o a single composi e sample pe plo and pu in labeled plas ic bags o anspo .
Upon a i al a he labo a o y, all soil samples we e s o ed a 4°C in a cold oom o p ese e hei
physiochemical p ope ies o subsequen analyses.
3.3 Li e collec ion and p ocessing in WP3
In each o he 150 exis ing plo s, wo li e samples we e aken, o which each sub-sample a 25x25
cm g id was placed on he o es loo and all li e ma e ial (lea es, wigs, seeds) we e collec ed and
pu in a plas ic bag. The bags we e s o ed a 4°C in a cold oom o u he p ocessing.
Li e samples we e sepa a ed in ac ions o lea es, wigs and seeds. A e sepa a ion, all sub
samples we e o en d ied (The mo Scien i ic He a he m OMS180) a 45°C o 48 hou s and weighed
a e wa ds o de e mine he d y weigh o each li e ac ion.
3.4 Ligh a ailabili y
Fo he 150 exis ing plo s ligh a ailabili y was measu ed using a densiome e (Baud y e al., 2013).
Th ee measu emen s we e aken pe plo o assess ee canopy densi y. The pe cen age o co e ed
canopy was de e mined by eco ding he p opo ion o squa es on he densiome e wi hou
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ege a ion, which was hen mul iplied by a co ec ion ac o (1.04) o es ima e o al canopy co e .
The h ee measu emen s we e a e aged o ob ain one canopy co e pa ame e o he plo .
3.5 Decomposi ion measu emen s
Due o p oblems in he supply o Be a-glucosidase enzymes o decomposi ion expe imen s, he Tea-
Bag Index (TBI) me hodology was applied o measu e in- ield decomposi ion a es. The TBI is a
s anda dized me hod used o assess ea ly-s age o ganic ma e decomposi ion and s abiliza ion in
soils and sedimen s (Keuskamp e al., 2013). I in ol es bu ying bags con aining g een and ooibos
ea— ep esen ing labile and ecalci an plan ma e ials, espec i ely— o a ixed pe iod. Changes in
mass o e ime a e used o calcula e decomposi ion a es and s abiliza ion ac o s.
The s anda d g een ea as used in he esea ch by Keuskamp e al. (2013) (Lip on Indonesian ea
Sencha adi ion, EAN8722700055525) is no longe a ailable, and al e na i e g een ea is only
a ailable in deg adable bags. We he e o e ans e ed he ea om deg adable bags o sel -made
mesh bags wi h he same mesh size as he o iginal ea bags as p esc ibed o he TBI (200 mic on). We
used an al e na i e g een ea ha is p esc ibed as a sui able al e na i e by he Tea Bag Index
o ganiza ion (Lip on Japanese Sencha: EAN 5063270101797). Howe e , he au ho s o he ea lie
men ioned TBI esea ch a e s ill de eloping sui able pa ame e s o de i e k and S alues om
decomposi ion da a o his ea. Fo he mos accu a e k and S calcula ions, hese pa ame e s can be
ecalcula ed using upda ed pa ame e s in he u u e. Howe e , due o a change in es ing ma e ial
(new ype o g een ea), a decomposi ion ime se ies es had o be applied.
G een and Rooibos mesh bags we e bu ied in duplo in each o he 150 exis ing ield ials (2 g een , 2
ooibos). In he new ield ials In Aus e li z whe e he saplings we e plan ed, one ooibos and one
g een ea mesh bag was bu ied in each o he 64 plo s whe e also soil measu emen s we e pe o med.
3.5.1 Tea Bag P epa a ion, deploymen , and p ocessing
Mesh bags we e cons uc ed using 200-mic on nylon mesh, which was cu in o uni o m squa es
(app oxima ely 5 cm x 5 cm), wi h h ee sides glued oge he using ho glue, lea ing one side open o
adding li e .
Each mesh bag con ained ca. 3 g o g een ea ( ep esen ing labile ma e ial) and one con aining ca. 3 g
o ooibos ea ( ep esen ing ecalci an ma e ial). Exac ea weigh s we e eco ded a-p io i using a
p ecision balance. Each bag was labeled wi h a unique iden i ie indica ing loca ion, plo , and ea ype
(“G” o g een ea, and “R” o ooibos ea). The weigh o each comple ed mesh bag was also
measu ed and eco ded.
In he ield, he eabags we e bu ied pe plo using s anda dized p o ocol. Teabags we e bu ied
app oxima ely 1-1.5 m No h om he plo ’s cen al ee. Bags we e bu ied a he soil su ace a a
uni o m dep h o app oxima ely 20 cm, and 15 cm apa in a squa e. Each bu ial loca ion was ma ked
wi h a skewe and he cen al ee was ma ked wi h a ibbon o acili a e e ie al. Li e bags emained
in si u o exac ly 91 days. Exac bu ial and e ie al da es can be ound in able 3.1.
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Table 3.1: bu ial and e ie al da es o mesh ea bags
IN: Mesh ea bags
OUT: Mesh ea bags
25-4-2025
25-7-2025
28-4-2025
28-7-2025
29-4-2025
29-7-2025
30-4-2025
30-7-2025
2-5-2025
1-8-2025
7-5-2025
6-8-2025
8-5-2025
7-8-2025
9-5-2025
8-8-2025
12-5-2025
11-8-2025
13-5-2025
12-8-2025
A e exac ly 91 days, mesh bags we e e ie ed in he same o de as hei deploymen o ensu e
empo al consis ency. Using a sho el, eabags we e ca e ully emo ed, and adhe ing soil pa icles
we e b ushed o in he ield. Re ie ed bags we e placed in sealed con aine s and anspo ed o he
lab o p e en con amina ion o u he decomposi ion du ing ansi .
In he lab, all soil pa icles we e emo ed om he ou side o he bags. Mesh bags we e hen ca e ully
opened and he con en s we e sp ead ou o e a la ge pape shee , allowing o iden i ica ion and
emo al o ing own oo ma e ial. we e placed in sepa a e pape cups o d ying and weighing. The
ea in pape cups was o en-d ied a 70°C o 48 hou s o s anda dize mois u e con en .
A e emo al ou o he o en, wa m ea a ac s mois om he ai . The inc ease in weigh due o
mois u e a ac ion wi hin he i s 10 minu es was conside ed negligible. The e o e, each pape cup
was emo ed om he o en sepa a ely and weighed immedia ely a e . The inal d y weigh o he
ma e ial was hen measu ed using a p ecision balance.
Two compensa ions ac o s a ec ing decomposi ion needed o ake place. Fi s , he ea be o e
deploymen was no d ied in he o en a 70°C o 48 hou s in ad ance, as was done wi h he ea
a e deploymen . We did ollow his p ocess o 5 ea samples o de e mine an a e age mois u e
con en in he g een ea. The a e age o his mois u e con en was used o compensa e he weigh s
be o e deploymen wi h.
𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷 𝑡𝑡𝐷𝐷𝑡𝑡 𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵 = 𝑀𝑀𝑀𝑀𝐷𝐷𝑀𝑀𝑡𝑡 𝑤𝑤𝐷𝐷𝐷𝐷𝑤𝑤ℎ𝑡𝑡 𝑡𝑡𝐷𝐷𝑡𝑡 𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵𝐵 ∗𝑡𝑡𝑎𝑎𝐷𝐷𝐷𝐷𝑡𝑡𝑤𝑤𝐷𝐷 𝑚𝑚𝑀𝑀𝐷𝐷𝑀𝑀𝑡𝑡𝑚𝑚𝐷𝐷𝐷𝐷 %
A second compensa ion was applied o possible in lux o e y small soil pa icles ha may ha e passed
h ough he 200 mic on mesh openings. In 15 plo s we he e o e bu ied a bag and e ie ed i di ec ly
a e . These bags we e b ushed o ca e ully, con en s we e emp ied in a pape cup, d ied a 70°C o
48 hou s and compensa ed o soil mois u e. Any inc ease in weigh ha was eco ded was deemed
in lux o soil pa icles in he mesh bags. These weigh di e ences we e a e aged o ob ain a
compensa ion ac o o soil in lux.
𝑆𝑆𝑀𝑀𝐷𝐷𝑆𝑆 𝑐𝑐𝑀𝑀𝑚𝑚𝑐𝑐𝐷𝐷𝑐𝑐𝑀𝑀𝑡𝑡𝑡𝑡𝐷𝐷𝐷𝐷 𝑡𝑡𝐷𝐷𝑡𝑡 𝐴𝐴𝐵𝐵𝐴𝐴𝐵𝐵𝐵𝐵 = 𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷𝐷 𝑤𝑤𝐷𝐷𝐷𝐷𝑤𝑤ℎ𝑡𝑡 𝑡𝑡𝐷𝐷𝑡𝑡 𝐴𝐴𝐵𝐵𝐴𝐴𝐵𝐵𝐵𝐵 ∗𝑡𝑡𝑎𝑎𝐷𝐷𝐷𝐷𝑡𝑡𝑤𝑤𝐷𝐷 𝑀𝑀𝑀𝑀𝐷𝐷𝑆𝑆 𝐷𝐷𝑐𝑐𝑖𝑖𝑆𝑆𝑚𝑚𝑖𝑖 %
3.5.2 Decomposi ion ime se ies
A e a ce ain amoun o ime all labile ac ions in g een ea ha e decomposed and he weigh
s abilizes, a e which he pa ame e s in sec ion 3.5.3. can be calcula ed. In o de o check whe he
he weigh loss o g een ea indeed s abilized o e ime, se e al ime se ies we e done. Teabags o
19
japanese sencha, indonesian sencha and ooibos we e bu ied in 5 di e en plo s. In each plo 5 bags
we e bu ied and e ie ed a e 7, 14, 29, 68 o 130 days. The weigh loss o each bag was eco ded.
The ime se ies se up was based on he esea ch by Keuskamp e al. (2013) and measu emen days
we e chosen acco dingly; eabags we e measu ed a 0, 4, 7, 14, 29, 68 and 130 days. Fo he g een
ea used in his expe imen , Japanese sencha, a s abiliza ion o decomposi ion ook place be ween
67 and 130 days as shown in able 3.2.
Figu e 3. 1 shows he ime se ies plo , showing he ac ion o emaining ea o e ime. Indi idual
poin s ep esen obse ed alues om each expe imen al plo . The lines ep esen he mono onic
means pe ea ype. The Japanese g een sencha ea as used in his expe imen mee s he necessa y
assump ion ha g een ea decomposi ion s abilizes a e 90 days, wi h no u he signi ican
decomposi ion. Wi h his assump ion me , decomposi ion pa ame e s can be calcula ed.
Table 3.2: Tea bag index ime se ies a e age esul s o emaining ea weigh ac ions o e ime o
di e en ea ypes.
Days Rooibos emaining ac ion
Japanese G een
emaining ac ion
Indonesian G een emaining
ac ion
0
1.00
1.00
1.00
4
1.00
0.94
0.92
7
0.98
0.89
0.89
14
0.91
0.71
0.67
29
0.85
0.53
0.54
68
0.80
0.35
0.39
130
0.63
0.32
0.30
Figu e 3.1: Tea bag index ime se ies esul s o di e en ea ypes. Indi idual poin s ep esen he
emaining ac ions in each o he 5 ime se ies plo s. Lines ep esen he mono onic means h ough
hese poin s.
20
3.5.3 Tea Bag Index pa ame e calcula ions
The TBI esul s in wo pa ame e s ha oge he desc ibe he decomposi ion in he soil: he
s abiliza ion ac o S and he decomposi ion a e k. The s abiliza ion ac o is calcula ed as ollowing:
𝑆𝑆= 1 −�𝑡𝑡𝑤𝑤
𝐻𝐻𝑤𝑤�
Whe e:
• S = S abilisa ion ac o
• ag = ac ion o decomposed g een ea
• Hg = hyd olysable ac ion o g een ea
The decomposi ion a e k is calcula ed as ollowing:
𝑘𝑘=𝐿𝐿𝑐𝑐�𝑡𝑡𝐷𝐷
𝑊𝑊𝑡𝑡−(1−𝑡𝑡𝐷𝐷)�
𝑡𝑡
In which =
𝑡𝑡𝐷𝐷 =𝐻𝐻𝐷𝐷∗(1 −𝑆𝑆)
Whe e:
• k = decomposi ion a e
• a = p edic ed labile ac ion ed ea
• W = ac ion emaining ed ea
• = bu ial pe iod
• H = hyd olysable ac ion o ed ea
• S = s abiliza ion ac o
I is impo an o no e ha he hyd olysable ac ion o bo h g een and ed ea o he ea ypes used
a e no ye up o da e, and based on he o iginal ea ypes as p esc ibed by Keuskamp e al. (2023).
When upda ed pa ame e s become a ailable h ough hei esea ch, he k and S can be upda ed wi h
he ea weigh loss da a ha is p o ided.
Also: in some plo s k o S could no be calcula ed. Acco ding o he au ho s o he Tea Bag Index his
is a esul o a as b eakdown o labile ma e ial in he ooibos eabags, in which he ea mo es om
he i s o he second phase o decomposi ion. The o mula will hen calcula e a nega i e k o no k a
all.
3.6 Soil auna eeding ac i i y
The bai lamina es was used o complemen decomposi ion expe imen s o he ex en o assess soil
aunal eeding ac i i y in each plo . This me hod in ol es illing s anda dized s ips wi h bai ma e ial
o measu e eeding ac i i y ia pe o a ion consump ion o e ime (K a z, 1998). Two s ips we e
deployed pe plo . This ac ion was pe o med wo imes, in 2024 and 2025, wi h some o e lap in plo s.
21
The bai ma e ial was p epa ed by mixing a s anda dized subs a e consis ing o 70% cellulose, 25%
whea b an, and 5% ac i a ed ca bon. Fo his s udy, oa s we e used as he whea b an sou ce and
we e g ound in o ine pa icles using a se ies o sie es wi h dec easing mesh sizes (0.5 mm, 0.2 mm,
and inally 0.12 mm). The ac i a ed ca bon was sou ced om No i pills, which we e c ushed in o a
ine powde using a spoon o achie e he desi ed consis ency. The p epa ed d y componen s we e
hen ho oughly mixed o ensu e uni o m dis ibu ion. Dis illed wa e was g adually added o he
mix u e o o m a pas e-like consis ency sui able o illing he bai lamina s ips. The bai ma e ial was
hen s o ed in an ai igh con aine o main ain i s mois u e and consis ency un il i was applied o he
s ips.
The bai lamina s ips we e cons uc ed using s anda dized poly inyl chlo ide (PVC) s ips wi h 16
e enly spaced pe o a ions (2 mm diame e ) along hei leng h. The emp y s ips we e aligned on an
aluminum shee and aped a he uppe po ion o each s ip (abo e he pe o a ions) o keep hem
in place. The p epa ed bai mix u e was applied di ec ly on o he pe o a ed a ea o he s ips using a
spa ula. The bai was p essed i mly agains he s ips o ensu e ha each pe o a ion was comple ely
illed. This p ocess was epea ed o all s ips. Excess bai on he su ace o he s ips was ca e ully
wiped away o p e en obs uc ion o con amina ion du ing deploymen .
Once all pe o a ions we e illed, he s ips we e placed in an o en se a 40°C o 48 hou s o d y he
bai wi hin he holes. A e d ying, any holes ha had los bai (as he ma e ial is agile and can all
ou du ing handling) we e e illed ollowing he same p ocedu e. The s ips we e hen d ied again o
secu e he bai in place. A e ully p epa ed, s ips we e s o ed in a labeled plas ic con aine o p o ec
hem om mois u e o damage un il deploymen .
In he ield, wo bai lamina s ips we e deployed pe plo , wi h s ips placed on opposi e sides o he
plo ’s cen al ee o accoun o spa ial a iabili y. S ips we e inse ed e ically in o he soil using a
small sli made wi h a owel. Inse ion dep h was such ha he opmos hole was jus below g ound
le el ensu ing ha he pe o a ions we e bu ied a uni o m and exposed o he same en i onmen al
condi ions. A ma ke was placed nea each s ip o iden i ica ion du ing e ie al. S ips emained in
he soil o wo weeks, allowing adequa e ime o soil auna o in e ac wi h he bai . Exac bu ial and
e ie al da es o he 2025 da ase can be ound in able 3.3.
Table 3.3: Bu ial and e ie al da es o bai lamina s ips in he 2025 measu ing se ies.
IN: Bai lamina 2025
OUT: bai lamina 2025
26-6-2025
10-7-2025
30-6-2025
14-7-2025
11-6-2025
25-6-2025
24-6-2025
8-7-2025
13-6-2025
27-6-2025
25-6-2025
9-7-2025
A e wo weeks, s ips we e e ie ed in he same o de as hei deploymen . S ips we e ca e ully
aken ou o he soil o a oid dis u bing he bai o al e ing he pe o a ions. Re ie al was pe o med
sys ema ically o ensu e consis ency, wi h each s ip being gen ly emo ed and immedia ely assessed
o bai consump ion. Fo each pe o a ion (16 in o al), da a was eco ded as ollows:
• “0” o an emp y hole (bai ully consumed)
• “0.5” o a hal - illed hole (bai pa ially consumed)
• “1” o a ull hole (no bai consumed)
22
Fo each plo , he s ip was di ided in ou dep h sec ions: hole 1-4-, hole 5-8, hole 9-12, hole 13-16.
Fo each g oups he p opo ion o non- illed/ illed was de e mined, which is a measu e o he
eeding ac i i y. This was also done o he whole s ip in o al. The esul s o bo h s ips we e
a e aged o ob ain an a e age o al eeding ac i i y pe plo as well as a e age eedings ac i i ies pe
dep h sec ion pe plo .
23
4. Soil and nu ien p ocessing
Soil and nu ien analysis was conduc ed in labo a o ies a he acul y o Geosciences and Na u al
Sciences, U ech Uni e si y, using s anda dized p o ocols o ensu e consis ency and eliabili y. An
o e iew o ypes o measu emen s, me hods and used equipmen can be ound in able 4.1.
Table 4.1: O e iew o applied me hods o soil, li e and decomposi ion analysis.
Measu emen
Me hod
Equipmen
Uni
pH
Measu emen o soil in
KCl ex ac
Me le Toledo
Se enCompac Duo
pH (KCl)
C:N a io
Analysis o d ied, g ound
and acidi ied soil samples
He o Powe D y
LL3000; SCALA
TFD5503;CN-analyze
(NA1500, Fisons
Ins umen s); He zog
HP-Ma au oma ic
pul e ize
Soil C/N a io, soil C
(%) and soil N (%)
Soil O ganic
Ma e
Calcula ed as weigh loss
a e 4 hou s hea ing a
550°C
He o Powe D y
LL3000; SCALA
TFD5503TGA701;
The mog a ime ic
Analyze
Soil o ganic Ma e
(%)
NH
4
-N, NH
3
-N,
NH2-N, NOx-N, PO4-
P
Sample in KCl in disc e e
analyze
The mo Fishe
Scien i ic Galle y
NOₓ-N (mg N/kg soil)
PO₄-P (mg P/kg soil)
Al, Ca, Fe, K, Mg,
Na
He o Powe D y
LL3000;SCALA
TFD5503;
He zog HP-Ma
au oma ic pul e ize ,
Pe kin-Elme A io 500
ICP-OES
Al, Ca, Fe, K, Mg, Na
(mg/L)
Li e
O en d ying
The mo Scien i ic
He a he m OMS180,
p ecision balance
Biomass (g/m2 su ace
a ea)
Teabags
O en d ying
The mo Scien i ic
He a he m OMS180,
p ecision balance
g
4.1 Soil Analyses
Soil samples we e analyzed o de e mine hei physical and chemical p ope ies using he ollowing
me hods:
4.1.1 Soil pH
Soil pH was measu ed using a 1 M KCl solu ion o assess exchangeable acidi y (Van Reeuwijk, 1992).
20 mL o 1M KCl was added o exac ly 8 g ams o soil in a 50 mL G eine ube. The mix u e was shaken
o 2 hou s using a ecip oca ing shake . The suspension was cen i uged and he elec ode was
24
imme sed in he supe na an . The soil pH (pH-KCl) was measu ed using a Me le Toledo
Se enCompac Duo pH me e . pH eadings we e eco ded once s abilized (±0.1 uni accu acy), wi h
luc ua ions no exceeding 0.1 uni pe 30 seconds.
4.1.2 Ano ganic ni ogen: NOx, NO3, NO2, NH4, PO4
Ni ogen and phospha e concen a ions we e measu ed using a 1 M KCl solu ion. 20 mL o 1M KCl was
added o exac ly 8 g ams o soil in a 50 mL G eine ube. The mix u e was shaken o 2 hou s using a
ecip oca ing shake . The supe na an esul ing om he p epa a ion o he pH measu emen was
il e ed h ough a 0,45 µm glas ibe sy inge il e . The concen a ions o ammonium (NH₄⁺), o al
ni ogen oxides (NOₓ) and ni i ie (NO₂⁻) we e measu ed using a disc e e analyze (The mo Fishe
Scien i ic Galle y). Ni a e (NO₃⁻) concen a ion was de i ed om he NOₓ and NO₂⁻ measu emen s.
The mois u e pe cen age in he soil ha was de i ed om he eeze-d ying p ocedu es a 4.1.4 was
used o calcula e NOx-N, NO3-N, NO2-N and NH4-N in g pe kg o d y soil using using he ollowing
o mula (simila o all measu emen s):
𝑁𝑁𝐻𝐻4−𝑁𝑁 𝑀𝑀𝐷𝐷 𝑁𝑁𝐵𝐵𝑖𝑖−𝑁𝑁 𝑀𝑀𝐷𝐷 𝑁𝑁𝐵𝐵3−𝑁𝑁 𝑀𝑀𝐷𝐷 𝑁𝑁𝐵𝐵2−𝑁𝑁 𝑀𝑀𝐷𝐷 𝑃𝑃𝐵𝐵4−𝑃𝑃 =𝑡𝑡∗𝑀𝑀
1000 ∗𝐿𝐿
𝑚𝑚∗(100 +𝑤𝑤)
100
Whe e:
• NH₄-N, NO₃-N, NO₂-N, NOₓ-N, PO4-P,= ni ogen o phospho
concen a ions in mg kg⁻¹ d y soil
• a = concen a ion o NH₄⁺, NO₃⁻, o NO₂⁻ measu ed in he dilu ed sample
(μmol/L)
• M = a omic mass o N o P in g/mol
• m = mass o we soil used in he suspension (g)
• L = olume o liquid used in suspension (ml)
• w = pe cen age o wa e con en based on eeze-d ied soil
4.1.3 Ca ion concen a ions
App oxima ely 20 g ams o soil was weighed using a p ecision balance in o a p e-weighed 50 mL
G eine ube. The ubes we e hen placed in one o he wo a ailable eeze d ye s (He o Powe D y
LL3000 and SCALA TFD5503) o 48 hou s. The samples we e subsequen ly g ound in he He zog HP-
Ma au oma ic pul e ize . Exac weigh s o samples o analysis we e eco ded using a p ecision
balance. The concen a ions o Ca, Al, K, Na, Mg and P we e measu ed using a ICP-OES analyze
(Pe kin-Elme A io 500).
4.1.4 Soil o ganic ca bon and C:N a io
App oxima ely 20 g ams o soil was weighed using a p ecision balance in o a p e-weighed 50 mL
G eine ube. The ubes we e hen placed in one o he wo a ailable eeze d ye s (He o Powe D y
LL3000 and SCALA TFD5503) o 48 hou s. The weigh was hen measu ed again o de e mine he
soil mois u e con en . The samples we e subsequen ly g ound in he He zog HP-Ma au oma ic
pul e ize . A small po ion o each sample was weighed using a p ecision balance and ans e ed o
sil e cups. In o de o measu e soil o ganic ca bon con en , po en ial CaCO3 was emo ed om he
sample h ough acidi ica ion by placing he samples in a desicca o wi h a small beake o 20ml 37%
25
uming HCl unde nea h i o 6 hou s. The pe cen age ca bon (C) and ni ogen (N) was subsequen ly
de e mined using a CN-analyze (NA1500, Fisons Ins umen s), and he C:N a io was calcula ed by
di iding hese wo alues.
No e ha in he esul s some alues ha e an * o wo imes ** be o e he alue. * indica es a
measu emen ha was in be ween he Limi o Quan i ica ion (LOQ) and he Below E ec i e
Concen a ion (BEC). In p ac ice his means ha hese alues ha e an e o o >10% and should hus
be in e p e a e as an es ima e. ** indica es a alue ha is below he LOQ and hus canno be
conside ed accu a e.
4.1.5 Soil O ganic Ma e Con en
App oxima ely 20 g ams o soil was weighed using a p ecision balance in o a p e-weighed 50 mL
G eine ube. The ubes we e hen placed in one o he wo a ailable eeze d ye s (He o Powe D y
LL3000 and SCALA TFD5503) o 48 hou s. The weigh was hen measu ed again o de e mine he
soil mois u e con en . The samples we e subsequen ly g inded in he He zog HP-Ma au oma ic
pul e ize . A po ion o hese soil samples we e used o de e mine he pe cen age o o ganic ma e ,
by measu ing he weigh loss a e ou hou s o hea ing a 450 deg ees Celsius (TGA701
The mog a ime ic Analyze ). The samples we e hen s epwise hea ed o 1000°C (550-800-1000°C),
which esul ed in addi ional o ganic ma e mass loss a each empe a u e. Dec ease in weigh was
eco ded a each s ep as a pe cen age o he ini ial sample weigh . To al soil o ganic ma e
pe cen age was calcula ed by summa ion o hese weigh losses.
