Environmental Risk AssessmentEnvironmental Risk AssessmentEnvironmental Risk AssessmentEnvironmental Risk Assessment
of Antimicrobialsof Antimicrobialsof Antimicrobialsof Antimicrobials
Hans-Christian Holten Lützhøft
Thesis for the degree of Philosophiae Doctor.
The defence takes place Thursday 31 August 2000 at 14.00 in the Benzon lecture room,
The Royal Danish School of Pharmacy, Universitetsparken 2, DK-2100 Copenhagen Ø.
The Royal Danish School of Pharmacy
Department of Analytical and Pharmaceutical Chemistry
Copenhagen 2000

ISBN 87-988069-0-4
Section of Environmental Chemistry
Department of Analytical and Pharmaceutical Chemistry
The Royal Danish School of Pharmacy
Universitetsparken 2
DK-2100 Copenhagen Ø
Tel: +45 35 30 60 00 direct: +45 35 30 64 59
Fax: +45 35 30 60 13
e-mail: hchl@dfh.dk
Web: www.dfh.dk

Preface • v
Preface
This Ph.D. thesis was performed at Section of Environmental Chemistry, Department of
Analytical and Pharmaceutical Chemistry, The Royal Danish School of Pharmacy, in order to
obtain the pharmaceutical Ph.D. degree. The project was undertaken from October 1996 to
July 2000, with Professor D.Sc. Sven Erik Jørgensen and Assoc. Professor Ph.D., M.Sc.
(Pharm.) Bent Halling-Sørensen as supervisors.
In the thesis 4 articles are enclosed which have been published or submitted to international
peer-reviewed journals. A bold Roman numeral will follow reference to one of mentioned
articles:
I Holten Lützhøft HC, Halling-Sørensen B, Guardabassi L, Ingerslev F and
Tjørnelund J. Submitted. Oxolinic acid in freshwater sediment – extraction
method and occurrence due to fish farm activities.
II Holten Lützhøft HC, Vaes WHJ, Freidig AP, Halling-Sørensen B, and
Hermens JLM. 2000. 1-Octanol/water distribution coefficient of oxolinic
acid: Influence of pH and its relation to the interaction with dissolved
organic carbon. Chemosphere, 40(7), 711-714.
III Holten Lützhøft HC, Vaes WHJ, Freidig AP, Halling-Sørensen B, and
Hermens JLM. Accepted. The influence of pH and other modifying factors
on the distribution behaviour of 4-quinolones to solid phases and humic
acids studied by “negligible-depletion” SPME-HPLC. Environ Sci Technol.
IV Holten Lützhøft HC, Halling-Sørensen B, and Jørgensen SE. 1999. Algal
toxicity of antibacterial agents applied in Danish fish farming. Arch Environ
Contam Toxicol, 36(1), 1-6.
Copenhagen, July 2000.

• Contentsvi
Contents
Preface v
Contents vi
Abbreviations ix
Figures x
Tables xi
1. General Introduction 1
1.1. Introduction 2
1.2. Selection of Chemicals 3
1.3. Objectives of This Ph.D. Thesis 4
1.4. Outline of This Ph.D. Thesis 7
2. Basic Data of Danish Fish Farms and Antimicrobials – Initial Environmental
Assessment 11
2.1. Fish Farm Characteristics 12
2.2. Chemicals 13
2.2.1. Antimicrobial Therapy in Fish Farming 13
2.2.2. Physical Chemical Properties 15
2.2.3. Environmental Distribution According to Fugacity 20
2.2.4. Pharmacology 22
2.2.4.1. Pharmacokinetics in Rainbow Trout 22
2.2.4.2. Pharmacodynamics 28
2.3. Initial Environmental Assessment 30
3. Environmental Occurrence of Antimicrobials 35
3.1. Introduction 36
3.2. Analytical procedures 38
3.2.1. Separation methods 38
3.2.2. Extraction Methods 43
3.2.2.1. Marine sediment 43
3.2.2.2. Freshwater sediment 43
3.3. Antimicrobials in Environmental Samples 49

Contents • vii
3.3.1. Marine occurrence 49
3.3.2. Freshwater Occurrence 50
4. Environmental Fate of Antimicrobials 57
4.2. Environmental Distribution 58
4.2.1. pH-dependent 1-octanol/water distribution 59
4.2.2. Experimental Distribution Coefficients 60
4.2.3. Complexation with metals 65
4.3. Degradability 67
4.3.1. Abiotic degradation 67
4.3.1.1. Hydrolysis 67
4.3.1.2. Photodegradation 68
4.3.2. Biotic degradation 71
4.3.2.1. Biodegradation 71
4.3.2.2. Enzymatic Degradation 73
4.4. Transport 73
5. Environmental Effects of Antimicrobials 75
5.2. Selection of Test Organisms 76
5.3. Toxicity on Various Trophic Levels 78
5.3.1. Micro-organisms – Bacteria and Micro-algae 80
5.3.2. Crustaceans 81
5.3.3. Fish 82
5.4. Factors Affecting Toxicity 82
6. Environmental Risk Assessment of Antimicrobials Applied in Danish Fish Farms 87
6.2. Predicted No Effect Concentrations 89
6.3. Exposure Scenarios 92
6.3.1. Main Settings 93
6.3.2. Definitions and Procedures to Derive Predicted Environmental Concentrations 94
6.3.2.1. Scenario 1 – Worst Case 94

• Contentsviii
6.3.2.2. Scenario 2 – Incorporation of Inevitable Processes 95
6.3.2.3. Scenario 3 – Incorporation of Inevitable Processes and Natural Dilution 96
6.4. Assessment of the Derived Risk Quotients 97
6.5. Risk Management Procedures 101
7. Conclusions 105
References 109
Summary 123
Resumé på dansk 127
Publications 130
Abstracts 131
Curriculum Vitae 132
Acknowledgements 133

Abbreviations • ix
Abbreviations
AMX Amoxicillin
AUC Area under the plasma concentration-time curve
DDOC Dissolved organic carbon/water distribution coefficient for an ionized
molecule
DF Dilution factor
DNA Deoxyribonucleic acid
DOC Dissolved organic carbon – a measure for humic acids
DOW 1-Octanol/water distribution coefficient for an ionized molecule
DSED Sediment/water distribution coefficient for an ionized molecule
EC50 The concentration that provokes effect in 50 % of the population
ERA Environmental risk assessment
F Bioavailability
FLU Flumequine
HPLC High performance liquid chromatography
i.a. Intra arterial administration
i.v. Intra venous administration
KCOM Metal complexation constant
ke Elimination rate constant
KOC Organic carbon/water partition coefficient for a neutral molecule
KOW 1-Octanol/water partition coefficient for a neutral molecule
LC50 The concentration that provokes lethality in 50 % of the population
LLE Liquid liquid extraction
MeOH Methanol
MIC Minimum inhibitory concentration
nd Negligible depletion
NOEC No observed effect concentration
OC Organic carbon
OTC Oxytetracycline
OXA Oxolinic acid
p.o. Oral administration
PEC Predicted environmental concentration
PNEC Predicted no effect concentration
QSAR Quantitative structure activity relationship
RP HPLC Reversed phase high performance liquid chromatography
RQ Risk quotient
SAF Sarafloxacin
SDZ Sulphadiazine
SPE Solid phase extraction
SPME Solid phase microextraction
THF Tetrahydrofuran
TMP Trimethoprim

• Figuresx
Figures
Figure 1.1 – Conceptual diagram over the processes that an antimicrobial encounters in the
aquatic environment. 4
Figure 1.2 – Conceptual diagram of the tasks performed in this Ph.D. thesis. 8
Figure 2.1 – A schematic drawing of a typical Danish fish farm. 13
Figure 2.2 – Fractional composition and experimental 1-octanol/water distribution
coefficients vs. pH for the antimicrobials. 18
Figure 2.3 – pH corrected DOW profiles for the neutral species of OXA and TMP. 19
Figure 2.4 – Plasma concentration-time profiles for FLU in rainbow trout. 24
Figure 3.1 – Anticipated exposure routes of antimicrobials applied in fish farming. 36
Figure 3.2 – Column material effect on the chromatography of OXA. 38
Figure 3.3 – SPME-HPLC analysis of FLU, OXA and SAF. 39
Figure 3.4 – Procedure to extract OXA from freshwater sediment. 44
Figure 4.1 – Experimental 1-octanol/water distribution for OXA. 59
Figure 4.2 – Experimental distribution coefficients to DOC for FLU, OXA and SAF. 62
Figure 4.3 – Experimental distribution coefficients vs. log DOW. 64
Figure 5.1 – Simplified characteristics of micro-organisms. 77
Figure 6.1 – Illustration of the constant assessment factor approach. 90
Figure 6.2 – Interpretation of the risk quotient in the context of this thesis. 98
Figure 6.3 – Graphical representation of the antimicrobial RQs according to scenario 3. 100

Tables • xi
Tables
Table 2.1 – Antimicrobial consumption in Danish fish farming during 1994-1997, kg. 14
Table 2.2 – Chemical structures and selected physical chemical properties of the
investigated antimicrobials. 16
Table 2.3 – Environmental distribution of antimicrobials according to fugacity
principles. 21
Table 2.4 – Pharmacokinetics in rainbow trout. 26
Table 2.5 – Dosing regimes and environmental load in Danish fish farms. 30
Table 2.6 – Experimental 1-octanol/water distribution coefficients. 32
Table 3.1 – HPLC methods for the investigated quinolones. 40
Table 3.2 – Eluents evaluated for the freshwater sediment extraction. 45
Table 3.3 – Recovery study for the extraction of OXA from freshwater sediment. 46
Table 3.4 – Sediment extraction methods for the investigated quinolones. 47
Table 3.5 – Environmental occurrence of the investigated antimicrobials. 52
Table 4.1 – Estimated and experimental distribution coefficients. 60
Table 4.2 – Influence of cations on the antimicrobial activity. 66
Table 4.3 – Antimicrobial stability under illumination. 70
Table 4.4 – Antimicrobial biodegradability. 71
Table 5.1 – Acute toxicity of antimicrobials, LC50 (NOEC), mg/L. 79
Table 5.2 – Chronic toxicity of antimicrobials, EC50 (NOEC), mg/L. 79
Table 6.1 – Antimicrobial PNECs in the aquatic environment. 91
Table 6.2 – Overview of exposure scenarios. 97
Table 6.3 – Antimicrobial risk quotients in the freshwater environment. 99

Chapter 1
General Introduction

• General Introduction2
1.1. Introduction
Until a few years ago, limited attention was paid to the possible risk associated with
application of antimicrobials in various environmental contexts (Schneider, 1994; Henschel et
al., 1997; Halling-Sørensen et al., 1998; Jørgensen et al., 1998; Pors, 1998; Montforts et al.,
1999; Ternes, 1999; Jørgensen and Halling-Sørensen, 2000). One of several applications is in
fish farming, although it is not the most significant. In this situation, the exposure is directly
to the aquatic environment. Aquaculture is a comprehensive industry, with production of e.g.
shrimp and trout in Asia and salmon and trout in America as well as Europe. Production water
undergoes only a simple wastewater treatment. In most cases the water only passes a
sedimentation pond in order to settle suspended solids. Sediment/sludge from fish farming is
often applied as fertilizer on arable land. If antimicrobials have high affinity for solids e.g.
sediment or soil, arable land will thus be indirectly contaminated with antimicrobials if
fertilized with fish farm sediment/sludge.
Thorough investigations are needed for the registration of pharmaceuticals to obtain a
marketing license. Some of these data are covered by the patent, but some can be found in the
open literature. Basic physical chemical and toxicological data for the antimicrobials can
therefore be achieved. However, an environmental risk assessment (ERA) has not yet been
required for the registration. Thus, only limited knowledge of the environmental impact of
antimicrobials used in aquaculture exists.
Antimicrobials are chemicals with a specific mode of action, designed to control pathogenic
bacterial infections. A few investigations show that depending on environmental conditions
some antimicrobials, e.g. oxytetracycline and oxolinic acid are persistent in marine
environments (Jacobsen and Berglind, 1988; Samuelsen et al., 1992b; Hektoen et al., 1995;
Lunestad et al., 1995). In Denmark, the major quantity of antimicrobials is applied in
agriculture (Halling-Sørensen et al., 1998). A few tonnes are applied in Danish fish farming,
whereas the consumption in fish farming in other countries is both less and more. Therefore,
application of antimicrobials in aquaculture creates a possible risk for the environment.
The environmental risks associated with application of antimicrobials in aquaculture are e.g.
acute toxicity (Harras et al., 1985), genotoxicity (Mamber et al., 1993; Couturier and

Chapter 1 • 3
Melderen, 1998), mutagenicity (Mamber et al., 1993) and development of bacterial resistance
(Nygaard et al., 1992; Samuelsen et al., 1992b; Guardabassi et al., 2000b). Especially bacteria
and related organisms, e.g. cyanobacteria may be affected in the environment. Not only fish
pathogenic bacteria, but also natural occurring bacteria may acquire resistance towards certain
antimicrobials. Resistance genes may be transferred to other (pathogenic) bacteria, with a
high extent of irreversible effects on the environment including mankind (Toranzo et al.,
1984; Sandaa et al., 1992; Kruse and Sørum, 1994). Consequently, even long time after the
chemical’s disappearance side effects as resistance may persist.
1 January 1998, the European Community adopted an environmental assessment guideline for
veterinary pharmaceuticals (EMEA, 1998b). Consequently, an environmental exposure
assessment, the so-called phase I assessment, is required for the registration of new veterinary
pharmaceuticals. However, due to the direct entry into the aquatic environment antimicrobials
applied in aquaculture have to undergo a more thorough investigation, the so-called phase II
assessment, compared to other veterinary pharmaceuticals. This assessment considers both
fate and effects studies (EMEA, 1998b).
In this thesis I will focus on specific elements, i.e. occurrence, distribution and selection of
target organisms, enabling an assessment of the environmental risks of currently used
antimicrobials in Danish fish farming in the context of said guideline.
1.2. Selection of Chemicals
The investigated chemicals represent different antimicrobial groups, see Chapter 2 for a
detailed description. Oxolinic acid, 5-ethyl-5,8-dihydro-8-oxo-1,3-dioxolo[4,5-g]quinoline-7-
carboxylic acid, (OXA); sulphadiazine, 4-amino-N-2-pyrimidinylbenzenesulfonamide,
(SDZ); trimethoprim, 5-[(3,4,5-trimethoxyphenyl)methyl]-2,4-pyrimidinediamine, (TMP);
amoxicillin, [2S-[2α,5α,6β(S*)]]-6-[[amino[4-hydroxyphenyl)acetyl]amino]-3,3-dimethyl-7-
oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid, (AMX) and oxytetracycline, [4S-
(4α,4aα,5α,5aα,6β,12aα)]-4-(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-
3,5,6,10,12,12a-hexahydroxy-6-methyl-1,11-dioxo-2-naphthacenecarboxamide, (OTC) are
currently applied in Danish fish farming.
Flumequine, 9-fluoro-6,7-dihydro-5-methyl-1-oxo-1H,5H-benzo[i,j]quinolizine-2-carboxylic
acid, (FLU) and sarafloxacin, 6-fluoro-1-(4-fluorophenyl)-1,4-dihydro-4-oxo-7-(1-

piperazinyl)-3-quinolinecarboxylic acid, (SAF) are included, as FLU is used and SAF is
contemplated to be used in other countries.
This selection means that the physical chemical properties are limited to the mentioned
antimicrobials; thus, properties for all possible chemicals will not necessarily be covered.
1.3. Objectives of This Ph.D. Thesis
When entering the aquatic environment, antimicrobials, similar to other xenobiotics,
encounter various processes, e.g. binding and degradation, as conceptualised in Figure 1.1.
Figure 1.1 – Conceptual diagram over the processes that an antimicrobial encounters in
the aquatic environmenta
.
a
: OXA is shown as example. “Sun”: photodegradation, Ca2+
: complexation with ions, T: temperature, :
biodegradation, and DOC: dissolved organic carbon.
The aim of this thesis is therefore to answer the following questions connected with the fate
and effects of antimicrobials:
1. Antimicrobials are used in fish farming to treat infections among fish. Knowing that
effluents from fish farming are discharged to the local stream, the exposure is directly
into the aquatic environment. Can antimicrobial residues therefore after treatment be
found in the sediment near Danish fish farms?
2. Antimicrobials are hydrophilic chemicals often with many functional groups (see
Chapter 2) affecting the fate of said chemicals, e.g. distribution between solids/organic

Chapter 1 • 5
phases and water. Often these groups are weak (carboxylic) acids or weak bases, which
make the distribution pH dependent. Furthermore, these groups contribute to e.g.
hydrogen bonds, dipole-dipole and ionic interactions. Humic acids, in this thesis
designated dissolved organic carbon (DOC), contains both hydrophobic sites and
functional groups similar to the antimicrobials, which make interactions likely to take
place. Consequently, the distribution is not straightforward and may therefore be
difficult to predict. It raises the following questions:
a) does the antimicrobial distribution coefficient between 1-octanol and water (DOW)
follow the understanding of distribution of ionized species?
b) to what extent do antimicrobials distribute to DOC and sediment?
c) can the distribution to DOC or sediment be predicted from the DOW?
3. In conventional ERA three trophic levels – algae, crustaceans and fish – are normally
proposed used to evaluate the effects of chemicals in the aquatic environment (OECD,
1992; EMEA, 1998b). Guidelines, e.g. ISO (1989), often suggest Selenastrum
capricornutum as test organism to represent micro-algae. Is this model target organism
an appropriate selection as the lowest trophic level for evaluation of antimicrobials?
4. Knowledge about environmental exposure, fate and effects of antimicrobials enable the
risk assessment of the application in Danish fish farming. Based on mentioned
properties, is it possible to rank the expected risk? Hence, which antimicrobials can be
recommended and which can not be recommended?
Answers to the above-mentioned questions all improve the reliability of the ERA of
antimicrobials; thus making it more realistic. The following investigations were performed to
answer the outlined questions; for a detailed scheme of the experimental work, see Figure 1.2:
Re. 1. In co-operation with a fish farm in Jutland, Denmark, sediment samples were taken
before and after a treatment with OXA. Samples were taken from the inlet, the
medicated pond, at the outlet and 300 m downstream the outlet. A sediment extraction
method was developed and the samples were analysed for the occurrence of OXA by
means of high performance liquid chromatography (HPLC). This subject is covered in
Holten Lützhøft et al. (Submitted) I.

Re. 2. The focus for the fate experiments was directed towards the quinolone antimicrobials.
Using the three selected quinolones – FLU, OXA and SAF – made it possible to
predict trends in their fate due to physical chemical properties.
a) In order to evaluate the distribution of antimicrobials between organic phases and
water, the OXA distribution between 1-octanol and water was studied. In order to
study the distribution of ionized species the DOW was investigated in the pH range
3 to 11. The DOW was evaluated according to the Henderson-Hasselbalch
principles. The results are published in Holten Lützhøft et al. (2000) II.
b) A newly developed technique involving solid phase microextraction (SPME)
coupled to HPLC was used to investigate the distribution behaviour between DOC
and water. Aldrich humic acids was used as DOC source. Samples were allowed to
equilibrate and the distribution coefficients between DOC and water (DDOC) for
FLU, OXA and SAF were investigated between pH 3 and 8. These experiments
are shown in Holten Lützhøft et al. (Accepted) III. A simpler set-up was used for
the sediment experiments. Flasks containing sediment, water (pH=7) and the
investigated chemical were allowed to equilibrate. Samples were taken, filtered
and analysed by HPLC. The distribution coefficient between sediment and water
(DSED) for FLU, OXA, SAF and TMP was established.
c) Prediction of binding to organic matter is often based on the partition coefficient
between 1-octanol and water (KOW) for the chemical. However, this approach
requires that the hydrophobicity of the chemical reflects its affinity for organic
matter. For ionizeable chemicals, the affinity for organic matter is expected to
decrease when the chemical is ionized, unless electrostatic interactions contribute
to the binding. In Holten Lützhøft et al. (2000) II an experimental DOW value for
OXA was used to predict the binding to organic carbon. This value was compared
with the DDOC value.
Re. 3. Antimicrobials are chemicals designed to prevent the growth of or kill micro-
organisms, i.e. bacteria. There is a distinct difference between micro-algae and bacteria
viz. micro-algae are eucaryotic and bacteria are procaryotic. However, the blue-green
algae or cyanobacteria are organisms sharing the oxygen producing properties of algae
and the procaryotic structure of bacteria. The usefulness of the green alga S.
capricornutum as test alga for antimicrobials was therefore evaluated using the

Chapter 1 • 7
cyanobacteria Microcystis aeruginosa and the cryptophycean Rhodomonas salina. The
growth inhibiting effects of FLU, OXA, SAF, SDZ, TMP, AMX and OTC were
investigated towards the three algae. This enabled the comparison of the procaryotic
cyanobacteria with the eucaryotic algae and the evaluation of the standard test
organism. These results are published in Holten Lützhøft et al. (1999) IV.
Re. 4. An ERA was performed for antimicrobials applied in Danish fish farming. Including
the knowledge acquired from the afore-mentioned investigations, the assessment
became more realistic. A risk quotient (RQ) viz. the ratio between the predicted
environmental concentration (PEC) and the predicted no effect concentration (PNEC),
PEC/PNEC, was calculated for each antimicrobial. Considering both exposure and fate
a PEC was calculated. As many relevant organisms as possible were considered when
calculating the PNEC. The RQs include currently available data. Based on the RQs the
antimicrobials and their applications were assessed.
Despite its importance, the aspect of bacterial resistance was not a subject for this thesis. All
investigations were limited to parent compounds, thus no biotransformation and degradation
products were considered. Only sediment from a freshwater fish farm was studied. Though
interesting, it is beyond the scoop of this thesis to investigate the situation in other countries –
consequently the situation will only be assessed in Denmark.
1.4. Outline of This Ph.D. Thesis
The various tasks performed in this thesis are conceptualised in Figure 1.2.

Figure 1.2 – Conceptual diagram of the tasks performed in this Ph.D. thesis.
Chapter 2 serves as a chapter with basic information about fish farming in Denmark.
Moreover, knowledge of the basic physical chemical and pharmacological properties of the
investigated antimicrobials are given. Based on said information an initial environmental
assessment is performed.
Chapters 3 to 5 mainly serve to provide answers to objective 1 to 3. They address the
occurrence, fate and effects of antimicrobials in an environmental context, respectively.
Additionally, Chapter 3 addresses analysis methods and extraction methods. Besides
distribution processes to environmental constituents, the fate processes discussed in Chapter
4 also cover abiotic and biotic degradation. Chapter 5 is not limited to the discussion of
proper test organisms; also, the toxicity of the antimicrobials towards different trophic levels
is discussed.
Along with data from Chapter 2, Chapters 3 to 5 also serve as input to the ERA – the
answer to objective 4.
Chapter 6 thus synthesises the basic information of the antimicrobials with the environmental
properties in an ERA. The way to derive PNECs as suggested by EMEA (1998b) is discussed.
Three exposure scenarios are presented and discussed. Finally, RQs are derived and
recommendations for the use of antimicrobials in fish farming are given.

Chapter 2
Basic Data of Danish Fish Farms
and Antimicrobials – Initial
Environmental Assessment

• Basic Data of Danish Fish Farms and Antimicrobials – Initial Environmental Assessment12
2.1. Fish Farm Characteristics
At present 470 freshwater fish farms are in operation in Denmark – mainly in the middle and
northern part of Jutland. In total, they produce abt. 35,000 tonnes of fish, of which the vast
majority is rainbow trout (Oncorhynchus mykiss formerly known as Salmo gairdneri). An
average fish farm in Denmark has a yearly production of 74 tonnes. Small fish farms have ca
15 ponds and large farms have ca 90 ponds. A typical pond measures an area of 150-200 m2
with a depth of 0.7 m. In total the yearly water flow is abt. 4⋅1012
L. Each pond contains up to
2,000 kg fish. A sediment layer of 5 cm is assumed.
The production of fry is abt. 610 tonnes, which takes place in either glass or concrete ponds.
The size of a fry pond is abt. 0.7 m in the width, abt. 6 m in the length, and 0.5 m in the depth.
The density of fry is 15-30 kg/m3
, which results in abt. 50 kg in each pond.
When the fry are transferred to the ponds they measure 4-5 cm and weigh abt. 2.5 g. 75 % of
the fish weigh 200-350 g and measure 30 cm.
A schematic drawing of a typical Danish fish farm is exhibited in Figure 2.1. The water to the
fry section is supplied from a well, whereas the water to the ponds is supplied from the local
stream. Presently fish farmers are allowed to utilize the total water flow of the stream, but
current legislation aims at reducing the utilization to half the median water flow by the year
2005. Water from the fry section and the ponds are led to a backchannel, which often also
contains fish. The water is further led to a sedimentation pond to allow particulate matter to
settle, but no active degradation procedures are undertaken. Finally, the water is often stripped
with air in order to increase the oxygen content and oxidize organic carbon before it is
discharged to the stream (Michelsen, Personal communication).
During the years 1993 to 1997 the average pH value and temperature of Danish streams, to
which fish farms effluents are discharged, were abt. 7.2 and 7.2ºC, respectively (Svendsen,
Personal communication).

Chapter 2 • 13
Figure 2.1 – A schematic drawing of a typical Danish fish farma
.
a
: arrows indicate water flow.
2.2. Chemicals
2.2.1. Antimicrobial Therapy in Fish Farming
In order to run an economically sound business the farmers themselves often seem compelled
to apply antimicrobials in the production (Dalsgaard and Bjerregaard, 1991). Factors that
cause diseases are e.g. intensive farming, poor water quality, inadequate feeding regimes
(McCracken et al., 1976; Dalsgaard and Bjerregaard, 1991). This can be optimized and
although vaccination programmes are used, antimicrobial treatment may be required
(Dalsgaard and Bjerregaard, 1991). Antimicrobials are either applied prophylactically or
therapeutically, however the only legal way of application is through a prescription from the
veterinarian (Tørnæs, 1990; Danmarks Apotekerforening, 1996; Andersen, 1999).
Bacterial diseases that usually occur in production-fish in Danish fish farming are enteric red
mouth disease (Yersinia ruckeri), furunculosis (Aeromonas salmonicida), and vibriosis
(Vibrio anguillarum). They are treated with either OXA or a combination of SDZ/TMP.
Under special circumstances, e.g. failure of previous treatment, AMX or OTC may be
prescribed. However, AMX and OTC are mainly used to treat fry mortality syndrome

(Cytophaga psychrophila). These two chemicals are only prescribed on dispensation from the
Danish Veterinary Directorate.
Antimicrobials are mainly administered as medicated food pellets, i.e. incorporated in the feed
pellets (Dalsgaard and Bjerregaard, 1991; Elema, 1995; Kilsgaard, 1996). However, it is
known that the food consumption is reduced among sick fish (Poppe, 1990; Samuelsen et al.,
1992a; Burka et al., 1997) and that medicated feed reduces feed intake (Hustvedt et al.,
1991b). Since the antimicrobials are administered directly to the water of the pond, a risk for
exposing the receiving stream exists. This will happen if the antimicrobials are not degraded
or if overfeeding takes place to circumvent anorexia (Samuelsen et al., 1992a).
Table 2.1 shows data for the consumption of antimicrobials in Danish fish farming from 1994
to 1997. As for human medicines, no reporting system for medicines applied in fish farming
exists yet. However, some voluntarily reporting from fish farmers to local authorities
(counties) and from manufactures to national authorities (The Danish Plant Directorate),
occur. It is therefore assumed that the data presented in Table 2.1 is the minimum
consumption of antimicrobials in Danish fish farming.
Table 2.1 – Antimicrobial consumption in Danish fish farming during 1994-1997, kg.
Year OXAa
SDZa
TMPa
AMXb
OTCb
Otherb
Total
1994 700 1,000 200 6 94 132 2,132
1995 906 1,241 248 78 67 242 2,782
1996 511 845 169 141 27 177 1,870
1997 587 1,677 335 132 16 181 2,928
a
: Viuf (Personal communication), b
: Data from 3 counties (Danske amter, Personal communication), Other:
dimetridazole (antiprotozoal), florfenicol, metronidazole, sulfamerazine, and antimicrobials as such.
An annual quantity of 2-3 tonnes is applied resulting in a usage of 57-86 mg per produced kg
fish. For comparison, both in Norway and Scotland, the usage of antimicrobials per kg
produced fish has decreased during the last years, mainly due to vaccination and improved
husbandry (Baird et al., Personal communication; Markestad and Grave, 1997). In Norway
the usage decreased from 885 mg/kg in 1987 to 7 mg/kg in 1994. In Scotland, the usage was
40 mg/kg in 1994 decreasing to 8 mg/kg in 1997. On the other hand, antimicrobial
concentrations of 4 mg/L was predicted in the waste water from American fish farming due to

Chapter 2 • 15
an annual usage of ca 11,700 tonnes (Vicari et al., Personal communication). A similar
measure for Danish conditions predicts concentrations reaching 0.7 mg/L in the effluents.
2.2.2. Physical Chemical Properties
Table 2.2 shows structures and selected physical chemical properties of the investigated
antimicrobials. They are grouped with respect to their chemical classes. FLU, OXA and SAF
belong to the 4-quinolones of which FLU and SAF also are classified as fluoroquinolones.
SDZ and TMP are so-called folate inhibitors. AMX belongs to the group of β-lactams and
OTC belongs to the tetracyclines.

Table 2.2 – Chemical structures and selected physical chemical properties of the investigated antimicrobials.
Group 4-quinolones Folate Inhibitors β-lactams Tetracyclines
Antimicrobial FLU OXA SAF SDZ TMP AMX OTC
Structure
N
F
CH3
COOH
O
N
O
COOH
CH3
O
O
N
F
O
COOH
N
HN
F
N
N NH
S
NH2
O O
OCH3
H3CO
H3CO
N
N NH2
NH2
HO
NH
O
NH2 N
S CH3
CH3
COOH
O
H H
O O
OH
OH OH
OH
OHHO CH3
N
CH3
HH
NH2
O
H3C
CAS # 42835-25-6 14698-29-4 98105-99-8 68-35-9 738-70-5 26787-78-0 79-57-2
Molecular formula C14H12FNO3 C13H11NO5 C20H17F2N3O3 C10H10N4O2S C14H18N4O3 C16H19N3O5S C22H24N2O9
Mw, g/mole 261.25 261.23 385.37 250.28 290.32 365.41 460.44
mp, °C 253-255b
314-316 (dec)b
275 (dec)i
252-256a
199-203a
194 (dec)r
200 (dec)v
S, mg/L 71c
4.1c
100j
74l
400a
4,000s
241x
pKa 6.4d
6.9f
4.1k
, 6.8k
or 6.0i
& 8.6i
2.0 & 6.5m
7.1p
2.7, 7.2 & 9.6t
3.3, 7.3 & 9.1y
Log KOW 1.7d
0.7g
- -0.1n
0.8q
-1.2u
-0.9z
Log DOW, pH=7.4 1.1e
0.4h
-1.2i
-1.0o
0.6p
-1.5u
-1.2g
KH, atm·m3
/molea
2.7⋅10-13
4.1⋅10-16
1.9⋅10-19
1.6⋅10-10
2.4⋅10-14
2.5⋅10-21
1.7⋅10-25
Mw: Molecular weight, mp: melting point, dec: decomposes, S: Aqueous solubility at pH 7, Log KOW: Distribution coefficient at the pH where the uncharged or most neutral species
dominates (see fractional composition according to Figure 2.2), a
: KH values estimated according to Howard and William (1992), b
: Budavari (1996), c
: Elema (1995), d
: Takács-Novák
and Avdeef /1996), e
: Hirai et al. (1986) pH=7.2, f
: Timmers and Sternglanz (1978), g
: Takács-Novák et al. (1992), h
: Bjørklund and Bylund (1991), i
: Renau et al. (1995), j
: Appears
more soluble than FLU and OXA, k
: Holten Lützhøft et al., (Accepted) III, l
: Stober and DeWitte (1982), m
: Koizumi et al. (1964), n
: Morishita et al. (1973), o
: Wang and Lien (1980),
p
: Seiler et al. (1982), q
: Dietrich et al. (1980), r
: Budavari (1996) β-naphthalenesulfonate trihydrate, s
: Budavari (1996) trihydrate, t
: Tsuji et al. (1978), u
: Smyth et al. (1981), v
:
Chapman & Hall (1998), x
: Parfitt (1999), y
: Stephens et al. (1956), z
: Schumacher and Linn (1978), -: no data found.

Chapter 2 • 17
The structures shown in Table 2.2 give the impression of complex chemicals with many
functional groups, e.g. carboxylic acids, amines, carbonyl- sulfur- and hydroxyl groups. These
groups are ionizeable and polar groups, which make the speciation dependent on various
conditions, e.g. complex forming ions as Mg2+
and Ca2+
and pH. According to the pKa values
the chemicals will to some extent be ionized at physiological and environmental relevant pH
values, viz. 5-8. When the chemicals get more ionized the likelihood of electrostatic
interactions increases. The combination of water-soluble chemicals, solubility (S) at neutral
pH ranges from 4 mg/L to several grams per litre, and hydrophilic chemicals, log KOW ranges
from –1.2 to 1.7, decreases the apparent risk for (bio)accumulation. The S and KOW data
therefore indicate that the chemicals have high affinity for the aquatic compartments. The
molecular weight makes absorption by passive diffusion possible. However, the absorption
rate will be affected as the chemicals are ionized due to changes in pH.
Judging the bare physical chemical properties the antimicrobials will be assessed to reach the
aquatic environment when discharged.
The pH effect mentioned above is for instance seen in the KOW/DOW values. Instead of the
partition coefficient to 1-octanol, KOW, the term distribution coefficient, DOW, should be used.
KOW is used for neutral molecules, whereas DOW is used for ionized molecules.
Table 2.2 gives both the true partition coefficient and the distribution coefficient at pH≈7. As
antimicrobials are ionizeable chemicals, their hydrophobicity decreases when they become
ionized. Whether they are weak (carboxylic) acids or weak bases, this happens at high and
low pH values, respectively.
Figure 2.2 represents the fractional composition and experimental DOW values as a function of
pH. For most of the chemicals the DOW is reflected in the degree of ionization – however, this
is not clear for SAF, which may be due to the few experimental data. The antimicrobial
activity is also reflected in the degree of ionization. OTC shows the highest activity between
pH 5.5 and 6 (Colaizzi and Klink, 1969), and the effect of FLU and OXA decreases abt. 10-15
times from pH 6 to pH 8 (Palmer et al., 1992). Figure 2.2 therefore shows that the
environmental pH is very important for the chemical speciation, accumulation and activity.

Figure 2.2 – Fractional composition and experimental 1-octanol/water distribution
coefficients vs. pH for the antimicrobialsa
.
FLU
0 2 4 6 8 10 12 14
0.0
0.2
0.4
0.6
0.8
1.0
0
5
10
15
20
25
30
35
40
45
50
pH
Fractionalcomposition
Distributioncoefficient
SDZ
0 2 4 6 8 10 12 14
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
pH
OXA
0 2 4 6 8 10 12 14
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
pH
TMP
0 2 4 6 8 10 12 14
0.0
0.2
0.4
0.6
0.8
1.0
0
1
2
3
4
5
6
pH
SAF
0 2 4 6 8 10 12 14
0.0
0.2
0.4
0.6
0.8
1.0
0.00
0.05
0.10
0.15
0.20
pH
AMX
0 2 4 6 8 10 12 14
0.0
0.2
0.4
0.6
0.8
1.0
0.00
0.01
0.02
0.03
0.04
0.05
0.06
pH
OTC
0 2 4 6 8 10 12 14
0.0
0.2
0.4
0.6
0.8
1.0
0.00
0.05
0.10
pH
a
: Fractional composition for neutral and charged species are represented by full and dashed lines, respectively.
Experimental 1-octanol/water distribution coefficients are represented by symbols. For each antimicrobial
individual symbols refer to different references, see Table 2.6.

Chapter 2 • 19
According to the Henderson-Hasselbalch principles, the hydrophobicity can be corrected for
pH effects. Using the KOW and the pKa values, while assuming no distribution of the ionized
species, the DOW profile for a monovalent weak (carboxylic) acid is expressed as:
apKpH
OW
OW
101
K
D
−
+
= Equation 2.1
and for a monovalent weak base as:
pHpK
OW
OW
a101
K
D
−
+
= Equation 2.2
These equations are valid for e.g. FLU, OXA and TMP while for SAF, SDZ, AMX and OTC
one can imagine that more complex equations are required due to the polyvalent structures. In
order to make similar distribution profiles the distribution coefficient for each species is
therefore needed (Winiwarter et al., 1998).
Figure 2.3 shows theoretical DOW profiles, i.e. distribution of the neutral species, for the weak
carboxylic acid OXA and the weak base TMP based on their KOW and pKa values, as
presented in Table 2.2.
Figure 2.3 – pH corrected DOW profiles for the neutral species of OXA and TMPa
.
4 5 6 7 8 9 10
0
1
2
3
4
5
6
7
pH
DOW
a
: Calculated according to Equation 2.1 (OXA – full line) and Equation 2.2 (TMP – dashed line) using pKa and
KOW from Table 2.2.
The DOW profiles shown in Figure 2.3, can be affected by the presence of complex forming
ions. For phenolate species, a relative increase in DOW was observed in the presence of cations

(K+
or Na+
) due to formation of ion pairs (Escher and Schwarzenbach, 1996). When the
phenolate species were allowed to distribute to liposomes, the relative increase was much less
outspoken. However, antibacterial activity was shown to decrease in presence of Mg2+
, see
Chapter 4, indicating a decreased uptake. The last mentioned is known from bioavailability of
OTC in humans; the presence of e.g. Ca2+
or Mg2+
decreases the bioavailability of OTC
(Jensen, 1993).
2.2.3. Environmental Distribution According to Fugacity
Mackay and Paterson (1981) presented an approach based on basic physical chemical
properties of chemicals in order to predict chemical behaviour in the environment. This
approach considers the chemical equilibrium distribution in the environment, by calculating
the fugacity, i.e. the chemical’s escaping tendency. Equilibrium is obtained when the fugacity
in one phase equals that from another. The fugacity approach assumes a well-mixed
environment in a steady state.
This approach enables the calculation of chemical distribution among several environmental
compartments, e.g. water, soil and air. The input data are: environmental temperature,
molecular weight, vapour pressure, solubility and log KOW. Assuming that hydrophobicity
reflects the (bio)accumulation KOW is used to calculate the distribution to soil (Ksoil =
0.02·KOC = 0.02·0.6·KOW), sediment (Ksediment = 0.04·KOC = 0.04·0.6·KOW) and biota (log Kbiota
= 0.85·log KOW – 0.70). Knowing that KOW is affected by pH the DOW at pH≈7 (see Table 2.2)
was used in this particular case.
However, one has to keep in mind that the fugacity approach uses a single DOW value to
estimate the chemical distribution to soil, sediment and biota. This single value is neither
corrected for effects due to pH or influence of complex forming ions. Nevertheless, if the
value for the neutral species is used, it will lead to overestimation of the (bio)accumulation,
which in fact accords to the precautionary principle. Still, under the assumption that
hydrophobicity reflects the (bio)accumulation potential.
As shown in figure 2.2 and figure 2.3 DOW for chemicals as the selected antimicrobials only
changes within less than two orders of magnitude. This is due to their inherent low KOW

Chapter 2 • 21
values and that distribution of the ionized species hardly takes place. This means that pH only
will have a minor effect on the estimated (bio)accumulation factors for said chemicals.
According to the fugacity approach (Mackay and Paterson, 1981), an exposure assessment
was performed using a computer programme (Mackay, 1991). The input data needed for the
program were taken from Table 2.2. An environmental temperature of 7°C was used. The
estimated values for Henrys constant, used to calculate the vapour pressure, are believed to be
acceptable, since the antimicrobials show high melting points and are therefore not expected
to be volatile.
Based on mole fractions, the results showed that the antimicrobial distribution to the aquatic
environment was ≥99.8% for each chemical, see Table 2.3. The computer programme can
only handle positive log DOW values. In case of negative log DOW, 10-10
was used instead. This
approximation seems to be acceptable, since the negative log DOW would only favour
distribution to the water, which already is ≥99.8%.
Table 2.3 – Environmental distribution of antimicrobials according to fugacity
principlesa
.
Air 0.00 0.00 0.00 0.00 0.00 0.00 0.00
Water 99.80 99.96 99.98 99.98 99.93 99.98 99.98
Soil 0.10 0.02 0.01 0.01 0.03 0.01 0.01
Bottom
sediment
0.10 0.02 0.01 0.01 0.03 0.01 0.01
Suspended
aqueous matter
0.00 0.00 0.00 0.00 0.00 0.00 0.00
Biota 0.00 0.00 0.00 0.00 0.00 0.00 0.00
a
: Calculated according to Mackay (1991). Figures represent percentage distribution to the various
compartments. Input parameters needed for the fugacity calculation: Mw, S, and DOW according to Table 2.2.
The vapour pressure was calculated from S and KH, see Table 2.2 (Berg et al., 1995). The environmental
temperature was set to 7ºC. Since the fugacity programme would not accept negative values, log DOW for SAF,
SDZ, AMX and OTC was set to 10-10
.
This confirms the apparent assumption made from the physical chemical properties, that these
antimicrobials probably will distribute to the aquatic environment and not (bio)accumulate.
Due to their high affinity for the aquatic environment, antimicrobials applied in Danish fish

farming will distribute widely to surface waters. Furthermore, the results would not change
significantly whether the KOW was corrected for pH.
2.2.4. Pharmacology
In order to ensure an optimal treatment and minimal environmental impact knowledge of the
pharmacokinetics and –dynamics of antimicrobials are required (Horsberg et al., 1995).
However, dosing regimes are often based on field results (Soltani et al., 1995).
During the last 20 years several authors have stated a lack of knowledge on pharmacokinetics
of antimicrobials in fish and expressed a need for thorough investigations (Bergsjø and
Søgnen, 1980; Sohlberg et al., 1990; Bjørklund, 1991; Ishida, 1992; Nouws et al., 1992;
Hustvedt, 1992; Luzzana et al., 1994; Martinsen et al., 1994; Tan and Wall, 1995). Table 2.4
and Table 2.5 address the pharmacokinetics in rainbow trout, the dosing regimes and the
environmental load of the investigated antimicrobials.
Data for other organisms have to be considered with care, since the pharmacokinetics seem to
vary from organism to organism (Grondel et al., 1989; Martinsen et al., 1994; Tan and Wall,
1995; Horsberg et al., 1995) and from mammals to fish (Tan and Wall, 1995).
2.2.4.1. Pharmacokinetics in Rainbow Trout
As seen from Table 2.4 not all requested data have been reported. Data from other organisms
could have been included, but due to the mentioned organism sensitivity, this was not done.
Especially data for AMX and to a certain extent SAF are lacking. For the widely used SDZ, it
is surprising that, to the best knowledge of the author, the latest relevant data was published in
the late seventies. However, studies can be found for related structures and other organisms,
e.g. Uno et al. (1993) and Samuelsen et al. (1995).
Qualitative and quantitative distribution data indicate that the investigated antimicrobials are
widely distributed in the fish body, see Table 2.4. This is based on measured concentrations in
various tissues, autoradiographic studies, viz. the study of a radioactive labelled chemical’s
distribution in a body, and volume of distribution at steady state. Despite the effective
distribution, the oral bioavailability is often poor, ranging from a few percent for OTC to as
much as 72 % for FLU.

Chapter 2 • 23
However the oral bioavailability is affected by external factors, e.g. OXA is dose dependent
(Cravedi et al., 1987), OTC is affected by way of administration and FLU is temperature
dependent (Sohlberg et al., 1990; Sohlberg et al., 1994). Plasma concentrations and sampling
times after both intra arterial (i.a.) and oral (p.o.) administration of FLU at 3ºC and 13ºC were
presented by Sohlberg et al. (1994).
The bioavailability (F) is defined as follows:
( )
( ) .a.i
.o.p
DAUC
DAUC
F = Equation 2.3
AUC is the area under the plasma concentration-time curve and D is the dose for p.o. and i.a.
administration, respectively.
For i.a. administration AUC is defined as:
e
0
p
k
C
AUC0
=∞ Equation 2.4
∞
0AUC is the area under the curve from time zero to infinite time. 0
pC is the plasma
concentration at time zero viz. at the moment of injection and calculated from the y-intercept
from linear regression of the first sampling points. ke is the elimination rate constant and
obtained as the slope in the linear regression of the curve which represents the elimination
phase.
For p.o. administration the AUC is defined as:
∞∞
+= T
T
00 AUCAUCAUC Equation 2.5
T
0AUC defines the area under the curve from time zero to the last sampling time, T. This part
is calculated by the trapezoidal rule. The residual area is then calculated as follows:
e
T
p
T
k
C
AUC =∞ Equation 2.6

T
pC is the plasma concentration at the last sampling time. It is recommended that the residual
area represents less than 10 % of the total area, since error in ke due to misinterpreted
distribution/elimination phase will underestimate the AUC.
The bioavailability for FLU presented in Table 2.4 was calculated from the above equations
and the plasma concentrations and sampling times given in Sohlberg et al. (1994). The plasma
concentration-time profiles are shown in Figure 2.4. It has to be mentioned that the residual
area represents more than 50 % of the total AUCp.o., which means that the bioavailability may
be underestimated.
Figure 2.4 – Plasma concentration-time profiles for FLU in rainbow trouta
.
0 250 500 750 1000 1250 1500
-2
-1
0
1
2
α=ke
α=ke
Cp
0
3°°°°C
Time, h
LnCp,mg/L
0 200 400 600
-3
-2
-1
0
1
2
α=ke
α=ke
Cp
0
13°°°°C
Time, h
LnCp,mg/L
a
: Data from (Sohlberg et al., 1994). The solid symbols represent i.a. administration and the transparent symbols
represent p.o. administration. Lines represent the linear regression of the initial and terminal phases. α represents
the slope for the respective parts of the curves. Full lines: i.a. administration and dashed lines: p.o.
administration.
The fraction of the antimicrobial that is absorbed in the fish may be exposed to
biotransformation. The antimicrobial may e.g. be hydrolysed or oxidized (phase I reactions)
or conjugated by e.g. glucuronidation or acetylation (phase II reactions). The quinolones are
mainly phase II biotransformed; this is most pronounced for OXA. Glucuronidation leads to
e.g. amide or ester bonds, which are hydrolytically unstable chemicals. Thus, some conjugates
may be cleaved when entering the aquatic compartment and thereby liberate the parent
compound. Moreover, conjugated antimicrobials were cleaved in manure by bacteria (Berger
et al., 1986). Only OTC is not biotransformed.
The literature suggests that most of the antimicrobials be excreted both in urine and bile.
Depending on environmental temperature, dose and way of administration, the elimination
half-lives presented in Table 2.4 differ as much as several hundred hours.

Chapter 2 • 25
The withdrawal times have therefore been differentiated with respect to temperature. The
wide distribution and long elimination half-lives therefore result in correspondingly long
withdrawal times.
See footnotes to Table 2.4 for specific information of the individual antimicrobials and
pharmacokinetic parameters.
The bioavailability of oral administered antimicrobials is low, meaning that a high fraction
passes the fish unabsorbed. Moreover, the absorbed fraction is hardly biotransformed;
resulting in excretion mainly as a parent compound. Thus, the pharmacokinetics in rainbow
trout predict that antimicrobials are expected to reach the aquatic environment as parent
compounds.

Table 2.4 – Pharmacokinetics in rainbow trout.
Bioavailability, % 36a1
, 72a2
14e1
, 14f1
, 38f2
incompletej
- - - 1.3m2
, 30n1
, 5.6e6
,
8.6f1
, 7.1f2
Distribution wellb
widelyg
, goode2
, well (>90
% outside plasma)h1
, wellb
wellb,j
wellk
wellk
, fast
and widelyl1
- goode2,o1
Vd,ss, L/kg 3.2a3
, 3.6a4
1.9e3
, 2.6g
, 2.9h2
- - 6.0l2
- 4.0m3
, 2.7m4
,
2.1p1
, 0.9n2
, 1.2e7
Biotransformation
products
noc
≤ 6 % -OHd
≤ 12.5 % -
GLUd
66 %i1
- - probablyl3
- nop2
Urinary excretion probablya5
- most likelyj
most
likelyk
most likelyk
,
14.4l4
- -
Biliary excretion probablya5
29 %i2
most likelyj
major
excretory
pathwayk
major
excretory
pathwayk
,
possiblel1
- yeso2
Elimination t½, h 569a6
, 137a7
,
736a8
, 285a9
20-43g
, 42.8e4
, 69.7e5
,
52.6h2
- - 8m1
, 36.1l2
- 130m5
, 76m6
,
150m7
, 89.5p1
,
94.2n2
, 479.4n3
,
60.3e8
, 74.9e9
Withdrawal timeq
≥10ºC: 40 d
<10ºC: 80 d
≥10ºC: 30 d
<10ºC: 60 d
≥10ºC: 40 d
<10ºC: 80 d
≥10ºC: 40 d
<10ºC: 80 d
≥10ºC: 40 d
<10ºC: 80 d
≥10ºC: 60 d
<10ºC: 120 d

Vd,ss: distribution volume at steady state,
a1
: Sohlberg et al. (1994) 3ºC, 5 mg/kg p.o. and i.a., see text for details, a2
: Sohlberg et al. (1994) 13ºC, 5 mg/kg p.o. and i.a., see text for details, a3
: Sohlberg et al. (1994) 13ºC, 5
mg/kg i.a., a4
: Sohlberg et al. (1994) 3ºC, 5 mg/kg i.a., a5
: Sohlberg et al. (1994) due to rapid decrease form plasma, a6
: Sohlberg et al. (1994) 3°C, 5 mg/kg i.a., a7
: Sohlberg et al.
(1994) 13°C, 5 mg/kg i.a., a8
: Sohlberg et al. (1994) 3°C, 5 mg/kg p.o., a9
: Sohlberg et al. (1994) 13°C, 5 mg/kg p.o.,
b
: Steffenak et al. (1991),
c
: Sohlberg et al. (1990),
d
: EMEA (1996) OH: hydroxy-FLU, GLU: FLU-glucuronide,
e1
: Bjørklund and Bylund (1991) 16ºC, 75 mg/kg p.o., 10 mg/kg i.v., e2
: Bjørklund and Bylund (1991), e3
: Bjørklund and Bylund (1991) 16ºC, 10 mg/kg i.v., e4
: Bjørklund and Bylund
(1991) 16ºC, 75 mg/kg p.o., e5
: Bjørklund and Bylund (1991) 16ºC 75 mg/kg p.o., 20 mg/kg i.v., e7
: Bjørklund and Bylund
(1991) 16ºC, 20 mg/kg i.v., e8
: Bjørklund and Bylund (1991) 16ºC, 75 mg/kg p.o.,
f1
: Cravedi et al. (1987) 14ºC, 100 mg/kg p.o., calculated from recovery from faeces, f2
: Cravedi et al. (1987)14ºC, 20 mg/kg p.o., calculated from recovery from faeces,
g
: Hustvedt (1992),
h1
: Hustvedt and Salte (1991a), h2
: Hustvedt and Salte (1991a) 8.5ºC, 10 mg/kg i.v.,
i1
: Ishida (1992) three glucuronides, 66 % present in the bile as OXA-glucuronide 24 hours after administration, i2
: Ishida (1992) 29 % present in the bile as OXA 24 hours after
administration,
j
: Martinsen et al. (1994) based on autoradiography studies,
k
: Bergsjø et al. (1979) based on autoradiography studies,
l1
: Tan and Wall (1995), l2
: Tan and Wall (1995) 10ºC, 10 mg/kg i.a., l3
: Tan and Wall (1995) high biotransformation anticipated, l4
: Tan and Wall (1995) percentage parent
compound recovered in urine,
m1
: Nouws et al. (1992) 12ºC, 10 mg/kg intra muscular, m2
: Nouws et al. (1992) 60 mg/kg p.o. and i.v., m3
: Nouws et al. (1992) 10ºC, 60 mg/kg i.v., m4
: Nouws et al. (1992) 19ºC, 60
mg/kg i.v., m5
: Nouws et al. (1992) 10ºC, 60 mg/kg intra muscular,
n1
: Abedini et al. (1998) 11ºC, 50 mg/kg i.a. and p.o. dissolved in MeOH and administered in a capsule, n2
: Abedini et al. (1998) 11ºC, 50 mg/kg i.a., n3
: Abedini et al. (1998) 11ºC,
p.o. 50 mg/kg dissolved in MeOH and administered in a capsule,
o1
: Rogstad et al. (1991), o2
: Rogstad et al. (1991) can be expected from the high liver/plasma ratio,
p1
: Grondel et al. (1989)12ºC, 60 mg/kg i.v., p2
: Grondel et al. (1989) OTC biotransformation in fish is presumed very small,
q
: Dalsgaard and Bjerregaard (1991),
-: no data available.

2.2.4.2. Pharmacodynamics
All antimicrobials investigated in this thesis interfere with the deoxyribonucleic acid (DNA)
or protein synthesis in bacteria (Lambert, 1992), thus preventing the bacterial disease from
developing further. Table 2.5 gives the dosing regimes, the environmental load and the
percentage of fish/fry treated with antimicrobials during one year.
The 4-quinolones FLU, OXA and SAF are broad-spectrum synthetic antimicrobials, acting
primarily on Gram negative bacteria. The chemicals block the chromosomal replication by
specific inhibition of DNA gyrase which catalyses supercoiling of DNA. The 4-quinolones
have much higher affinity for prokaryotic than human DNA gyrase. Cross-resistance is often
observed among quinolones (Smith, 1995). Resistance is rarely mediated by plasmids. Mostly
the enzyme that 4-quinolones block has altered polarity, or the production of the proteins that
mediate the transport of 4-quinolones has decreased (Franklin, 1992; Smith, 1995).
The combination of the folate antagonists SDZ and TMP gives a synthetic broad-spectrum
antimicrobial, acting by interfering with two steps in the folate synthesis by substrate
competition and enzyme inhibition respectively. The TMP affinity for the bacterial enzyme is
more than 104
times that for the human (Jensen, 1993; Rang and Dale, 1993). Applied as
single substances they only act bacteriostatically, whereas applied in combination they act
bactericidally. The co-administration also results in one tenth of the doses that would be
needed if the chemicals were applied as single chemicals (Dalsgaard and Bjerregaard, 1991;
Rang and Dale, 1993). The resistance mechanism is plasmid mediated. The plasmid genes
code for alterations in the target enzymes, either by favouring the natural substrate or by being
resistant to the inhibitor (Franklin, 1992).
The ββββ-lactam AMX is a broad-spectrum semisynthetic antimicrobial, belonging to the group
of penicillins, acting on Gram positive as well as Gram negative bacteria. AMX is interfering
with the biosynthesis of peptidoglycan of the bacterial cell. AMX binds covalently to the
enzyme that cross-links the peptidoglycan. The enzyme is inactivated because AMX simulate
the natural substrate. Resistance is mediated through production of an enzyme, β-lactamase,
which cleave the β-lactam ring of the chemical. Cross-resistance towards cephalosporins is
often seen (Franklin, 1992).

Chapter 2 • 29
The tetracycline OTC is a broad-spectrum antimicrobial that is actively taken up by the
bacterial cell. OTC mediates its bacteriostatic effect on the protein synthesis by inhibiting
bacterial ribosome function through binding to the 30S subunit (Lambert, 1992). The
resistance is mediated by plasmid genes encoding for proteins that promotes the efflux of
tetracyclines (Franklin, 1992).
Depending on the antimicrobial the environmental load results in fish farm effluents
concentrations in the range 0.5-19 mg/L assuming no dilution, biotransformation or
degradation and application of the treatment in one lot. This is a worst case scenario enabling
one to assess the maximal exposure to the environment.
Equation 2.7 is used to calculate the percentage of fish/fry treated with antimicrobials. The
numerator represents the actual consumption of the particular antimicrobial and the
denominator represents a single treatment of the total biomass.
%100kgmg10
mTD
m
%reated,Fish/fry t 6
Fish/fry
A
⋅⋅⋅⋅⋅⋅⋅⋅
⋅⋅⋅⋅⋅⋅⋅⋅
==== Equation 2.7
ma is the consumption of the respective antimicrobials in kg, see Table 2.1. D is the dose in
mg/kg bw/d and T is the duration in days, see Table 2.5. mFish/fry is the mass of total produced
biota in kg, see Fish Farm Characteristics.
Roughly estimated 55% of production fish and 30-50% of fry undergo antimicrobial treatment
once a year! In other words, nearly all fish can be considered treated with antimicrobials once
in their life time!

Table 2.5 – Dosing regimes and environmental load in Danish fish farms.
Dose, mg/kg bw/d 15d
10e
10f
25e,g
5e,g
50-80h
80e
Duration, d 7d
10e
5f
5e,g
5e,g
10h
8e
Environmental load, ga
fish 210 200 100 250 50 1,000-1,600 1,280
fry - - - - - 25-40 32
Cmax in pond, mg/La, b
fish 2 1.9 1 2.4 0.48 9.5-15 12
fry - - - - - 12-19 15
Fish treated, %c
- 17 - 38 0.5-0.8 0.1
Fry treated, %c
- - - - 27-43 4
bw: fish body weight, d: days, a
: based on specifications in Fish Farm Characteristics, b
: one treatment applied in
one lot assuming no dilution, biotransformation or degradation, c
: based on specifications in Fish Farm
Characteristics, Table 2.1 and Equation 2.7, d
: Schneider (1994), e
: Kilsgaard (1996), f
: EMEA (1998a), g
:
Dalsgaard and Bjerregaard (1991), h
: Alderman and Michel (1992), -: not applied in Denmark.
2.3. Initial Environmental Assessment
In the previous sections physical chemical and pharmacological properties from the literature
along with fugacity calculations of the antimicrobials were presented. Based on that
knowledge the following properties are attributed the antimicrobials:
• Low oral bioavailability and low biotransformation results mainly in excretion of a
parent compound.
• The principal exposure is to the aquatic environment, however the distribution is
widely pH dependent.
• Environmental exposure of biological active chemicals may affect non-target
organisms.
Due to the antimicrobials’ affinity for the aquatic compartment (high aqueous solubility and
low hydrophobicity) surface waters and maybe ground water will consequently be the natural
recipient.

Chapter 2 • 31
Generally, the antimicrobials suffer from low bioavailability; resulting in low uptake in the
fish and direct excretion of active parent compound to the environment. Furthermore, the part,
which is absorbed in the fish, is hardly biotransformed, e.g. OTC. If the antimicrobial is
biotransformed, e.g. OXA, mainly glucuronide conjugates are formed. Since glucuronide
conjugates often are hydrolytically unstable chemicals, parent compounds may be liberated
upon cleavage when entering the environment. However, the absorbed part of the
antimicrobial is widely distributed in the fish body and stay there for long periods. This means
that, if antimicrobials enter the aquatic environment, the wild fauna may be exposed to and
absorb the antimicrobials.
Since the antimicrobials are biologically active chemicals, especially bacteria and similar
organisms, e.g. micro-algae, may be affected. The biodiversity among micro-organisms may
therefore be affected. Bacteria may develop resistance towards the antimicrobials. This
property is to a certain extent considered as an irreversible effect, since it may persist after the
disappearance of the resistance-provoking chemical. Moreover, resistance may be transferred
from target organisms to human pathogenic bacteria and environmental bacteria.
Therefore, the application of antimicrobials in quantities of tonnes in fish farming has to be
considered with caution, since they are directly exposed to the aquatic environment. However,
specific environmental research regarding fate and effects of antimicrobials is required in
order to make a proper ERA of said chemicals.

Table 2.6 – Experimental 1-octanol/water distribution coefficients.
Chemical Log DOW pH Ionic strength, M Buffer, M Method Ref.
FLU 1.60 < 4.4 ns 0.04 acetate SF a
FLU 1.72 < 4.4 0.1 and 0.15 - pH-m a
FLU 1.11 7.2 ns 0.1 phosphate m-SF b
OXA 0.68 4.0 ns 0.04 acetate SF c
OXA 0.35 7.2 ns 0.1 phosphate m-SF b
OXA 0.38 7.4 ns 0.1 phosphate SF d
SAF -1.18 7.4 ns ns RML e
SAF -0.71 7.4 ns phosphate SF f
SDZ -0.09 3.0-3.5 0.1 acetate ns g
SDZ -0.29 5.5 ns ns SF h
SDZ -0.69 6.4 ns phosphate ns i
SDZ -0.27 6.9 ns H2O SF j
SDZ -1.00 7.4 ns 0.01 phosphate SF j
SDZ -1.00 7.4 ns 0.01 TRIS SF j
SDZ -1.26 7.5 ns phosphate SF k
SDZ -2.12 9.2 ns 0.01 bicarbonate SF j
TMP -1.55 1 ns 0.1 HCl SF l
TMP 0.64 7.4 ns 0.05 phosphate SF m
TMP 0.82 13 ns 0.1 NaOH SF l
AMX -1.70 2 ns 0.1 KCl-HCl SF n
AMX -1.70 3 ns 0.1 citrate SF n
AMX -1.22 6 ns 0.1 phosphate SF n
AMX -1.52 7.4 ns 0.1 phosphate SF n
AMX -1.52 8 ns 0.1 phosphate SF n
OTC -2.46 2.1 0.1 phosphate SF o
OTC -0.89 5.5 0.15 phosphate ns p
OTC -0.92 6.6 0.1 phosphate SF q
OTC -1.60 7.0 ns phosphate SF r
OTC -1.22 7.4 ns 0.1 phosphate SF d
OTC -1.60 7.5 ns ns ns s
ns: not specified, TRIS: tris(hydroxymethyl)aminomethan, SF: shake flask principle, pH-m: pH-metric, m-SF:
modified version of shake flask, RML: Robertson Microlit Laboratories, a: Takács-Novák and Avdeef (1996), b:
Hirai et al. (1986), c: Takács-Novák et al. (1992), d: Bjørklund and Bylund (1991), e: Renau et al. (1995), f:
Jürgens et al. (1996), g: Morishita et al. (1973), h: Ehlert et al. (1998), i: Yamazaki et al. (1970), j: Wang and
Lien (1980), k: Abel et al. (1975), l: Dietrich et al. (1980), m: Seiler et al. (1982), n: Smyth et al. (1981), o:
Colaizzi and Klink (1969), p: Schumacher and Linn (1978), q: Miller et al. (1977), r: Kellaway and Marriott
(1978), s: Toon and Rowland (1979).

Chapter 3
Environmental Occurrence of
Antimicrobials

• Environmental Occurrence of Antimicrobials36
3.1. Introduction
Due to human activities antimicrobials are found in various environmental matrices, e.g. SDZ
in groundwater (Holm et al., 1995), TMP in river and surface waters (Hirsch et al., 1998) and
sewage treatment plant effluents (Hirsch et al., 1999) and ciprofloxacin (a fluoroquinolone) in
hospital waste water (Hartmann et al., 1999). Detailed discussions of the environmental
exposure routes of pharmaceuticals in general are found in Halling-Sørensen et al. (1998) and
Jørgensen and Halling-Sørensen (2000). However, only the environmental exposure and
occurrence of antimicrobials in connection to fish farm application is discussed in this
chapter.
Figure 3.1 represents a schematic drawing of the antimicrobial exposure routes due to
application in a traditional Danish fish farming.
Figure 3.1 – Anticipated exposure routes of antimicrobials applied in fish farminga
.
a
: Modified from Jørgensen and Halling-Sørensen (2000), full lines: flow, dashed lines: transport, dotted lines:
interaction.

Chapter 3 • 37
Antimicrobials are applied as a consequence of intensive farming (Dalsgaard and Bjerregaard,
1991). They are conveniently applied directly to the water phase through medicated feed
pellets (Dalsgaard and Bjerregaard, 1991; Elema, 1995). The extent of distribution to the
sediment depends on the physical chemical properties of the antimicrobial. If the
antimicrobial has high affinity for the sediment it has the potential to accumulate in the
sediment, see Chapter 4. The sediment may be used as fertilizer on arable land if e.g.
cadmium content do not exceed 0.8 mg/kg dry weight (Holm Sørensen and Landsfeldt, 1997;
Jørgensen and Halling-Sørensen, 2000). In that case, there is a risk for indirect exposure of
soil organisms. However, if the sediment is not removed it will serve as an antimicrobial
reservoir. The antimicrobial then distributes to the water according to its particular
distribution coefficient, see Chapter 4. If the antimicrobial has high affinity for the water, it is
discharged to the stream and further to the ecosystem. In the latter case, there is a risk for
indirect exposure of aquatic organisms.
According to the pharmacokinetic data presented in Chapter 2, it is likely that antimicrobials
from treatment in fish are excreted to the environment. Furthermore, they are applied in a
restricted area and according to Figure 3.1, they may consequently be discharged either to the
terrestrial or the aquatic environment.
It is therefore expected that antimicrobial residues can be found in the sediment near fish
farms, whereas Hirsch et al. (1999) state that the antimicrobial application in non-human
treatment is of minor importance.
In order to quantitate antimicrobials in the environment, both separation and extraction
methods are required. In this thesis, the work was focused on quinolones, and therefore the
discussion of separation and extraction methods is limited to those chemicals. Moreover,
discussion of the extraction methods is limited to sediment. Following the analytical
procedures, the occurrence is discussed.

• Environmental Occurrence of Antimicrobials38
3.2. Analytical procedures
3.2.1. Separation methods
Table 3.1 gives a range of reversed phase (RP) HPLC methods for the analysis of FLU, OXA
and SAF. They are mainly applied to fish and sediment samples. All isocratic procedures
present an asymmetric, i.e. tailing peak shape of the quinolone. This is a well-known feature
of the chromatography of polar chemicals, e.g. amine and carboxylic acid containing analytes.
The problem is the interaction between the column and the ionizable group(s) of the analyte.
An RP column consists of silica substituted with alkyl chains, e.g. octadecylsilyl (C18) chains.
However, the substitution is rarely, if ever, complete. Not more than 50 % of the silanol
groups are substituted. The remaining free silanol groups are therefore active for interactions
with e.g. amine groups. The result of such an interaction is a broadening of the peak, which is
often seen as a tailing peak, Figure 3.2A. The results of tailing are decreased sensitivity,
efficiency and separation. However, many commercial columns are partly deactivated by
further substitution with shorter alkyl chains, so-called end capped columns (Smith, 1988).
Figure 3.2 – Column material effect on the chromatography of OXAa
.
A
0 2 4 6 8 10
0.00
0.05
0.10
0.15
0.20
0.25
0.30
Time, min
UVresponse
B
0 2 4 6 8 10 12 14 16 18 20
0.00
0.05
0.10
0.15
0.20
0.25
0.30
Time, min
UVresponse
a
: Analysis of a 20 mg/L (400 ng injected) solution of OXA. A: Conventional end capped C18-column, isocratic
elutions; MeOH:2 mM H3PO4/KH2PO4 pH 2.9 70:30, 60:40 and 50:50, respectively. B: Discovery C18 end
capped column, isocratic elutions; MeOH:2 mM H3PO4/KH2PO4 pH 2.9 60:40, 50:50, 40:60 and 30:70,
respectively. Detection: UV 260 nm. Flowrate: 2 mL/min. From Holten Lützhøft et al. (1999).
A gradient mobile phase can sometimes diminish tailing, however it is more a result of the
analyte’s increasing affinity for the mobile phase. To circumvent problems with tailing a
polymer column can be used or masking agents can be added the mobile phase, i.e. chemicals
that have similar properties as the analyte (Smith, 1988). It is actually seen from Table 3.1

Chapter 3 • 39
that often a polymer column is used or that most of the applied mobile phases are rather
complex, e.g. multiple organic modifiers and organic acids, although still providing tailing
peaks.
The problem is effectively eliminated by using a column that does not possess the ability to
interact in the above-mentioned way. This was demonstrated by Holten Lützhøft et al. (1999).
An RP Discovery C18 end capped column was used providing symmetric non-tailing peaks
although the quantity of analyte injected on the column was 400 ng, equal to a sample
concentration of 20 mg/L, Figure 3.2B.
Furthermore, a very simple mobile phase consisting of methanol (MeOH) and H3PO4/KH2PO4
pH 2.9 was used. Figure 3.2 represents a comparison of the chromatography of OXA on a
conventional end capped RP column and the RP Discovery C18 end capped column,
respectively. Using the Discovery column, complex mobile phases containing acetonitrile and
tetrahydrofuran (THF) can be avoided. Holten Lützhøft et al. (1999) applied the column in
connection with SPME-HPLC analysis. In these kinds of analyses it is especially required that
the analytes are chromatographed well, due to the nature of the SPME-HPLC application. In
this case, the column also served its purpose, see Figure 3.3.
Figure 3.3 – SPME-HPLC analysis of FLU, OXA and SAFa
.
0 1 2 3 4 5 6 7
0.000
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
Time, min
UVresponse
a
: Chromatographic conditions: Discovery C18 end capped column, gradient elution: MeOH:2 mM
H3PO4/KH2PO4 pH 2.9 from 20:80 to 90:10 in 7 min. tR=3.6 min.: SAF, tR=4.9 min.: OXA and tR=6.0 min.:
FLU. Dashed line: SPME injection. Full line: Autosampler injection. Detection: UV 260 nm. Flowrate: 2
mL/min. From Holten Lützhøft et al. (1999).
Holten Lützhøft et al. (1999) recommended said column for future analysis of chemicals
containing problematic groups, e.g. the HPLC analysis of freshwater sediment extracts was
performed using mentioned column (Holten Lützhøft et al., Submitted I).

Table 3.1 – HPLC methods for the investigated quinolones.
Antimicrobial Stationary phase Mobile phase Detectiona
tR, min Peak shape m, pgb
Matrixc
LOD, pg ref.
FLU 5 µm PLRP-S polymer adsorbent
150×4.6 mm
2mM
H3PO4:MeCN:
THF 65:20:15
F, 260, 380 8.4 asymmetric 5,000 fish tissues 200 A
FLU 3 µm ODS-Hypersil 100×5 mm 0.1 M pH 3.2
C6H8O7:MeOH:
MeCN:THF
60:30:5:5
F, 324, 363 6.0 asymmetric 2,000 fish muscle 400 B
FLU 3 µm MOS-Hypersil (C8)
150×4.6 mm
25 mM
(COOH)2 pH
3.2:MeCN:Me
OH:THF
F, 325, 360 8.9 nc nc fish plasma 250 C
FLU 5 µm Ultrabase octacecyl
250×4.6 mm
MeCN:2.7 mM
(COOH)2 pH
2.5 gradient
F, 252, 356 20 nc nc pig tissue 3,000 D
FLU 3 µm phenyl YMC/3-4-5
cartridge columns 50×4 mm
MeCN:20 mM
HCOOH pH
2.75 gradient
MS 6.7 nc nc fish muscle 250 E
FLU 5 µm Supelco Discovery C18 end
capped column 150×4.6 mm
MeOH:2 mM
H3PO4/KH2PO4
pH 2.9 gradient
UV, 260 6.0 nc nc buffer 162 F1
OXA 5 µm PLRP-S polymer adsorbent
150×4.6 mm
2mM
H3PO4:MeCN:
THF 65:20:15
F, 260, 380 4.8 asymmetric 1,000 fish tissues 50 A

OXA 3 µm MOS-Hypersil (C8)
150×4.6 mm
25 mM
(COOH)2 pH
3.2:MeCN:Me
OH:THF
F, 325, 360 6.6 nc nc fish plasma 150 C
OXA 5 µm ISRP 150×4.6 mm MeCN:0.1 M
KH2PO4 pH 2.0
10:90
UV, 254 9.4 asymmetric 10,000 sediment 5,000 G
OXA 5 µm LiChroSorp 100 RP-18E
125×4.6 mm
20 mM
H3PO4:MeCN
76:24
UV, 262 6.6 asymmetric 25,000 sediment 1,000 H
OXA 5 µm LiChroSorp 100 RP-18E
125×4.6 mm
20 mM
H3PO4:MeCN
76:24
UV, 262 - - - mud
sandy mud
sand
11,000
10,000
10,000
I
OXA 5 µm HISEP shielded
hydrophobic phase column
150×4.6 mm
50 mM
C6H8O7, 200
mM Na2HPO4
pH 2.5 in 10
mM (n-C4H9)4-
NH4Br:MeCN
85:15
UV, 265 13.5 asymmetric 100,000 fish serum 2,500 J
OXA 3 µm phenyl YMC/3-4-5
MeCN:20 mM
HCOOH pH
2.75 gradient
MS 5.3 nc nc fish muscle 250 E
OXA 5 µm Supelco Discovery C18 end
MeOH:2 mM
H3PO4/KH2PO4
pH 2.9 gradient
OXA 5 µm Supelco Discovery C18 end
60 % MeOH
30 % MeOH
UV, 260
UV, 260
2.7
15.4
symmetric
symmetric
400,000
400,000
buffer
buffer
-
-
F2

SAF 5 µm PLRP-S polymer adsorbent
150×4.6 mm
2 mM
H3PO4:MeCN:
MeOH 73:19:8
F, 278, 440 5.9 asymmetric 5,760 fish tissues 115 K
SAF 3 µm phenyl YMC/3-4-5
MeCN:20 mM
HCOOH pH
2.75 gradient
MS 4.9 nc nc milk 20 E
SAF 5 µm Supelco Discovery C18 end
MeOH:2 mM
H3PO4/KH2PO4
pH 2.9 gradient
tR: retention time, LOD: limit of detection, nc: not comparable, -: not reported,
a
: detection: F: fluorescence, excitation wavelength, emission wavelength; MS: mass spectrometry; UV: ultraviolet, wavelength, b
: injected quantity for the mentioned retention time
and peak shape, c: target analysis matrix,
MeCN: acetonitrile, THF: tetrahydrofuran, C6H8O7: citric acid, MeOH: methanol, (COOH)2: oxalic acid, HCOOH: formic acid, ISRP: internal surface reversed-phase,
A: Rogstad et al. (1989), B: Samuelsen (1989b), C: Samuelsen (1990) mobile phase: I: 25 mM (COOH)2 pH 3.2:MeCN:MeOH:THF (80:2.5:15:2.5), II: (COOH)2 pH
3.2:MeCN:MeOH:THF (50:20:25:5); t0: I:II 100:0; t5: 0:100; 5 min isocratic and 5 min calibration, D: Guyonnet et al. (1996) mobile phase: I: MeCN, II: 2.7 mM (COOH)2 pH 2.5; t0:
I:II 10:90; t20: I:II 70:30; 5 min isocratic and 5 min calibration, E: Volmer et al. (1997) mobile phase: I: MeCN, II: 20 mM HCOOH pH 2.75; t0: I:II 2:98; t10: I:II 57:43, F1: Holten
Lützhøft et al. (1999) mobile phase: I: MeOH, II: 2mM H3PO4/KH2PO4 pH 2.9; t0: I:II 20:80; t8: I:II 100:0; 2 min isocratic and 8 min calibration, F2: Holten Lützhøft et al. (1999)
mobile phase: accomplished by 2 mM H3PO4/KH2PO4 pH 2.9 to 100 %, G: Bjørklund (1990) and Bjørklund et al. (1991), H: Pouliquen et al. (1994b), I: Pouliquen et al. (1994a), J:
Ueno and Aoki (1996), K: Hormazabal et al. (1991).

Chapter 3 • 43
3.2.2. Extraction Methods
Sediment extraction methods for quinolones, i.e. FLU and OXA that can be found in the
literature are presented in Table 3.4. Three methods are described for extraction from marine
sediment and one for freshwater sediment.
3.2.2.1. Marine sediment
In all three methods the chemical is extracted from the sediment into an aqueous phase – two
using NaOH and one KH2PO4 pH 7. Samuelsen et al. (1994) actually just analyse the NaOH
extract after centrifugation. This gives a recovery of 95.7 and 92.6 % for FLU and OXA,
respectively. Bjørklund et al. (1991) extract with KH2PO4 and concentrate the filtered extracts
on a C18 solid phase extraction (SPE) cartridge. The eluate is further reduced to 0.5 mL and
analysed. Pouliquen et al. (1994b) extract the sediment with NaOH, acidify and extract OXA
into an organic phase by liquid liquid extraction (LLE). After centrifugation, the organic
phase is evaporated to dryness, dissolved in 0.5 mL, and analysed. The latter two methods
both give recoveries of ca 70 %.
Pouliquen et al. (1994a) used the LLE to extract more specific sediment types. The result was
that OXA tends to be more easily extracted from sand/sandy sediment than from mud/muddy
sediment. Recoveries went from 60 % to 90 % when mud was replaced by sand.
Based on these results it is tempting to conclude that the sediment used by Samuelsen et al.
(1994) must have contained a large sand fraction! The method is essentially the same as the
method described and used by Pouliquen et al. (1994a) and Pouliquen et al. (1994b).
3.2.2.2. Freshwater sediment
The methods of Samuelsen et al. (1994) and Pouliquen et al. (1994b) were adopted for the
extraction of OXA from freshwater sediment. However, recoveries less than 50 % were
judged futile (Holten Lützhøft et al., Submitted I). Various attempts using water and NaOH
containing MeOH did not improve results. Instead an approach using sediment filled SPE
tubes was applied. Figure 3.4 outlines the steps in the extraction procedure.

44 • Environmental Occurrence of Antimicrobials
Figure 3.4 – Procedure to extract OXA from freshwater sedimenta
.
a
: Holten Lützhøft et al. (Submitted) I.
SPE tubes were filled with contaminated sediment. The strategy was to use an eluent that was
strong enough to extract OXA and at the same time was able to be removed in a simple way.
As was the case for chromatography on RP columns, polar interactions between OXA and the
sediment were expected. To circumvent this, eluents consisting of various agents were
evaluated.
Table 3.2 gives an overview of the variety in eluent composition evaluated for the freshwater
sediment extraction. MeOH or THF were used as the main organic modifier. These were in
some cases accomplished by either dimethylsulfoxide or triethylamine in order to enhance
extraction. Both acidic and alkaline buffers were used as aqueous phase. Furthermore, the
fraction of organic modifier was varied.

Chapter 3 • 45
Table 3.2 – Eluents evaluated for the freshwater sediment extractiona
.
Agents v/v % No. of aliquotsb
Volume of aliquot, mL Recovery, %c
MeOH 100 1 6 0
THF 100 0 6 0
THF:MeOH 80:20 0 6 0
THF:MeOH:DMSO 79:19:2 0 6 0
MeOH:NaHCO3 90:10 1 6 5
MeOH:H3PO4 90:10 5 6 24
THF:H3PO4:DMSO 70:20:10 2 10 31
THF:NaHCO3 90:10 5 6 38
THF:H3PO4 95:5 5 6 49
THF:H3PO4:Et3N 80:20:5mM 5 6 54
THF:H3PO4:DMSO 40:50:10 5 10 67
THF:H3PO4:DMSO 79:19:2 5 6 77
THF:H3PO4 80:20 4 10 95
THF:H3PO4 50:50 5 10 99
v/v %: volume/volume %, THF: Tetrahydrofuran, MeOH: Methanol, DMSO: Dimethylsulfoxide, H3PO4: 10
mM pH 2.5, Et3N: Triethylamine, NaHCO3: 10 mM pH 10,
a
: Holten Lützhøft et al. (Submitted) I, b
: The figures represent the number of aliquots where detection of OXA
was possible. For the 6 mL aliquots, the sediment was always extracted five times. For the 10 mL aliquots, the
sediment was always extracted seven times. c
: Extractions were performed in duplicate.
THF was selected as the organic modifier, since eluents including THF showed the most
efficient extracting capacity. Furthermore, THF could easily be evaporated under N2 at
elevated temperature. 10 mM H3PO4 pH 2.5 was selected as the buffer, in a concentration of
20 % in THF, allowing a five time concentration step due to removal of THF. Three aliquots
of 10 mL were selected, since the fourth extraction did not improve the recovery significantly.
The following recovery study was performed, see Table 3.3 for details. 50 mL buffer pH 7
added 5 g dry sediment was spiked with OXA in three levels. The flasks were thoroughly
mixed and stored three days in the dark at 4°C. Sediment was extracted in triplicate using the
procedure described above. The aqueous phase was analysed in duplicate in order to establish
a mass balance. The obtained recovery was 98 % with a relative standard deviation of 36 %.
This was attributed to the heterogeneity of the sediment.

Table 3.3 – Recovery study for the extraction of OXA from freshwater sedimenta
.
Recovery
VAQ, mL mS, g mOXA spiked, µg mOXA(AQ), µg mOXA(S), µg Totalb
, % Sedimentc
, %
50 5.45 25.34 5.05 18.46 93 91
50 5.43 50.67 9.55 30.24 79 74
50 5.85 101.34 23.40 99.81 122 128
98±28d
98±36d
a
: Holten Lützhøft et al. (Submitted) I, VAQ: volume of the aqueous phase, mS: mass of sediment, mOXA spiked:
mass of OXA spiked to the sediment sample of the former column, mOXA(AQ): mass of OXA in the aqueous
phase, mOXA(S): mass of OXA in the sediment,
b
:
( )
spikedOXA
OXA(S)AQOXA
m
mm
100recoveryTotal
+
⋅= , c
:
( )AQOXAspikedOXA
OXA(S)
mm
m
100recoverySediment
−
⋅= , d
: Overall
recovery±relative standard deviation (n=9).

Table 3.4 – Sediment extraction methods for the investigated quinolones.
Antimicrobial Sediment mass, g Spike, µg/g Equilibration, h Extraction Concentration step Recovery±RSD ref.
FLU marine 1 25 - 4, 4 and 2 mL 0.1 M NaOH
Centrifugation
Combine supernatants
Centrifugation
no 95.7±2.8a
A
OXA marine 5 - - 3×20 mL 0.1 M KH2PO4 pH 7
Homogenization
Centrifugation
Filtration
Combine filtrates
C18 SPE
Elute with 5 mL
MeOH:1 M H3PO4
90:10
Evaporation at 35ºC
at reduced pressure
to 0.5 mL
70.9±5.1b
B
OXA marine 1 25 - 4, 4 and 2 mL 0.1 M NaOH
Centrifugation
Centrifugation
no 92.6±3.1a
A
OXA marine 1 0.1-2.5 - 3×4 mL 0.2 M NaOH
Homogenization
Centrifugation
Add 2.5 mL 1 M HCl
Extract with 4 mL
EtOAc:CHCl3 1:1
Homogenization
Centrifugation
Evaporation of the
organic phase under
N2 at 35ºC
Dissolve in 0.5 mL
mobile phase
68.1±1.7 (n=30) C

OXA mud
sandy mud
sand
1 0.05-0.8 1 h do do 58.3±5.2 (n=15)
70.9±4.7 (n=15)
90.2±4.0 (n=15)
D
OXA freshwater 0.1 4,900-19,600 72 h Extract sediment filled SPE
tubes with 3×10 mL 20 % 10
mM H3PO4 pH 2.5 in THF
Reduction to 6 mL
under N2 at 36ºC
Filtration
98±36 (n=9) E
RSD: relative standard deviation, SPE: solid phase extraction, MeOH: methanol, EtOAc: ethylacetate, THF: tetrahydrofuran, -: not reported,
a
: mean±standard deviation, b
: mean±coefficient of variation
A: Samuelsen et al. (1994), B: Bjørklund (1990) and Bjørklund et al. (1991), C: Pouliquen et al. (1994b), D: Pouliquen et al. (1994a) calcium, magnesium, zinc, iron, aluminium,
fraction < 63 µm, organic matter significant correlate negatively with recovery, E: Holten Lützhøft et al., (Submitted) I

Chapter 3 • 49
3.3. Antimicrobials in Environmental Samples
Environmental occurrence of antimicrobials has mainly been studied in Norway and Finland,
though studies in Italy and Ireland have also been conducted. An extensive simulation study
was performed in France. The occurrence has so far only been reported in the marine
environment. Table 3.5 lists the literature on the environmental occurrence of the
antimicrobials investigated in this thesis. With nine references, OTC has been in focus. Four
investigations report the finding of OXA and only two addresses the finding of FLU.
3.3.1. Marine occurrence
The vast majority of experiments have been undertaken with sediment, though a few studies
address the occurrence in wild fauna and one the experimental determination in water.
Near or around fish farms OTC sediment concentrations up to 10 µg/g are frequently reported
(Jacobsen and Berglind, 1988; Bjørklund et al., 1990a; Bjørklund et al., 1991; Pouliquen et
al., 1992; Coyne et al., 1994). However, one study reports up to 281 µg/g (Samuelsen et al.,
1992b). OTC is shown to distribute to deeper layers (Samuelsen et al., 1992b; Coyne et al.,
1994). Usually OTC persists for up to several weeks (Jacobsen and Berglind, 1988; Bjørklund
et al., 1990a; Coyne et al., 1994), but in the case of 281 µg/g still 15 µg/g was found 18
months after (Samuelsen et al., 1992b). A few studies also reported the half-life in sediment
(Bjørklund et al., 1990a; Pouliquen et al., 1992; Samuelsen et al., 1992b; Coyne et al., 1994).
It ranges from 9 to 419 days. It was concluded that the half-life was mainly affected by water
current, exposure, ratio that reach sediment, area and how deep OTC distributes in the
sediment (Bjørklund et al., 1990a; Coyne et al., 1994). Moreover, disappearance is mainly due
to leakage and out washing and to a minor extent degradation, since no biotransformation
products could be detected (Bjørklund et al., 1990a).
In a study performed in France OTC medication was simulated in a tank system. One tank
was medicated and the water flew to other tanks containing sediment and shellfish. Water
concentrations up to 250 µg/L was reported 14 days after medication (Pouliquen et al., 1993).
Sediment concentrations at the same time were up to 1.96 µg/g (Pouliquen et al., 1992). After
14 days, the shellfish still contained up to 0.7 µg/g, the highest concentration was reported to
1.42 µg/g (Le Bris et al., 1995).

A comparative study calculated the aqueous concentration 1 cm above the sediment to be 16
and 110 µg/L, one day after medication (Smith and Samuelsen, 1996). However, under the
marine circumstances the bioavailable concentrations would be diminished due to complex
formation and would not be harmful to the micro-fauna.
In one study, OTC has been reported in wild fish. At the last day of medication up to 1 µg/g
was found, however, 13 days after only traces could be found (Bjørklund et al., 1990a).
OXA has been reported in both wild fauna and sediment. In one case 0.12 µg/g was reported
in wild fish two days before medication of the farmed fish. In another case, up to 4.89 µg/g
was found in wild fish, post medication. The wild fish were sampled up to 50 m from the fish
farm (Ervik et al., 1994). Another investigation reported OXA concentrations up to 4.4 µg/g
in wild fish, 0.65 µg/g in blue mussels and 0.81 µg/g in crab sampled up to 400 m from the
medicated location (Samuelsen et al., 1992a). However, compared to OTC lower levels are
found in sediment. Concentrations up to 0.2 µg/g are reported and OXA seems to disappear
fast from sediment, since OXA could not be detected 6 days post medication. As for OTC,
OXA is suggested to leave the sediment rather than being degraded (Bjørklund et al., 1991).
Ervik et al. (1994) also found FLU concentrations of 0.95 µg/g in wild fish 1 day after
medication. At the outflow of a pond in Italy, sediment concentrations in ng/g were found,
whereas aqueous concentrations up to 49.82 µg/L were reported (Migliore et al., 1996).
As seen from Table 3.5 the quantity applied for a treatment with OTC is higher than for the
quinolones. Additionally, the bioavailability of OTC is much lower than for OXA. This may
explain that OTC concentrations found in sediment are accordingly higher.
3.3.2. Freshwater Occurrence
One investigation reports the environmental occurrence in a freshwater habitat (Holten
Lützhøft et al., Submitted I). In co-operation with a Danish fish farm in Jutland, sediment was
sampled before and after an OXA treatment. Samples were taken at the inlet, within the fish
farm, at the outlet and 300 m downstream the fish farm outlet. The analysis and extraction
methods described above were applied to the sediment samples.

Chapter 3 • 51
There was no clear correlation between sampling time/location and sediment concentration.
However, 21 days post treatment a sediment sample was taken 300 m downstream the fish
farm. An OXA concentration of 1.6 µg/g was found. From the results two conclusions was
drawn:
• It is very important that the sediment is sampled at the same location from time to
time, and that the depth of the sample is measured.
• After antimicrobial treatment OXA can be found in sediment samples nearby the fish
farm – even 300 m downstream the outlet.
This indicates that medication of Danish fish farms may result in antimicrobial residues in the
environment near the farm site.
The result, that OXA was present 21 days post treatment, is in contradiction to the findings in
the Baltic (Bjørklund et al., 1991). At that location, OXA could not be detected 6 days post
treatment. Approximately the same quantity was applied, but two differences are obvious:
sampling location and sediment type. In the study by Bjørklund et al. (1991) the sediment was
from the Baltic and therefore of marine origin, whereas the sediment in the study of Holten
Lützhøft et al. (Submitted) I was from a small freshwater stream. Due to the location, a much
higher dilution effect can be expected in the Baltic. According to Pouliquen et al. (1994a)
sediment of sandy character is better extracted than muddy sediment. However, neither
sediment was further described, but sediment from the Baltic appears to be more sandy than
freshwater sediment, and may therefore easier release OXA.

Table 3.5 – Environmental occurrence of the investigated antimicrobials.
Antimicrobial Total load, kg Compartmenta
Concentration, µg/g or Lb
Days after treatment Distance from fish farm ref.
FLU 6 wild fishNO
0.95c1
1 up to 50 m A
FLU - marine sedimentIT
0.00002-0.00618 - outflow from pond B
FLU - salt waterIT
0.01-49.82 - do B
OXA 34 wild fishNO
4.4d1
1.8d2
0.09d3
0.01d4
0
4
7
13
up to 400 m C
OXA do blue musselsNO
0.65d5
0.05d6
0d7
0
4
7
do C
OXA do crabNO
0.81d8
0.45d9
0.08d10
0.03d11
0
4
7
13
do C
OXA 9.84 wild fishNO
0.32d12
0.004d13
0
7
up to 400 m C
OXA do crabNO
0.005d14
0.047d15
0.002d16
0
4
7
do C
OXA 0.36 marine sedimentFI
0.2
0
0
6
nearby D
0.05
0
0
6
do D
0.2
0
during treatment
6
do D

OXA 34 wild fishNO
4.89c2
0 up to 50 m A
OXA 10 wild fishNO
0.58c3
0 do A
OXA 26 wild fishNO
2.41c4
1 do A
OXA 20 wild fishNO
0.12c5
1.00c6
-2
1
do A
OXA 1.4 wild fishNO
1.02c7
1 do A
OXA 0.595 freshwater sedimentDK
1.6 21 300 m downstream E
OTC - marine sedimentNO
0.13e
70 under cage F
4.19f
84 do F
0f
1.2g
7 do F
2.4 and 3.6f
28 do F
0.46 and 1.0g
28 do F
OTC 1.6 wild fish – bleakNO
0.2-1.3
0.06h
traces
0
1
7
nearby G
OTC do marine sedimentFI
0.1
0
0
8
around treated pen G
OTC 1.78 wild fish – BleakFI
wild fish – RoachFI
traces
0.06
0.05
traces
1
1
2
13
nearby G
OTC do marine sedimentFI
1-3.8
16i
1-4.4
1
8
308
around treated pen G
OTC 2.6 marine sedimentFI
6.4
2.6
7
12
nearby D

4.4
3.5
during treatment
12
do D
1.9
0.8
1
12
do D
OTC 0.8625 marine sedimentNO
192j
1.7j
10
560
under cage H
OTC 1.6875 marine sedimentNO
281j
15j
75
560
do H
OTC not treated marine sedimentNO
30j
1.7j
75
560
do H
OTC 1.6875 salt waterNO
110k
1 do I
OTC 0.315 marine sedimentFR
3.47-4.19
1.50-1.96
0
14
tank systeml
J
OTC do salt waterFR
2,000-2,500
125-250
0
14
do K
OTC do Crassostrea gigasFR
1.42
0.7
0
14
do L
OTC do Ruditapes philippinarumFR
0.5
0.4
during treatment
14
do L
OTC do Scrobicularia planaFR
1.0
0.62
during treatment
14
do L
OTC 47 marine sedimentIR
9.9m
2.3m
1.6i,m
3
32
66
under cage M
OTC 169.5 marine sedimentIR
10.9m
3.3m
1.6m
during treatment
19
33
do M
OTC do salt waterIR
16k
1 do N

a
: NO: Norway, IT: Italy, FI: Finland, DK: Denmark, FR: France, IR: Ireland, b
: g for fish and sediment. L for water, c:
Percent positive samples of one or several of the following fish:
Saithe, Cod, Ling, Ballan wrasse, Pollack, Haddock, Mackerel, Whiting, Flounder or Salmon, c1
: 77 % of 31, c2
: 100 % of 32, c3
: 87 % of 15, c4
: 88 % of 42, c5
: 17 % of 24, c6
: 69 % of
39, c7
: 77 % of 30, d
: Percent positive muscle samples of several of the following fish: Coalfish, Ballan wrasse, Ling, Haddock, Salmon, Cod or Pollack unless otherwise stated. At
sampling location d1-d11
additionally 660 tonnes of fish were treated in the same area during the sampling. This may explain the relatively higher concentrations compared to sampling
location d12-d16
, d1
: 100 % of 42, d2
: 97 % of 32, d3
: 93 % of 27, d4
: 58 % of 33, d5
: 100 % positive homogenate samples of 5 Blue mussels, d6
: 60 % positive homogenate samples of 5
Blue mussels, d7
: 0 % positive homogenate samples of 3 Blue mussels, d8
: 100 % of 5, d9
: 85 % of 13, d10
: 60 % of 10, d11
: 33 % of 12, d12
: 74 % of 19, d13
: 20 % of 5, d14
: 50 % of 4, d15
:
36 % of 22, d16
: 9 % of 4, e
: Water level 10 m, f
: Water level 20 m, g
: Water level 40 m, h
: 1 sample out of 8, i
: One sample, j
: Not specified but most likely the upper 2 cm. Profiles
were made and OTC was detected 12 cm down in the sediment 245 days after treatment. The profile indicated OTC to move downwards due to time. This may be do to further
sedimentation of particulate matter, e.g. faeces, k
: In the water 1 cm above the sediment, l
: Simulation of a fish farm. Water from a polluted tank flew to three other tanks from which
samples were taken, m
: In the upper 2 cm. Profiles were made and showed OTC concentrations in deeper layers to increase until 19 days after treatment, data from Smith and
Samuelsen (1996),
-: not reported,
A: Ervik et al. (1994) Probably obtained by eating surplus medicated feed from the farm, B: Migliore et al. (1996), C: Samuelsen et al. (1992a), D: Bjørklund et al. (1991), E: Holten
Lützhøft et al. (Submitted) I, F: Jacobsen and Berglind (1988), G: Bjørklund et al. (1990a), H: Samuelsen et al. (1992b), I: In Smith and Samuelsen (1996) calculated from Samuelsen
et al. (1992b), J: Pouliquen et al. (1992), K: Pouliquen et al. (1993), L: Le Bris et al. (1995), M: Coyne et al. (1994), N: In Smith and Samuelsen (1996) calculated from Coyne et al.
(1994).

Chapter 4
Environmental Fate of
Antimicrobials

58 • Environmental Fate of Antimicrobials
4.1. Introduction
The environmental fate of chemicals is an important factor in the ERA procedure (Berg et al.,
1995) and is required for veterinary pharmaceuticals that have direct entry into the aquatic
environment (EMEA, 1998b). The physical chemical properties of the chemicals determine
the distribution between solids/organic phases and the aqueous phase. Additionally the
inherent properties contribute to whether the chemicals will be degraded or not. The physical
chemical properties therefore affect both the occurrence and to what extent the chemicals will
show effects on organisms in the environment.
Since the majority of the work in this thesis has been carried out on the 4-quinolones, FLU,
OXA and SAF will be in focus, especially in the section describing the distribution properties.
The chapter will focus on the antimicrobial interaction with environmental constituents, e.g.
humic acids and sediment, and the likelihood of antimicrobial degradation in the environment,
e.g. biodegradation, hydrolysis and photolysis. A conceptual diagram of
mentioned/interfering processes was shown in Figure 1.1.
4.2. Environmental Distribution
KOW is often used to predict chemical behaviour e.g. Geyer et al. (1984), Di Toro (1985) and
Nendza and Hermens (1995). Quantitative structure activity relationships (QSARs) have been
developed to predict/estimate the interaction with solids, organic carbon (OC) or DOC using
the KOW for the chemical. Equation 4.1 and Equation 4.2 are examples of such QSARs.
Log KOC = 0.983·log KOW + 0.00028
Equation 4.1
Log KOC = 0.52·log KOW + 0.64
Equation 4.2
Equation 4.1 was developed for pesticides with log KOW values in the range 1 to 7 (Di Toro,
1985) and Equation 4.2 was developed for various chemicals with log KOW values in the
range –0.6 to 7.4 (Briggs, 1981).
This makes sense only if the chemicals' affinity for 1-octanol, i.e. its hydrophobicity, reflects
its affinity for DOC. The equations moreover assume a hydrophobic nature of the interaction
(Berg et al., 1995), which for several organic pollutants have shown to work (McCarthy and

Chapter 4 • 59
Jimenez, 1985; Day, 1991; Kukkonen and Oikari, 1991). However, it is advised not to apply
mentioned equations to ionizable chemicals due to more complex interactions, i.e. various
electrostatic interactions (Nendza and Hermens, 1995; EMEA, 1998b).
4.2.1. pH-dependent 1-octanol/water distribution
As discussed in Chapter 2, the hydrophobicity of antimicrobials is pH dependent and
decreases when they become ionized. To evaluate the distribution of ionized species the
influence of pH on DOW was investigated for OXA (Holten Lützhøft et al., 2000 II). The
experimental data appeared to fit Equation 2.1, which only uses the ionization constant and
the true partition coefficient. This means that only the neutral species distribute to 1-octanol,
and indicates that DOW may be a good predictor for DOC interaction, under the assumption
that only hydrophobic interactions govern the binding to DOC.
Figure 4.1 shows the pH-dependent DOW for OXA including data from literature.
Figure 4.1 – Experimental 1-octanol/water distribution for OXAa
.
2 4 6 8 10 12
0
2
4
6
8
10
12
pH
DOW
a
: Holten Lützhøft et al., (2000) II, curve represents the fitting of individual measurements ( ) to Equation 2.1.
data from Takács-Novák et al. (1992), data from Hirai et al. (1986), and data from Bjørklund and Bylund
(1991).
These results confirm that the hydrophobicity of antimicrobials decreases when the chemicals
become ionized and that the DOW for OXA follows the understanding of distribution of
ionized species. For weak (carboxylic) acids, this happens when pH increases and vice versa
for weak bases. It is therefore expected that the ionized species will not interact with DOC

60 • Environmental Fate of Antimicrobials
and due to their inherent low KOW values, the interaction of the neutral form will furthermore
be negligible.
Using the QSARs outlined above to predict partition to OC (KOC) at neutral pH for the
investigated antimicrobials produces values of the same magnitude as their log KOW values,
see Table 4.1 and Table 2.2. When pH increases, weak carboxylic acids, e.g. FLU and OXA,
become increasingly ionized. In theory, when pH increases this should result in less and less
interaction with DOC, due to the decreasing part of the neutral molecule.
Table 4.1 – Estimated and experimental distribution coefficients.
QSAR estimated Log KOC
a
Antimicrobial Equation 4.1 Equation 4.2 Log DDOC
b
Log DSED
c
FLU 1.1 1.2 4.2 2.3
OXA 0.4 0.8 4.5 2.7
SAF -1.2 0.0 4.7 2.7
SDZ -1.0 0.1 4.5d
2.7d
TMP 0.6 1.0 4.3d
2.3
AMX -1.5 -0.1 4.6d
2.8d
OTC -1.2 0.0 4.4-5.0e
2.7f
a
: Estimated according to Equation 4.1 (Di Toro, 1985) and Equation 4.2 (Briggs, 1981) using log DOW from
Table 2.2, b
: Holten Lützhøft et al. (Accepted) III pH 7, c
: Holten Lützhøft (Unplublished) freshwater sediment
pH 7, d
: based on linear regression (log DDOC or log DSED vs. log DOW from Table 2.2) of the other values of that
column, see Figure 4.3, e
: Rabølle and Spliid (2000) soil experiments pH 5.6-6.3, f
: Lai et al. (1995) freshwater
sediment pH 7.7.
Nevertheless, the use of KOW to predict interaction with DOC works for hydrophobic
chemicals but applying this estimation method to antimicrobials results in estimates deviating
from experimental data, see below.
4.2.2. Experimental Distribution Coefficients
The major parts of natural occurring DOC are fulvic and humic acids, of which humic acids
is the major constituent (Masini, 1993), and also possesses the highest binding capacities (De
Paolis and Kukkonen, 1997). These macromolecules contain hydrophobic cavities enabling
hydrophobic interactions. On the other hand, antimicrobials are chemicals with a variety of
functional groups enabling other interactions than just hydrophobic interactions, for chemical
structures see Table 2.2.

Here are the key points made in the introduction:- Until recently, limited attention was paid to the potential environmental risks of antimicrobial use, including in fish farming. - Fish farming involves direct exposure of antimicrobials to the aquatic environment, as production water undergoes only simple wastewater treatment.- This PhD thesis will focus on antimicrobial use in Danish fish farms and evaluate the environmental risks. - The main antimicrobials used in Danish fish farms will be investigated to understand their environmental fate and effects.- An environmental risk assessment will be conducted to evaluate potential risks to the aquatic environment from antimicrobial use in fish farms.- The objectives are to study the environmental occurrence, fate

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