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Faculty of Bioscience Engineering
Academic year 2014 – 2015
Acute aquatic toxicity test of caffeine with
Daphnia magna Straus and biomonitoring of
PRB and Intensive Green Filter wastewater
treatment systems
Kim Driesen
Promotor: Prof. dr. ir. Diederik Rousseau
Tutor: Dr. Isabel Martín García
Master’s dissertation submitted in partial fulfillment of the
requirements for the degree of
Master of Science in Engineering Technology in Environmental
Sciences
Faculty of Bioscience Engineering
Academic year 2014 – 2015
Acute aquatic toxicity test of caffeine with
Daphnia magna Straus and biomonitoring of
PRB and Intensive Green Filter wastewater
treatment systems
Kim Driesen
Promotor: Prof. dr. ir. Diederik Rousseau
Tutor: Dr. Isabel Martín García
Master’s dissertation submitted in partial fulfillment of the
requirements for the degree of
Master of Science in Engineering Technology in Environmental
Sciences
ii
Copyrights
The author, the promotor and the tutor give their permission to put this master’s thesis at
disposal for consultation and to copy parts of it for personal use. Every other use is
subject to the restrictions of copyright, in particular with the obligation to explicitly
mention the source when quoting results of this master's thesis.
Seville, January 2015
The author,
The promotor,
iv
Acknowledgments
To complete the “Master of Science in Engineering Technology in Environmental Sciences”
at Ghent University, Belgium, I accomplished an internship of 14 weeks at the Foundation
Center of New Water Technologies, CENTA, in the Experimental Center on R&D&I of
Carrión de los Céspedes (Seville, Spain). I was accompanied by dr. Isabel Martín García,
project coordinator at the CENTA Foundation. My Belgian promotor was prof. dr. ir.
Diederik Rousseau.
Before I start describing the situation, I would like to express my gratitude to the people
who helped me to complete this thesis.
This research was made possible due to the support of prof. dr. ir. Diederik Rousseau
(Ghent University) and dr. Isabel Martín García (CENTA) for giving me this opportunity to
finish my thesis at CENTA. I would also like to express my gratitude for their support
during my stay.
Finally, I would like to thank my boyfriend Michaël Baguet, my mother Greet Embrechts
and her friend Jef Vervecken, for supporting me psychologically and financially at all times
when I spent 4 months in Seville, Spain.
Thanks to all these people, I experienced an amazing way to enlarge my knowledge
about possible solutions for environmental problems in the world and to enrich my view of
the Andalusian culture.
Thank you Kim Driesen
vi
Table of Contents
List of Abbreviations ................................................................................................. vii
List of Figures .......................................................................................................... viii
List of Tables ............................................................................................................ ix
Abstract .................................................................................................................... x
Dutch abstract ......................................................................................................... xii
1 Introduction ........................................................................................................ 1
2 Literature review ................................................................................................. 3
2.1 Project: Water reuse ..................................................................................... 3
2.2 Treatment technologies ................................................................................. 4
2.3 Microcontaminants ...................................................................................... 10
2.4 Daphnia magna .......................................................................................... 12
2.5 Defining the microcontaminants to be tested ................................................. 15
2.6 Relevant researches relating Daphnia magna ................................................ 17
3 Methodology ..................................................................................................... 19
3.1 Culturing method ........................................................................................ 19
3.2 Reagents and materials ............................................................................... 23
3.3 Acute aquatic toxicity test with Daphnia magna Straus ................................... 24
3.4 Procedure for caffeine ................................................................................. 25
3.5 Procedure for influents and effluents ............................................................ 27
3.6 Interpretation and validity of the results........................................................ 27
4 Results ............................................................................................................. 29
4.1 Acute aquatic toxicity test of caffeine ............................................................ 29
4.2 Acute aquatic toxicity test of the influents/effluents ....................................... 32
4.3 Validity of the results ................................................................................... 37
5 Discussion ......................................................................................................... 41
5.1 Acute aquatic toxicity test of caffeine ............................................................ 41
5.2 Acute aquatic toxicity test of the influents/effluents ....................................... 41
5.3 Validity of the results ................................................................................... 42
6 Conclusions ....................................................................................................... 45
7 Recommendations ............................................................................................. 47
8 References ........................................................................................................ 49
Appendices .................................................................................................................
vii
List of Abbreviations
BOD5 Biological Oxygen Demand during five days of incubation
CENTA Foundation Center of New Water Technologies
COD Chemical Oxygen Demand
DOC Dissolved Organic Carbon
EC50 Median Effective Concentration
EPA Environmental Protection Agency
IGF Intensive Green Filter
ITIS Integrated Taxonomic Information System
NSAID Non-steroidal anti-inflammatory drugs
NT Total nitrogen
PhACs Pharmaceutically active compounds
PRB Permeable Reactive Barrier
PT Total phosphorus
R&D&I Research & Development & Innovation
REAGUAM Reuse of treated urban wastewaters for environmental uses
TOC Total Organic Carbon
TSS Total Suspended Solids
WWTP Wastewater treatment plants
viii
List of Figures
Figure 1: Experimental Center on R&D&I of Carrión de los Céspedes (Seville, Spain) ....... 1
Figure 2: Flow sheet REAGUAM-project: Intensive Green Filter and Permeable Reactive
Barrier ....................................................................................................................... 5
Figure 3: Overview of the Intensive Green Filter with poplars (Populus alba) ................... 6
Figure 4: Intensive Green Filter plot at CENTA, Seville (de Miguel, 2014) ....................... 6
Figure 5: Permeable Reactive Barrier (PRB) with horizontal flow [12] ............................. 8
Figure 6: Layers of the Permeable Reactive Barrier at CENTA ......................................... 9
Figure 7: Overview of the Permeable Reactive Barrier at CENTA .................................... 9
Figure 8: Daphnia magna [29] .................................................................................. 13
Figure 9: Neonates of Daphnia magna ....................................................................... 20
Figure 10: Daphnia magna - Post-abdominal claw ...................................................... 20
Figure 11: Back-up aquarium of ± 100 liter ................................................................ 21
Figure 12: 3-liter-aquaria for the culturing of Daphnia magna ...................................... 21
Figure 13: Test plate with test containers ................................................................... 24
Figure 14: Test containers - The test wells in each column are labelled A, B, C and D and
the rows are labelled X (controls), 1, 2, 3, 4 and 5 for the five toxicant dilutions. [51] ... 25
Figure 15: Logistic regression of response by log(dose) for caffeine after 24 h .............. 30
Figure 16: Logistic regression of response by log(dose) for caffeine after 48 h .............. 31
Figure 17: Logistic regression of response by log(dose) for influent IGF after 24 h ......... 33
Figure 18: Logistic regression of response by log(dose) for effluent IGF after 24 h ........ 34
Figure 19: Logistic regression of response by log(dose) for influent PRB after 24 h ........ 35
Figure 20: Logistic regression of response by log(dose) in the validity test of culture A .. 38
Figure 21: Logistic regression of response by log(dose) in the validity test of culture B .. 39
Figure 22: Logistic regression of response by log(dose) in the validity test of culture C .. 40
ix
List of Tables
Table 1: Average concentrations of the wastewater applied to the Intensive Green Filter
at CENTA and the groundwater ................................................................................... 7
Table 2: Layers of the Permeable Reactive Barrier at CENTA .......................................... 9
Table 3: Average concentrations of the wastewater applied to the Permeable Reactive
Barrier at CENTA and the groundwater [11] ................................................................. 9
Table 4: PhACs in the wastewater of the Experimental Center of Carrión de los Céspedes
(Seville, Spain) ......................................................................................................... 10
Table 5: Optimal culturing conditions for Daphnia magna [30] ..................................... 14
Table 6: Review of several studies about acute aquatic toxicity tests with Daphnia magna
concerning the relevant PhACs .................................................................................. 15
Table 7: Results of the acute aquatic toxicity test of 4-FAA and 4-AAA [22] ................... 15
Table 8: Pharmaceutically active compounds present in the wastewater of the
Experimental Center of Carrión de los Céspedes (Seville, Spain) – Cost price [45].......... 16
Table 9: Results preliminary test on caffeine after 24 h –Immobilized Daphnia magna ... 29
Table 10: Results preliminary test on caffeine after 48 h – Immobilized Daphnia magna 29
Table 11: Results definitive test on caffeine after 24 h – Immobilized Daphnia magna ... 30
Table 12: Probability analysis for caffeine after 24 h – determination of EC50 ................. 30
Table 13: Results definitive test on caffeine after 48 h – Immobilized Daphnia magna ... 31
Table 14: Probability analysis for caffeine after 48 h – determination of EC50 ................. 32
Table 15: Results preliminary test of non-diluted influents and effluents after 24 and 48 h
– Immobilized Daphnia magna .................................................................................. 32
Table 16: Results preliminary test of 1:2 diluted influents and effluents after 24 and 48 h
– Immobilized Daphnia magna .................................................................................. 32
Table 17: Results definitive test influent IGF after 24 h – Immobilized Daphnia magna .. 33
Table 18: Probability analysis influent IGF – determination of EC50 ............................... 34
Table 19: Results definitive test effluent IGF after 24 h – Immobilized Daphnia magna .. 34
Table 20: Results definitive test influent PRB after 24 h – Immobilized Daphnia magna .. 35
Table 21: Probability analysis influent PRB – determination of EC50 ............................... 36
Table 22: Results definitive test effluent PRB after 24 h – Immobilized Daphnia magna . 36
Table 23: Sampling data Intensive Green Filter, 28/10/2014 ........................................ 36
Table 24: Sampling data Permeable Reactive Barrier, 28/10/2014 ................................ 37
Table 25: Results validity test of culture A after 24 h – Immobilized Daphnia magna...... 37
Table 26: Probability analysis validity test of culture A – determination of EC50 .............. 38
Table 27: Results validity test of culture B after 24 h – Immobilized Daphnia magna ...... 38
Table 28: Probability analysis validity test of culture B – determination of EC50 .............. 39
Table 29: Results validity test of culture C after 24 h – Immobilized Daphnia magna...... 39
Table 30: Probability analysis validity test of culture C – determination of EC50 .............. 40
Table 31: EC50-values for caffeine – 24 and 48 h......................................................... 41
Table 32: EC50–24 h for influents/effluents ................................................................. 42
x
Abstract This thesis was written in connection with an internship of 14 weeks which was performed
at the Foundation Center of New Water Technologies (CENTA). The study site of CENTA is
located in the South East of Spain, in the Experimental Center on R&D&I of Carrión de los
Céspedes (Seville, Spain).
One current project carried out by CENTA is the project “REAGUAM”. This project
investigates the use of reclaimed water deriving from treated wastewater by two non-
conventional technologies:
The first treatment system represents the Intensive Green Filter with short-
rotation coppice of poplars (Populus alba). The wastewater derives from CENTA’s
office building and passes an Imhoff tank which reduces its total organic load with
20-30 %.
The second treatment system is a Permeable Reactive Barrier with horizontal
layers, from top to bottom: palygorskite, activated carbon, zeolite and sand. This
treatment is preceded by a preliminary treatment that consists of screening,
degritting and degreasing, followed by an extended aeration treatment and a sand
filter.
The main goal of the project is to evaluate the capability of the medium to regenerate the
quality of the treated wastewater that is applied, in both the Intensive Green Filter and
the Permeable Reactive Barrier. Within this objective, the impact of the irrigated
wastewater on the groundwater quality has to be assessed. Therefore an ecotoxicological
evaluation has to be made to determine the toxicity of the most important
microcontaminants in the wastewater.
One of the most important microcontaminant in the wastewater of Carríon de los
Céspedes is caffeine. Caffeine appeared an adequate microcontaminant to be tested,
because of its high presence in the wastewater, its low cost price and because not a lot of
studies were published before about the acute toxicity of caffeine with Daphnia magna
after 24 hours.
In this thesis, an acute aquatic toxicity test with Daphnia magna is performed on caffeine.
Because one analyzed microcontaminant cannot represent the mixture of
microcontaminants that are present in the wastewaters of the IGF and the PRB, an
additional acute toxicity test with Daphnia magna is performed on the influents and
effluents of both treatment technologies to monitor the water quality before and after the
treatment method.
The acute toxicity tests are performed in accordance to the International Standard ISO
6341. For the tests, 20 Daphnia magna neonates were exposed to several concentrations
for 24 h/48 h. Each concentration consisted of four groups of test containers with five
Daphnia magna neonates in 10 ml of the corresponding concentration. The test was
performed in an atmosphere controlled at 20 °C ± 2 °C and the vessels were kept in an
incubator with 16 hours of light and 8 hours of darkness. During the culturing, the
Daphnia magna were fed once daily with a suspension of freeze-dried Chlorella vulgaris
xi
algae in aged tap water. The algae food is supplemented with 0.5 ml per culture per day
of a 100 mg/l stock suspension of dry baker’s yeast.
The toxicity test with Daphnia magna on caffeine showed that the EC50 – 24 h for caffeine
is 207.525 mg/l with a 95 % confidence interval of 124.764 to 287.895 mg/l and the EC50
– 48 h for caffeine is 112.884 mg/l with a 95 % confidence interval of 52.856 to 166.423
mg/l. With this information, it can be concluded that the present concentration of caffeine
in the wastewater of 5.199 mg/l is far below the obtained EC50 – 24 h and even far below
the obtained EC50 – 48 h.
The EC50 – 24 h for the influent of the Intensive Green Filter is 0.573 ml influent/ml test
volume with a 95 % confidence interval of 0.546 to 0.596 ml influent/ml test volume. The
EC50 – 24 h for the influent of the Permeable Reactive Barrier is 0.878 ml influent/ml test
volume with a 95 % confidence interval of 0.853 to 0.903 ml influent/ml test volume.
With this information it can be concluded that the acute toxicity of the influent of the
Intensive Green Filter is higher than the acute toxicity of the influent of the Permeable
Reactive Barrier.
A calculation of the EC50 – 24 h of both effluents was not possible because the amount of
immobilized Daphnia magna was too low. The Permeable Reactive Barrier shows no effect
of immobilization, while the Intensive Green Filter shows a low acute toxicity of 10 % for
Daphnia magna after 24 hours. The culturing method and the test results are considered
to be valid as they meet all validity criteria.
The obtained results are found to be as expected, because: i) the quality of the influent of
the Permeable Reactive Barrier is better than the quality of the influent of the Intensive
Green Filter, and ii) a Permeable Reactive Barrier with three layers (palygorskite, activated
carbon and zeolite) that are specialized to remove a wide spectrum of pollutants, is more
likely to have a better performance than a vegetation filter with soil and poplars.
The results of the acute aquatic toxicity tests on the effluents of the Intensive Green Filter
and the Permeable Reactive Barrier indicate that the Intensive Green Filter is not able to
remove all toxicological risks towards Daphnia magna. A small acute toxicity was found in
the groundwater, which suggests that a chronic toxicity is possible that has even more
environmental risks. The groundwater underneath the Permeable Reactive Barrier shows
no acute toxicity towards Daphnia magna. This indicates that the environmental risks are
lower. However, a chronic toxicity test may expose other important impacts on the
groundwater quality.
The obtained results only represent the acute toxicity and do not include chronic toxicities
towards the reproduction of the Daphnia magna. Performing a chronic study could be
interesting to further investigate the small immobilization effects of the effluent of the
Intensive Green Filter and the non-observed effect of the Permeable Reactive Barrier. To
assess an accurate representation of the environmental risks of the present substances
(e.g. caffeine), these chronic studies are needed.
xii
Dutch abstract Deze masterproef is geschreven met betrekking tot een 14-weken-durende stage bij het
Centrum voor Nieuwe Watertechnologieën (CENTA). Het studiegebied van CENTA is
gelegen in het zuidoosten van Spanje, in het experimentele R&D&I centrum van Carrión
de los Céspedes (Sevilla, Spanje).
Één huidig project, uitgevoerd door CENTA, is het project “REAGUAM”. Dit project
onderzoekt het gebruik van teruggewonnen water afkomstig van gezuiverd afvalwater
door twee non-conventionele technologieën:
Het eerste behandelingssysteem vertegenwoordigt een Intensieve Vegetatiefilter
met korte-omloop houtteelt van populieren (Populus alba). Het afvalwater is
afkomstig van CENTA’s kantoorgebouw en passeert een Imhoff tank die de totale
organische belasting reduceert met 20-30 %.
Het tweede behandelinssysteem is een Permeabele Reactieve Barrière met
horizontale lagen, van boven naar beneden: palygorskiet, actieve koolstof, zeoliet
en zand. Deze behandeling wordt voorafgegeaan door een voorbehandeling die
bestaat uit het afscheiden met behulp van roosters, het breken en verkleinen van
de vaste bestanddelen en het ontvetten van het afvalwater, gevolg door een actief
slibbehandeling met langdurige beluchting en een zandfilter.
Het hoofddoel van het project is om een evaluatie te maken van het vermogen van het
medium om de kwaliteit van het behandelde afvalwater dat wordt toegepast te
regenereren, dit zowel in the Intensieve Vegetatiefilter als in de Permeabele Reactieve
Barrière. Binnen deze doelstelling dient de impact van het geïrrigeerde afvalwater op de
kwaliteit van het grondwater te worden ingeschat. Daarom moet er een ecotoxicologische
evaluatie gemaakt worden om de toxiciteit te bepalen van de belangrijkste
microcontaminanten in het afvalwater.
Één van de belangrijkste microcontaminanten in het afvalwater is caffeine. Caffeine blijkt
een geschikt microcontaminant te zijn voor de ecotoxicologische evaluatie door zijn hoge
aanwezigheid in het afvalwater, zijn lage kostprijs en omdat er niet veel eerder
uitgevoerde studies naar de acute toxiciteit van caffeine met Daphnia magna na 24 uur
zijn teruggevonden.
In deze masterproef is er een acute aquatische toxiciteitstest met Daphnia magna
uitgevoerd op caffeine. Omdat één geanalyseerde microcontaminant het mengsel van
microcontaminanten aanwezig in de afvalwaters van de Intensieve Vegetatiefilter en de
Permeabele Reactieve Barrière niet kan vertegenwoordigen, werd er een extra acute
toxiciteitstest met Daphnia magna uitgevoerd op de influenten en de effluenten van de
twee behandelingstechnologieën om de waterkwaliteit voor en na de behandeling te
controleren.
De acute toxiciteitstest werd uitgevoerd in overeenstemming met de Internationale
Standaard ISO 6341. Voor deze test werden 20 Daphnia magna neonaten blootgesteld
aan verschillende concentraties gedurende 24 u/48 u. Elke concentratie bestond uit vier
groepen van testcontainers met vijf Daphnia magna neonaten in 10 ml van de
xiii
overeenkomstige concentraties. De test werd uitgevoerd in een gecontroleerde atmosfeer
van 20 °C ± 2 °C en de testplaten werden geincubeerd bij een cyclus van 16u licht/8u
donker. Tijdens het kweken werden de Daphnia magna eenmaal per dag gevoed met een
suspensie van gevriesdroogde Chlorella vulgaris-algen in verouderd kraantjeswater. Het
algenvoedsel werd aangevuld met 0.5 ml per cultuur per dag van een 100 mg/l stock-
oplossing van droge bakkersgist.
Uit de toxiciteitstest met Daphnia magna op caffeine bleek dat de EC50 – 24 u voor
caffeine 207.525 mg/l is met een 95 % betrouwbaarheidsinterval van 124.764 tot
287.895 mg/l en de EC50 – 48 u voor caffeine is 112.884 mg/l met een 95 %
betrouwbaarheidsinterval van 52.856 tot 166.423 mg/l. Met deze informatie kan er
geconcludeerd worden dat de aanwezige concentratie van caffeine van 5.199 mg/l in het
afvalwater veel lager is dan de verkregen EC50 – 24 u en zelfs lager is dan de verkegen
EC50 – 48 u.
De EC50 – 24 u van het influent van de Intensieve Vegetatiefilter is 0.573 ml influent/ml
test volume met een 95 % betrouwbaarheidsinterval van 0.546 tot 0.596 ml influent/ml
test volume. De EC50 – 24 u van het influent van de Permeabele Reactieve Barrière is
0.878 ml influent/ml test volume met een 95 % betrouwbaarheidsinterval van 0.853 tot
0.903 ml influent/ml test volume. Met deze informatie kan er geconcludeerd worden dat
de acute toxiciteit van het influent van de Intensieve Vegetatiefilter hoger is dan de acute
toxiciteit van het influent van de Permeabele Reactieve Barrière.
Een berekening van de EC50 – 24 u van de twee effluenten was niet mogelijk omdat het
aantal geimmobiliseerde Daphnia magna te laag was. De Permeabele Reactieve Barrière
toont geen effect van immobilisatie, terwijl de Intensieve Vegetatiefilter een lage acute
toxiciteit van 10 % toont voor Daphnia magna na 24 uur.
De kweekmethode en de test resultaten worden als geldig beschouwd omdat ze voldoen
aan alle validatiecriteria.
De verkregen resultaten zijn zoals verwacht, omdat: i) de kwaliteit van het influent van de
Permeabele Reactieve Barrière beter is dan de kwaliteit van het influent van de Intensieve
Vegetatiefilter, en ii) een Permeabele Reactieve Barrière met drie lagen (palygorskiet,
actieve kool en zeoliet) die gespecialiseerd zijn om een wijd spectrum aan
verontreinigende stoffen te verwijderen, heeft een grotere kans om een betere prestatie
te hebben dan een vegetatiefilter met aarde en populieren.
Uit de resultaten van de acute toxiciteitstest op de effluenten van de Intensieve
Vegetatiefilter en de Permeabele Reactieve Barrière blijkt dat de Intensieve Vegetatiefilter
niet in staat is om alle toxicologische risico’s voor Daphnia magna te voorkomen. In het
grondwater werd een lage acute toxiciteit aangetoond, wat suggereert dat een chronische
toxiciteit ook mogelijk is die mogelijks nog grotere milieurisico’s met zich meebrengt. Het
grondwater onder de Permeabele Reactieve Barrière toont geen acute toxiciteit voor
Daphnia magna. Dit wijst op lagere milieurisico’s. Hoewel, een chronische toxiciteitstest
kan ook hier andere belangrijke effecten op de grondwaterkwaliteit blootleggen.
xiv
De verkregen resultaten gaan alleen maar over de acute toxiciteit en houden geen
informatie in over de chronische toxiciteit ten opzichte van de voortplanting van Daphnia
magna. Het uitvoeren van een chronische studie kan interessant zijn om het lage
immobilisatie-effect van het effluent van de Intensieve Vegetatiefilter en het niet-
waargenomen effect van het effluent van de Permeabele Reactieve Barrière op hun
grondwaters verder te onderzoeken. Om een juiste weergave van de milieurisico’s van de
aanwezige stoffen (vb. caffeine) te kunnen geven, zijn verdere chronische studies vereist.
1
1 Introduction This thesis was written in connection with an internship of 14 weeks which was performed
at the Foundation Center of New Water Technologies (CENTA). The study site of CENTA is
located in the South East of Spain, in the Experimental Center on R&D&I of Carrión de los
Céspedes (Seville, Spain). Carrión de los Céspedes is a small village with 2,500 inhabitants
that produces an average urban wastewater flow of 400 m3/d. This wastewater is treated
in the Experimental Center by different conventional and non-conventional wastewater
treatment technologies.
Figure 1: Experimental Center on R&D&I of Carrión de los Céspedes (Seville, Spain)
CENTA is a private research center that is supported by the Ministries of Agriculture,
Fisheries, Environment and Innovation of the Governance of Andalusia and other entities
and enterprises related with the water sector. CENTA currently occupies a prominent
position in the water sector. Their aim is to promote a better management of water
resources through an innovative, sustainable and fair approach. With their twenty years of
research experience in the field of purification of wastewaters of small communities and
rural areas, CENTA has become a reliable reference at an international level. [1]
One current project carried out by CENTA is the project “REAGUAM”, supported by the
Spanish Ministry of Economy and Competitiveness in the frame of water reuse for aquifer
recharge and environmental application for energy production. This project investigates
the use of reclaimed water deriving from treated wastewater by two non-conventional
technologies: i) an Intensive Green Filter (IGF), preceded by a primary treatment (Imhoff-
tank) and ii) a Permeable Reactive Barrier (PRB) with horizontal layers, preceded by a
preliminary treatment, an extended aeration and a sand filter. This thesis, appertaining in
this project, studies the ecotoxicological water status of water percolating through both
systems.
To make the ecotoxicological evaluation, a toxicity test has to be performed to determine
the toxicity of the most important microcontaminants in the wastewater. To determine the
2
toxicity of a substance or a water flow to the aquatic environment, several bioindicators
such as invertebrates, fish and algae can be used. The project applies the acute aquatic
toxicity test with the Cladocera Daphnia magna, as Daphnia magna have proven to be a
good study object for toxicity tests and as they are recommended by the Environmental
Protection Agency (EPA) [2]. Benefits that contributed to the selection of Daphnia magna
are the ease and the low cost of culturing in the laboratory and their sensitivity to a
variety of pollutants.
At first, the most important microcontaminants are being determined. When these
microcontaminants are known, an acute toxicity test with Daphnia magna is performed on
these microcontaminants present in the highest concentrations. Furthermore, a toxicity
test with Daphnia magna is performed on the influents and effluents of both treatment
technologies to monitor the environmental risk of the treated effluents and to evaluate
the treatment of the influent waters. The acute toxicity tests are performed in accordance
to the International Standard ISO 6341 [3].
3
2 Literature review
2.1 Project: Water reuse
As the world population continues to increase, the demand for water of a certain quality
keeps on growing, especially in countries with dry seasons. Subsequently the use of
groundwater grows, which causes a depletion of the available groundwater. During the
last decades, more research is carried out about the recycling and the reuse of water.
Accomplished methods are the recovery of aquifer storages during times when there is
sufficient water and the irrigation of agricultural lands by using wastewater treatment
effluents [4]. Nowadays, effluents are reused for irrigation purposes all over the world
[5].
The Mediterranean climate in Spain suffers from a non-equally distributed precipitation. A
lot of money is spent to transport the available water to the places where it is needed at a
specific time. The main water consumer in Spain is the irrigated agriculture. About 75 %
of the total water consumption consists of irrigation water in order to provide the
agriculture with enough water [6]. The consumption particularly occurs during the dry
season and has often been solved in the last decades by pumping up the groundwater.
Subsequently this caused an overexploitation of the groundwater, which in turn has
caused a limit to the availability of groundwater.
The project “REAGUAM”, in the frame of “Water reuse” investigates the use of reclaimed
water deriving from a preliminary treatment followed by an extended aeration and a sand
filter as water for aquifer recharge. This reuse of water is considered to be a technically
and economically feasible solution to cope with the water scarcity and the growing water
demand [6]. Not only will it provide a high and constant volume of reclaimed water that
can be used for irrigation, it would also be beneficial for the limited availability in water
resources [6].
The current project is carried out by the Foundation Center of New Water Technologies
(CENTA). The study is a continuation of co-operations started in the Consolider Program
TRAGUA: “Treatment and Reuse of Wastewaters for Sustainable Management” and
REAGUAM: “Reuse of treated urban wastewaters for environmental uses: aquifer recharge
through permeable reactive beds and forestry for power production”. [7]
The study site of CENTA is located in the South East of Spain, in the Experimental Center
of Carrión de los Céspedes (Seville, Spain). Carrión de los Céspedes is a small village with
2,500 inhabitants that produces an average urban wastewater flow of 400 m3/d [6]. The
wastewater is treated in the Experimental Center of Carrión de los Céspedes (managed by
CENTA [1]).
The study considers two reclamation technologies for treated wastewater effluents based
on soil application [7]. The first one is crop irrigation and involves wastewater reuse in an
Intensive Green Filter (IGF) planted with poplars (Populus alba) and an irrigated plot with
sun flowers (Helianthus annuus). The second water reclamation technology is a
Permeable Reactive Barrier (PRB) with horizontal layers [7].
4
The main goal of the project is to evaluate the capability of the medium to regenerate the
quality of the treated wastewater that is applied, in both the Intensive Green Filter and
the Permeable Reactive Barrier. This evaluation needs to be done on environmental
sustainability, health protection and on economic and financial balance. In this purpose,
two particular goals are implicit: i) to estimate the capacity of the crop irrigation and the
PRB to regenerate the wastewater effluents, and ii) to assess the impacts of the irrigated
wastewater on the groundwater quality [7]. Within the second part of the objective, this
thesis appertains to evaluate the ecotoxicological water status.
Because it is shown that wastewater treatment plants are not able to eliminate all the
pharmaceuticals completely by traditional treatment processes [8], it can be assumed that
the pretreated water still contains certain concentrations of microcontaminants. These
waters, containing microcontaminants, are irrigated to the IGF and the PRB. The
microcontaminants can cause certain effects on the microorganisms present in the soil
and in the groundwater. Therefore, their toxicity for the aquatic environment must be
examined.
To determine the toxicity of a substance or a water flow to the aquatic environment,
several bioindicators such as invertebrates, fish and algae can be used. In this project,
the study object is chosen to be Daphnia magna, because of the ease and low cost of
culturing in the laboratory and their sensitivity to a variety of pollutants. Furthermore, the
use of Daphnia magna for acute toxicity tests is recommended by the Environmental
Protection Agency (EPA) [2].
To evaluate the water status, an acute toxicity test is performed to determine the acute
toxicity for Daphnia magna (Cladocera, Crustacea) of the most important
microcontaminants in the wastewater. Furthermore, the toxicity for Daphnia magna of the
influent and effluent waters of the Intensive Green Filter and the Permeable Reactive
Barrier is being monitored.
2.2 Treatment technologies
The influents and effluents of two different wastewater treatment systems were analyzed
for their acute toxicity for Daphnia magna. This chapter presents a description of both
treatment systems.
The first treatment system represents the Intensive Green Filter with short-rotation
coppice of poplars. The wastewater derives from CENTA’s office building, and passes an
Imhoff tank which reduces its total organic load with 20-30 %. (Figure 2)
The second treatment system is a Permeable Reactive Barrier with horizontal layers, from
top to bottom: palygorskite, activated carbon, zeolite and sand. This treatment is
preceded by a preliminary treatment that consists of screening, degritting and degreasing,
followed by an extended aeration treatment and a sand filter. (Figure 2)
5
Figure 2: Flow sheet REAGUAM-project: Intensive Green Filter and Permeable Reactive Barrier
2.2.1 Intensive Green Filter (IGF)
Small municipalities often experience difficulties to connect their wastewaters to the
sewage networks. Nature-based wastewater treatment systems have been reported as a
feasible solution in these situations. The advantages are known to be their low
management and maintenance costs and their low sludge production. [9]
One specific type is the vegetation filter. The vegetation filter consists of a vegetated soil
surface on which the pre-treated and/or treated wastewater is applied. The applied
wastewater is partially evaporated, and the rest is taken up by the roots of the vegetation
and filtered through the soil [1]. Potential wastewater contaminants are removed from
the irrigation water by the attenuation capacity of the soil and the plant uptake of the
crops. [9]
The last decade, vegetation filters with short-rotation coppices showed to be very suitable
to improve wastewater quality when irrigated with wastewater [10]. Short-rotation
coppices are fast-growing species that are able to resprout from stumps after being
harvested at short intervals. This intensive biomass production strategy allows biomass to
be an alternative for fossil fuels for renewable energy production [10]. The most common
species used are poplars and willows, because of their high transpiration rate.
A 3-year research was conducted at CENTA (Seville, Spain) in which a vegetation filter,
based on short-rotation coppice of poplars (Populus alba), was applied to treat the
wastewater produced by CENTA’s office building (Figure 3). The effluent wastewater had
a capacity of 20 workers who produce an average wastewater volume of 0.5 m³/day [9].
6
The wastewater was previously treated in an Imhoff tank with a volume of 2.5 m³ before
it was irrigated to the vegetation filter. The treatment through the Imhoff tank was able
to remove about 20-30 % of the total organic load. Effluent was applied once per week.
Figure 3: Overview of the Intensive Green Filter with poplars (Populus alba)
The purpose of the project line was to evaluate the pollutant removal capacity of the
vegetation filter taking into account the lack of wastewater storage facilities and highly
variable amounts and qualities of the irrigated wastewater [9]. To assess the potential
effects of wastewater irrigation on the groundwater quality, a piezometer (filter at a depth
of 10 meter), located 4 meter down gradient of the vegetation filter, was monitored.
(Figure 4)
Figure 4: Intensive Green Filter plot at CENTA, Seville (de Miguel, 2014)
7
The experimental plot has a gentle slope directed towards the South. The loamy soils
consist of 20.4 % clay, 46.8 % sand and 32.8 % silt [1]. The vegetation filter consists of
10 lines of poplars with a distance of 1 meter in between. The density of the plantation is
10,000 plants/ha. [9]
The water use of the office building is mostly for flushing toilets and hand washing [1].
Based on 3-year data of the project, the effectiveness of the vegetation filter to remove
wastewater contaminants was analyzed by comparing the data of the Imhoff tank effluent
and the leachate collected by a lysimeter (Table 1). The average removal percentage
calculated in terms of concentration was about 85 % for COD and DOC. The average
removal percentage of the Intensive Green Filter for NT and PT was respectively 73 % and
90 %.
Table 1: Average concentrations of the wastewater applied to the Intensive Green Filter at CENTA
and the groundwater
COD (mg/l) DOC (mg/l) NT (mg/l) PT (mg/l)
Imhoff tank effluent (= influent IGF)
269.6 88.0 154.9 16.1
Piezometer groundwater (= effluent IGF)
40.1 12.7 41.9 1.5
The low concentrations of contaminants in the groundwater represent a good water
quality. The 3-year data analysis showed that no significant differences were detected in
pH, EC and in the total P, COD, total N and NO3-N concentrations before the vegetation
filter operation was started and during the 3-year application. [9]
At the end of the project it was concluded that the vegetation filter system could be a
suitable wastewater treatment strategy for small populations. [9]
2.2.2 Permeable Reactive Barrier (PRB)
A Permeable Reactive Barrier (PRB) is a passive zone of in-situ treatment that consists of
reactive materials that transform or immobilize a pollutant when the water flows through
(Figure 5) [11]. A PRB acts as a filter where water passes through, withholding or
adsorbing chemical pollutants, and hereby obtaining an improvement of the water quality.
PRB technology has been applied to a wide spectrum of pollutants. It has shown its
effectiveness in removing both organic compounds and inorganic substances. Most of the
times a PRB is a temporary or permanent vertical barrier perpendicular to the flow of the
plume (Figure 5). To eliminate a large variability of compounds in the wastewater (such
as pharmaceuticals and personal care products), a PRB can consist of different layers of
materials. Materials are chosen which interact with the water flowing through, so that the
contaminant is removed from the water and retained on the solid phase by physical,
chemical and/or biological processes, including precipitation, adsorption, oxide-reduction
and degradation [13].
8
Figure 5: Permeable Reactive Barrier (PRB) with horizontal flow [12]
At CENTA, an experimental Permeable Reactive Barrier with horizontal layers is applied in
order to evaluate the sorption capacity of different substrates. The used materials in the
layers are, from top to bottom: palygorskite1, activated carbon2, zeolite3 and sand. The
installation consists of a circular tank (diameter = 5 m) with four horizontal layers. The
influent wastewater is distributed on top of the installation and infiltrates with a natural
speed. At the bottom of the installation, the treated water infiltrates into the ground.
The materials of the layers are chosen in order to retain a wide spectrum of pollutants.
Activated carbon is a very porous material and has the characteristics to be a good
adsorbent due to its large internal surface area (between 500 - 1500 m2/g) [15], its
microporous structure and its large surface reactivity. Palygorskite and zeolite, both
minerals, have a high cation exchange capacity enabling the retention of several heavy
metals (Ba2+, Cd2+
, Cu2+, Fe2+,Pb2+…) [17]. They can also form complexes that withhold
some anions such as phosphates. Underneath, a layer of sand is applied to improve the
water flow.
The influent of the PRB is wastewater of a small village with 2,500 inhabitants, Carrión de
los Céspedes. The wastewater is treated in CENTA’s Experimental Center. Approximately
0.8 m³/d of the wastewater is applied to the PRB after being treated by a preliminary
treatment that consists of screening, degritting and degreasing, an extended aeration and
a sand filter. The sample of the influent was taken at the distribution tank of CENTA,
located between the preliminary treatment and the extended aeration, where the
wastewater of Carrión de los Céspedes gets distributed to all CENTA’s different treatment
technologies.
1 Palygorskite = Magnesium aluminum phyllosilicate Mg(Al0.5-1, Fe0-0.5)Si4O10(OH). 4H2O [14] 2 Activated carbon = A form of carbon processed specifically to achieve a very big internal surface that is available for adsorption or chemical reactions [15] 3 Zeolite = Microporous, aluminosilicate minerals (often referred to as molecular sieves) commonly used as commercial adsorbents and catalysts [16]
9
A detailed description of the different layers is given in Figure 6 and Table 2 :
Figure 6: Layers of the Permeable Reactive Barrier at CENTA
Figure 7: Overview of the Permeable Reactive Barrier at CENTA
Table 2: Layers of the Permeable Reactive Barrier at CENTA
Layer Material Thickness of layer Size
1 Sand 10 cm 2 Zeolite 20 cm 2 - 5 mm 3 Active carbon 30 cm 0.6 – 2.36 mm 4 Palygorskite 10 cm 0.074 – 4.76 mm
Inside the PRB, three piezometers were installed, respectively with a depth of 2, 6 and 10
meters. During the project, only the piezometer with a depth of 6 meters presented a
stable presence of water. Therefore this piezometer was chosen for the sampling of the
groundwater for this experiment (effluent PRB).
The effectiveness of the Permeable Reactive Barrier to remove wastewater contaminants
was analyzed by comparing physic-chemical properties of the influent of the PRB and the
groundwater (Table 3) [11].
Table 3: Average concentrations of the wastewater applied to the Permeable Reactive Barrier at
CENTA and the groundwater [11]
COD (mg/l) TOC (mg/l) NO2- (mg/l) NO3
- (mg/l) PO43- (mg/l)
Influent PRB 55.15 22.2 0.5 177.9 2.75 Piezometer groundwater
<0.4 - 15.4 <0.5 – 5.7 / 0 – 7.0 /
The result of the PRB with the use of four different layers point to a selective adsorption,
allowing a good water quality that will not deteriorate the groundwater quality [11].
10
2.3 Microcontaminants
2.3.1 Microcontaminants in the wastewater
The wastewater contains a certain contamination of pharmaceutically active compounds
(PhACs). One sampling was performed during the project whereof the concentrations of
several PhACs were analyzed by Institutos Madrileños de Estudios Avanzados (IMDEA) in
Madrid. The results of this analysis illustrate the microcontaminants with the highest
concentrations present in the wastewater:
Table 4: PhACs in the wastewater of the Experimental Center of Carrión de los Céspedes (Seville,
Spain)
Pharmaceutically active compounds
Concentration (µg/l)
4-AAA
9 967
4-FAA 3 634
Caffeine
5 199
Carbamazepine
221
Citalopram HBr 5 950
Diclofenac
70
Ibuprofen
-
Naproxen
153
Paraxanthine
1 672
Primidone
260
The problem with these PhACs is that they are not easily removed with traditional
treatment systems. Some of these PhACs are very persistent and they have a capacity to
bioaccumulate. Therefore treated effluents often contain amounts of PhACs [5]. Although
the concentrations in treated effluents are smaller than when used as a drug or a
personal care product, the PhACs can contain certain toxicities that could lead to
consequences (especially long-term) in the aquatic eco-systems [18]. One long-term
impact caused by the release of PhACs to the environment, is their potentially feminizing
and masculinizing effects on the aquatic organisms [18]. Additionally there is also the
concern that low-level contamination by certain PhACs can develop an antibiotic
resistance in soil and water organisms [4]. This is especially a problem when human
pathogens get more resistant to antibiotics [18].
11
2.3.2 Description of the selected microcontaminants
1. Caffeine
Caffeine is a pharmaceutical that is widely present in the environment [19]. According to
Benowitz (1995), the effects of caffeine in humans include: “mental stimulation, systemic
catecholamine release, and sympathetic neural stimulation, including an increase in blood
pressure and lipolysis with an increase in plasma free fatty acid concentrations” [20].
2. Paraxanthine
Paraxanthine (1,7-dimethylxanthine) is a psychoactive central nervous system stimulant
which is structurally related to caffeine. Nearly 84% of caffeine is metabolized to
paraxanthine [21].
3. 4-AAA and 4-FAA
Dipyrone is an analgesic and antipyretic drug. The drugs is taken in orally, after which it is
rapidly hydrolyzed to 4-methylaminoantipyrine (4-MAA). 4-MAA undergoes enzymatic
reactions that cause the absorption and the bio-transformation of the substance.
Subsequently 4-MAA is metabolized in the liver to 4-aminoantipyrine (4-AA) via
demethylation which in turn gets acetylated to acetylaminoantipyrine (4-AAA). An
oxidation of the n-methyl group causes another metabolite to be formed, 4-
formylaminoantipyrine (4-FAA). 4-MAA, 4-AA, 4-AAA and 4-FAA are not fully eliminated by
the biological system which causes them to be present in sewage treatment plants
effluents and surface water at high concentrations. [22]
4. Carbamazepine
Carbamazepine is one of the most frequently detected pharmaceuticals in the aquatic
environment. Carbamazepine is an antiepileptic drug used to treat seizures, for the
alleviation of neuralgia and for several mental disorders. [23]
5. Naproxen
Naproxen is a nonsteroidal anti-inflammatory drug (NSAID) used in conditions as in
painful and inflammatory rheumatic and in conditions with significant stomachic
irritations. [24]
6. Ibuprofen
Ibuprofen is a NSAID which is commonly used as an antipyretic and an analgesic agent.
Ibuprofen is used widespread as a medicine for alleviating pain, to help with fevers ad to
reduce inflammation. [25]
7. Diclofenac
Diclofenac is another one of the most frequently detected pharmaceuticals in the aquatic
environment. It is a NSAID used to reduce inflammation and to alleviate pain. Diclofenac
works as an analgesic in cases like an acute injury, arthritis or menstrual pain. [23]
12
8. Primidone
Primidone is an anticonvulsant. The drug is used to treat movement disorders such as
tremors. In the liver, Primidone is metabolized to another anticonvulsant drug,
phenobarbital, which is excreted in the urine. [26]
9. Citalopram HBr
Citalopram (1-[3-(dimethylamino)propyl]-1-(4-fluorophenyl)-1,3-dihydroisobenzofuran-5-
arbonitrile) is part of the group of the selective serotonin reuptake inhibitors (SSRIs). It is
an antidepressant drug used for the treatment of depression. [27]
2.4 Daphnia magna
Daphnia, commonly known as water fleas, belong to the Crustacea. The taxonomic
hierarchy is reported by the Integrated Taxonomic Information System (ITIS) as follows
[28]:
Kingdom: Animalia
Subkingdom: Bilateria
Infrakingdom: Protostomia
Superphylum: Ecdysozoa
Phylum: Arthropoda
Subphylum: Crustacea
Class: Branchiopoda
Order: Diplostraca
Suborder: Cladocera
Infraorder: Anomopoda
Family: Daphniidae
Genus: Daphnia
Species: Daphnia magna Straus
The bodies of the Cladocera are enclosed by an uncalcified shell, known as the carapace
[29]. The carapace is often transparent, and this makes Daphnia an excellent subject for
heart studies. Bioassays can be conducted to figure out if Daphnia show signs of stress.
For example, the heart rate can be observed with a microscope, or it can be observed
whether they have been eating. [30]
The genus Daphnia includes more than 100 known species of freshwater plankton
organisms found around the world. One of these species is Daphnia magna, which is used
in this experiment.
13
Figure 8: Daphnia magna [29]
Daphnia magna have six thoracic appendages and two sets of long and doubly branched
antennae [31]. These parts are held inside of the carapace and are the reason why
Daphnia magna are filter-feeding animals. The appendages help to produce a current of
water which carries the food and oxygen to their mouths. They also have two large claws
to clean their carapace. Clearly visible as a dark spot is their one compound eye. [31]
During the growth season Daphnia magna reproduce asexually. The female produces
parthenogenetic4 eggs [32], which are incubated in a brood pouch located underneath the
carapace. Embryos develop directly and are brooded as fully extended individuals. After
their birth, they immediately molt and look like a smaller version of the adults. [33]
Guilhermino’s study [34] showed that Daphnia magna can be applied for acute aquatic
toxicity tests as a prescreening method in toxicity testing. Their asexual self-reproduction,
transparent carapace and relatively stable presence in good conditions make them a good
study object for several experiments.
Nowadays, Daphnia magna is a standard aquatic test specie for toxicity tests,
recommended by the Environmental Protection Agency (EPA) [2]. The species are often
used in bioassays and in the environmental monitoring of the aquatic environment
because of the ease and the low cost price culturing and their sensitivity to a variety of
pollutants. A lot of literature exists on the response of Daphnia magna to different types
of toxins [35] and both the acute as the chronic tests with Daphnia magna are frequently
performed studies in aquatic toxicology.
2.4.1 Life cycle
The duration of Daphnia magna’s lives depends heavily on environmental conditions such
as oxygen levels, food availability and temperature. Daphnia typically live 40 to 56 days,
varying according to environmental conditions [30]. At 20 °C, Daphnia magna reach
sexual maturity in 6 to 8 days. Usually 6 to 10 parthenogenetic eggs complete their
development into embryos inside the brood chamber and are born as free-swimming
neonates at day 8-10 [36]. Subsequently, the mature females release a brood of neonates
4 Parthenogenicity = the ability to self-replicate without fertilization of any form (a type of asexual reproduction). The parthenogenetic eggs are exact genetic replicas of the parent animals. [32]
14
every 2 to 3 days. When the adult Daphnia are getting older, the time between broods
will increase and the size of the brood will decrease. [36]
A healthy population of Daphnia consists mostly of females that have been produced
asexually. This population can be obtained by keeping certain conditions stable, in order
to avoid the Daphnia of experiencing stress. For example, the population density must be
maintained stable. If the population density gets too high, the culture could crash down.
Other requirements are a sufficient food concentration in the water, a good water quality
and no extreme conditions as in temperature shocks. Under stressful conditions, Daphnia
switch to sexual reproduction which makes them produce more male embryos and
subsequently dormant eggs (ephipia) [36]. Thus, production of males may be used as an
indicator of changing conditions.
2.4.2 Optimal culture conditions
Daphnia are typically freshwater organisms and are mostly found in lakes or ponds.
Therefore culture conditions must be similar to those of standing freshwater.
Daphnia can be cultured in a standardized medium. This medium consists of distilled
water with an addition of essential minerals and nutrients needed for growth. Another
possibility is the use of tap water that was left for more than 24 hours so that chlorine
was able to evaporate.
Daphnia are sensitive to dissolved oxygen, temperature, conductivity, pH and chemical
contaminants.
For optimal culture growth, the following conditions are recommended [30]:
Table 5: Optimal culturing conditions for Daphnia magna [30]
Factor Optimal Range
pH 7-8.6 Temperature 20-25°C Dissolved oxygen > 6 mg/L Hardness 160-180 mg CaCO3/L Lighting cycle 16 h light/8 h dark
To maintain a good growing culture and a good reproduction, the culture water should
contain certain hardness (170 mg carbonate hardness). Daphnia need calcium and other
minerals in their chitinous carapaces. [32]
Slight aeration must be provided to make sure all chlorine is evaporated. Additionally, it
helps to increase the gaseous exchange at the surface of the water and it also helps to
stabilize the water conditions.
If cultures are maintained under these optimal conditions, a 1.2-L-vessel stocked with 30
Daphnia will produce approximately 120 neonates per week [36].
15
2.4.3 Nutrition
The best foods for culturing Daphnia are algae, yeasts and bacteria or a combination.
Feeding algae supplemented with yeast seems to have the most success [32]. However,
the culture of fresh algae is time consuming and it takes some effort to produce a stable
culture that matches the demand of the Daphnia.
In 1992, a research by Naylor & Bradley at the University of Sheffield in the United
Kingdom stated as follow: “Freeze-dried Chlorella was adequate as a food for D. magna
over a number of generations – the EC validity criteria of less than 10 % mortality and
greater than 60 neonates in 21 days were always met. However, the fecundity of animals
was always significantly poorer than when fed the fresh algal diet.” [37]
2.5 Defining the microcontaminants to be tested
2.5.1 Reported results of acute aquatic toxicity tests with Daphnia magna
Several studies reported results of acute aquatic toxicity tests concerning these relevant
pharmaceutically active compounds (PhACs) with Daphnia magna (Table 6). The EC50
values represent the concentration where 50 % of Daphnia magna were immobilized after
24 or 48 hours. Immobilization was considered to have happened if no movement was
detected for 15 seconds after gentle shaking of the test vehicle.
Table 6: Review of several studies about acute aquatic toxicity tests with Daphnia magna
concerning the relevant PhACs
Pharmaceutical compound
Test results on Daphnia magna
EC50 (mg/l) Reference
Caffeine EC50 - 24 h 161.28 Lilius (1995) [38] Carbamazepine EC50 - 48 h > 100 Cleuvers (2003) [39] EC50 - 48 h > 13.8 Ferrari (2004) [40] Citalopram HBr EC50 - 48 h 30.14 Minguez (2014) [41] Diclofenac EC50 - 48 h 68 Cleuvers (2003) [39] EC50 - 48 h 22.4 Ferrari (2004) [40] Ibuprofen EC50 - 24 h > 45 Kim (2010) [42] EC50 - 48 h 108 Cleuvers (2003) [39] EC50 - 48 h 9.06 to 11.5 Halling-Sørensen (1998) [43] Naproxen EC50 - 48 h 66.4 Fent (2006) [44] Primidone / / /
Gómez (2008) presented the following results in percentages for the inhibition of Daphnia
magna of 4-FAA and 4-AAA: [22]
Table 7: Results of the acute aquatic toxicity test of 4-FAA and 4-AAA [22]
Pharmaceutical compound
% of inhibition of Daphnia magna – 24 h
% of inhibition of Daphnia magna – 48 h
4-FAA (10 mg/L) 20 40 4-AAA (10 mg/L) 20 20
16
2.5.2 Consideration of the microcontaminants to be tested
The research presented in this paper will start with the acute aquatic toxicity test of one
microcontaminant. If good results are obtained and all the validity criteria are met, the
research will be continued with other relevant microcontaminants.
For the selection of the first microcontaminant to be researched, the price is taken into
account (Table 8):
Table 8: Pharmaceutically active compounds present in the wastewater of the Experimental Center
of Carrión de los Céspedes (Seville, Spain) – Cost price [45]
Pharmaceutically active compounds
Price (in €) – Sigma-Aldrich
4-AAA
90.50 / 100 mg
4-FAA 107 / 10 mg
Caffeine
18.30 / 5 g
Carbamazepine
69.40 / 5 g
Citalopram HBr 158.00 / 10 mg
Diclofenac
75.20 / 10 g
Ibuprofen
210.50 / 5 g
Naproxen
51.80 / 250 mg
Paraxanthine
213.50 / 100 mg
Primidone
37.20 / 5 g
Furthermore, scientific papers and articles were scanned for microcontaminants whereof
the acute aquatic toxicity already is determined repeatedly. Microcontaminants were
chosen of which not a lot of studies were published and which had a relatively low price.
Because of this, and also because of its presence in high concentrations in the
wastewater, caffeine appeared an adequate microcontaminant to be tested.
The acute aquatic toxicity test was performed on caffeine. The solubility of caffeine in
water is approximately 16 mg/ml at room temperature and 200 g/L at 80 °C, which
satisfies for this experiment [46]. The price is very low compared to other
microcontaminants, and only one research was found that reflected EC50-values (Table 6).
This research dated from 1995, so a new determination of the acute toxicity could be
usefull to compare the results.
Also carbamazepine is taken into consideration because of the relatively low price and the
absence of exact values of EC50 – 48 h. According to Borisover (2010) [47], the solubility
of carbamazepine in water is 126.1 ± 3 mg/L, which is very low [48]. This could induce
some problems with the acute aquatic toxicity test, because its EC50-value was earlier
presented as higher than 100 mg/L (Table 6). Also for primidone, the poor solubility (60
17
mg per 100 mL at 37 °C) [49] is the reason why primidone is not adequate for the acute
aquatic toxicity test.
4-AAA, 4-FAA, citalopram HBr, ibuprofen, naproxen and paraxanthine were not considered
to be an option because of their high cost.
Diclofenac was chosen as a second microcontaminant to be tested. It is relatively cheap,
and its solubility in water is 50 mg/ml which is adequate. Other researches already
presented EC50-values for 48 h, but no value for 24 h was found. Furthermore, diclofenac
can represent the NSAIDs (e.g. ibuprofen, naproxen…) in this research. Ultimately, the
acute toxicity of diclofenac was not determined due to a late arrival of the product and a
lack of time.
2.6 Relevant researches relating Daphnia magna
The effects of several pharmaceuticals for humans were shown in the previous chapter
(2.3.2). These pharmaceuticals however are dispersed in the environment, where they
cause biological effects and physicochemical behaviors towards organisms they come into
contact with [8].
Last century, not much attention has been paid to these potential risks of pharmaceuticals
as toxic contaminants in aquatic environments [8]. As earlier described in 2.3.1, it is
known that wastewater treatment plants (WWTPs) are not able to eliminate all the
pharmaceuticals completely by traditional treatment processes [8].
The last decade, a lot of research is performed to clarify the ecological influence and
occurrence of these pharmaceutical contaminants. At first, studies were performed to
detect the potential risk of contaminants individually. However, there is still not much
known about the potential risks of complex chemical mixtures of pharmaceuticals.
Cleuvers (2003) presented that the toxicity effects were stronger when testing
combinations of various pharmaceuticals as they were when testing the pharmaceuticals
separately [39]. Also, research is being performed to demonstrate the effect of
metabolites of several pharmaceuticals. Some metabolites are proven to be more
lipophilic and more persistent than the drugs they originally derive from [8].
Daphnia magna have been used in several kinds of experiments. Daphnia magna turns
out to be one of the most popular herbivorous Cladocerans to use in culture experiments.
Additionally, Chlorella vulgaris is frequently used in Daphnia growth experiments [50]. For
example, Daphnia magna are the most commonly used crustacean test species for
determination of the effects of xenobiotics on organisms in the aquatic environment [51].
Han (2006) [8] presented a study of the overall ecotoxicological effect of PhACs detected
in the effluents of Korean WWTPs to Daphnia magna. Results of the study showed slight
synergistic effects for the combined toxicity of pharmaceuticals, compared to the toxicity
of the individual pharmaceuticals [8]. Another research was performed by Wollenberger
(2000) [52] to observe the toxicity of veterinary antibiotics to Daphnia magna. The results
indicated that only oxolinic acid, which is commonly used as a feeding additive in fish
farms, could possibly cause adverse effects on the aquatic environment.
18
Another way how Daphnia magna can be used to assess environmental risks is in
bioassays where they are used in situ to determine pollutant effects. This deals with the
problem that chemical analyses often take a lot of time and can be expensive. Also, the
prediction of the combined toxic effects is very hard. Bioassays seem to be a cheaper and
faster method to give an idea of the overall effects of toxic chemicals, including the
synergistic and antagonistic effects of mixtures [53].
For example, Barata (2007) [54] studied the toxicity effects of pesticides in the aquatic
environment by using in situ bioassays with Daphnia magna. Hereby, Daphnia magna
were in situ present in a cage during 24 hours. By combining several biochemical
biomarkers and toxicological responses to pollutants, the impact of specific pesticides
could be estimated [54]. In 2008, this result was confirmed by a subsequent study of
Damásio [55], where a similar research was performed on effluent discharges of sewage
treatment plants in surface waters in Spain. The results of the study emphasized the
importance of combining biomarkers and in situ responses to identify ecological effects of
effluent discharges. [55]
Furthermore, research has been performed to develop biomonitoring methods to detect
abnormal activity of Daphnia magna. These methods can trigger an alarm when the water
quality changes which makes it possible to react faster.
Jeon (2008) presented the Grid Counter device [53]. This method uses changes in the
movement of Daphnia magna, induced by stress-situations, as an indicator of
ecotoxicological risks. The swimming activity of each Daphnia magna was automatically
monitored in six chambers every 5 minutes for more than 3 hours. Jeon (2008) concludes
that a new biological early warning system with multiple channels has been developed to
detect unusual events in the behavior of Daphnia magna [53].
Another method, presented by Ren (2007) [35], is the online monitoring of behavioral
changes of Daphnia magna. Here, the movement behavior of Daphnia magna is studied
as a bio-indicator of organophosphorous pesticide contamination with the Multispecies
Freshwater Biomonitor. The Multispecies Freshwater Biomonitor is an online instrument
for continuous water control that consists of two pairs of electrodes on the walls of a test
chamber. One pair sends a high frequency signal of an alternating current. The second
pair of electrodes receives the current. The amplitude of the generated sinus single of this
current resembles the movements of the Daphnia magna individuals [35]. Ren (2007)
[35] finally constructed a behavioral change model for Daphnia magna as an early
warning system of aquatic organophosphorous contamination.
In the research presented in this thesis, an acute toxicity test is performed to determine
the acute toxicity for Daphnia magna (Cladocera, Crustacea) of one of the most important
microcontaminants in the wastewater presented to the Intensive Green Filter and the
Permeable Reactive Barrier at CENTA. Because one analyzed microcontaminant cannot
represent the mixture of microcontaminants that are present in the wastewaters, an
additional acute toxicity test with Daphnia magna is performed on the influents and
effluents of the Intensive Green Filter and the Permeable Reactive Barrier to monitor the
water quality before and after the treatment method.
19
3 Methodology
3.1 Culturing method
3.1.1 Materials
3.1.1.1 Collection and determination
Sampling bottle
Filter 0.043 mm
Microscope
Plastic Pasteur pipettes with the end point cut off at ± 3 centimeters opening
big enough to capture Daphnia adults.
3.1.1.2 Culture set-up
Aquarium 100 L
2 aquaria 3 L
Aged tap water
Incubator at 20 °C ± 2 °C with a photoperiod of 16 h light/8 h darkness
Thermometer
pH-meter
Conductivity-meter
Aeration: airline tube with air stone
Sieves from 0.1 mm to 2 mm
Plastic Pasteur Pipettes
3.1.1.3 Feeding
Plastic Pasteur Pipettes
Magnetic stirrer
Medicura Naturprodukte – Bio Chlorella 100 % pure
Dry baker’s yeast
Mortar and pestle
Aged tap water
3.1.1.4 Maintenance
Plastic Pasteur Pipettes
Sieves from 0.1 mm to 2 mm
Aged tap water
3.1.2 Collection of Daphnia magna Straus
Daphnia magna Straus were present in a freshwater reservoir for irrigation in the
Experimental Center of Carrión de los Céspedes, CENTA. The stored water consists of
effluent water of different types of constructed wetlands, installed at the Experimental
Center. To cultivate a culture in the laboratory, 4 liters of water from the reservoir were
sampled and were brought over a filter of 0.043 mm. The Daphnia captured on the filter,
were as quickly as possible transferred to a big aquarium of ± 100 liter in CENTA’s
laboratory. The obtained Daphnia magna individuals were observed in the laboratory for
several weeks, to make sure that they were free of bacterial or parasitic infections [2].
20
The Daphnia captured in the reservoir were identified as Daphnia magna Straus by
observing the typical structure of the post-abdominal claw.
Using Daphnia for bioassays requires advance planning in order to make sure that it is a
healthy, non-stressed population that can be used as test organisms. To minimize clonal
variations such as age at maturity and brood size in acute toxicity tests, Daphnia magna,
from at least the third generation and deriving from one single female should be used
[36]. Therefore, five adult Daphnia which contained eggs in their brood chamber were set
aside each in a separate aquarium of 0.1 liter. When 15 neonates where present in the
second generation of the single female, the 15 neonates were transferred to 3-liter-
aquaria to produce a culture of 15 Daphnia magna individuals.
3.1.3 Culture set-up
The big aquarium of ± 100 liter, where the Daphnia were transferred to, was located in
the laboratory of CENTA. This aquarium was maintained to be a back-up aquarium in case
something went wrong with the culturing. The aquarium was filled with tap water. The
tap water was aged (> 24 h) so that chlorine was able to evaporate out of the water. The
aquarium water temperature was set at 20 °C ± 2 °C and experienced a cycle of
approximately 12 hours of daylight followed by 12 hours of darkness. The water was
equipped with aeration by an air stone connected to an airline tube. The aeration was set
on a low level so the fine bubbles did not endanger the Daphnia.
Figure 9: Neonates of Daphnia magna Figure 10: Daphnia magna - Post-abdominal claw
21
Figure 11: Back-up aquarium of ± 100 liter
To produce cultures of Daphnia magna deriving from one single female, 3-liter-aquaria
were used. These aquaria were kept in an incubator where temperature was set on 20 °C
± 2 °C and the light cycle was 16 h light/8 h darkness. In these aquaria, no aeration was
provided. The aquaria were filled with 1.2 liter of > 24 h aged tap water.
Figure 12: 3-liter-aquaria for the culturing of Daphnia magna
For the first culture, aquarium A was set up with 1.2 L aged tap water and 15 neonates.
Since Daphnia magna populations tend to crash under the best of conditions for no
apparent reason [32], a back-up culture B was set up in this experiment. The neonates of
aquaria A and B derived from the same second generation of one female adult Daphnia.
3.1.4 Feeding
Because of the fulfilment of the EC validity criteria and the easy accessibility of the freeze-
dried Chlorella vulgaris, freeze-dried and subsequently rehydrated Chlorella vulgaris was
provided to Daphnia magna in this experiment. The algae food was supplemented with
small amounts of dry baker’s yeast. The Daphnia magna used in the experiment had been
acclimated to the experimental diet for two generations.
The Daphnia magna were fed once daily (Monday-Friday) with a suspension of freeze-
dried Chlorella vulgaris algae in aged tap water. The amount of algae food was increased
until the Daphnia magna reached adulthood: 0.5 mg carbon on day 1-2; 0.75 mg carbon
on day 3-7; 1 mg carbon on day 8+ [36] per Daphnia magna.
22
Several researchers like Ebeling (2006) [56], Yang (2011) [57] and Shurin (2014) [58] all
used the following general structural formula for green algae: C106H263O110N16P. In this
thesis, this structural formula is used for the green algae Chlorella vulgaris. This brings
the ration of Chlorella vulgaris and its carbon content to 3,550:1,272 or in other words,
Chlorella vulgaris has a carbon content of 35.83 %.
The algae used for feeding were Medicura Naturprodukte – Bio Chlorella 100 % pure.
These algae consisted of 6 g tablets. For feeding, one tablet was grounded with a mortar
and a pestle and was suspended in 1 liter of aged water and mixed with a magnetic
stirrer.
1 L H2O + 6 g Chlorella vulgaris 6 g * 35.83
100 = 2.1498 g C 2 150 mg C
: 4 300 : 4 300
0.2326 ml 0.50 mg C
In a culture of 15 Daphnia magna, the total of algae suspension needed per day was
0.2326 ml * 15 = 4.4884 ml 4.5 ml
The algae food is supplemented with 0.5 ml per culture per day of a 100 mg/l stock
solution of dry baker’s yeast. The amount of feeding of algae and yeast was multiplied by
the amount of days where feeding was not possible.
3.1.5 Maintenance
Neonates were removed daily before feeding to avoid crowding and to ensure that the
founding adults obtained a constant level of food. Neonates were removed with a plastic
pipette. The production of neonates was noted in order to monitor the health of the
founding adults.
The water where the Daphnia magna were cultured in was changed twice a week for 50
% with fresh aged tap water. Adults were transferred with a plastic pipette to an
aquarium with fresh aged tap water containing the right amount of algae and yeast. 50 %
of the old aquarium water was passed through a sieve with a mesh size as big as possible
to remove residual waste (molts).
3.1.6 Datasheets of culturing
During the research, three cultures were maintained. All data of the culturing was
recorded on datasheets with excel. These datasheets can be found in Appendices 1 & 2.
On 10/11/2014, 2 cultures were set up with each 15 neonates respectively in aquarium A
and B. On 26/11/2014, a new culture C was set up with 15 neonates deriving from the
adult Daphnia magna in aquarium B.
In the datasheets, information can be found about the amount of adult Daphnia magna
and the amount of neonates produced. Also, information about the feeding and the
maintenance (water removal) is recorded. Furthermore, the performed tests are noted in
the final columns.
23
3.2 Reagents and materials
All solutions are prepared and stored in a controlled atmosphere of 20 °C ± 2 °C and all
procedures are performed within this atmosphere. The atmosphere needs to be free from
vapors and dusts toxic to Daphnia magna.
3.2.1 Test organism
In the procedure the organism Daphnia magna Straus (Cladocera, Crustacea) of at least
the third generation is used. The used individuals need to derive from one single female
and need to be less than 24 hours old (neonates).
Two hours before the start of the test, 0.5 mg C of Chlorella vulgaris per Daphnia magna
neonate was added to the recipient containing the neonates. This feeding is necessary to
provide the Daphnia magna with an energetic reserve and this makes sure that no
mortality occurs due to starvation, which would bias the test results, during the test [51].
3.2.2 Dilution water
The dilution water was prepared according to the International Standard ISO 6341. [3]
1. The following solutions were prepared:
Calcium chloride solution:
11.76 g of calcium chloride dehydrate (CaCl2.2H20) was dissolved in water and
made up to 1 liter with distilled water.
Magnesium sulfate solution:
4.93 g of magnesium sulfate heptahydrate (MgSO4.7H2O) was dissolved in
water and made up to 1 liter with distilled water.
Sodium bicarbonate solution:
2.59 g of sodium bicarbonate (NaHCO3) was dissolved in water and made up
to 1 liter with distilled water.
Potassium chloride solution:
0.23 g of potassium chloride (KCl) was dissolved in water and made up to 1
liter with distilled water.
2. 25 ml of each of the four solutions were mixed and the total volume was made up
to 1 liter by adding distilled water.
The dilution water was stored for maximum one month in the refrigerator at 4 °C in
darkness [36]. Before use, the cooled medium was gradually brought back to room
temperature.
The dilution water was aerated until the dissolved oxygen concentration had reached
saturation and the pH had stabilized (at least 15 minutes). If necessary, the pH to 7.8 ±
24
0.2 was adjusted by adding sodium hydroxide (NaOH) or hydrochloric acid (HCl). The
dilution water prepared in this way did not get aerated further before use.
3.2.3 Potassium dichromate (K2Cr2O7)
ITW Companies: AppliChem – Panreac
MM = 294.19 g/mol
3.2.4 Multimeter to measure dissolved oxygen, pH and temperature
YSI Incorporated 556 MPS
3.2.5 Test containers
Test plates with test containers of chemically inert material. Before use, the test
containers were carefully washed and rinsed with water and with the dilution water.
Figure 13: Test plate with test containers
3.2.6 Other instruments
Light box
Plastic Pasteur pipettes with the end point cut off at ± 3 centimeters, to adjust the
opening to the body size of the Daphnia.
Parafilm
3.3 Acute aquatic toxicity test with Daphnia magna Straus
The acute aquatic toxicity test is performed according to the International Standard ISO
6341. [3]
For a statistically acceptable evaluation of the effects, each test concentration as well as
the control, was assayed in four replicates.
In the first row on the test plate, each test container was filled with 10 ml dilution water.
These containers served as the control row. The next rows were each filled with 10 ml of
increasing concentrations of the test water.
In the first cup of each row, which serve as rinsing wells, 20 actively swimming Daphnia
magna neonates were placed. These rinsing wells serve to prevent dilution of the toxicant
in the test containers during the transfer of the test organisms from the aquarium to the
test plate. Exactly five Daphnias from each rinsing well were transferred into the four test
wells of the corresponding row. For each concentration and each control, a minimum of
20 Daphnia magna were used. [51]
25
Figure 14: Test containers - The test wells in each column are labelled A, B, C and D and the rows are labelled X (controls), 1, 2, 3, 4 and 5 for the five toxicant dilutions. [51]
During the transfer, the tips of the Pasteur pipettes were always placed in the medium. It
was always made sure that no organisms were dropped at the surface of the medium.
Otherwise the Daphnia magna could have captured air, what would have caused surface
floating.
During the test, the vessels were kept in an incubator at a temperature of 20 °C ± 2 °C
with 16 hours of light and 8 hours of darkness. The test plate was covered with parafilm
to prevent contamination.
At the end of the test period of 24 and 48 hours, the immobile Daphnia magna in each
container were counted. Hereby, the test plate was placed above a light box. Daphnia
magna which were not able to swim in the 15 seconds that follow gentle agitation of the
liquid were considered to be immobilized, even if they could still move their antennae.
Immediately after counting the immobilized Daphnia magna, the dissolved oxygen
concentration was measured in the test containers.
The concentration range giving 0 to 100 % immobilization was determined and any
anomalies in the behavior of the Daphnia magna were noted.
3.4 Procedure for caffeine
3.4.1 Test solutions
The substance tested was caffeine.
Caffeine
Sigma-Aldrich
Powder, ReagentPlus
1,3,7-Trimethylxanthine C8H10N4O2
MM = 194.19 g/mol
Solubility: H2O: soluble 15 mg/mL
26
Zhang (2013) concluded that the loss of caffeine by photodegradation was negligible in
his research to determine the uptake, the accumulation and the translocation of caffeine
in Scirpus validus [59]. Buerge (2003) also had a similar finding. He concluded that the
photodegradation in sunlight incubation experiments with lake water was 0.3 %/day.
Because of these results, it can be assumed that the removal of caffeine by
photodegradation is negligible in the 2-day-long experiment that is presented in this
research. Caffeine only undergoes a very slow photochemical degradation in the
environment [60].
3.4.1.1 Stock solutions
The stock solution of caffeine was prepared by dissolving a known quantity of caffeine in
a specified volume of dilution water in a glass container. The stock solution was prepared
at the moment of use.
For this test, a stock solution of 1 000 ppm was made.
3.4.1.2 Preparation of the test solutions
The test solutions were prepared by making dilutions of the stock solution with dilution
water in the following concentrations:
1:10 100 ppm
1:100 10 ppm
1:1 000 1 ppm
1:10 000 0.1 ppm
3.4.2 Preliminary test
This test enables the determination of the range of concentrations over which the
definitive test was to be carried out.
The preliminary test was carried out over the following test concentrations:
1:1 1 000 ppm
1:10 100 ppm
1:100 10 ppm
1:1 000 1 ppm
1:10 000 0.1 ppm
3.4.3 Definitive test
This test gives the percentages of Daphnia magna which were immobilized for every
concentrations tested. This enabled the determination of the EC50 – 24 h and the EC50 –
48 h.
The range of concentrations were chosen so three or four percentages of immobilization
between 10 % and 90 % were obtained. They span the range of the lowest concentration
producing 100 % effect and the highest concentration producing less than 10 % effect in
the preliminary test.
27
3.5 Procedure for influents and effluents
3.5.1 Test waters (influents and effluents)
The test waters were the influents and effluents of the PRB and the IGF. Water samples
were collected from the Imhoff tank (influent IGF), the piezometer that controlled the
groundwater of the IGF (effluent IGF), the distribution tank (influent PRB) and the
piezometer that controlled the groundwater of the PRB (effluent PRB). Analyses of COD,
BOD5 and TSS were performed at CENTA Foundation laboratories.
The toxicity test was carried out as soon as possible after collection. The samples were
cooled (+ 4 °C) at the place of collection and the bottles were completely filled to exclude
air. There were no chemical preservatives used.
Because of the possibility of Daphnia magna to get stuck in sediments, the samples were
brought over a sieve with a mesh size of 0.100 mm before the test was performed.
3.5.2 Preliminary test
This test enabled determination of the range of concentrations over which the definitive
test was to be carried out. For this purpose, the test was performed on the four non-
diluted influent and effluent samples and on their 1:2 dilutions to see what would happen.
Five Daphnia magna individuals were added to 10 ml of each sample and dilution, and the
results were observed after 24 and 48 hours.
3.5.3 Definitive test
This test enabled determination of the percentages of Daphnia magna which were
immobilized by different concentrations and determination of the EC50 – 24 h and the EC50
– 48 h.
The following concentrations were tested for each sample.
Concentration 1: Influent 1:1
Concentration 2: Influent 9:10
Concentration 3: Influent 8:10
Concentration 4: Influent 7:10
Concentration 5: Influent 6:10
Because the concentration of contaminants in the influent and effluent waters is
unknown, EC50 – 24 h and the EC50 – 48 h were expressed as the percentage of dilution
of the samples or as milliliters per liter, instead of the concentration.
3.6 Interpretation and validity of the results
3.6.1 Estimation of the EC50 – 24 h and the EC50 – 48 h
At the end of the test, the percentage immobilization for each concentration in relation to
the total number of Daphnia magna used was calculated. The EC50 – 24 h and the EC50 –
48 h were estimated with the “Dose effect analysis” tool of XLSTAT-Dose. XLSTAT is a
statistical analysis add-in that is compatible with Microsoft Excel. It offers a wide variety
28
of functions to do statistical data-analyses and it has a specific tool for “Dose effect
analysis”.
It is a dose effect analysis that is represented as a probit model. The model takes into
account that in each acute toxicity test no immobilized Daphnia magna were present in
the control containers, by setting the natural mortality parameter at zero. The amounts of
immobilized Daphnia magna per concentration were set as the response variable, the
total of 20 Daphnia magna was set as the observation weight and the different doses
were selected as the quantitative explanatory variable. The probit analysis was
represented with the log of the dose, because this usually gives a better fitted model
[61].
The dose effect analysis tool is also used to calculate the probability of the concentration
of the sample where 50 % of all Daphnia magna are immobilized.
3.6.2 Sensitivity check of Daphnia magna
The EC50 – 24 h of potassium dichromate was determined to check the sensitivity of the
Daphnia magna.
The check was carried out as described in the definitive test of caffeine, but with
concentrations between 0.1 mg/l and 4 mg/l. The EC50 – 24 h of the potassium
dichromate should fall inside the range 0.9 mg/l to 2.0 mg/l for the test results to be
valid.
The definitive test was carried out over the following test concentrations:
0.1 mg/l
1 mg/l
1.5 mg/l
2 mg/l
4 mg/l
At the end of the test period of 24 hours, the immobile Daphnia magna in each container
were counted and the EC50 – 24 h was estimated.
3.6.3 Other validity criteria
The results of the tests were considered as valid if the following requirements were
fulfilled:
The dissolved oxygen concentration at the end of the test (measured as indicated
in the definitive test) is greater than or equal to 2 mg/l;
The percentage of immobilization of the controls is less than or equal to 10 %;
Furthermore, the cultures need to fulfill the following requirements during the culturing:
The mortality of the adults may not exceed 20 % at the end of the test.
After 21 days, the mean offspring per adult is ≥ 60.
29
4 Results
4.1 Acute aquatic toxicity test of caffeine
4.1.1 Preliminary test
The results of the preliminary test are observed after 24 (Table 9) and 48 hours (
Table 10). The amounts of immobilized Daphnia magna are as followed:
Table 9: Results preliminary test on caffeine after 24 h –Immobilized Daphnia magna
Control 0.1 ppm 1 ppm 10 ppm 100 ppm 1 000 ppm
A 0 0 0 0 2 5
B 0 0 0 0 0 4
C 0 0 0 0 1 5
D 0 0 0 0 2 3
Total 0 0 0 0 5 17
Table 10: Results preliminary test on caffeine after 48 h – Immobilized Daphnia magna
Control 0.1 ppm 1 ppm 10 ppm 100 ppm 1 000 ppm
A 0 0 0 0 2 5
B 0 0 0 0 1 5
C 0 0 0 0 3 5
D 0 0 0 0 3 5
Total 0 0 0 0 9 20
The results presented in Table 9 and Table 10 show that the EC50-value after 24 h and 48
h is located between 10 ppm and 1 000 ppm. Therefore the concentrations for the
definitive test are selected between these two concentrations.
4.1.2 Definitive test
The definitive test on caffeine is performed on the following concentrations: 100 ppm,
250 ppm, 500 ppm, 750 ppm, 1 000 ppm. Every concentration is tested with 4 replicate
containers with each containing five Daphnia magna. The results are observed after 24
and 48 hours.
4.1.2.1 EC50 after 24 hours
The results of the acute aquatic toxicity test with Daphnia magna on caffeine are
observed after 24 hours (Table 11).
30
Table 11: Results definitive test on caffeine after 24 h – Immobilized Daphnia magna
Control 100 ppm 250 ppm 500 ppm 750 ppm 1 000 ppm
A 0 0 3 5 5 4
B 0 2 3 3 2 4
C 0 2 3 4 5 5
D 0 1 2 5 5 5
Total 0 5 11 17 17 18
% 0% 25% 55% 85% 85% 90%
By use of the dose effect analysis tool of XLSTAT-Dose, a probit model is presented
(Figure 15).
Figure 15: Logistic regression of response by log(dose) for caffeine after 24 h
The probability results of the dose effect analysis tool (Table 12) show that the EC50 – 24
h for caffeine is 207.525 mg/l with a 95 % confidence interval of 124.764 to 287.895
mg/l.
Table 12: Probability analysis for caffeine after 24 h – determination of EC50
Probability Dose (mg/l) Lower bound 95%
Upper bound 95% 0.10 48.762 12.845 90.116
0.50 207.525 124.764 287.895
0.90 883.205 596.779 1 867.667
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
0 0,5 1 1,5 2 2,5 3 3,5
Resp
on
se
Log(Dose)
Logistic regression of response by log(Dose)
Active Model Natural mortality
Lower bound (95%) Upper bound (95%)
31
4.1.2.2 EC50 after 48 hours
The results of the acute aquatic toxicity test with Daphnia magna on caffeine are
observed after 48 hours (Table 13) as follows:
Table 13: Results definitive test on caffeine after 48 h – Immobilized Daphnia magna
Control 100 ppm 250 ppm 500 ppm 750 ppm 1 000 ppm
A 0 2 4 5 5 5
B 0 2 5 4 4 5
C 0 3 4 4 5 5
D 0 2 3 5 5 5
Total 0 9 16 18 19 20
% 0% 45% 80% 90% 95% 100%
By use of the dose effect analysis tool of XLSTAT-Dose, a probit model is presented
(Figure 16).
Figure 16: Logistic regression of response by log(dose) for caffeine after 48 h
The probability results of the dose effect analysis tool (Table 14) show that the EC50 – 48
h for caffeine is 112.884 mg/l with a 95 % confidence interval of 52.856 to 166.423 mg/l.
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
0 0,5 1 1,5 2 2,5 3 3,5
Resp
on
se
Log(Dose)
Logistic regression of response by log(Dose)
Active Model Natural mortality
Lower bound (95%) Upper bound (95%)
32
Table 14: Probability analysis for caffeine after 48 h – determination of EC50
Probability Dose (mg/l) Lower bound 95%
Upper bound 95% 0.10 29.563 5.316 60.091
0.50 112.884 52.856 166.423
0.90 431.030 301.067 804.520
4.2 Acute aquatic toxicity test of the influents/effluents
4.2.1 Preliminary test
The results of the preliminary test are observed after 24 and 48 hours. The amounts of
immobilized Daphnia magna are as follows:
Table 15: Results preliminary test of non-diluted influents and effluents after 24 and 48 h – Immobilized Daphnia magna
Exposure time (h) Control Influent IGF 1:1
Effluent IGF 1:1
Influent PRB 1:1
Effluent PRB 1:1
24 0 5 0 5 0
48 0 5 2 5 0
The results of the non-diluted samples (Table 15) show that all Daphnia magna are
immobilized after 24 hours in the influents of the IGF and the PRB. Therefore a dilution is
needed to obtain more information. The effluent of the IGF shows no effect after 24
hours, but after 48 hours 40 % of Daphnia magna were immobilized. The effluent of the
PRB shows no effect of toxicity for Daphnia magna. However, the effluents are further
examined in the definitive test.
Table 16: Results preliminary test of 1:2 diluted influents and effluents after 24 and 48 h –
Immobilized Daphnia magna
Exposure time (h) Control Influent IGF 1:2
Effluent IGF 1:2
Influent PRB 1:2
Effluent PRB 1:2
24 0 0 0 0 0
48 0 0 0 0 0
The results presented in Table 16 show that no Daphnia magna were immobilized when
the samples were diluted by 1:2. Therefore the dilutions for the definitive tests are
selected between 1:1 and 1:2.
4.2.2 Definitive test
The definitive tests of the samples are performed on the following dilutions: 5:10 (=1:2),
6:10, 7:10, 8:10, 9:10 and 10:10 (=1:1). Every concentration is tested with four replicate
33
containers with each containing five Daphnia magna. Results are observed after 24 hours.
Due to time management, an observation after 48 hours was not possible.
4.2.2.1 Influent IGF
The results of the acute aquatic toxicity test with Daphnia magna on the influent of the
Intensive Green Filter (IGF) are presented in Table 17. Results indicate that the minimum
concentration corresponding to 100 % immobilization is the dilution of 8:10. The
maximum concentration corresponding to 0 % immobilization is the dilution of 5:10.
Table 17: Results definitive test influent IGF after 24 h – Immobilized Daphnia magna
Control 5:10 6:10 7:10 8:10 9:10 10:10
A 0 0 4 5 5 5 5
B 0 0 3 5 5 5 5
C 0 0 5 4 5 5 5
D 0 0 5 5 5 5 5
Total 0 0 17 19 20 20 20
% Immobilized 0% 0% 85% 95% 100% 100% 100%
By use of the dose effect analysis tool of XLSTAT-Dose, a probit model is presented
(Figure 17).
Figure 17: Logistic regression of response by log(dose) for influent IGF after 24 h
The probability results of the dose effect analysis tool (Table 18) show that the EC50 – 24
h for the influent of the IGF is 0.573 ml influent/ml test volume with a 95 % confidence
interval of 0.546 to 0.596 ml influent/ml test volume.
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
-0,35 -0,3 -0,25 -0,2 -0,15 -0,1 -0,05 0
Resp
on
se
Log(Dose)
Logistic regression of response by log(Dose)
Active Model Natural mortality
Lower bound (95%) Upper bound (95%)
34
Table 18: Probability analysis influent IGF – determination of EC50
Probability Dose (# ml influent/ml test volume) Lower bound 95%
Upper bound 95% 0.10 0.516 0.468 0.543
0.50 0.573 0.546 0.596
0.90 0.636 0.609 0.685
4.2.2.2 Effluent IGF
The results of the acute aquatic toxicity test with Daphnia magna on the effluent of the
Intensive Green Filter (IGF) are presented in Table 19. Results indicate that the maximum
concentration corresponding to 0 % immobilization is the dilution of 8:10. At the non-
diluted sample, only 10 % of all Daphnia magna are immobilized. Therefore, the minimum
concentration corresponding to 100 % immobilization cannot be determined.
Table 19: Results definitive test effluent IGF after 24 h – Immobilized Daphnia magna
Control 6:10 7:10 8:10 9:10 10:10
A 0 0 0 0 0 1
B 0 0 0 0 1 1
C 0 0 0 0 0 0
D 0 0 0 0 0 0
Total 0 0 0 0 1 2
% Immobilized 0% 0% 0% 0% 5% 10%
By use of the dose effect analysis tool of XLSTAT-Dose, a probit model is presented
(Figure 18).
Figure 18: Logistic regression of response by log(dose) for effluent IGF after 24 h
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
-0,25 -0,2 -0,15 -0,1 -0,05 0
Resp
on
se
Log(Dose)
Logistic regression response by log(Dose)
Active Model Natural mortality
Lower bound (95%) Upper bound (95%)
35
Because only 10 % of Daphnia magna are immobilized at the non-diluted sample (Table
19), a calculation of the EC50 – 24 h is not possible for the effluent of the IGF.
4.2.2.3 Influent PRB
The results of the acute aquatic toxicity test with Daphnia magna on the influent of the
Permeable Reactive Barrier (PRB) are presented in Table 20. Results indicate that the
minimum concentration corresponding to 100 % immobilization is the non-diluted sample
(=10:10). The maximum concentration corresponding to 0 % immobilization is the
dilution of 7:10.
Table 20: Results definitive test influent PRB after 24 h – Immobilized Daphnia magna
Control 5:10 6:10 7:10 8:10 9:10 10:10
A 0 0 0 0 0 2 5
B 0 0 0 0 0 3 5
C 0 0 0 0 0 4 5
D 0 0 0 0 1 4 5
Total 0 0 0 0 1 13 20
% Immobilized 0% 0% 0% 0% 5% 65% 100%
By use of the dose effect analysis tool of XLSTAT-Dose, a probit model is presented
(Figure 19).
Figure 19: Logistic regression of response by log(dose) for influent PRB after 24 h
The probability results of the dose effect analysis tool (Table 21) show that the EC50 – 24
h for the influent of the IGF is 0.878 ml influent/ml test volume with a 95 % confidence
interval of 0.853 to 0.903 ml influent/ml test volume.
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
-0,35 -0,3 -0,25 -0,2 -0,15 -0,1 -0,05 0
Resp
on
se
Log(Dose)
Logistic regression response by log(Dose)
Active Model Natural mortality
Lower bound (95%) Upper bound (95%)
36
Table 21: Probability analysis influent PRB – determination of EC50
Probability Dose (# ml influent/ml test volume) Lower bound 95% Upper bound 95%
0.10 0.820 0.770 0.846
0.50 0.878 0.853 0.903
0.90 0.941 0.913 0.999
4.2.2.4 Effluent PRB
The results of the acute aquatic toxicity test with Daphnia magna on the effluent of the
Permeable Reactive Barrier (PRB) are presented in Table 22. Results indicate that no
Daphnia magna are immobilized. Therefore, a dose effect analysis can’t be done and the
EC-value can’t be calculated.
Table 22: Results definitive test effluent PRB after 24 h – Immobilized Daphnia magna
Control 6:10 7:10 8:10 9:10 10:10
A 0 0 0 0 0 0
B 0 0 0 0 0 0
C 0 0 0 0 0 0
D 0 0 0 0 0 0
Total 0 0 0 0 0 0
% Immobilized 0% 0% 0% 0% 0% 0%
4.2.3 Sampling data
Analyses of COD, BOD5 and TSS are performed at CENTA Foundation laboratories on
0ctober 28, 2014, on the samples of the influents and effluents of the IGF and the PRB.
4.2.3.1 Intensive Green Filter (IGF)
Water samples are collected from the Imhoff tank effluent (influent IGF) and the
piezometer that controlled the groundwater (effluent IGF). The lysimeter did not contain
any leachate at the time of sampling.
On October 28, 2014, the following results are obtained (Table 23):
Table 23: Sampling data Intensive Green Filter, 28/10/2014
COD (mg/l) BOD5 (mg O2/l) TSS (mg/l)
Imhoff tank effluent (= influent IGF)
202 150 66
Piezometer groundwater (= effluent IGF)
5 9 14
The results of the IGF (Table 23) show that the groundwater quality only contains low
concentrations of COD, BOD5 and TSS. The concentrations for COD, BOD5 and TSS of the
37
effluent, diluted in the already present groundwater, are respectively ± 98 %, ± 94 %
and ± 78 % lower than the corresponding concentrations in the influent of the IGF.
4.2.3.2 Permeable Reactive Barrier (PRB)
Water samples are collected from the distribution tank (influent PRB) and the piezometer
that controlled the groundwater (effluent PRB). Analyses of COD, BOD5 and TSS were
performed at CENTA Foundation laboratories.
On October 28, 2014, the following results are obtained (Table 24):
Table 24: Sampling data Permeable Reactive Barrier, 28/10/2014
COD (mg/l) BOD5 (mg O2/l) TSS (mg/l)
Distribution tank (= influent PRB)
384 220 158
Piezometer groundwater (= effluent PRB)
16 15 13
As with the IGF, the results of the PRB (Table 24) show that the groundwater quality only
contains low concentrations of COD, BOD5 and TSS. The concentrations for COD, BOD5
and TSS of the effluent, diluted in the already present groundwater, are respectively ± 96
%, ± 93 % and ± 92 % lower than the corresponding concentrations in the influent of
the PRB.
4.3 Validity of the results
4.3.1 Sensitivity check of the Daphnia magna – culture A
The results of the validity test of culture A with K2Cr2O7 are observed after 24 hours. The
amounts of immobilized Daphnia magna are as followed:
Table 25: Results validity test of culture A after 24 h – Immobilized Daphnia magna
Concentrations (mg/l) Control 0.5 0.75 1 1.5 2
A 0 0 1 2 3 5
B 0 0 0 4 4 5
C 0 0 0 3 5 4
D 0 0 1 0 2 4
Total 0 0 2 9 14 18
% 0% 0% 10% 45% 70% 90%
By use of the dose effect analysis tool of XLSTAT-Dose, a probit model is presented
(Figure 20).
38
Figure 20: Logistic regression of response by log(dose) in the validity test of culture A
The probability results of the dose effect analysis tool (Table 26) show that the EC50 – 24
h for the validity test of culture A is 1.161 mg/l with a 95 % confidence interval of 1.000
to 1.333 mg/l.
Table 26: Probability analysis validity test of culture A – determination of EC50
Probability Dose (mg/l) Lower bound 95% Upper bound 95%
0.50 1.161 1.000 1.333
4.3.2 Sensitivity check of the Daphnia magna – culture B
The results of the validity test of culture B with K2Cr2O7 are observed after 24 hours. The
amounts of immobilized Daphnia magna are as followed:
Table 27: Results validity test of culture B after 24 h – Immobilized Daphnia magna
Concentrations (mg/l) Control 0.5 0.75 1 1.5 2
A 0 0 1 2 2 5
B 0 0 1 2 5 4
C 0 0 0 2 3 4
D 0 0 0 2 4 4
Total 0 0 2 8 14 17
% 0% 0% 10% 40% 70% 85%
By use of the dose effect analysis tool of XLSTAT-Dose, a probit model is presented
(Figure 21).
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
-0,2 -0,1 0 0,1 0,2 0,3 0,4
Resp
on
se
Log(Dose)
Logistic regression of response by log(Dose)
Active Model Natural mortality
Lower bound (95%) Upper bound (95%)
39
Figure 21: Logistic regression of response by log(dose) in the validity test of culture B
The probability results of the dose effect analysis tool (Table 28) show that the EC50 – 24
h for the validity test of culture B is 1.205 mg/l with a 95 % confidence interval of 1.034
to 1.397 mg/l.
Table 28: Probability analysis validity test of culture B – determination of EC50
Probability Dose (mg/l) Lower bound 95% Upper bound 95%
0.50 1.205 1.034 1.397
4.3.3 Sensitivity check of the Daphnia magna – culture C
The results of the validity test of culture C with K2Cr2O7 are observed after 24 hours. The
amounts of immobilized Daphnia magna are as followed:
Table 29: Results validity test of culture C after 24 h – Immobilized Daphnia magna
Concentrations (mg/l) Control 0.5 0.75 1 1.5 2
A 0 0 0 2 3 4
B 0 0 1 1 4 5
C 0 0 1 2 3 5
D 0 0 1 2 3 4
Total 0 0 3 8 13 18
% 0% 0% 15% 40% 65% 90%
By use of the dose effect analysis tool of XLSTAT-Dose, a probit model is presented
(Figure 22).
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
-0,2 -0,1 0 0,1 0,2 0,3 0,4
Resp
on
se
Log(Dose)
Logistic regression of response by log(Dose)
Active Model Natural mortality
Lower bound (95%) Upper bound (95%)
40
Figure 22: Logistic regression of response by log(dose) in the validity test of culture C
The probability results of the dose effect analysis tool (Table 32) show that the EC50 – 24
h for the validity test of culture C is 1.177 mg/l with a 95 % confidence interval of 1.005
to 1.368 mg/l.
Table 30: Probability analysis validity test of culture C – determination of EC50
Probability Dose (mg/l) Lower bound 95% Upper bound 95%
0.50 1.177 1.005 1.368
4.3.4 Other validity criteria
Concerning the test results:
During all tests, the dissolved oxygen concentration at the end of the test is
greater than 2 mg/l.
The percentage of immobilization in the controls is 0 % in all the tests.
Concerning the culturing:
In all the cultures, the mortality of the adults is found to be 0 %. No adults died
during the culturing (Appendices 1 & 2).
After 21 days, cultures A, B and C have a mean offspring per Daphnia magna of
respectively 75, 81 and 72 (Appendices 1 & 2). All cultures have a mean offspring
per Daphnia magna that was higher than 60.
0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1
-0,2 -0,1 0 0,1 0,2 0,3 0,4
Resp
on
se
Log(Dose)
Logistic regression of response by log(Dose)
Active Model Natural mortality
Lower bound (95%) Upper bound (95%)
41
5 Discussion
5.1 Acute aquatic toxicity test of caffeine
The toxicity test with Daphnia magna on caffeine shows that the EC50 – 24 h for caffeine
is 207.525 mg/l with a 95 % confidence interval of 124.764 to 287.895 mg/l and the EC50
– 48 h for caffeine is 112.884 mg/l with a 95 % confidence interval of 52.856 to 166.423
mg/l (Table 31).
Table 31: EC50-values for caffeine – 24 and 48 h
Probability Dose (mg/l) Lower bound 95%
Upper bound 95%
EC50–24 h 207.525 124.764 287.895
EC50–48 h 112.884 52.856 166.423
In 1995, Lilius [38] defined the EC50 – 24 h of caffeine as 161.28 mg/l (Table 6). This
value has a deviation of 46.245 mg/l which is 22.28 % lower than the obtained result in
this study. However, the EC50 – 24 h by Lilius is located in between the 95 % confidence
interval obtained in this study.
The estimated concentration of caffeine in the wastewater influent of the Experimental
Center of Carrión de los Céspedes, Seville, was analyzed with one sample. The
concentration was 5 199 µg/l or 5.199 mg/l (Table 4). With this information, it can be
concluded that the present concentration of caffeine in the wastewater is far below the
obtained EC50 – 24 h and even far below the obtained EC50 – 48 h.
5.2 Acute aquatic toxicity test of the influents/effluents
Because one analyzed microcontaminant cannot represent the mixture of
microcontaminants that are present in the wastewaters of the IGF and the PRB, an
additional acute toxicity test with Daphnia magna is performed on the influents and
effluents of the two water flows to monitor the water quality before and after the
treatment method.
The EC50 – 24 h for the influent of the IGF is 0.573 ml influent/ml test volume with a 95 %
confidence interval of 0.546 to 0.596 ml influent/ml test volume. In the effluent of the
IGF, only 10 % of the Daphnia magna are immobilized after 24 hours in the non-diluted
sample. Therefore, a calculation of the EC50 – 24 h is not possible for the effluent of the
IGF.
The EC50 – 24 h for the influent of the PRB is 0.878 ml influent/ml test volume with a 95
% confidence interval of 0.853 to 0.903 ml influent/ml test volume. In the effluent of the
PRB, no Daphnia magna are immobilized after 24 hours in the non-diluted sample.
Therefore, no EC-value can be calculated for the effluent of the PRB.
42
Table 32: EC50–24 h for influents/effluents
Sample EC50 – 24 h (# ml influent/ml test volume)
Lower bound 95%
Upper bound 95%
Influent IGF 0.573 0.546 0.596
Effluent IGF / / /
Influent PRB 0.878 0.853 0.903
Effluent PRB / / /
These results show that the EC50 – 24 h of the influent of the IGF is lower than the EC50 –
24 h of the influent of the PRB. So, the concentration where 50 % of the Daphnia magna
are immobilized is more diluted for the IGF than for the PRB. With this information it can
be concluded that the acute toxicity of the influent of the IGF is higher than the acute
toxicity of the influent of the PRB. These results are found to be as expected, because the
influent of the IGF only precedes an Imhoff-tank, while the influent of the PRB is the
outlet of a reclamation treatment: preliminary treatment, extended aeration and sand
filter.
Where the effluent of the PRB does not show any acute toxicity after 24 hours, the
effluent of the IGF shows a small percentage of immobilized Daphnia magna which results
in an acute toxicity of 10 % after 24 hours. It can be concluded that the acute toxicity of
the effluent of the IGF is higher than the acute toxicity of the effluent of the PRB. These
results are also found to be as expected, because: i) the quality of the influent of the PRB
is better than the quality of the influent of the IGF, and ii) a Permeable Reactive Barrier
with three layers (palygorskite, activated carbon and zeolite) that are specialized to
remove a wide spectrum of pollutants, is more likely to have a better performance than a
vegetation filter with soil and poplars.
When comparing the influents with their corresponding effluents, it can be concluded that
in both wastewater treatment systems the acute toxicity for Daphnia magna decreases
enormously. While all Daphnia magna are immobilized after 24 hours in the non-diluted
influents, almost no acute effect is observed after 24 hours in the effluent.
5.3 Validity of the results
5.3.1 Validity criteria
The EC50 – 24h of the potassium dichromate test of the four cultures are respectively
1.161; 1.205 and 1.177 mg/l. These values all fall in the range of 0.900 mg/l to 2.000
mg/l and confirm the validity of the tests.
Additionally the dissolved oxygen concentration at the end of the test never proceeded
under 2 mg/l and the percentage of immobilization of the controls never exceeded 10 %.
Therefor the results of the tests were considered to be valid.
Furthermore, the mortality of the adults was 0 % and the mean offspring per adult always
exceeded 60. These values confirm the validity of the culturing method.
43
5.3.2 Culturing conditions
During the culturing it was noticed that all food had sunk to the bottom of the aquaria
after the days where no feeding was possible. This however did not seem to give any
problems for the Daphnia magna. It was observed that the Daphnia magna were
swimming close to the bottom of the aquarium. After feeding, their normal swimming
behavior resumed.
Also, the feeding of 0.5 to 1 mg C of Chlorella vulgaris per Daphnia magna in combination
with 0.5 ml per culture of 15 Daphnia magna per day of a 100 mg/l stock suspension of
dry baker’s yeast seems to be sufficient. No mortality of the adult Daphnia magna
occurred and their mean offspring was higher than the required amount of 60 in the first
21 days.
In the 25th day of culture A and B, neonates were found to be stillborn. The adult Daphnia
magna were still alive. This probably occurred due to a temperature shock in the previous
water renewal. After renewing the water again and providing a sufficient amount of food,
the adult Daphnia magna resumed their normal movement behavior. A couple of days
later new healthy offspring was born. After this occurred, neonates were only used for the
tests of the effluents of the IGF and the PRB. During the test of the effluent of the PRB,
no neonates were immobilized so no effects of the earlier temperature shock to their
parent animals were observed. The effluent of the IGF show a small percentage of
immobilized Daphnia magna, but this could be explained by the influent water of the IGF
being more contaminated than the influent water of the PRB. However, a level of
uncertainty must be taken into consideration for this result.
5.3.3 Reflection on the test method
The obtained result only represents the acute aquatic toxicity of caffeine for Daphnia
magna and does not include the chronic toxicity towards the reproduction of the Daphnia
magna. An acute toxicity test has the advantages to be fast, and thereby relatively cheap.
A chronic toxicity test is more accurate because it shows the result of repeated exposures,
often at lower levels of contamination over a longer time. It also indicates the effects of
the contamination on reproduction and growth of the studied animals. This test however
includes feeding and needs more attention, what makes the test more expensive.
To assess an accurate representation of the environmental risks of the present substances
(e.g. caffeine), a chronic study is needed. A chronic study will probably show a
concentration, lower than the EC50 with the effect of immobilization of the Daphnia
magna, where reproduction or growth experience adverse effects.
45
6 Conclusions
The toxicity test with Daphnia magna on caffeine shows that the EC50 – 24 h for caffeine
is 207.525 mg/l with a 95 % confidence interval of 124.764 to 287.895 mg/l and the EC50
– 48 h for caffeine is 112.884 mg/l with a 95 % confidence interval of 52.856 to 166.423
mg/l. The present concentration of caffeine in the wastewater is far below the obtained
EC50 – 24 h and even far below the obtained EC50 – 48 h.
Because one analyzed microcontaminant cannot represent the mixture of
microcontaminants that are present in the wastewaters of the Intensive Green Filter and
the Permeable Reactive Barrier, an additional acute toxicity test with Daphnia magna is
performed on the influents and effluents of the two water flows to monitor the water
quality before and after the treatment method.
The EC50 – 24 h for the influent of the Intensive Green Filter is 0.573 ml influent/ml test
volume with a 95 % confidence interval of 0.546 to 0.596 ml influent/ml test volume. The
EC50 – 24 h for the influent of the Permeable Reactive Barrier is 0.878 ml influent/ml test
volume with a 95 % confidence interval of 0.853 to 0.903 ml influent/ml test volume.
With this information it can be concluded that the acute toxicity of the influent of the
Intensive Green Filter is higher than the acute toxicity of the influent of the Permeable
Reactive Barrier.
A calculation of the EC50 – 24 h of both effluents was not possible because the amounts of
immobilized Daphnia magna were too low. The Permeable Reactive Barrier shows no
effect of immobilization, while the Intensive Green Filter shows a low acute toxicity of 10
% for Daphnia magna after 24 hours.
The obtained results are found to be as expected, because: i) the quality of the influent of
the Permeable Reactive Barrier is better than the quality of the influent of the Intensive
Green Filter, and ii) a Permeable Reactive Barrier with three layers (palygorskite, activated
carbon and zeolite) that are specialized to remove a wide spectrum of pollutants, is more
likely to have a better performance than a vegetation filter with soil and poplars.
When comparing the influents with their corresponding effluents, it can be concluded that
in both the Intensive Green Filter and the Permeable Reactive Barrier, the acute toxicity
for Daphnia magna decreases enormously. Furthermore, in both wastewater treatment
systems, the concentrations for COD, BOD5 and TSS of the effluent, diluted in the already
present groundwater, were respectively ± 96 %, ± 93 % and ± 78 % lower than the
corresponding concentrations in their influent waters.
The culturing method and the test results are considered to be valid as they meet all
validity criteria.
These results however only represent the acute toxicity and do not include chronic
toxicities towards the reproduction of Daphnia magna.
47
7 Recommendations To avoid a contamination of the aquatic environment, the ecotoxicological status of the
groundwater underneath the Intensive Green Filter and the Permeable Reactive Barrier is
assessed. It is important that the irrigated wastewater that is applied to both treatment
technologies has a minimal impact on the groundwater quality to prevent environmental
risks.
The results of the acute aquatic toxicity tests on the effluents of the Intensive Green Filter
and the Permeable Reactive Barrier indicate that the Intensive Green Filter is not able to
remove all toxicological risks towards Daphnia magna. A small acute toxicity was found in
the groundwater, which suggests that a chronic toxicity is possible that has even more
environmental risks. The groundwater underneath the Permeable Reactive Barrier showed
no acute toxicity towards Daphnia magna. This indicates that the environmental risks are
lower. However, a chronic toxicity test may expose other important impacts on the
groundwater quality.
The obtained results only represent the acute toxicity and do not include chronic toxicities
towards the reproduction of the Daphnia magna. Performing a chronic study could be
interesting to further investigate the small immobilization effects of the effluent of the
Intensive Green Filter and the non-observed effect of the Permeable Reactive Barrier. To
assess an accurate representation of the environmental risks of the present substances
(e.g. caffeine), these chronic studies are needed.
Furthermore, additional research for the acute toxicity of caffeine is possible in order to
reduce the upper and lower boundary of the 95 % confidence interval to have a more
accurate result. Also, a research for the acute toxicity of more microcontraminants, e.g.
diclofenac as a representation of the NSAIDs, could give a broader picture of the toxicity
of the wastewater.
Also, the feeding of 0.5 to 1 mg C of Chlorella vulgaris per Daphnia magna in combination
with 0.5 ml per culture of 15 Daphnia magna per day of a 100 mg/l stock suspension of
dry baker’s yeast seems to be a sufficient feeding for the Daphnia magna to grow a
healthy culture. No mortality of the adult Daphnia magna occurred and their mean
offspring was higher than the required amount of 60 in the first 21 days. Furthermore, it
is highly recommended to watch out for temperature shocks, as this causes the Daphnia
magna culture to experience a large level of stress.
49
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Appendices
1. Datasheet culturing method – Culture A and B
Date Day
Culture (# adults) # offspring Food
Water renewal Remarks Tests A B A B
Algae (ml)
Yeast (ml)
10/11/14 1 15 15 0 0 7 1 100% Food x 2
11/11/14 2
12/11/14 3 15 15 0 0 7 1
Food x 2
13/11/14 4
14/11/14 5 15 15 0 0 15.75 1.5 50% Food x 3 for the weekend
15/11/14 6
16/11/14 7
17/11/14 8 15 15 0 0 7 0.5 50%
18/11/14 9 15 15 0 0 7 0.5
Preliminary test influents/effluents
19/11/14 10 15 15 0 0 7 0.5
20/11/14 11 15 15 9 208 7 0.5
1st brood aquaria A & B Validation with K2Cr2O7 – culture B
21/11/14 12 15 15 140 0 21 1.5 50% Food x 3 for the weekend; 1st brood aquaria A
22/11/14 13
23/11/14 14
24/11/14 15 15 15 310 341 7 0.5 50% 2nd brood aq. A, B
25/11/14 16 15 15 0 0 7 0.5
26/11/14 17 15 15 0 137 7 0.5
3th brood aq. B - new culture C started Preliminary test caffeine
27/11/14 18 15 15 254 223 7 0.5
3th brood aq. A & B Test influent 1 + influent 2
+ Validation with K2Cr2O7 - culture A
28/11/14 19 15 15 119 2 21 1.5 50% Food x 3 for the weekend
29/11/14 20
30/11/14 21
1/12/14 22 15 15 294 307 7 0.5 50% 4th brood aq. A & B
2/12/14 23 15 15 0 0 7 0.5
3/12/14 24 15 15 0 0 7 0.5 50%
4/12/14 25 15 15 >100 >100 7 0.5 100% Aq. A & B: 5th brood - dead neonates. Temperature shock?
5/12/14 26 15 15 0 0 28 2
Food x 4 for the weekend
6/12/14 27
7/12/14 28
8/12/14 29
Holiday
9/12/14 30 15 15 98 87 7 0.5 100%
10/12/14 31 15 15 154 137 7 0.5
Test effluent 1 + effluent 2
11/12/14 32 15 15 0 0
Total offspring in 21 days 1 126 1 218
Offspring per adult 75 81
2. Datasheet culturing method – Culture C
Date Day
Culture (# adults) # offspring Food
Water renewal Remarks Tests C C Algae (ml) Yeast (ml)
26/11/2014 1 15 0 3.5 0.5
27/11/2014 2 15 0 3.5 0.5
28/11/2014 3 15 0 15.75 1.5 50% Food x 3 for the weekend
29/11/2014 4
30/11/2014 5
1/12/2014 6 15 0 5.25 0.5 50%
2/12/2014 7 15 0 5.25 0.5
3/12/2014 8 15 0 7 0.5
4/12/2014 9 15 0 7 0.5
5/12/2014 10 15 0 28 2 50% Food x 4 for the weekend
6/12/2014 11
1st brood
7/12/2014 12
8/12/2014 13
Holiday
9/12/2014 14 15 502 7 0.5 50% 2nd brood
10/12/2014 15 15 0 7 0.5
11/12/2014 16 15 0 28 2 50% Food x 4 for the weekend
12/12/2014 17
3rd brood
13/12/2014 18
14/12/2014 19
15/12/2014 20 15 304 7 0.5 50%
16/12/2014 21 15 275 7 0.5
4th brood Definitive test caffeine + Validation with K2Cr2O7
17/12/2014 22 15
7 0.5