Acute aquatic toxicity test of caffeine with Daphnia...

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


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


Magnetic stirrer

Medicura Naturprodukte – Bio Chlorella 100 % pure

Dry baker’s yeast

Mortar and pestle

Aged tap water

3.1.1.4 Maintenance


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.


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.


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





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


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.


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%


(Figure 16).

Figure 16: Logistic regression of response by log(dose) for caffeine after 48 h


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)




32

Table 14: Probability analysis for caffeine after 48 h – determination of EC50


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


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.


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%


(Figure 17).

Figure 17: Logistic regression of response by log(dose) for influent IGF after 24 h


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)




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%


(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)



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%


(Figure 19).

Figure 19: Logistic regression of response by log(dose) for influent PRB after 24 h


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)



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%


(Figure 20).

38

Figure 20: Logistic regression of response by log(dose) in the validity test of culture A


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


Table 27: Results validity test of culture B after 24 h – Immobilized Daphnia magna


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%


(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)




39

Figure 21: Logistic regression of response by log(dose) in the validity test of culture B


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


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


Table 29: Results validity test of culture C after 24 h – Immobilized Daphnia magna


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%


(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)




40

Figure 22: Logistic regression of response by log(dose) in the validity test of culture C


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


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)




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



mg/l (Table 31).

Table 31: EC50-values for caffeine – 24 and 48 h


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


microcontaminants that are present in the wastewaters of the IGF and the PRB, an


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



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.


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

8 References

[1] CENTA. (2014). Fundación Centro de las Nuevas Tecnologías del Agua. Available: http://www.centa.es

[2] EPA. (2002). Methods for measuring the acute toxicity of effluents and receiving waters to freshwater and marine organisms. Available: http://www.epa.gov/region6/water/npdes/wet/wet_methods_manuals/atx.pdf

[3] ISO, "Water Quality - Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea)," in Evironment - Water Quality. vol. 3, I. 6341, Ed., ed Genève: International Organization for Standardization - ISO 6341, 1989, pp. 63-70.

[4] J. T. Yu, E. J. Bouwer, and M. Coelhan, "Occurrence and biodegradability studies of selected pharmaceuticals and personal care products in sewage effluent," Agricultural water management, vol. 86, pp. 72-80, 2006.

[5] S. Toze, "Reuse of effluent water—benefits and risks," Agricultural water management, vol. 80, pp. 147-159, 2006.

[6] A. De Miguel, V. Martínez-Hernández, M. Leal, V. González-Naranjo, I. De Bustamante, J. Lillo, et al., "Short-term effects of reclaimed water irrigation: Jatropha curcas L. cultivation," Ecological Engineering, vol. 50, pp. 44-51, 2013.

[7] CENTA, "Project: Reutilización de aguas: mas allá del Real Decreto 1620/2007 - Memoria técnica," M. d. E. y. Competitividad, Ed., ed, 2012.

[8] G. H. Han, H. G. Hur, and S. D. Kim, "Ecotoxicological risk of pharmaceuticals from wastewater treatment plants in Korea: occurrence and toxicity to Daphnia magna," Environmental Toxicology and Chemistry, vol. 25, pp. 265-271, 2006.

[9] A. de Miguel, R. Meffe, M. Leal, V. González-Naranjo, V. Martínez-Hernández, J. Lillo, et al., "Treating municipal wastewater through a vegetation filter with a short-rotation poplar species," Ecological Engineering, vol. 73, pp. 560-568, 2014.

[10] C. Pistocchi, W. Guidi, E. Piccioni, and E. Bonari, "Water requirements of poplar and willow vegetation filters grown in lysimeter under Mediterranean conditions: results of the second rotation," Desalination, vol. 246, pp. 137-146, 2009.

[11] M. Leal, R. G. Pacheco, J. Lillo, I. d. Bustamante, Á. d. Miguel, F. Carre, et al., "Horizontal permeacle reactive barriers for groundwater recharge with wastewater: kinetic and adsorption assays.," 2013.

[12] US-EPA, "Permeable reactive barrier with horizontal flow," F1.large, Ed., ed, 1998. [13] L. Geranio, "ETH Zürich," 2007. [14] IPCS, International, P. on, and C. Safety. (2012). International Chemical Safety

Card 1321. Available: http://www.inchem.org/documents/icsc/icsc/eics1321.htm [15] L.-W. T. Solutions. (2015). Adsorption/active carbon. Available:

http://www.lenntech.nl/bibliotheek/adsorption.htm [16] W. R. G. a. Co.-Conn. (2014). Synthetic Non-fibrous Zeolites. Available:

www.grace.com [17] A. Woinarski, G. Stevens, and I. Snape, "A natural zeolite permeable reactive

barrier to treat heavy-metal contaminated waters in Antarctica: kinetic and fixed-bed studies," Process Safety and Environmental Protection, vol. 84, pp. 109-116, 2006.

[18] M. M. Huber, A. GÖbel, A. Joss, N. Hermann, D. LÖffler, C. S. McArdell, et al., "Oxidation of pharmaceuticals during ozonation of municipal wastewater effluents: a pilot study," Environmental Science & Technology, vol. 39, pp. 4290-4299, 2005.

[19] C. Fernández, M. González-Doncel, J. Pro, G. Carbonell, and J. Tarazona, "Occurrence of pharmaceutically active compounds in surface waters of the

http://www.centa.es/

http://www.epa.gov/region6/water/npdes/wet/wet_methods_manuals/atx.pdf

http://www.inchem.org/documents/icsc/icsc/eics1321.htm

http://www.lenntech.nl/bibliotheek/adsorption.htm

http://www.grace.com/

50

Henares-Jarama-Tajo river system (Madrid, Spain) and a potential risk characterization," Science of the total environment, vol. 408, pp. 543-551, 2010.

[20] N. L. Benowitz, P. Jacob, H. Mayan, and C. Denaro, "Sympathomimetic effects of paraxanthine and caffeine in humans*," Clinical Pharmacology & Therapeutics, vol. 58, pp. 684-691, 1995.

[21] S. Guerreiro, D. Toulorge, E. Hirsch, M. Marien, P. Sokoloff, and P. P. Michel, "Paraxanthine, the primary metabolite of caffeine, provides protection against dopaminergic cell death via stimulation of ryanodine receptor channels," Molecular pharmacology, vol. 74, pp. 980-989, 2008.

[22] M. J. Gómez, C. Sirtori, M. Mezcua, A. R. Fernández-Alba, and A. Agüera, "Photodegradation study of three dipyrone metabolites in various water systems: Identification and toxicity of their photodegradation products," Water research, vol. 42, pp. 2698-2706, 2008.

[23] Y. Zhang, S.-U. Geißen, and C. Gal, "Carbamazepine and diclofenac: removal in wastewater treatment plants and occurrence in water bodies," Chemosphere, vol. 73, pp. 1151-1161, 2008.

[24] G. G. Liversidge and P. Conzentino, "Drug particle size reduction for decreasing gastric irritancy and enhancing absorption of naproxen in rats," International journal of pharmaceutics, vol. 125, pp. 309-313, 1995.

[25] R. L. Thomas, G. C. Parker, B. Van Overmeire, and J. V. Aranda, "A meta-analysis of ibuprofen versus indomethacin for closure of patent ductus arteriosus," European journal of pediatrics, vol. 164, pp. 135-140, 2005.

[26] F. J. Real, F. J. Benitez, J. L. Acero, J. J. Sagasti, and F. Casas, "Kinetics of the chemical oxidation of the pharmaceuticals primidone, ketoprofen, and diatrizoate in ultrapure and natural waters," Industrial & Engineering Chemistry Research, vol. 48, pp. 3380-3388, 2009.

[27] T. S. H. ü. S. Yöntemle, "Spectrofluorometric Determination of Citalopram HBr in Tablets," FABAD J. Pharm. Sci, vol. 32, pp. 73-77, 2007.

[28] ITIS. (2014). ITIS Report Daphnia magna Straus, 1820. Available: http://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&search_value=83884

[29] D. Ebert. (2005). Introduction to the Ecology, Epidemiology, and Evolution of Parasitism in Daphnia. Available: http://www.ncbi.nlm.nih.gov/books/NBK2042/

[30] C. U. a. P. S. U. Environmental Inquiry. (2009). Bioassays - Daphnia. Available: http://ei.cornell.edu/toxicology/bioassays/daphnia/

[31] M. Elenbaas. (2013). Daphnia magna. Available: http://animaldiversity.ummz.umich.edu/accounts/Daphnia_magna/

[32] J. Clare. (2002). Daphnia: an aquarist's guide. Available: http://www.caudata.org/daphnia/

[33] B. Mittmann, P. Ungerer, M. Klann, A. Stollewerk, and C. Wolff, "Development and staging of the water flea Daphnia magna (Straus, 1820; Cladocera, Daphniidae) based on morphological landmarks," EvoDevo 2014, vol. 5, p. 20, 2014.

[34] L. Guilhermino, T. Diamantino, M. Carolina Silva, and A. Soares, "Acute Toxicity Test with Daphnia magna: An Alternative to Mammals in the Prescreening of Chemical Toxicity?," Ecotoxicology and Environmental Safety, vol. 46, pp. 357-362, 2000.

[35] Z. Ren, J. Zha, M. Ma, Z. Wang, and A. Gerhardt, "The early warning of aquatic organophosphorus pesticide contamination by on-line monitoring behavioral changes of Daphnia magna," Environmental monitoring and assessment, vol. 134, pp. 373-383, 2007.

http://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&search_value=83884

http://www.itis.gov/servlet/SingleRpt/SingleRpt?search_topic=TSN&search_value=83884

http://www.ncbi.nlm.nih.gov/books/NBK2042/

http://ei.cornell.edu/toxicology/bioassays/daphnia/

http://animaldiversity.ummz.umich.edu/accounts/Daphnia_magna/

http://www.caudata.org/daphnia/

51

[36] R. C. Lars-Henrik Heckmann. (2007). Culturing of Daphnia magna - Standard Operating Procedure. Available: http://www.reading.ac.uk/web/FILES/biosci/Culturing_Daphnia_201KB.pdf

[37] C. Naylor, M. C. Bradley, and P. CALow, "Freeze-Dried Chlorella vulgaris as Food for Daphnia magna Straus in Toxicity Testing," Ecotoxicology and environmental safety, vol. 25, pp. 166-172, 1993.

[38] H. Lilius, T. Hästbacka, and B. Isomaa, "Short Communication: A comparison of the toxicity of 30 reference chemicals to Daphnia Magna and Daphnia Pulex," Environmental Toxicology and Chemistry, vol. 14, pp. 2085-2088, 1995.

[39] M. Cleuvers, "Aquatic ecotoxicity of pharmaceuticals including the assessment of combination effects," Toxicology letters, vol. 142, pp. 185-194, 2003.

[40] B. Ferrari, R. Mons, B. Vollat, B. Fraysse, N. Paxēaus, R. L. Giudice, et al., "Environmental risk assessment of six human pharmaceuticals: are the current environmental risk assessment procedures sufficient for the protection of the aquatic environment?," Environmental toxicology and chemistry, vol. 23, pp. 1344-1354, 2004.

[41] L. Minguez, E. Farcy, C. Ballandonne, A. Lepailleur, A. Serpentini, J.-M. Lebel, et al., "Acute toxicity of 8 antidepressants: What are their modes of action?," Chemosphere, vol. 108, pp. 314-319, 2014.

[42] J. Kim, J. Park, P.-G. Kim, C. Lee, K. Choi, and K. Choi, "Implication of global environmental changes on chemical toxicity-effect of water temperature, pH, and ultraviolet B irradiation on acute toxicity of several pharmaceuticals in Daphnia magna," Ecotoxicology, vol. 19, pp. 662-669, 2010.

[43] B. Halling-Sørensen, S. Nors Nielsen, P. Lanzky, F. Ingerslev, H. Holten Lützhøft, and S. Jørgensen, "Occurrence, fate and effects of pharmaceutical substances in the environment-A review," Chemosphere, vol. 36, pp. 357-393, 1998.

[44] K. Fent, A. A. Weston, and D. Caminada, "Ecotoxicology of human pharmaceuticals," Aquatic toxicology, vol. 76, pp. 122-159, 2006.

[45] Sigma-Aldrich. (2014). Available: http://www.sigmaaldrich.com [46] Sigma-Aldrich. (2014). Product information: caffeine (anhydrous). Available:

https://www.sigmaaldrich.com [47] M. Borisover, M. Sela, and B. Chefetz, "Enhancement effect of water associated

with natural organic matter (NOM) on organic compound–NOM interactions: A case study with carbamazepine," Chemosphere, vol. 82, pp. 1454-1460, 2011.

[48] S. Prajapati, M. Gohel, and L. Patel, "Studies to enhance dissolution properties of carbamazepine," Indian journal of pharmaceutical sciences, vol. 69, p. 427, 2007.

[49] Drugs.com. (2014). Primidone. Available: http://www.drugs.com/pro/primidone.html

[50] J.-Y. Choi, S.-K. Kim, K.-H. Chang, M.-C. Kim, G.-H. La, G.-J. Joo, et al., "Population Growth of the Cladoceran, Daphnia magna: A Quantitative Analysis of the Effects of Different Algal Food," PloS one, vol. 9, p. e95591, 2014.

[51] MicroBioTests. (2013). Daphtoxkit Ftm magna - Crustacean Toxicity Screening Test for Freshwater - SOP. Available: http://www.microbiotests.be/SOPs/Daphtoxkit%20magna%20F%20SOP%20-%20A5.pdf

[52] L. Wollenberger, B. Halling-Sørensen, and K. O. Kusk, "Acute and chronic toxicity of veterinary antibiotics to Daphnia magna," Chemosphere, vol. 40, pp. 723-730, 2000.

[53] J. Jeon, J. H. Kim, B. C. Lee, and S. D. Kim, "Development of a new biomonitoring method to detect the abnormal activity of Daphnia magna using automated Grid Counter device," Science of the total environment, vol. 389, pp. 545-556, 2008.

http://www.reading.ac.uk/web/FILES/biosci/Culturing_Daphnia_201KB.pdf

http://www.sigmaaldrich.com/

http://www.sigmaaldrich.com/

http://www.drugs.com/pro/primidone.html

http://www.microbiotests.be/SOPs/Daphtoxkit%20magna%20F%20SOP%20-%20A5.pdf

http://www.microbiotests.be/SOPs/Daphtoxkit%20magna%20F%20SOP%20-%20A5.pdf

52

[54] C. Barata, J. Damasio, M. A. Lopez, M. Kuster, M. L. De Alda, D. Barceló, et al., "Combined use of biomarkers and in situ bioassays in Daphnia magna to monitor environmental hazards of pesticides in the field," Environmental Toxicology and Chemistry, vol. 26, pp. 370-379, 2007.

[55] J. Damásio, R. Tauler, E. Teixidó, M. Rieradevall, N. Prat, M. C. Riva, et al., "Combined use of Daphnia magna in situ bioassays, biomarkers and biological indices to diagnose and identify environmental pressures on invertebrate communities in two Mediterranean urbanized and industrialized rivers (NE Spain)," Aquatic Toxicology, vol. 87, pp. 310-320, 2008.

[56] J. M. Ebeling, M. B. Timmons, and J. Bisogni, "Engineering analysis of the stoichiometry of photoautotrophic, autotrophic, and heterotrophic removal of ammonia–nitrogen in aquaculture systems," Aquaculture, vol. 257, pp. 346-358, 2006.

[57] G. Yang, J. Wang, Y. Mei, and Z. Luan, "Effect of Magnetic Field on Protein and Oxygen-production of Chlorella Vulgaris," 数理水産科学, vol. 9, pp. 116-126, 2011.

[58] J. B. Shurin, R. L. Abbott, M. S. Deal, G. T. Kwan, E. Litchman, R. C. McBride, et al., "Industrial‐strength ecology: trade‐offs and opportunities in algal biofuel production," Ecology letters, vol. 16, pp. 1393-1404, 2013.

[59] D. Q. Zhang, T. Hua, R. M. Gersberg, J. Zhu, W. J. Ng, and S. K. Tan, "Fate of caffeine in mesocosms wetland planted with Scirpus validus," Chemosphere, vol. 90, pp. 1568-1572, 2013.

[60] I. J. Buerge, T. Poiger, M. D. Müller, and H.-R. Buser, "Caffeine, an anthropogenic marker for wastewater contamination of surface waters," Environmental science & technology, vol. 37, pp. 691-700, 2003.

[61] XLSTAT. (2014). Tutorial: Running a dose effect analysis with XLSTAT-Dose. Available: http://www.xlstat.com/en/learning-center/tutorials/dose-effect-analysis-with-xlstat-dose.html#

http://www.xlstat.com/en/learning-center/tutorials/dose-effect-analysis-with-xlstat-dose.html

http://www.xlstat.com/en/learning-center/tutorials/dose-effect-analysis-with-xlstat-dose.html

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

18/12/2014 23 15

7 0.5

19/12/2014 24

Total offspring in 21 days 1 081

Offspring per adult 72

Acute aquatic toxicity test of caffeine with Daphnia...

Documents

Transcript of Acute aquatic toxicity test of caffeine with Daphnia...