Arzneirückstände in der aquatischen Umwelt
Green Week 2012
PILLS Final Conference19th / 20th September 2012
Ecotoxicological assessment of
hospital wastewater
Dr. Nafo Folie 1
Monika Hammers-Wirtz, Henner Hollert, Issa Nafo, Sven Lyko, Axel Magdeburg, Daniel
Stalter, Jörg Oehlmann, Wilko Verweij, Evert-Jan van den Brandhof, Marja Wouterse,
Herman Evenblij Jessica Richard, Melanie Gerhards, Elke Dopp, Jochen Türk, Cornelia
Kienle, Christa S. McArdell, Colin Hunter, Dainis Sudmalis and Adrien Pilandon
Arzneirückstände in der aquatischen Umwelt
• Introduction
• Objectives for bioassays in PILLS ?
• Which bioassays ?
• Which WWTP are investigated ?
Content
Dr. Nafo Folie 2
• Which WWTP are investigated ?
• Exemplary results
• Summary of results
• Conclusions
Arzneirückstände in der aquatischen Umwelt
Introduction
• Hospital wastewater contains micropollutants
• Analytical detection does not allow conclusions about the potential effects on the environment
• Ecotoxicological test systems are able to integrate effects of all compounds in the sample
• Each test system gives a specific response
Dr. Nafo Folie 3
• Each test system gives a specific response
A biotest battery with different test organisms
is recommended
T47D
Arzneirückstände in der aquatischen Umwelt
Objectives for bioassays in PILLS
1. Characterisation of the hospital wastewater
Comparison between hospital wastewater and municipal wastewater
2. Determination of the efficiency of the pilot plants
Comparison between raw hospital wastewater and treated hospital wastewater
Comparison between the different treatment techniques of the pilot
Dr. Nafo Folie 4
Comparison between the different treatment techniques of the pilot plants
3. Assessment of possible toxic effects induced by the advanced treatment with ozone or UV
Evaluation of effects due to by-products after ozonation or UV treatment
Arzneirückstände in der aquatischen Umwelt
How are bioassays used in PILLS ?
Bioassays for specific toxicity Bioassays for non-specific toxicity
testing ...
• different mode of action
• using original and enriched samples
testing ...
• organisms of different trophic levels
• short-term and long-term exposure
• static/semistatic and flow-through design
• using original and diluted samples
Test 1...test n Test 1...test n
Dr. Nafo Folie 5
Test 1...test n Test 1...test n
Impacts on the environment
Arzneirückstände in der aquatischen Umwelt
Specific toxicity
Proteins
Enzymes
Luciferase
Toxicity
Light
Dioxin
Add substrate (luciferin)
Estrogen Proteins
Enzymes
Luciferase
Toxicity
Light
Dioxin
Add substrate (luciferin)
Proteins
Enzymes
Luciferase
Toxicity
Light
Dioxin
Add substrate (luciferin)
Estrogen
• Cytotoxicity
• Mutagenicity
• Estrogenicity
• Antibiotic resistance
Which bioassays ?
Dr. Nafo Folie 6
Nucleus
Dioxinbinding
Cytosol
TranscriptionDiox inResponsiveElement(CRE)
Transport protein
Hsp
Diox in receptor
Estrogen
Estrogen
Nucleus
Dioxinbinding
Cytosol
TranscriptionDiox inResponsiveElement(CRE)
Transport protein
Hsp
Diox in receptor
Estrogen
Nucleus
Dioxinbinding
Cytosol
TranscriptionDiox inResponsiveElement(CRE)
Transport protein
Hsp
Diox in receptor
Estrogen
Estrogen
T47D
Arzneirückstände in der aquatischen Umwelt
Which bioassays ���� Specific toxicity
Bioassays Endpoints PILLS Objectives
NL* DE* CH* UK*
MTT test (with T47D cells)
Cytotoxicity (viability)
Characterisation, treatment efficiency, (oxidation by-products)
IWW with original and pre-concentrated samples
Ames test (Salmonella typhimurium,
strain YG7108)
(Reifferscheid et al. 2011)
Mutagenicity
(No. of histidine revertants)
Characterisation, treatment efficiency, (oxidation by-products)
BIK-F with original and pre-concentrated samples
Dr. Nafo Folie 7
ER Calux (with T47D cells)
Estrogenicity
(EE2 equivalent)
Characterisation, treatment efficiency, (oxidation by-products)
IWW with original and pre-concentrated samples
A-YES test (AQUA 1.0)
Estrogenicity
(Phytase activity) (EE2 equivalent)
Characterisation, treatment efficiency, (oxidation by-products)
IUTA with original and pre-concentrated samples
Waterscan (antibiotics test)
Inhibition of bacterial growth
(concentration factor EC50)
Characterisation, treatment efficiency, (oxidation by-products)
RIVM with pre-concentrated samples; Inhibition after 24 hr
*PILLS-partner countries: Germany (DE), The Netherlands (NL), Scotland (UK), Switzerland (CH)
Arzneirückstände in der aquatischen Umwelt
Which bioassays ?
Non-specific toxicity ���� Different trophic levels
Primary producers Secondary consumersPrimary consumers
Filter feeders
Shredders
Dr. Nafo Folie 8
DecomposersDetritus feeders
Arzneirückstände in der aquatischen Umwelt
Which bioassays ?
Non-specific short-term toxicity
Bioassays Endpoints PILLS Objectives
NL* DE* CH* UK*
Bacteria test (Vibrio fischeri)
Inhibition of luminescence (concentration factor EC50)
Characterisation, treatment efficiency
RIVM with pre-concentrated samples; Inhibition after 30 min
Ecotox Centre with pre-con-centrated samples; Inhibition after 30 min
GCU with pre-concentrated samples; Inhibition after 30 min
Algal photo-synthesis test (Selenastrum capricornutum)
Inhibition of photosynthetic efficiency (concentration factor EC50)
Characterisation, treatment efficiency, (oxidation by-products)
RIVM with pre-concentrated samples;
GCU with pre-concentrated samples;
Dr. Nafo Folie 9
capricornutum) EC50) products) d samples; inhibition after 4.5 and 24 hr
samples; inhibition after 4.5 hr
Algae growth test (Desmodesmus subspicatus)
(ISO-NWIP)
Growth rate inhibition after 72 hr
Characterisation, treatment efficiency, oxidation by-products
GAIAC with original and diluted samples
Test with Gammarus fossarum (scuds)
Mortality after 4, 24, 48, 72 hr
Characterisation Ecotox Centre with original samples
Fish embryo test (Danio rerio)
(DIN EN ISO 15088, 2009)
Mortality (coagulation of embryos, non-detachment of the tail, lack of heart-beat)
Treatment efficiency, oxidation by-products
GAIAC 48 hr, with original and diluted samples
*PILLS-partner countries: Germany (DE), The Netherlands (NL), Scotland (UK), Switzerland (CH)
Arzneirückstände in der aquatischen Umwelt
Which bioassays ?
Non-specific long-term toxicity
Bioassays Endpoints PILLS Objectives
NL* DE* CH* UK*
Daphnia magna population test (water flea)
(Hammers-Wirtz et Ratte 2003)
Population growth inhibition (population abundance and structure, ephippia) after 21 d
Treatment efficiency, oxidation by- products
GAIAC semi-static in laboratory with original samples
Online test with Potamopyrgus antipodarum
Mortality, shell size, number of embryos per female after 28 d
Oxidation by-products
BIK-F flow through with original
Dr. Nafo Folie 10
antipodarum (snail)
(Duff et al. 2007)
with original samples
Online test with Lumbriculus variegatus (worm)
(OECD 2007)
Reproduction, biomass, biomass per worm after 28 d
Oxidation by- products
BIK-F flow through with original samples
*PILLS-partner countries: Germany (DE), The Netherlands (NL), Scotland (UK), Switzerland (CH)
Arzneirückstände in der aquatischen Umwelt
Country Municipal wastewater
Hospital wastewater
Germany wwtp Bad Sassendorf Marienhospital in
Gelsenkirchen
The
Netherlands
wwtp Zwolle
wwtp Hessenport
Isala Clinics, Zwolle
Wastewater treatment plants
Dr. Nafo Folie 11
Switzerland wwtp Laufäcker Hospital Baden
UK wwtp Galashiels
wwtp Shieldhall
Borders General Hospital
Western Infirmary
Arzneirückstände in der aquatischen Umwelt
Results ���� Municipal vs hospital wastewater
Specific effects
Effect classification
Bioassay Endpoints Raw municipal wastewater
Effluent of municipal
wastewater treatment plant
Raw hospital
wastewater
MTT test (with T47D cells) [DE]
Cytotoxicity (viability) 88.3%
(original sample)1
66.6%
(original sample)
2
A-YES test (AQUA 1.0) [DE]
Estrogenicity (EE2 equivalent)
19.7 ng/L (origninal sample)
1
43 ng/L (original sample)
2
Ames test
Dr. Nafo Folie 12
no negative effects
weak or moderate effects
strong effects
Effect classificationAmes test (Salmonella thyphimurium, strain YG7108) [DE]
Mutagenicity (No. of histidine revertants)
2
Waterscan (antibiotics test) [NL]
Sulfanomides (concentration factor EC50)
3
Waterscan (antibiotics test) [NL]
Macroliden/ ß-lactam (concentration factor EC50)
3
Origin of the samples: 1 Influent of the municipal wwtp Bad Sassendorf, Germany ( n=1, 24h-composite sample); 2
Raw
wastewater of
Marienhospital in Gelsenkirchen, Germany (n=1-4, 24h-composite sample); 3
wwtp Zwolle and wwtp
Hessenpoort, The Netherlands (n=6, grab samples)
Arzneirückstände in der aquatischen Umwelt
Results ���� Municipal vs hospital wastewater
Non-specific short-term effects – Bacteria test (30 min)
Dr. Nafo Folie 13
Arzneirückstände in der aquatischen Umwelt
Results ���� Municipal vs hospital wastewater
Non-specific short-term effects – Algal growth inhibition test (72h)
Inhib
itio
n o
f gro
wth
rate
aft
er
72
h [%
]
60
80
100
Raw hospital wastewater
Raw hospital wastewater
Raw municipal wastewater
Dr. Nafo Folie 14
G8 (12.5%) G4 (25%) G2 (50%) G1(80%)
Inhib
itio
n o
f gro
wth
rate
aft
er
72
h [%
]
-20
0
20
40
Dilution (% of wastewater)
Arzneirückstände in der aquatischen Umwelt
Results ���� Municipal vs hospital wastewater
Non-specific short-term effects
Bioassay Endpoints Raw municipal wastewater
Effluent of municipal wastewater treatment
plant
Raw hospital
wastewater
Bacteria test (Vibrio fischeri) [NL]
Inhibition of luminescence (concentration factor EC50)
3
Bacteria test (Vibrio fischeri) [CH]
Inhibition of luminescence (concentration factor EC50)
1.26 fold 4 0.84 fold
5
Bacteria test (Vibrio fischeri) [UK]
Inhibition of luminescence (concentration factor EC50)
0.75 fold 6 0.26 fold
7
Algal photosynthesis test
Inhibition of photosynthetic
Dr. Nafo Folie 15
no negative effects
weak or moderate effects
strong effects
Effect classification
photosynthesis test (Selenastrum capricornutum) [UK]
photosynthetic efficiency (concentration factor EC50)
12.07 fold 6 1.97 fold
7
Algal photosynthesis test (Selenastrum capricornutum) [NL]
Inhibition of photosynthetic efficiency (concentration factor EC50)
3
Algae growth test (Desmodesmus subspicatus) [DE]
growth rate inhibition, average for dilutions 80% and 50% wastewater
34 % 1 64-88 %
2
Test with Gammarus fossarum [CH]
Mortality after 48hr 100% 4 > 50%
5
Origin of the samples: 1 Influent of the municipal wwtp Bad Sassendorf, Germany ( n=1, 24h-composite sample); 2
Raw
wastewater of
Marienhospital in Gelsenkirchen, Germany (n=1-4, 24h-composite sample); 3
wwtp Zwolle and wwtp
Hessenpoort, The Netherlands (n=6, grab samples); 4
Raw wastewater of hospital Baden, Switzerland; 5
Influent of the
municipal wwtp Laufäcker, Switzerland ; 6 Average of influents of wwtp Galashiels (n= 9, 7d-comp. sample) and wwtp Shieldhall
(n=13, 7d-comp. sample), UK; 7 Average of raw wastewater of Borders General Hospital (n= 46, 7d-comp. sample) and Western
Infirmary (n=35, 7d-comp. sample), UK
Arzneirückstände in der aquatischen Umwelt
Summary ���� Objective 1
� Raw hospital wastewater was moderately cytotoxic and estrogenic, but acutely toxic to various tests organisms like bacteria, algae and invertebrates.
� In general hospital wastewater was more toxic compared to municipal wastewater.
Characterisation of hospital wastewater
Dr. Nafo Folie 16
wastewater.
Arzneirückstände in der aquatischen Umwelt
Sampling points in the wwtp
Ozonation
MBR
+ O3
Sampling point
MBR
PAC reactor
Sand filter (DE)
or Moving bed
bioreactor (CH)
MBR + O3
+ SF
Pilot plants in
Gelsenkichen (DE)
and Baden (CH)
MBR
+ PAC/SFSand filter (DE)
or Moving bed
bioreactor (CH)
MBR
Dr. Nafo Folie 17
Pilot plant in
Zwolle(NL)
MBR ozonation
GAC filter
MBR + O3
+GAC
Sampling point MBR
UV/H2O2
MBR + UV
+ GAC
MBR + GAC
GAC filter
MBR
+ UV
GAC filter
MBR
+ O3
Arzneirückstände in der aquatischen Umwelt
Results ���� Efficiency of pilot plants
Specific effects – Ames test (No. of revertants Strain YG7108)
75
100
Mu
tag
en
icit
y [
% o
f m
ax
. re
sp
on
se
]
Dr. Nafo Folie 18N
C PC
MBR
MBR
+O3
MBR
+O3+
SF
0
25
50
Mu
tag
en
icit
y [
% o
f m
ax
. re
sp
on
se
]
� �
Arzneirückstände in der aquatischen Umwelt
Results ���� Efficiency of pilot plants
Specific effectsBioassays Endpoints Effluent of
MBR MBR + O3
MBR + O3 + SF
MBR + PAC/SF
MBR + O3 + GAC
MBR + GAC
MBR + UV
MBR + UV + GAC
MTT test (with T47D cells)
1
Cytotoxicity (viability)
↓ ↗ ↗
ER Calux (with T47D cells)
1
Estrogenicity (EE2 equivalent)
0.1 ng/L < LOD < LOD
A-YES test (AQUA 1.0)
1
Estrogenicity (EE2 equivalent)
0.235 ng/L 0.261 ng/L
0.176 ng/L
0.079 ng/L
Ames test
Dr. Nafo Folie 19
no negative effects
weak or moderate effects
strong effects Classification
↑ ↓ in/decreasing toxicity > 20% ↗ ↘ slightly in/decreasing toxicity < 20%
Ames test (Salmonella typhimurium, strain YG7108)
1
Mutagenicity (No. of histidine revertants)
↓ ↑ ↘ ↓
Waterscan (antibiotics test)
2
Sulfonamides (concentration factor EC50)
Waterscan (antibiotics test)
2
Macrolides/ß-lactam (concentration factor EC50)
Origin of samples: 1 pilot plant in Gelsenkirchen, Germany;
2 pilot plant in Zwolle, The Netherlands
Arzneirückstände in der aquatischen Umwelt
Results ���� Efficiency of pilot plants
Non-specific short-term effects
Bioassays Endpoints Effluent of MBR
MBR + O3
MBR + O3 + SF
MBR + PAC/SF
MBR + O3 + GAC
MBR + GAC
MBR + UV
MBR + UV + GAC
Bacteria test (Vibrio fischeri)
2
Inhibition of luminescence (concentration factor EC50)
↓ ↓ ↓
Bacteria test (Vibrio fischeri)
3
Inhibition of luminescence (concentration factor EC50)
↓ ↓ ↘ ↓
Algal photosynthesis test
2
Inhibition of photosynthetic efficiency (concentration
↓ ↓
Dr. Nafo Folie 20
no negative effects
weak or moderate effects
strong effects Classification
↑ ↓ in/decreasing toxicity > 20% ↗ ↘ slightly in/decreasing toxicity < 20%
test (4.5 and 24hr)
2
(concentration factor EC50)
Algae growth test (72hr)
1
growth rate inhibition, average for dilutions 80% and 50% wastewater
↓ ↗
Test with Gammarus fossarum
3
Mortality after 48hr
variable effects
variable effects
variable effects
variable effects
Fish embryo test (48hr, DIN)
1 lethal mortality
Origin of samples: 1 pilot plant in Gelsenkirchen, Germany;
2 pilot plant in Zwolle, The Netherlands;
3 pilot plant in
Baden, Switzerland
Arzneirückstände in der aquatischen Umwelt
Results ���� Efficiency of pilot plants
Non-specific long-term effects
Bioassays Endpoints Effluent of MBR
MBR + O3 MBR + O3 + SF
MBR + PAC/SF
mean abundance ↗ ↘
mean of sectional growth ↗ ↗
sum of neonates
sum of dead animals
sum of aborted eggs
sum of ephippial eggs
body length on day 21
Daphnia population test (21d, semistatic)
Dr. Nafo Folie 21
no negative effects
weak or moderate effects
strong effects Classification
↑ ↓ in/decreasing toxicity > 20% ↗ ↘ slightly in/decreasing toxicity < 20%
body length on day 21
portion of adult daphnids ↗ ↘
Mortality ↗ ↗ ↓
Shell size ↑ ↓
Online test with Potamopyrgus antipodarum (28d, flow through) number of embryos/female ↑ ↘ ↑
Reproduction ↗ ↘
Biomass ↑ ↘
Online test with Lumbriculus variegatus (28d, flow though)
biomass per worm ↑ ↘
All tests performed on samples from the pilot plant in Gelsenkirchen, Germany, Evaluation is performed on average values.
Arzneirückstände in der aquatischen Umwelt
Summary ���� Objective 2
Efficiency of pilot plants
� The biological treatment in a MBR considerably decreased cytotoxic and estrogenic effects in raw hospital wastewater.
� Non-specific bioassays demonstrate also a decrease in toxicity, but MBR permeate still affected some organisms like bacteria, algae and snails.
� Advanced treatment by PAC addition or GAC filtration reduced
Dr. Nafo Folie 22
� Advanced treatment by PAC addition or GAC filtration reduced ecotoxicological effects of raw wastewater, but the effluent of this treatment process still contained algal toxic compounds.
Arzneirückstände in der aquatischen Umwelt
Results ���� Effects after ozonation
Static test over 72h – Algal growth rate (Mean of 3 samplings)
Gro
wth
ra
te a
fte
r 7
2 h
[1
/d]
1,2
1,4
1,6
1,8
�
Dr. Nafo Folie 23
Gro
wth
ra
te a
fte
r 7
2 h
[1
/d]
0,0
0,2
0,4
0,6
0,8
1,0
MBR+O3
MBR+O3+SF
MBRControl
• MBR: 17% inhibition
• MBR+O3: 25% inhibition
• MBR+O3+SF: 24% inhibition
Arzneirückstände in der aquatischen Umwelt
Online test – Reproduction of Potamopyrgus antipodarum
Results ���� Effects after ozonation
15
20
��
�
Nu
mb
er
of
em
bry
os [
me
an
]
Dr. Nafo Folie 24
• Increase in number of embryos after
sand filter
• OS at similar level compared to EE2
C
MBR
MBR
+O3
MBR
+O3+
SF
EE2
0
5
10
�
�
�
�
�
�
Nu
mb
er
of
em
bry
os [
me
an
]
Arzneirückstände in der aquatischen Umwelt
Online test – Biomass of Lumbriculus variegatus
Results ���� Effects after ozonation
80
100
��
�
Dry
bio
ma
ss [
mg
]
Dr. Nafo Folie 25
• 49% reduced biomass after ozonation
• 32% after sand filter
• adverse effects in accordance with
previous studies (Stalter et al. 2011;
Magdeburg et al., 2012)
C
MBR
MBR
+O3
MBR
+O3+
SF
0
20
40
60
Dry
bio
ma
ss [
mg
]
Arzneirückstände in der aquatischen Umwelt
Summary ���� Objective 3
� Ozonation reduced considerably antibiotic and estrogenic effects of hospital wastewater.
� Increasing toxicity is detected after the oxidation processes by ozonation for algae and invertebrates like daphnids, snails and worms.
Effects of ozonation by-products
Dr. Nafo Folie 26
Arzneirückstände in der aquatischen Umwelt
Conclusions ���� 1
� This set of bioassays is a useful tool to assess the potential effects on the environment.
� The biological treatment in a MBR decreased toxic effects considerably in raw hospital wastewater. But, MBR permeate may still affect some organisms like bacteria, algae and snails.
Dr. Nafo Folie 27
algae and snails.
� Advanced treatment by PAC addition or GAC filtration reduced the toxicity of raw wastewater, but the effluent of this treatment process may still contain algal toxic compounds.
Arzneirückstände in der aquatischen Umwelt
Conclusions ���� 2
� Ozonation considerably reduced antibiotic and estrogenic effects of hospital wastewater. However, increasing toxicity is detected after ozonation.
� A post-treatment of the ozonated effluent in a biofilter (e.g. sand filter with biological activity) reduced the adverse effects of the advanced treatment by oxidation significantly but did not remove it totally (e.g. toxic effects on algae).
Dr. Nafo Folie 28
but did not remove it totally (e.g. toxic effects on algae).
� A post-treatment of the UV treatment effluent by GAC filtration removed the adverse effects of the oxidation process.
� In the present study, the sand filter was not as efficient as observed at previously evaluated treatment plants to reduce oxidation product induced toxicity. Further studies on oxidation product removal after the ozonation process are desirable.
Arzneirückstände in der aquatischen Umwelt
Thanks to
• Issa Nafo and Sven Lyko (Emschergenossenschaft)
• Axel Magdeburg (BiK-F), Daniel Stalter (BiK-F), Jörg Oehlmann (BiK-F), Wilko Verweij (RIVM), Evert-Jan van den Brandhof (RIVM), Marja Wouterse (RIVM), Herman Evenblij (WGS), Jessica Richard (IWW), Melanie Gerhards (IWW), Elke Dopp (IWW), Jochen Türk (IUTA), Linda Gehrmann (IUTA), Cornelia Kienle (Ecotox Centre, Eawag/EPFL), Christa S. McArdell (Eawag), Colin Hunter (GCU), Dainis Sudmalis
Dr. Nafo Folie 29
Christa S. McArdell (Eawag), Colin Hunter (GCU), Dainis Sudmalis (GCU), Adrien Pilandon (GCU) and Henner Hollert (gaiac)
• my colleagues Dennis Becker, Rachel Conrad and Melanie Grolms-Aal (gaiac)
... and you for your attention!... and you for your attention!
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