Effects of fly ash and stablized
ash on wheat, Daphnia magna,
radish and lettuce in laboratory
tests
Evgeny Krakow
Uppsats för avläggande av naturvetenskaplig magisterexamen i
Miljövetenskap 30 hp
Institutionen för växt- och miljövetenskaper, Göteborgs universitet
Juni 2010
Summary
Increasing use of biofuels, especially logging residues, will contribute to acidification and loss
of nutrients, unless wooden ash is returned to the forest. Wooden ash contains valuble
nutrients and alkalies, but also potentially toxic heavy metals.The aim of this study has been
to investigate the toxicity and to identify the toxic agents in fresh and stabilized fly ash
originating from combustion of biofuels at Västermalmsverket, Falun.Ash toxicity was tested
using a growth test on wheat (Triticum aestivum). The toxicity of leacahate was determined
for Daphnia magna and seeds of lettuce (Lactuca sativa) and raddish (Raphanus sativus).
Chemical analysis of leachate was made along with a TIE procedure to identify its chemical
composition and the toxic agents. Soils pH increased with time and amount of added wooden
ash, probably due to presence of alkali metal hydroxides in leachate. Ash leachates were toxic
to Daphnia magna at doses exceeding 24 tons of ash per hectare. Statistical analyses showed
a significant dependency of toxicity on ash type, number of sampling week, exposure time in
hours and ash +week. Fresh fly ash was more toxic to daphnids than stabilized ash, and both
ashes showed the highest toxicity at the beginning of the sampling period. Growth of wheat
was not negatively affected by moderate ash concentrations. Seeds of radish and lettuce were
less sensitive to leachate toxicity than Daphnia magna. Attempts were made to identify
substances responsible for leachate toxicity by performing a TIE with columns neutralizing
lipophilic compounds, cations and anions. However, none of the tests showed any clear
results. Chemical anlysis of ash leachate showed significantly elevated concentrations of
copper, magnesium, manganese and sulphate compared to controll. Samples with the highest
concentrations were also most toxic to Daphnia.
Key words: wooden ash, biofuels, ash recycling, toxicity, Daphnia, growth test, metal,
Raphanus, Lactuca, TIE, root elongation, Triticum.
Sammanfattning
Ökad användning av biobränslen, i synnerhet avverkningsrester, kan bidra till försurningen
och förlusten av näringsämnen om askan inte återförs till skogsmarken. Vedaskan har
alkaliska egenskaper och är rik på näringsämnen, men innehåller även potentiellt toxiska
tungmetaller. Syftet med denna studie har varit att undersöka toxiciteten och identifiera de
toxiska komponenterna hos lakvattnet från flygaska och stabiliserad aska som uppstår vid
förbränning av biobränslen. Askans toxicitet undersöktes med tillväxttester med vete
(Triticum aestivum). Lakvattnet testades på Dapnia magna samt frön av sallad (Lactuca
sativa) och rädisa (Raphanus sativus). TIE användes för att identifiera de toxiska elementen.
Markens pH steg med tiden och med ökade koncentrationer av aska, troligtvis på grund av
ökade halter av alkaliska metalhydroxider i lakvattnet. Lakvattnet var toxisk för D. magna vid
doser överstigande 24 ton per hektar. Statistisk analys (ANOVA) visade att toxiciteten var
signifikant kopplad till typen av aska, provtagningsveckan, exponeringstiden i timmar samt
asktypen –provtagningveckan. Flygaskan var mer toxisk för D. magna än den stabiliserade
aska och både typer av aska var mer toxiska i början av provtagningsperioden. Tillväxten av
vete påverkades inte negativt av askkoncentrationen på upp till 24 ton per hektar. Sallads–och
rädisfrön visade sig vara mindre känsliga för lakvattnets toxicitet jämfört med D. magna.
Försök gjordes att identifiera de toxiska komponenterna i lakvattnet genom att använda TIE
med selektiva kolonner som absorberade lipofila ämnen, metaliska anjoner respektive
katjoner. TIE-testerna gav dock inga entydiga svar. Kemisk analys av lakvattnet från askan
visade på förhöjda halter av koppar, magnesium, mangan och sulfat jämfört med kontrollerna.
Proverna med de högsta metall– och sulfatkoncentrationerna var även mest toxiska för
Daphnia.
Nyckelord: biobränslen, askåterföring, bioaskan, toxicitet, tillväxttest, metaller,
rotförlängning, Daphnia, Raphanus, Lactuca, Triticum, TIE.
Table of contents
Summary................................................................................................................................1
Sammanfattning .....................................................................................................................2
Table of contents ....................................................................................................................3
1 Introduction .........................................................................................................................1
1.1 Renewable energy.........................................................................................................1
1.2 Ashes and recycling ......................................................................................................2
1.3 Characteristics of wooden ash .......................................................................................3
1.4 Stabilization of wooden ash ..........................................................................................3
1.5 Effects of wooden ash on soil and plants .......................................................................4
1.6 Aim of the present study ...............................................................................................4
2 Materials and methods.........................................................................................................4
2.1 Origins and chemical composition of wooden ash .........................................................4
2.2 Growth test with wheat (Triticum aestivum) .................................................................5
2.3 Collection of ash leachate .............................................................................................6
2.4 Identification of samples ...............................................................................................6
2.4 Immobility of Daphnia magna.......................................................................................6
2.5 Germination and root elongation of lettuce (Lactuca sativa) and raddish (Raphanus
sativus) seeds......................................................................................................................6
2.6 TIE – toxicity identification procedure..........................................................................7
2.7 Statistical analysis.........................................................................................................7
2.8 Chemical analysis .........................................................................................................7
3 Results.................................................................................................................................7
3.1 Variation of pH.............................................................................................................8
3.3 Growth rate of Triticum aestivum .................................................................................9
3.4 Immobility of Daphnia magna.....................................................................................11
3.5 Germination and growth of Lactuca sativa ..................................................................15
3.6 Germination and growth of Raphanus sativus .............................................................18
3.7 TIE of leachate............................................................................................................20
3.8 Chemical analysis .......................................................................................................21
4 Discussion .........................................................................................................................22
4.1 Effect on pH ...............................................................................................................22
4.2 Effect on wheat growth ...............................................................................................23
4.3 Effect on D. magna .....................................................................................................23
4.4 Effect on R. sativa and L. sativus ................................................................................23
4.5 Chemical analysis .......................................................................................................23
5 Conclusions.......................................................................................................................24
Acknowledgements ..............................................................................................................24
6 References.........................................................................................................................25
Annex A. Primary data from week 1.....................................................................................28
Annex B. Primary data from week 2 .....................................................................................30
Annex C. Primary data from week 3 .....................................................................................31
Annex D. Primary data from week 4.....................................................................................32
Annex E. Primary data on wheat growth...............................................................................33
Annex F. Data from ANOVA for effects on Daphnia ...........................................................34
1 Introduction
Annually, 1.3 millions of tonnes of ash are produced in Sweden, mainly as byproducts in heat
and power generation (Värmeforsk 2008). Ash contains important nutrients and trace
elements essential for plants growth, but also a small amount of heavy metals. The challenge
is to close the cycle, to use the ash as a source of nutrients to increase growth without causing
harmful effects on enviroment.
1.1 Renewable energy
The definition of sustainable development is that it “meets the needs of the present without
compromising the ability of future generations to meet their own needs” (UN, 1987).
Implementation of this vision in Swedish environmental policies is manifested by the decision
of the Parliament to solve or minimize all the greatest national environmental problems within
one generation. This visionary goal of the sustainable society is characterized by 16 National
Environmental Objectives, of which all but one should be reached by 2020.
The only exception is goal 1 “Reduced Climate Impact” which should be achieved by 2050
(Miljömålsrådet, 2010). This objective is accompanied with ambitions of European Union to
cut its emissions of greenhouse gases by 20% by 2020 compared to 1990 in order to halt
climate change. According to EU’s goals Swedish national emissions which are not part of
Emission Trading Scheme (ETS) for example from transportation, agriculture and household
are bond to decrease by 17% by 2020 compared to 1990 (Swedish Energy Agency, 2009).
One of the adopted action plans to achieve these reduction considers an increase of the total
amount of renewable energy to 50 % of the total energy supply by 2020. Renewable energy
already contributes for 44.1% of the total 170 TWh used 2008, an increase from 33.9% in
1990 (Swedish Energy Agency, 2009).
Biofuels contributed up to 20 % of the total energy supply in Sweden 2008. Biofuels are
produced from the following organic fuels:
• Unprocessed wood: energy forest, bark, shavings, logging residues
• Processed wood: pellet, briquettes
• White and black liquors from pulp mills
• Biofuels from crops such as corn
• Peat
• Biogas
• Ethyl Alcohol
Processed and unprocessed wood products are used as fuel in a variety of different sectors,
like forest industry, pulp industry, district heating and power generation. Wood products
contributed up to 25.5 TWh to heat production 2008, a 5-fold increase since 1990.
Biofuel’s part of the total energy supply is increasing by 3-4 TWh annually (Swedish Energy
Agency, 2009).
The goal of 50 % of energy coming from renewable sources can be achieved by the increasing
use of biofuels, primarily wood products. Calculations show a potential resource of 130 TWh
1
wood products compared to 50 TWh which are used today. The main part of those resources
(64TWh) might be provided logging residues (Parikka, 1997)
1.2 Ashes and recycling
Increased removal of logging residues from forests is not unproblematic as it may enhance
such problems as acidification, enrichment of heavy metals and depletion of nutrients in soil
as well as removing vital habitats for many species (Dahlberg, Stockland, 2004).
Trees take up nutrients from soil as positively charged ions of K, Mg, Ca, leaving hydrogen
ions in return, and a hydroxyl ions for the negatively charged ones. Since the uptakes of
positively charged ions are greater than that of anions the soil will gradually undergo a natural
acidification. Balance will be restored when dead wood is decomposed, because most of the
hydrogen ions will be depleted during the process and nutrients return to the soil. Logging is
disturbing this balance by removing the nutrients, especially when residues with high content
of nutrients are removed.
The increased use of logging residues as biofuels will put an additional stress on forest
ecosystem. Studies have shown a decrease in pH by 0.0-0.4 units (Egnell et al., 1998) and the
soil base ion saturation down by 10-20 % with residues removed (Olsson et al., 1996).
Removing biomass from forest has lead to a decrease in forest growth, probably due to
limitation of nitrogen supply (Egnell, Valinger, 2003; Egnell, Leijon 1999). Compensation
measures need to be taken to counter the net losses of nutrients (Akselsson, 2005)
In the recommendations from Swedish Forest Agency is stated:
“Removal of logging residues should be compensated with bioash if:
• Total removal of other tree parts than stem are corresponding to more than half ton of
ash per hectare
and
• The greatest part of needles is not left equally spread on the ground
• Ash should always be added even if removal equivalent is below half ton per hectare
when done on strongly acidified soils or when forest is grown on a peat land.
Recommendations also point out the need to treat the ash in order to increase its stability. The
benchmark is that ash should dissolve in 5-25 years.
• Ash should be stabilized to avoid damage on sensitive species
Recommendations also set the upper limit on the total amount of ash added to the forest
during a certain time:
• To avoid unwanted effects should no more than 3 tons DS ash per hectare be added
during a 10 –year period and no more than 6 tons per hectare during a life cycle of
the forest.
• The addition of heavy metals with bioash should not exceed the amount which is
removed with logging residues.
Required upper and lower limits of nutrients and metal concentrations in ash are
corresponding to the concentration of these subjects in the tree parts and on empirical
knowledge from previous spreading of wooden ash. Small deviations from those limits are
accepted if the source is proven to be from the combustion of biofuels (Skogsstyrelsen, 2008).
2
1.3 Characteristics of wooden ash
More than 80 % of wooden ash is composed of particles <1.0 mm, and the rest of nonincinerated wood (Etiegni et al., 1991a; Etiegni, Campbell, 1991). Bulk density varies from 0,
27 g cm-3 for wooden ash (Huang et al., 1992) to 0, 51 g cm-3 for ash from pulp and paper
waste (Muse, Mitchell, 1995).
The most abundant elements in wooden ash are Ca, K, and Mg which are particularly present
in ash as oxides, hydroxides, carbonates and bicarbonates (Someswhar 1996). High
combustion temperatures oxides C and N into gaseous forms, leaving only small quantities in
the ash.
Alkalinity of wooden ash depends on its content of oxides, hydroxides, bicarbonates and
carbonates. Ratio of these substances depends on combustion temperature. Carbonates and
bicarbonates dominate at temperatures up to 500oC while oxide formation is predominant
above 1000oC, an operational temperature for the majority of commercial boilers (Etiegni,
Campbell, 1991).
1.4 Stabilization of wooden ash
In order to avoid a rapid release of nutrients and potentially toxic compounds it is desirable to
stabilize the ash before its release into the ecosystem. This can be achieved by forming large
agglomerates or transforming ash’s content into minerals with low water solubility (Steenari,
Lindqvist 1997). Wood ash with a low (<10 %) percentage of unburned material can
spontaneously form large agglomerates when wetted, in so called self-hardening process
transforming calcium (CaO) into portlandite (Ca (OH)2).
(1) CaO + H2O→Ca (OH) 2
In contact with atmospheric CO2 portlandite transforms into calcite, a process known as
carbonization:
(2) Ca (OH) 2+ CO2→Ca CO3 + H2O
Further reactions leads to formation of gypsum and ettringite:
(3) CaSO4+ 2 H2O→ CaSO4* 2 H2O
(4) Ca3Al2O6+ 3 CaSO4* 2 H2O+ 26 H2O→Ca6 Al2 (SO4) 3(OH)12*26 H2O
Altogether, self hardening of ash significantly lowers its water solubility; calcite is for
instance 100 times less soluble in water than calcium oxide. These factors will contribute to
slower leaching rate of metals and other potential toxic compounds from stabilized ash. In
addition with a lower alkali effect and a more moderate change in soil pH (Steenari et al.,
1999) decreased toxicity of stabilized ash compared to untreated ash could be expected.
3
1.5 Effects of wooden ash on soil and plants
Effects of addition of wooden ash to soil are increased respiration and microbial activity
(Shahid et al., 2002; Fritze et al., 1993), increasing pH (Mandre et al., 2006; Perkiömäki,
Fritze, 2002; Arvidsson, Lundkvist, 2003). Addition of wooden ash increases P, Ca, Mg and
especially K in soil (Unger and Fernandez, 1990; Ohno, Erich, 1990; Meiwes, 1995).
Plants have been shown to benefit from nutrients in wooden ash. Studies on among others oat
(Avena sativa L.), winter wheat (Triticum aestivum L.) and corn (Zea mays L.) showed
increasing yield and growth (Erich, 1991; Huang et al., 1992; Krejsl, Scanlon, 1996).
Studies on trees have not given any conclusive answers,and positive as well as negative
effects have been observed (Ferm et al., 1992). The mainly factors behind increased growth
are attributed to be higher availability of K, P and B in soil, while negative effects depend on
decreased supply of N (Bramryd, Fransman, 1995). Addition of ash from biofuels can
sometimes increase growth rate when spread in growing forest. This is especially the case in
nutrient rich soils (Jacobson, 2003; Thelin, 2006).
Several differences between effects of fly ash and stabilized ash have been shown. Stabilized
ash doesn’t increase pH as much as fly ash (Kahl et al., 1996). Release of Mn and Zn is lower
in stabilized ash compared to fly ash (Steenari et al., 1999). Respiration and concentrations of
Ca and Mg increase more rapidly with addition of fly ash compared to stabilized ash
(Perkiömäki,Fritze, 2002)
1.6 Aim of the present study
The aim of this study was to determine leachability and possible toxic effects of metals and
other compounds from stabilized and fresh fly ash.
2 Materials and methods
2.1 Origins and chemical composition of wooden ash
Samples of wooden ash were received from Västermalmsverket, Falun. The plant uses 2000
tones of biofuels (bark, shavings, logging residues) annually to generate 300 GWh heat and
60 GWh of electricity. Two types of ash were used in the experiments (stabilized (hardened)
ash, SA, and fresh fly ash, FA). Chemical analysis of the ash were made at Eurofins
Lidköping, an laboratory accredited by SWEDAC. Chemical composition of samples (Table
1) shows that the metal concentrations do not exceed the national recommendation limits.
Concentrations of P, Mg and Ca are below the minimum limit.
4
Variable
pH
Conductivity
Cl
S
Al
Al2O3
Fe
Fe2O3
MgO
Mg
K
Ca
P
P2O5
Mn
MnO2
As
Pb
Co
Cd
Cu
Hg
Cr
Ni
Zn
N
B
V
Unit
mS m-1
% TS
% TS
mg kg-1 Ts
mg kg-1 Ts
mg kg-1 Ts
mg kg-1 Ts
mg kg-1 Ts
mg kg-1 Ts
mg kg-1 Ts
mg kg-1 Ts
mg kg-1 Ts
mg kg-1 Ts
mg kg-1 Ts
mg kg-1 Ts
mg kg-1 Ts
mg kg-1 Ts
mg kg-1 Ts
mg kg-1 Ts
mg kg-1 Ts
mg kg-1 Ts
mg kg-1 Ts
mg kg-1 Ts
mg kg-1 Ts
mg kg-1 Ts
mg kg-1 Ts
mg kg-1 Ts
Ash
Flower pot soil
10,7
1617
0.29
1.4
22700
43000
6900
9900
21000
12400
45500
124000
8200
19000
9300
15000
13
56
7,3
5,9
70
0,21
43
18
1400
150
14
6
40
39
1670
700
24
19
11
1400
-
Limit values
min
max
20000
30000
12500
10000
30
300
30
400
3
100
70
7000
500
70
Table 1. Chemical composion of ash and flower pot soil, compared to recommended Swedish limit values.
2.2 Growth test with wheat (Triticum aestivum)
Tests on spring wheat ( Triticum aestivum) were used to determine the impact of wooden ash
on growth rate. Five seeds of wheat were placed in each of the plastic pots filled with flower
pot soil at a depth of 1 cm, and covered either by stabilized or fresh fly ash.
The amount of ash corresponded to the addition of 3, 6, 12, 24, 48 and 96 tons of wooden ash
per hectare, which should be compared to limits of 3 tons per hectare recommended by
Swedish Forest Agency. There were four replicates for each concentration and four negative
replicates were made with no addition of ash.
The number of seeds that germinated in each pot was recorded, and 7 days after germination
all but the tallest plant in each pot were removed. The remaining plants were left to grow for
5
additional 4 weeks, then harvested and measured for root and shoot length. During this period
temperature ranged between 20-25oC. Light intensity ranged between 600-900 lux. Light
regime was 16 hours of light and 8 hours of darkness. Irrigation was made with deionized
water on Monday, Wednesday and Friday using a spray bottle. 100 ml of water was added to
each pot on every occasion.
2.3 Collection of ash leachate
Underneath each pot a plastic breaker was placed in order to collect the leachate. A filter
paper was placed at the bottom of each pot, preventing contamination of leachate by soil
particles. Leachate was collected once a week for 4 weeks and tested for toxicity on Daphnia
magna (water fleas), Lactuca sativa (lettuce) and Raphanus sativus (radish).
2.4 Identification of samples
Every pot and its leachate were codified in order to facilitate identification of the test
parameters specific for each pot. The name of each replicate begins with digits; 3, 6, 12, 24,
48 or 96 which correspondes to the amount of ash in tones added to one hectare. Letters SA
(stabilized ash) and FA (fresh fly ash) describes the structural properties of the ash. Last
digits; 1,2,3 or 4 represent the number of the replicate.
2.4 Immobility of Daphnia magna
Dapnia magna was used in bioassay to determine possible acute toxicity tested with 50 ml
leachate according to standardized procedures (ISO 1996). Each leachate sample was tested in
a Petri dish with 10 newborn neonate of D. magna. The number of immobilized daphnids was
recorded after 24 and 48 hours.
A positive control test using potassium dichromate (K2Cr7O7) was also made. The 24 EC50
value of potassium dichromate for Daphnia magna was determined in each test run.
2.5 Germination and root elongation of lettuce (Lactuca sativa) and
raddish (Raphanus sativus) seeds.
Tests on seed germination and root elongation of different seeds were performed to evaluated
the effect of ash leachate on the early development of plants. Radish and lettuce were used
because they are known to be sensetive to metal contamination (Beltrami et al., 1999; Renoux
et al., 2001).
Five seeds of Latuca sativa (Econova garden, KRAV, No: 8355) and Raphanus sativus
(Econova, Weibull Trädgård AB, No: 8363) respectively were put on a 90 mm filter paper
,(Munktell Filter Paper, ash content 0,007 %) and put into a Petri dish with 5 ml of leachates.
The number of germinated seed as well as root elongation after 96 hours were recorded.
Deionized water was used in negative controls (Beltrami et al., 1989).
6
2.6 TIE – toxicity identification procedure
TIE – toxicity identification evaluation was used to identify the toxic compounds causing the
toxic effect in Daphnia tests (Norberg-King et al., 1991). Samples with the two highest
leachate concentrations were poured into 5 out of 6 wells on a Nunc plate after the following
TIE treatments:
•
•
•
•
•
QMA-resin filtering which reduces toxicity of negative ions.
CM –resin filtering which reduces toxicity of positive ions.
C18 –resin filtering which reduces toxicity of lipophilic compounds.
One sample left untreated.
Control with SRW water.
After 1 hour 5 newborn Dapnia magna were added to each well. Immobility of daphnids in
each sample was determined after 24 hours and compared to the untreated one.
2.7 Statistical analysis
Moving average method was used for determination of 24- and 48- hours EC50 for Daphnia
magna. Differences in toxicity due to ash type, number of sampling week, exposure time in
hours, ash –week, ash –time, and week-hours was tested by analysis of variances (ANOVA)
using a statistical software ( Crunch ver. 4, Crunch, Software Corp., Oakland, USA)
2.8 Chemical analysis
ICP-OES (Inductively Coupled Plasma Optical Emission Spectrometry) was used to
determine concentration of metals and trace elements in leachate water (PE/Sciex ELAN
6000).
Ion chromatografy was used to analyse concentrations of negative ions as chloride and
sulphate in leachate water (ICS-90, Dionex, IonPac AS4A-SC 4 mm column).
3 Results
The effects from wooden ash on pH in soil’s leachate as well as the concentration of metals
are presented. Toxicity of ash is shown on growth rate of Triticum aestivum, immobilization
of Daphnia magna together with germination and root elongation of seeds from Lactuca
sativa and Raphanus sativus. Results of TIE of leachates are shown in Table 2.
7
3.1 Variation of pH
The recorded pH values during the 4 weeks are shown in Figure 1 (fresh ash) and 2 (stabilized
ash).
Figure 1. Variation of pH in leachate from pots with addition of fresh fly ash doses corresponding to 3-96
t/ha during three weeks.
Figure 2. Variation of pH in leachate from pots with addition of stabilized fly ash doses corresponding to
3-96 t/ha during three weeks.
Addition of wooden ash to soil affects its pH (Fig. 1, Fig. 2). Altogether pH levels were
higher with ash, and pH increased by 0,3-1,4 during the first week and by 0,1-1,0 the second
8
week with addition of stabilized ash compared to control. Corresponding numbers for fresh
fly ash were 0,3-1,3 and 0,4-0,9, respectivly. In general pH increased with time and higher
concentration of wooden ash at least up to the dose of 48 tons stabilized ash per hectare. pH
increased more rapidly with addition of fly ash compared to stabilized ash during the first two
weeks of testing.
3.3 Growth rate of Triticum aestivum
The effects of stabilized ash at the highest tested dose (96 tons per hectare) on wheat plants
fter 4 weeks are shown in Picture 1.
Picture 1. Wheat plants sampled from pots with stabilized ash. Ash concentration was 96 tons per hectare.
3.3.1 Effects on root length
Root growth was reduced by 23-27 % by stabilized ash and by 11-21 % by fresh fly ash
compared to control (Figure 3). In contrast concentration of 48 tons ash per hectare increased
root growth by 41 % with fly ash and by 65 % with stabilized ash.
9
Figure 3. Root length of wheat measured after 4 weeks. Mean values were calculated from 4 replicates for
each concentration.
3.3.2 Effects on shoot length
Shoot length of spring wheat was not equaly affected as the roots ( Figure 4). Slightly
negative effect on shoot length compared to control was found only at the two highest
concentrations of fresh fly ash, while the highest concentration of stabilized ash had an
opposite effect.
Figure 4. Shoot length of wheat measured after 4 weeks. Mean values are calculated from 4 replicates for
each concentration.
10
3.4 Immobility of Daphnia magna
Wooden ash leachate was toxic to newborn daphnids ( Figure 5 and 6). In general the toxicity
increased with concentration and decreased with time.
Figure 5. Percentage of immobilized daphnids. Leachate was taken once a week from pots with stabilized
ash. Values are showed as percentage of immobilized daphnids in 4 replicates.
Figure 6. Percentage of immobilized daphnids. Leachate was taken once a week from pots with fly ash.
Values are calculated as total proportion of immobilized daphnids in 4 replicates.
11
3.4.1 Immobility after 24 hours
Fresh fly ash was more toxic than stabilized ash.Toxicity appeared at doses of 12 tons of fly
ash per hectare during the first week of sampling, 24 tons the second week and 48 tons third
week.
Toxicity of leachate from stabilized wooden ash was lower compared to fresh fly ash. The
toxic effect starts to increase above a concentration of 24 tons of stabilized ash per hectare
during first week and at 48 tons all other weeks, however at a slower rate than for fresh ash..
3.4.2 Immobility in 48 hours
Similar trends were seen after 48 hours. Immobility increased with concentration and
decreased with time (1 week to 4 weeks). It was also higher for leachate of fresh fly ash
(Figure 8) compared to leachates from stabilized fly ash (Figure 7). Only a slightly increased
immobility was found after 48 hours compared to 24 hours, except for leachate from
stabilized ash sampled at week 3 and 4. Still it was about half as toxic as fresh fly ash leachate
from the same sampling time and concentration.
Figure 7. Percentage of immobilized daphnids. Leachate was taken once a week from pots with stabilized
ash. Values are calculated as total proportion of immobilized daphnids in 4 replicates.
12
Figure 8. Percentage of immobilized daphnids. Leachate was taken once a week from pots with fly ash.
Values are calculated as total proportion of immobilized daphnids in 4 replicates.
3.4.3 EC50 and statistic analysis
Calculated EC50 values (expressed as tons per hectare) for fresh fly ash and stabilized ash are
shown in Figure 9 and 10, respectively.
Figure 9. EC50 values for daphnids exposed to leachates from fresh fly ash. Maximum and minimum
values represent the 95 % confidence limits.
13
Figure 10. EC50 values for daphnids exposed to leachates from stabilized ash. Maximum and minimum
values represent the 95 % confidence limits.
EC50 values were slightly lower for 48 hours compared to 24 hours because of a longer
exposure time (Figure 9 and 10). EC50 increased for both ashes during the sampling period,
showing a diminshed toxicity with time. Fresh fly ash had lower EC50 compared to stabilized
ash, indicating a higher toxicity to daphnids. No EC50 values could be obtained for sampling
week 4 for both ashes, and week 3 for stabilized ash, due to low immobilization percentage of
daphnids.
Statistical analysis of variance (ANOVA) was made to reveal any dependency of leachate
toxicity to D. magna from variables such as ash type, number of sampling week, exposure
time in hours, and 2 factorial interactions of ash –week, ash –time, and week-hours (Table 2).
Toxicity to D. magna was found to depend on ash type, number of sampling week, exposure
time and ash-week (P< 0.05).
14
Source of variation
Between Subjects
A (ASH)
W (WEEK)
H (HR)
AW
AH
WH
Error 1
DF
15
1
3
1
3
1
3
3
Sums of squares
based on unique
variance
SS (U)
205752
16002
168043
49
21642
0
11
5
MSS
16002
56014
49
7214
0
4
2
F
10668
37343
33
4809
0
2
0
P
0,0000
0,0000
0,0106
0,0000
1,0000
0,2523
Table 2. ANOVA, analysis of variance.
3.5 Germination and growth of Lactuca sativa
3.5.1 Effect on seed germination
Ash leachate produced a variable effect on seeds of Lactuca sativa. During the first week of
sampling leachate reduced the germination rate of seeds to 83 % compared to 95 % in control
group, and 100 % in reference solution (deionized water). This effect was most obvious at
high concentrations, germination rate of seeds in leachate from 96 tons of stabilized ash per
hectare was 30 % and the corresponding figure was10 % for fly ash. No significant effect on
seed germination was observed during any of the other weeks.
3.5.2 Effect on root length
Effect of leachate on root elongation of Lactuca sativa varied with time and concentration
(Figure 11 and 12).
15
Figure 11. Root length of Lactuca sativa grown in leachate from stabilized fly ash, measured once a week.
Mean values are calculated from germinated seeds.
Figure 12. Root length of Lactuca sativa grown in leachate from fresh fly ash, measured once a week.
Mean values are calculated from germinated seeds.
Generally the root length was stable or decreased with higher concentrations compared to
control, but increased during sampling week 3 for both fly ash and stabilized ash. Highest root
length was shown during the third sampling week.
16
3.5.3 Effect on shoot length
Wooden ash leachate has a negative effect on shoot rate of growth during the first week
(Figure 13 and 14).
Figure 13. Shoot length of Lactuca sativa grown in leachate from stabilized ash, measured once a week.
Mean values are calculated from germinated seeds.
Figure 14. Shoot length of Lactuca sativa grown in leachate from fly ash, measured once a week. Mean
values are calculated from germinated seeds.
Shoot length at the highest dose ( 96 tons/ha) was 49 % for stabilized fly ash and 33 % for
fresh fly ash compared to control. A general increase in lenght with concentration and time
was found during week 2-4 for both stabilized and fly ash.
17
3.6 Germination and growth of Raphanus sativus
3.6.1 Effect on seed germination
Germination rate of Raphanus sativus seeds grown in leachates during the first week was the
same for control and reference solution (90 %). Growth rate for seeds in leachate improved
slightly during the sampling weeks, while it varied for control and reference solutions.
3.6.2 Effect on root growth
Stabilized ash and fresh fly ash were found to have a different effect on root elongation of
Raphanus sativus seeds (Figure 15 and 16). Increased concentration of stabilized ash had
none or a small positive effect during the first week, and a negative effect week 2-4. Time had
a positive effect on root elongation, but it was countered by increasing concentration. A
distinct toxic effect was observed at concentration of 96 tons per hectare at week 4, where
root length was about 50 % of control sample.
Figure 15. Effect on root length of Raphanus sativus grown in leachate from stabilized ash, determined
once a week. Mean values are calculated from germinated seeds.
18
Figure 16. Effects on root length of Raphanus sativus grown in leachate from fresh fly ash, determined
once a week. Mean values are calculated from germinated seeds.
Also fresh fly ash leachate had a toxic effect on root elongation, but only during the first two
weeks of sampling (Figure 16). Effects occurred at concentrations exceeding 24 tons per
hectare week 1 and 48 tons week 2. Increasing concentration of leachate durign week 3 and 4
did not exhibit any obvious trends.
3.6.3 Effect on shoot growth
Low doses of stabilized ash and fresh fly ash leachate showed generally a positive effect on
shoot growth (Figure 17 and 18)
Figure 17. Effect on shoot length of Raphanus sativus grown in leachate from stabilized ash, determined
once a week. Mean values are calculated from germinated seeds.
19
Figure 18. Effect on shoot length of Raphanus sativus grown in leachate from fresh fly ash, determined
once a week. Mean values are calculated from germinated seeds.
Higher doses of fresh fly ash leachate had a negative effect on shoot growth during the first
two weeks at doses above 12 tons ha -1.
3.7 TIE of leachate
Results of the TIE treatment with Daphnia magna were inconclusive (Table 2). Toxicity
increased with concentration and diminished with time, but the TIE treatment could not
connect lipophilic compounds, cations or anions to the toxic effect displayed by untreated
leachate. Controls were generally acceptable.
Sample
48SA1
48SA2
48SA3
96SA1
96SA2
96SA3
48FA1
48FA2
48FA3
96FA1
96FA2
96FA3
C18
80
40
0
100
100
40
100
60
0
100
100
100
Immobility (%) of D. magna1
CM
QMA
Untreated
60
60
80
0
0
20
0
0
0
100
100
100
80
80
100
80
40
80
100
100
100
60
60
40
0
0
0
100
100
100
80
80
100
100
100
100
Table 2. TIE of leachate, showed as percentage of immobilized daphnids
1
Five daphnids were added to each well.
20
Control
0
0
0
40
0
0
0
0
0
0
0
0
3.8 Chemical analysis
High concentrations of sulphate were found in leachate, exceeding the controll by 40 times at
the highest ash doze(Figure 19). Concentration fell with time by around 50 % for both
stabilized and fly ash. Fly ash had overall a higher content of sulphate compared to stabilized
ash.
Figure 19. Concentration of sulphate in leachate.
Samples contained very low concentrations of chloride, which was found in only 3 samples,
at the highest ash dozes, showing a decreasing trend (Figure 20).
Figure 20. Concentration of chloride in leachate.
Results of spectrometry show a distinct variation of concentration of metals and trace
elements in leachate samples (Table 3).
21
Sample no
O3
48SA1
48FA1
96SA1
96FA1
48SA2
48FA2
96SA2
96FA2
48SA3
48FA3
96SA3
96FA3
Unit
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
mg L-1
Al
0,2
0,4
0,6
0,8
0,9
0,2
0,2
4
0,6
0,1
0,2
0,2
0,5
Cu
0,3
1,2
2,2
1,5
3,1
1,3
1,7
2,2
2,2
1,5
1,8
1,8
2,4
Mg
17,8
146,8
321,6
349,3
289,1
58,1
103,9
101,9
180
41,3
75,7
47,7
147,6
Mn
0,2
3,8
10
6,8
13,3
1,5
3,9
14,4
4
0,2
0,1
0,8
0,6
P
32,5
23,4
43,9
59,1
78
10,2
10,1
18
26
1,8
0,6
12
3,8
Table 3. Chemical composition of leachate. Sample number designate dose (48 or 96 t ha-1), type of ash
(Stabilized Ash or Fresh Ash), week of experiment (1, 2 and 3). O3 is the control without ash.
Concentration of all alalyzed elements increased with ash dose, and decreased with time.
Leachates of fly ash contained higher concentrations of Cu, Mg and initially also Mn and P
than stabilized ash. Highest metal concentrations in ash exceeded the control (O3) by 8 times
for Cu and Mg and 4 times for Mn. Concentration of P in leachates was lower in leachates
compared to the control during the last week.
4 Discussion
The effects on pH (4.1), wheat growth (4.2), Daphnia magna (4.3), R. sativa and L. sativus
(4.4) and leachate chemistry (4.5) are discussed below.
4.1 Effect on pH
Many studies show an increase of pH in soil as a result of addition of the wooden ash
following dissolution of oxides, hydroxides and carbonates from the ash (Mandre et al., 2006;
Perkiömäki, Fritze, 2002; Arvidsson, Lundkvist, 2003). Increasing doses of wooden ash on
soil resulted in increased pH in leachate by 0.3-1.3 compared to control values during the first
week. Similar effects, but of decreased magnitude,were observed during week 2 and 3.
No clear difference in pH increase between fresh fly ash and stabilized ash was found. Fresh
fly ash has a higher content of CaO compared to stabilized ash, where Ca is mostly presented
as Ca (OH)2 and CaCO3 . Since calcite and portlandite are less soluble, a more moderate
increase of pH should be expected when stabilized ash is added. Absence of any significant
differences between the two ashes may depend on the short time of leaching. Long-term tests
can possiblyresult in a higher difference.
22
4.2 Effect on wheat growth
Wooden ash had only an insignificant negative effect on root growth for concentrations up to
24 tons per hectare. Increasing concentrations lead to doubling of root length, only to return to
previous levels as concentration exceeded 48 tons per hectare. Since no corresponding effects
were observed on the shoot length, this is difficult to interprete.
Previous studies has demonstrated a generally positive effect of wooden ash on plants growth,
among them wheat (Erich, 1991; Huang et al., 1992; Krejsl, Scanlon, 1996). Absence of such
effects during these tests might depend on a too short growth period. It may also result from
chemical characteristics of soil used in pot tests. The soil was already nutrient rich, therefore
removing this limiting factor to plants growth. Additional nutrients from the ash were then
redundant.
4.3 Effect on D. magna
Bioassays showed a toxic effect of wooden ash on Daphnia magna. Immobility increased
with concentration, exposure time, but decreased for each sampling week. Fresh fly ash was
more toxic than stabilized ash.
A slightly decreased EC50 values for 48 hours compared to 24 hours of exposure were
expected considering the longer time daphnids were exposed to leachate. EC50 values are also
in all aspects lower for fresh fly ash in comparison to stabilized ash, indicating higher toxicity
to D. magna. EC50 of both ashes increased during sampling weeks. This means that toxic
constituents leached easily, and especially from the fresh ash.
Since no difference in pH was shown between fresh fly ash and stabilized ash there must be
some other source of toxicity. TIE were made in order to reveal if cations, anions or lipophilic
compounds could be connected to the leachate toxicity, but failed to give any clear answers.
4.4 Effect on R. sativa and L. sativus
Radish and lettuce seeds were not as sensitive as daphnids to leachate toxicity. Usualy an
insignificant negative effect on shoot length was observed during the first sampling week.
Root length showed no general trends in response to increasing concentrations or sampling
weeks, except a slightly increasing root length during week 2 to 4 compared to week 1.
4.5 Chemical analysis
Chemical analysis revealed high concentrations of several metals and sulphate in leachates,
often many times higher than control sample. This must be a consequence of the high content
of sulphur and metals in wooden ash subsequently leached out by watering. Concentrations of
metals and sulphur were lower in leachates from stabilized ash compared to fresh fly ash,
which is in line with previous research (Steenari et al., 1999).
23
High metal and sulphur concentrations in leachate might also explain the initially high
toxicity to daphnids during the first two sampling weeks. Ash doses which had the highest
toxicity on D. magna were also shown to have the highest concentrations of metals and
sulphate. Presence of both positive and negative ions in leachate may also had disrupted TIE.
Either metals or sulphate could cause immobilization, since none of the filters could remove
both cations and anions simultaneously.
5 Conclusions
Results from bioassays with stabilized and fresh wooden ash on Daphnia magna, Triticum
aestivum and seeds of Raphanus sativus and Lactuca sativa have showed the following:
•
•
•
•
•
•
•
•
Growth of wheat was not negativly affected by ash concentrations of up to 24 tons per
hectare.
Wooden ash increase pH in soil from 0,1 to 1,4 in proportion to ash dose.
Leachates were toxic to daphnids at doses exceeding 24 tons per hectare. EC50 values
for leachates from fresh fly ash were lower in comparison to stabilized ash which
indicates higher toxicity.
EC50 for 24 hours exposure time was slightly higher for both ashes compared to 48
hours, which means that toxicity increased with exposure time.
Toxicity of both ashes to daphnids decreased for every week, which sugestest that
leaching of toxic components was initially rapid.
Analysis of variance (ANOVA) showed a significant relationship between toxicity of
ash to daphnids and variables such as ash type (fresh or stabilized), number of
sampling weeks (1-4), exposure time in hours (24 and 48) and ash type+week.
Effects of ash leachates on radish and lettuce seeds were not as distinct as on
daphnids. Negative effects were observed on root and shoot length during the first
week of sampling.
Attempts were made to identify substances responsible for toxicity by performing a
TIE with columns neutralizing lipophilic compounds, cations and anions. However,
none of the tests showed any clear results.
Acknowledgements
A lot of people have a share in this work, some directly, by guiding me through the whole
process and giving me valuable ideas and recommendations. Others by releaving me of some
of my daily duties as coaching, cooking et al or simply giving me a kick in the butt whenever
I needed one. Thank you all.
First of all I would like to thank my suprevisor Göran Dave for his support, advices and of
course his great patience, especially with my constant lateness!
Thanks to Britt-Marie Steenari for useful hints and approaches in the search for the toxic
agents.
Also a big hug to Vivian Aldén for assisting me with laboratory procedures.
Thanks to Arvid Ödegård Jensen, Dongmei Zhao and Kristian Larsson for helping me with
chemical analyses.
24
Last but not least I would like to thank Elisabet Sjökvist, Yelena Manakova and Boris Krakov
for pre reading and commenting on this thesis.
6 References
Akselsson, C. 2005. Regional nutrient budgets in forest soils in a policy perspective. Doctoral
thesis. Department of Chemical Engineering, Lund University, Lund.
Arvidsson, H., Lundkvist, H., 2003. Effects of crushed wood ash on soil chemistry in young
Norway spruce stands. Forest Ecology and Management. 176, 121-132.
Bramryd, T., Fransman, B., 1995. Silvicultural uses of wood ashes –effects on the nutrient
and heavy metal balance in a pine (Pinus sylvestris L.) forest soil. Water Air Soil Pollut. 85,
1039-1044.
Beltrami, M., Rossi, D., Baudo, R. 1999. Phytotoxicity assessment of Lake Orta sediments.
Aquatic Ecosystem Health and Management 2:391-401.
Dahlberg, A., Stokland, J., N., 2004. Vedlevande arters krav på substrat –sammanställning
och analys av 3600 arter. Rapport 2004:7 Skogsstyrelsen, Jönköping.
Egnell. G., Leijon, B. 1999. Survival and growth of planted seedlings of Pinus sylvestris and
Picea abies after different levels of biomass removal in clear-felling. Scandinavian Journal of
Forest Research 14: 303-311.
Egnell, G., Nohrstedt, H., O., Weslien, J., Westling, O., Örlander, G. 1998.
Miljökonsekvensbeskrivning (MKB) av skogsbränsleuttag, asktillförsel och övrig
näringskompensation. Rapport 1998:1 Skogsstyrelsen, Jönköping.
Egnell, G., Valinger, E. 2003. Survival, growth and growth allocation of planted Scots pine
trees after different levels of biomass removal in clear-felling. Forest Ecology and
Management 177: 65-74.
Energimyndigheten, 2009. Energiläget 2009. Statens energimyndighet. ET 2009:28
Erich, M. S., Ohno, T., 1992b. Phosphorus availability to corn from wood ash amended soils.
Water Air Soil Pollut. 64, 475-485.
Etiegni, L., Campbell, A.G. 1991. Physical and chemical characteristics of wood ash. Biores.
Technol. 37, 173-178.
Etiegni, L., Campbell, A.G., Mahler, R.L., 1991a. Evaluation of wood ash disposal on
agricultural land. I. Potential as soil additive and liming agent. Commun. Soil Sci. Plant
Anal. 22, 243-256.
Ferm, A., Hokkanen, T., Moilanen, M., Issakainen, J., 1992. Effects of wood bark ash on the
growth and nutrition of a Scots pine afforestation in central Finland. Plant and Soil 147, 305316.
25
Fritze, H., Smolander, A., Levula, T., Kitunen, V., Mälkönen, E., 1994. Wood-ash
fertilization and fire treatments in a Scots pine forest stand: Effects on the organic layer,
microbial biomass, and microbial activity. Bio Fertil Soils, 17:57-63.
Huang, H., Capmbell, A.G., Folk, R., Mahler, R. L., 1992. Wood ash as a soil additive and
liming agent for wheat. Field studies. Commun. Soil Sci. Plant Anal. 23, 25-33.
ISO, 1996. Water quality –determination of the inhibition of the mobility of Dapnia magna
Straus ( Cladocera, Crustacea) –acute toxicity test. ISO 6341:1996. International
Organisation for Standardisation, Geneva, Switzerland.
Jacobson, S. 2003. Addition of stabilized wood ashes to Swedish coniferous stands on mineral
soils –Effects on stem growth and needle nutrioent concentrations. Silva Fennica 37: 437-450.
Kahl, J., S., Fernandez, I., J., Rustad, L., E., Peckenam, J., 1996. Threshold application rates
of wood ash to an acidic forest soil. J. Environ. Qual. 25, 220-227.
Krejsl, J., A., Scanlon, T., M., 1996. Evaluation of beneficial use of wood-fired boiler ash on
oat and bean growth. J. Environ. Qual. 25, 950-954.
Mandre, M., Pärn, H., Ots, K., 2006. Short-term effects of wood ash on the soil and the lignin
concentration and growth of Pinus sylvestris L. Forest Ecology and Management. 223, 349357.
Meiwes, K., J., 1995. Application of lime and wood ash to decrease acidification of forest
soils. Water Air Soil Pollut. 85, 143-152.
Miljömålsrådet, 2010. Hämtad 2010-03-04 från: http://www.miljomal.nu/Undre-meny/OmMiljomalsradet/
Muse, J.K. Mitchell, C. C., 1995. Paper mill boiler-ash and lime by-products as soil liming
materials. Agron. J. 87, 432-438.
Ohno, T., Erich, M., S., 1990. Effect of wood ash application on soil pH and soil test nutrient
levels. Agric. Ecosyst. Environ. 32, 223-239.
Olsson, B., A., Bengtsson, J., Lundkvist H. 1996. Effects of different forest harvest intensities
on the pools of exchangable cations in coniferous forest soils. Forest Ecology and
Management 84:135-137.
Parikka, M. 1997. Biosims - a method for the estimation of woody biomass
for fuel in Sweden. Swedish Universityof Agricultural Sciences. Doktorsavhandling Silvestria
no. 27. Uppsala
Perkiömäki, J., Fritze, H., 2002. Short and long-term effects of wood ash on the boreal forest
humus microbial community. Soil Biology & Biochemistry 34, 1343-1353.
Renoux, A. Y., Tyagi, R. D., Samson, R. 2001. Assesment of toxicity reduction after metal
removal in bioleached sewage sludge. Water Research, 35:1415-1424.
26
Mahmood, S., Finlay, R. D., Fransson, A-M., Wallander, H., 2003. Effects of hardened wood
ash on microbial activity, plant growth and nutrient uptake by ectomycorrhizal spruce
seedlings. FEMS, Microbiology Ecology. 43, 121-131.
Norberg-King, T., D. Mount, E. Durhan, G. Ankley, L. Burkhard, J. Amato & L. AndersonCarnahan, 1991. Methods for aquatic toxicity identification evaluations: Phase I Toxicity
Characterization Procedures. (Second Edition), EPA-600/6-91/003, U.S. Environmental
Protection Agency, Duluth, MN.
Someshwar, A. V., 1996. Wood ash and combination wood-fired boiler ash characterization.
J. Environ. Qual. 25, 962-972.
Skogsstyrelsen 2008. Rekommendationer vid uttag av avverkningsrester och askåterföring.
Meddelande 2008*2. Skogsstyrelsens förlag.
Steenari, B.-M., Karlsson, L. G., Lindqvist, O., 1999. Evaluation of the leaching
characteristics of wood ash and the influence of ash agglomeration. Biomass and Bioenergy.
16, 119-136.
Steenari, B.-M., Lindqvist, O., 1997. Stabilization of biofuel ashes for recycling to forest soil.
Biomass and Bioenergy. 13, 39-50
Steenari, B.-M., Schelander, S., Lindqvist, O., 1999. Chemical and leaching characteristics of
ash from combustion of coal, peat and wood in a 12 MW CFB – a comparative study. Fuel.
78, 249-258.
Thelin, G. 2006. Askåterföring till gran- och bokbestånd – effekter på näring, tillväxt,
kvävedynamik och kolbalans. Rapport 965. Värmeforsk, Stockholm.
UN, 1987. Report of the World Commission on Environment and Development. General Assembly
Resolution 42/187. Hämtad 2010-05-10 från: http://www.un.org/documents/ga/res/42/ares42-187.htm
Unger, Y., L., Fernandez, I., J., 1990. The short-term effects of wood-ash amendmend on
forest soils. Water Air Soil Pollut. 49, 299-314.
Värmeforsk, 2008. Miljövänlig användning av askor 2009-2011 – Sammanfattning. Hämtad
2010-05-03 från: http://www.askprogrammet.com/inbjudan2009-11.htm.
27
Annex A. Primary data from week 1
Sampling leachate, week 1
Sample
pH
3SA1
3SA2
3SA3
3SA4
3FA1
3FA2
3FA3
3FA4
6SA1
6SA2
6SA3
6SA4
6FA1
6FA2
6FA3
6FA4
12SA1
12SA2
12SA3
12SA4
12FA1
12FA2
12FA3
12FA4
24SA1
24SA2
24SA3
24SA4
24FA1
24FA2
24FA3
24FA4
48SA1
48SA2
48SA3
48SA4
48FA1
48FA2
48FA3
48FA4
96SA1
96SA2
96SA3
96SA4
96FA1
96FA2
96FA3
96FA4
Control1
Control2
Contol3
Control4
Reference1
Reference2
6.2
6.0
6.1
6.2
6.1
6.2
6.0
6.2
6.1
6.1
6.2
6.3
6.8
6.3
6.3
6.6
6.8
6.3
6.6
6.6
6.7
6.6
6.9
7.0
6.9
7.1
6.9
6.9
6.7
7.1
7.4
7.1
7.1
7.3
7.2
7.0
7.2
6.9
7.0
7.1
6.7
6.7
6.9
7.0
6.8
6.8
6.7
6.9
5.8
5.8
5.8
5.8
2
3
4
Mobility of D. magna2
24h
48h
10
10
10
10
10
10
10
10
9
10
10
10
10
10
10
9
10
10
10
10
8
9
9
9
10
10
9
9
3
8
6
8
4
4
7
6
0
0
0
0
0
0
0
0
0
0
0
0
10
10
10
10
10
10
10
10
10
10
10
10
9
10
10
9
10
10
10
9
10
10
10
10
8
8
9
9
10
10
8
9
3
7
4
3
4
2
5
6
0
0
0
0
0
0
0
0
0
0
0
0
10
10
10
10
L. sativa3
Shoot Root
44
46
49
52
49
58
52
59
52
53
49
51
51
50
42
46
54
59
51
53
52
47
48
53
54
55
51
56
39
46
47
47
37
47
54
51
41
25
34
38
2
0
2
3
0
0
2
0
55
52
50
40
19
22
20
30
28
27
31
38
36
39
36
28
33
34
22
27
24
20
27
27
32
26
25
28
20
19
26
23
24
30
23
24
23
20
13
23
26
25
14
18
20
17
1
0
2
2
0
0
5
0
32
34
27
26
12
17
R. sativus4
Shoot Root
52
64
43
43
60
35
47
49
54
39
46
45
35
41
51
33
36
32
50
39
52
51
62
48
62
31
30
33
39
55
43
48
62
52
46
58
34
37
36
29
38
35
33
41
19
19
36
27
47
37
58
52
23
26
59
84
57
53
82
64
56
61
81
79
67
59
39
50
72
56
70
58
55
42
61
58
65
72
82
52
50
35
73
56
62
65
69
70
68
84
50
59
50
35
45
60
51
74
34
27
67
43
62
56
62
56
60
58
Number of living daphnids after 24 and 48 hours of exposure. Ten dahpnids were added for each sample
Measured in mm.
Measured in mm.
28
Reference3
Reference4
23
24
12
12
29
25
24
61
72
Annex B. Primary data from week 2
Sampling leachate, week 2
Sample
pH
3SA1
3SA2
3SA3
3SA4
3FA1
3FA2
3FA3
3FA4
6SA1
6SA2
6SA3
6SA4
6FA1
6FA2
6FA3
6FA4
12SA1
12SA2
12SA3
12SA4
12FA1
12FA2
12FA3
12FA4
24SA1
24SA2
24SA3
24SA4
24FA1
24FA2
24FA3
24FA4
48SA1
48SA2
48SA3
48SA4
48FA1
48FA2
48FA3
48FA4
96SA1
96SA2
96SA3
96SA4
96FA1
96FA2
96FA3
96FA4
Control1
Control2
Contol3
Control4
Reference1
Reference2
Reference3
Reference4
6.8
6.9
6.7
6.6
7.1
7.1
7.0
7.0
6.9
7.0
6.9
6.9
7.1
7.5
7.5
7.4
7.1
7.3
7.2
7.4
7.6
7.6
7.3
7.3
7.5
7.4
7.2
7.5
7.3
7.5
7.4
7.4
7.7
7.6
7.5
7.8
7.4
7.5
7.4
7.5
7.7
7.6
7.7
7.4
7.5
7.6
7.7
7.4
6.5
6.6
6.7
6.9
5
6
7
Mobility of D. magna
24h
48h
10
10
10
10
10
10
10
10
10
10
10
10
10
8
10
10
10
10
10
10
8
10
10
10
10
10
10
10
9
10
9
9
8
7
10
9
4
4
5
3
3
0
2
0
0
0
0
0
8
10
8
10
10
9
10
10
10
10
9
9
10
10
10
10
10
7
9
10
10
10
10
10
8
10
10
10
9
10
10
10
9
10
9
9
8
5
10
8
4
4
3
3
3
0
2
0
0
0
0
0
8
10
6
10
5
6
L. sativa
Shoot Root
46
46
40
33
45
48
41
44
43
54
35
38
38
36
38
24
50
45
47
42
31
44
39
39
47
43
49
49
42
33
47
49
46
48
47
49
49
47
35
45
41
44
30
38
40
49
51
45
40
39
40
30
29
30
25
31
67
67
53
58
30
31
32
38
39
66
37
60
61
56
43
37
59
55
46
45
38
63
60
60
55
59
42
56
34
43
51
46
44
49
64
55
42
45
42
52
35
33
28
33
22
24
32
17
44
54
61
42
41
36
35
34
7
R. sativus
Shoot Root
33
57
34
46
48
43
32
39
53
45
44
40
39
43
35
29
45
50
39
41
54
59
47
48
47
42
52
36
52
54
53
44
37
47
47
55
39
52
43
44
30
43
50
43
38
33
36
37
40
27
18
37
27
25
27
30
61
125
121
110
84
140
68
111
87
78
100
88
108
159
80
111
87
105
52
98
147
103
132
114
93
107
62
73
77
147
106
87
53
60
110
77
87
147
130
96
78
53
69
64
56
49
57
40
120
89
93
105
57
121
119
113
Number of living dahpnids after 24 and 48 hours of exposure. Ten dahpnids were added for each sample
Measured in mm.
Measured in mm.
30
Annex C. Primary data from week 3
Sampling leachate, week 3
Sample
pH
3SA1
3SA2
3SA3
3SA4
3FA1
3FA2
3FA3
3FA4
6SA1
6SA2
6SA3
6SA4
6FA1
6FA2
6FA3
6FA4
12SA1
12SA2
12SA3
12SA4
12FA1
12FA2
12FA3
12FA4
24SA1
24SA2
24SA3
24SA4
24FA1
24FA2
24FA3
24FA4
48SA1
48SA2
48SA3
48SA4
48FA1
48FA2
48FA3
48FA4
96SA1
96SA2
96SA3
96SA4
96FA1
96FA2
96FA3
96FA4
Control1
Control2
Contol3
Control4
Reference1
Reference2
Reference3
Reference4
7.3
7.2
7.4
7.4
7.4
7.5
7.6
7.5
7.5
7.6
7.7
7.7
8.0
7.5
7.6
7.7
7.7
8.1
8.1
7.8
7.9
8.0
8.1
8.5
8.3
8.5
8.0
8.2
8.1
8.3
8.3
8.2
8.2
8.2
8.0
8.1
8.0
7.9
-
8
9
Mobility of D. magna 8
24h
48h
10
10
8
10
10
10
10
10
10
10
10
10
10
10
8
10
10
8
9
7
10
9
10
10
10
10
9
10
10
9
10
10
10
10
9
10
7
9
10
10
7
7
7
9
6
1
7
0
10
10
10
10
10
10
7
10
10
10
10
10
10
10
10
9
10
10
8
10
10
8
9
0
8
8
10
10
10
10
9
10
10
6
10
6
10
10
9
10
3
9
10
6
5
7
7
9
5
1
5
0
10
10
10
9
L. sativa9
Shoot Root
31
35
41
40
48
42
47
44
44
48
48
44
47
48
47
43
48
49
51
48
48
47
45
50
47
47
46
41
50
46
47
48
47
51
50
52
44
45
43
50
51
49
50
51
45
49
44
44
42
41
45
35
37
35
41
39
48
56
54
74
60
64
70
70
55
57
61
54
69
72
51
68
52
51
52
57
72
54
69
72
52
76
65
75
77
59
60
73
52
69
65
69
63
61
78
71
60
50
45
58
48
49
61
40
88
76
88
74
61
49
57
55
R. sativus10
Shoot Root
45
32
26
41
33
31
31
29
44
47
40
38
34
35
37
29
42
46
42
49
42
46
38
41
44
44
33
41
43
35
49
38
48
37
47
45
47
46
37
42
52
52
42
36
48
54
51
54
40
37
41
16
30
35
38
32
134
120
115
162
97
122
68
89
112
157
107
72
187
118
101
131
78
124
117
130
133
128
79
75
123
103
69
112
102
105
118
81
99
101
117
134
133
148
135
89
125
87
48
94
102
119
104
107
135
111
133
52
106
90
137
103
Number of living daphnids after 24 and 48 hours of exposure. Ten dahpnids were added for each sample
Mesuared in mm.
Mesuared in mm.
10
31
Annex D. Primary data from week 4
Sampling leachate, week 4
Sample
3SA1
3SA2
3SA3
3SA4
3FA1
3FA2
3FA3
3FA4
6SA1
6SA2
6SA3
6SA4
6FA1
6FA2
6FA3
6FA4
12SA1
12SA2
12SA3
12SA4
12FA1
12FA2
12FA3
12FA4
24SA1
24SA2
24SA3
24SA4
24FA1
24FA2
24FA3
24FA4
48SA1
48SA2
48SA3
48SA4
48FA1
48FA2
48FA3
48FA4
96SA1
96SA2
96SA3
96SA4
96FA1
96FA2
96FA3
96FA4
Control1
Control2
Contol3
Control4
Reference1
Reference2
Reference3
Reference4
11
12
13
pH
Mobility of D. magna 11
24h
48h
10
10
10
10
10
10
10
10
10
10
10
10
10
9
10
10
10
10
10
9
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
9
7
10
10
8
7
9
8
10
9
10
9
10
10
9
10
10
10
10
10
10
10
9
10
10
9
10
10
10
10
10
0
9
10
10
10
10
10
10
10
10
9
10
10
10
9
9
10
8
9
10
10
9
7
10
10
8
6
9
7
10
8
10
9
L. sativa12
Shoot Root
38
42
42
40
36
35
34
33
37
39
39
31
36
36
38
34
40
41
40
43
36
37
37
35
38
44
43
46
39
34
39
39
45
44
42
43
37
43
38
41
52
51
51
45
46
49
44
47
29
33
30
31
23
21
23
20
51
54
52
40
41
41
46
45
38
48
47
34
46
46
44
44
45
43
43
47
38
43
48
35
41
48
50
41
47
42
44
43
46
45
44
45
45
47
46
39
37
38
32
42
37
36
42
33
42
40
39
39
24
22
21
18
R. sativus13
Shoot Root
45
35
45
48
32
33
28
29
52
40
36
42
35
26
23
31
41
37
29
43
32
39
24
26
32
45
51
37
43
31
35
34
38
40
31
33
48
39
38
42
44
47
45
45
44
40
39
49
31
35
36
36
24
23
26
28
168
107
150
160
73
112
117
120
133
158
89
141
113
80
67
119
160
97
96
147
105
89
85
68
55
135
101
122
122
74
128
84
87
82
70
81
112
67
91
103
69
67
81
94
108
143
88
113
155
162
150
174
67
54
50
34
Number of living daphnids after 24 and 48 hours of exposure. Ten dahpnids were added for each sample
Mesuared in mm
Mesuared in mm
32
Annex E. Primary data on wheat growth
Growth of wheat measured after 4 weeks
14
15
Replicate
Shoot14
Root15
3SA1
3SA2
3SA3
3SA4
3FA1
3FA2
3FA3
3FA4
6SA1
6SA2
6SA3
6SA4
6FA1
6FA2
6FA3
6FA4
12SA1
12SA2
12SA3
12SA4
12FA1
12FA2
12FA3
12FA4
24SA1
24SA2
24SA3
24SA4
24FA1
24FA2
24FA3
24FA4
48SA1
48SA2
48SA3
48SA4
48FA1
48FA2
48FA3
48FA4
96SA1
96SA2
96SA3
96SA4
96FA1
96FA2
96FA3
96FA4
Contol1
Contol2
Contol3
Contol4
462
419
415
385
465
381
410
397
390
414
378
392
421
384
370
326
445
380
428
386
379
378
349
457
405
395
393
400
435
405
456
365
411
423
394
415
400
375
405
360
492
377
503
395
391
500
454
167
421
432
344
439
195
443
175
460
210
130
745
170
257
346
241
208
595
177
275
164
171
125
663
160
244
160
502
304
235
293
276
571
200
391
556
192
719
720
640
185
155
473
783
513
137
257
421
178
218
205
200
400
336
297
225
510
Measured in mm.
Measured in mm.
33
Annex F. Data from ANOVA for effects on Daphnia
Statistical analysise of EC50 values, using moving average method
95% confidence limits
EC5016
Week 1
FA
Week 2
Week 3
Week 1
SA
Week 2
Week 317
24 h
21,88928
min
19,198
48 h
24 h
19,35434
40,26009
17,00242
35,1479
22,19875
46,61631
48 h
24 h
38,25366
80,78469
33,39987
71,10683
44,14538
96,82062
48 h
24 h
70,5107
44,69576
60,31694
39,5751
83,83122
51,15876
48 h
24 h
41,62199
60,84266
36,91333
53,56932
47,39886
70,61092
48 h
24 h
66,44061
-
58,72845
-
77,119
48 h
16
17
-
-
Measured in tons of ash per hectare
No EC50 values could be calculated for week 3, due to low toxicity.
34
max
25,32582
-