Aquaculture Nutrition

doi: 10.1111/anu.12121 2014 20; 712–721

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1 1 1 2 3

1 4
1 2 3 4
NIFES, Bergen, Norway; Nofima, Sunndalsøra, Norway; EFPRA, Brussels, Belgium; EWOS Innovation AS, Dirdal,
Norway

Concern of fish meal and fish oil availability to supply a

Residue levels of the antibacterials enrofloxacin and cipro-
rapidly growing aquaculture industry has led to the devel-
floxacin were analysed in 15 commercially relevant animal
opment of aquafeeds in which marine resources are
by-products (ABPs). Enrofloxacin was detected in all
replaced with alternative feed ingredients (Tacon &
ABPs, and ciprofloxacin was detected in 11 of 15 ABP
Metian 2008; Torrissen et al. 2011). On a global basis,
samples. Feed to muscle and skin carry –over of low back-
animal by-products (ABPs) from the rendering industry
ground enro- and ciprofloxacin levels were assessed by
constitute one of the largest source of high-quality animal
applying a simple toxicokinetic model. The muscle and skin
protein and lipid available for animal feed production
uptake and elimination rates were established in Atlantic
(Toldra et al. 2012). Examples of such products are
salmon (Salmo salar L.) fed enrofloxacin enriched diets
feather meal, poultry by-product meal, meat and bone
(100 lg kg
1 ‘low’ and 4000 lg kg
1 ‘high’) in triplicate
meal and blood meal. Several studies have shown that
for 41 days followed by a 90 days depuration period. The
there is a large potential for partially replacing fish meal
terminal half-lives were 17  0.4 and 18  0.7 days, and
and fish oil with ABPs in Atlantic salmon (Salmo salar)
uptake rates were 9.3  3.3 and 11  3.1 (day
1) for the
and rainbow trout (Oncorhynchus mykiss; Bransden et al.
‘low’ and ‘high’ groups, respectively. Only fish fed high
2001; Rosenlund et al. 2001; Yanik et al. 2003; Rahnema
background levels had quantifiable levels of the metabolite
& Borton 2007; Wilson et al. 2007; Friesen et al. 2008;
ciprofloxacin with a formation of 0.25  0.01% day
1.
Poppi et al. 2011; Burr et al. 2012). However, following
The toxicokinetic carry-over model predicted muscle and
the peak outbreaks of transmissible spongiform encephal-
skin steady state levels of 1.8 lg kg
1 when fed theoreti-
opathies in the UK in the early 1990s, the use of all pro-
cally high enrofloxacin levels (158 lg kg
1), which is below
cessed animal proteins (PAPs) in animal feeds was banned
the EU limit of 100 lg kg
1 for enrofloxacin in finfish food
in the EU (EC 2001, 2003). Following a bovine spongi-
products. The antibacterial residue levels could however be
form encephalopathies risk assessment by the European
detected in EU food surveillance programmes.
Food Safety Authorities (EFSA 2011), the EU has
recently set out a working plan for the re-authorization of
KEY WORDS: animal by-products, antibacterials, carry-over
the use of non-ruminant PAPs in animal feeds, initially
model, farmed fish, toxicokinetics
for aquafeeds (EC 2013).
The use of ABPs in salmon feeds can potentially intro-
Received 26 July 2013; accepted 21 August 2013
duce new chemical undesirables that previously have not
Correspondence: M.H.G. Berntssen, NIFES, N-5817 Bergen, Norway.
E-mail: marc.berntssen@nifes.no
been associated with farmed Atlantic salmon. Of special
interest are therapeutic agents such as antibacterials, which
are used in poultry and swine farming (Toldra et al. 2012).
Enrofloxacin is a member of the fluoroquinolone antimi-
crobial family and is widely used to treat various systemic
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ª 2014 John Wiley & Sons Ltd

 bacterial infections in terrestrial (Glisson & Hofacre 2006)
and aquatic animals (della Rocca et al. 2004; Samuelsen
2006; Xu et al. 2006; Koc et al. 2009; Fang et al. 2012;
Liang et al. 2012). Enrofloxacin is de-ethylated to cipro-
floxacin, a metabolite that contributes to the activity of
enrofloxacin (Intorre et al. 2000; Glisson & Hofacre 2006)
and legislation on upper limits in food products is based
on the sum of these two antibacterials (EC 2009a). A spe-
cific characteristic of both enrofloxacin and ciprofloxacin is
that it is deposited in high concentrations and persists in
cartilaginous tissues such as poultry feathers (Martin et al.
2007). Accordingly, enrofloxacin and ciprofloxacin residues
in feather meal can be a source of food antimicrobial con-
tamination, when fed to food producing animals (Martin
et al. 2007; Love et al. 2012).
In the US, the use of enrofloxacin was banned in 2005
with a legal limit of ‘absence of enrofloxacin’ in food prod-
ucts (FDA 2005). In the European Union (EU), the use of
enrofloxacin in food producing animals is still permitted
and a maximum residue limit (MRL) for enrofloxacin plus
ciprofloxacin residues in finfish muscle plus skin has been
set at 100 lg kg
1 (EC 2009a). In Norwegian salmon aqua-
culture, enrofloxacin has previously been used in small
quantities, but its use has not been reported in the last dec-
ade (Grave et al. 2008). The control of unintended back-
ground levels of enrofloxacin and ciprofloxacin in salmon
feed ingredients, as well as the potential carry-over to edi-
ble parts of the fish, are important to ensure compliance
with food safety legislation. In addition, concern has been
raised for the development of enrofloxacin resistant patho-
genic microorganisms in fish farming even when antibacte-
rials are present in low and non-clinical doses (Blazquez
et al. 2012; Gillings 2013).
The carry-over assessment of contaminants from feed to
animal food products is essential for establishing acceptable
feed residue levels that guarantees food safety compliance
(Leeman et al. 2007). In addition, such studies are impor-
tant for the harmonization of contaminant legislation
throughout the food production chain (van Raamsdonk
et al. 2009). Toxicokinetic models are used to assess the
feed-to-fillet carry-over of low feed residue contaminant
levels over extended periods, including farming conditions
such as feeding rate and fish growth (Van Eijkeren et al.
2006; Berntssen et al. 2007; Balshaw et al. 2008). Tissue
levels in these feed-to-fillet models are described as a func-
tion of dietary contamination and feed intake (FI), uptake
and biotransformation-elimination rates, and the degree of
growth dilution (Opperhuizen & Sijm 1990; Sijm et al.
1992). Pharmacokinetic properties of enrofloxacin during
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Aquaculture Nutrition, 20; 712–721 ª 2014 John Wiley & Sons Ltd
high-dose and short-term (≤10 days) clinical treatments
have been well studied in several farmed fish species,
describing factors such as relative bioavailability and tissue
levels in relation to pathogen inhibition and resistance
(reviewed by Samuelsen 2006). Clinically administered
enrofloxacin is relatively persistent in muscle plus skin
(Stoffregen et al. 1993, 1997; Lucchetti et al. 2004), with
relatively longer depletion time and higher oral bioavail-
ability compared with other quinolones (Martinsen &
Horsberg 1995). These characteristics indicate that muscle
and skin together are a ‘drug sink’ for enrofloxacin and the
metabolite ciprofloxacin (Stoffregen et al. 1997) when fish
are fed low background enrofloxacin levels over extended
periods of time.
This study investigates the long-term (>1.5 months) accu-
mulation and elimination (>3 months) of low background
levels of dietary enrofloxacin and the metabolite ciprofloxa-
cin in Atlantic salmon. Uptake and elimination rate con-
stants for enrofloxacin and biotransformation into
ciprofloxacin are estimated and are applied in a simple
accumulation model to predict levels in fillet and skin from
known feed levels at different growth and feeding rates
during continuous long-term exposures. This article is part
of a series of experiments that address the use of ABPs in
Atlantic salmon farming as alternatives to fish oil and fish
meal in fish feed.
A total of 15 commercially available none ruminant ABP
products were provided by the European Fat Processors
and Renderers Association (EFPRA). The ABPs were
obtained from 13 different producers located within the
European Union. The producers were located in Italy, Eng-
land, Denmark, the Netherlands, Germany, France,
Belgium, Sweden and Spain. All ABPs were produced
according the EU regulation for ABPs intended for use as
feed ingredients in animal feed (EC 2001, 2009b). The
ABPs are category 3 products that are fit for human con-
sumption at the point of slaughter (EC 2009b). The tempo-
rary ban of certain animal proteins in farmed animal diets
(EC 2001) was in the meantime lifted for non-ruminant
processed animal protein (PAP) in aquaculture (EC 2013)
The ABPs sourced included poultry feather meal (n = 3),
poultry and pork PAP low ash (n = 1 and n = 2,
respectively), poultry and pork high ash(n = 3 and 2,
respectively), blood meal (n = 1), pork greaves (n = 2), and
poultry fat (n = 1). All samples were taken from the indus-
trial production process and not produced only for this
project. Samples 100 g were collected in plastic bags and
stored in the dark at 4 °C until analysis within 2–3 months
of collection.
Locally bred Atlantic salmon (S. salar) postsmolt were ran-
domly distributed among six fibreglass tanks (500 L;
approximately 100 fish per tank) at EWOS Innovation
research station in Dirdal, N-4335 Norway. The experiment
was performed in the period September 2011–January
2012. At the beginning of the experiment, the weight and
length (fork-tail) of fish were 288  39 g, and
29.7  1.38 cm, respectively (mean  standard deviation;
n = 600). The fish were acclimated to the holding facilities
for 1 month while being fed a commercial diet that had no
detectable levels of enrofloxacin or ciprofloxacin [limit of
detection (LOD) < 0.03 lg kg
1]. After the acclimatization
period, fish in randomly selected triplicate tanks were fed
enrofloxacin enriched diets with nominal levels of 100 and
4000 lg kg
1 for 41 days. The feed concentrations repre-
sented an oral dose of approximately 0.65 and 25 lg kg
1
body weight per day. Following the dietary enrofloxacin
exposure period, the fish were given enrofloxacin-free diets
during and depuration period of 90 days. As no detectable
enro- or ciprofloxacin levels were present in the acclima-
tized fish or the basal diet, no control group was needed to
compensate for potential background enro- and ciprofloxa-
cin levels. Fish were reared under a 12 h light : 12 h dark
regime during the exposure period and fed by automatic
feeders in three meals a day to a level approximating 0.70
g kg-1 of body weight per day. The feeding rate was
adjusted for growth biomass increase, which was assessed
by measured average weight gain of the sampled fish per
sampling time point. Uneaten pellets were collected in a
flow-over system and registered daily thus providing exact
FI values daily. Water temperature, salinity and oxygen
saturation were monitored continuously and varied over
the course of the trial from 8 to 10 °C, 33–35 g L
1 and
75–84%, respectively. To avoid potential water contamina-
tion of enrofloxacin from the diet and faeces, a high water
flow (20–25 L min
1) through the tanks was maintained.
During the accumulation period, three fish from each tank
(n = 9 per sampling point per dietary treatment) were sam-
pled at day 0, 0.5, 1, 2, 3, 4, 5, 6, 13, 24 and 41. During
the depuration period, three fish per tank (n = 9 per
sampling point per dietary treatment) were sampled at day
0, 0.5, 1, 2, 3, 4, 7, 10, 21, 30, 39, 50, 70 and 90. At fish
sampling, the fish were anaesthetized with 3-amino benzoic
acid ethylester (MS-222; approximately 1 g L
1) before
body weight and fork-tail length were measured. Fish were
stored at
28 °C, and at the end of the experiment all fish
were filleted (whole fillet muscle plus skin on the left
side of the salmon) and analysed for enrofloxacin and
ciprofloxacin.
The enrofloxacin fortified diets were produced by top coat-
ing (1%) enrofloxacin-free commercial salmon feeds with
enrofloxacin (Sigma-Aldrich ≥98% HPLC graded, Eichenz-
ell, Germany) enriched fish oil. The lowest level of enro-
floxacin supplementation (100 lg kg
1) was chosen to give
detectable levels of enrofloxacin in fish muscle plus skin
while low enough to represent the upper range of what
could be expected in ABP feeds. The higher enrofloxacin
level (4000 lg kg
1) was also chosen to provide quantifi-
able data of newly formed ciprofloxacin in the edible part
of the fish. Immediately after production, six subsamples
were taken and analysed for enrofloxacin and ciprofloxacin
to test homogeneity of the feed concentrations. Measured
levels (mean  SD, n = 6) were 118  9 and 4089 
25 lg kg
1 for the nominal 100 and 4000 lg kg
1 feed,
respectively. The fortified feeds were stored for 2 weeks at
4 °C before and during the dietary enrofloxacin exposure
period. Feed samples were taken each week and analysed
for enrofloxacin and ciprofloxacin, and the analysis showed
no enrofloxacin degradation during the cold storage.
All fish, feed and ABP samples were analysed for enroflox-
acin and ciprofloxacin by LC-MS/MS after modification of
earlier published methods (VanTran et al. 2006). Briefly,
homogenized samples (1 g) were spiked with internal stan-
dards (D5-enrofloxacin; Sigma-Aldrich, St. Louis, MO,
USA, 13C3,15N-ciprofloxacin; Cambridge Isotope Laborato-
ries, Andover, MA, USA) and extracted with 1% (v/v) ace-
tic acid in methanol during ultrasound treatment (20 min).
The extracts were purified using a column system (ASPEC
XL 4; Gilson, Middleton, WI, USA) equipped with a solid
phase extraction column (Oasis-MCX, 3 cc, 60 mg, 30 lm;
Waters, Milford, MA, USA). The extracts were loaded to
the column and washed with aquadest (2 mL) and metha-
nol (2 mL) before the purified extracts were eluted with
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Aquaculture Nutrition, 20; 712–721 ª 2014 John Wiley & Sons Ltd
 10% (v/v) NH4OH in methanol (1 mL). After elution, the
purified extracts were concentrated, and formic acid added
(98–100%, v/v) was added prior to analysis by rapid reso-
lution liquid chromatography/tandem mass spectrometry
(RRLC-MS/MS; Agilent 1200 RRLC coupled to Agilent
6410B MS/MS, Santa Clara, CA, USA). The RRLC-MS/
MS was set in positive electrospray mode and equipped
with a reverse phase C18-column (150 mm 9 2.1 mm i.d.,
1.8 lm particle size, Zorbax C18; Agilent). The analytes
were separated on the column by isocratic elution
(0.35 mL min
1, 25 °C) by mixing 82% of 1% formic acid
in water (v/v) with 18% acetonitrile as mobile phase with a
run time of 5 min. Capillary voltage was set at 1 kV, and
gas temperature was set at 350 °C. The native enrofloxacin
was monitored using the following MRM transitions;
360.2 m/z?316.2 m/z (quantifier) and 360.1 m/z?245.1 m/
z (qualifier), and the mass labelled internal standard was
monitored using the 365.1 m/z?321.3 m/z transition. Cip-
rofloxacin was monitored using the following MRM transi-
tions; 332.1 m/z?314.2 m/z (quantifier) and 332.1 m/z?
231.1 m/z (qualifier), and the mass labelled internal stan-
dard was monitored using the 336 m/z?318.2 m/z transi-
tion. Quantification was carried out using the internal
standard method with analyte-specific relative response fac-
tors obtained from a linear analyte-specific standard curve
relative to the internal surrogate standard. The method
LOD was set at the analyte concentration giving a peak to
signal of three times the background noise. The method
limit of quantification (LOQ) was determined using three
times the LOD. Recovery was evaluated by spiking sample
matrix with standards for both analytes at 1, 10 and
100 lg kg
1 concentrations. The trueness of the method
was evaluated using recovery experiments and was found
to be between 87% and 120% and the linear in the range
of 0.1–100 lg kg
1 ww. Spiked control samples with native
and mass labelled analyte were analysed in each series to
monitor the quality of the method. Matrix blanks, proce-
dural blanks and instrument blanks were also determined
in each series.
Fish more than tripled their weight during the experiment,
and all fillet concentration data were growth corrected. The
fish growth rates were calculated by fitting fish weight to
the equation; ln fish, weight = a + b*t, where a is a con-
stant, b the growth rate (g day
1) and t the time of experi-
ment. All fillet concentrations (Cfillet) were multiplied by
the factor (1 + b*t) to correct for growth dilution. Long-
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Aquaculture Nutrition, 20; 712–721 ª 2014 John Wiley & Sons Ltd
term accumulation during continuously exposure is mainly
described as a process of overall uptake, terminal (slowest)
elimination, and growth and FI rates (Sijm et al. 1992).
The terminal elimination rate constant (kel) was estimated
from the non-linear least square regression of the terminal
phase of the concentration–time profile. Terminal elimina-
tion half-lives (t1/2) are ln2/kel. The enrofloxacin muscle
plus skin uptake rates were calculated by non-linear
custom fitting (Graphpad Prism software Inc., San Diego,
CA, USA), the muscle plus skin concentration data to the
following integrated form of the kinetic rate Eq. (1)
(Bruggeman et al. 1981):
Cfish ðtÞ  kel
a¼ (1)
F  Cfeed ½1
expð
kel  tÞ
where Cfeed are the total drug concentration (lg g
1 wet
weight) in feed, a is the muscle plus skin uptake rate con-
stant, and F is feeding rate (g feed g
1 fish d
1). Muscle
plus skin uptake rate is the combined outcome of absorp-
tion from intestine to blood, hepatic metabolization of
enrofloxacin into ciprofloxacin, and following distribution
to muscle and skin The uptake rate for the newly formed
ciprofloxacin was used as the biotransformation of enro-
floxacin into ciprofloxacin and the muscle plus skin redis-
tribution of ciprofloxacin.
Fillet accumulation concentrations were modelled using
formula (1) rewritten as Eq. (2), which is a simple model
based on the following equation (Sijm et al. 1992):
aFt
Cfillet ðtÞ ¼ Cfeed ð1
e
ðkel þbÞt Þ þ Cfillet0 e
ðkel þbÞt (2)
Kel þ b
where b is the growth rate and Cfillet0 is the fillet level at
the start of the exposure.
Specific growth rate (SGR) was calculated as SGR = (ln
(W2)
ln(W1))/(t2
t1); where W2 and W1 are weights on
day t2 and t1, respectively.
Feed intake was calculated as FI = (feed intake (g)*fish
weight gain (g)
1) 9 SGR.
The kinetic parameters were calculated on the basis of
observed muscle enrofloxacin and ciprofloxacin concentra-
tions over time. The uptake and elimination rates and
their standard errors were assessed by non-linear fitting,
by least-squares method, using the program Graph pad
Prism 6.0 (Graphpad Prism software Inc., 2012). Statisti-
cal differences for parameters between treatments were
assessed by t-test at a significance level of 0.05 (Zar
1984).
 day
1 and the final weights 986  207 and 929  191 g for
the high and low dietary groups, respectively. No signifi-
cant differences were observed among the two dietary
groups in any of the production parameters measured. No
The levels of enrofloxacin and ciprofloxacin in 15 selected
enrofloxacin or ciprofloxacin was detected (LOD <
ABPs are given in Table 1. Enrofloxacin was positively
0.03 lg kg
1) in the fillets at the start of the trial. Fig-
identified (greater than LOD of 0.03 lg kg
1) in all ABPs
ure 1a–c shows the muscle plus skin levels of enrofloxacin
while ciprofloxacin was positively identified in 11 of the 15
and ciprofloxacin in Atlantic salmon fed either 100 or
ABPs. No ciprofloxacin was observed in pork high ash
4000 lg kg
1 enrofloxacin for 41 days followed by 90 days
meal or pork greaves meal. Both enrofloxacin and cipro-
depuration period (total 131 days). For fish fed, both die-
floxacin were observed in poultry and pork products (for
tary levels of enrofloxacin no apparent steady state in mus-
ciprofloxacin only in pork low ash meal) with highest levels
cle and skin enrofloxacin levels were observed but only fish
in poultry products and in particular feather products.
fed the highest enrofloxacin levels had detectable levels of
Enrofloxacin was the dominant form, and the maximum
ciprofloxacin in muscle plus skin (Fig. 1c). Highest
proportion of ciprofloxacin compared with enrofloxacin
observed levels (mean  SD) for enrofloxacin and cipro-
was 8% and observed in feather meal samples. Highest
floxacin were 30  3.1 and 0.24  0.01 lg kg
1, respec-
levels of enrofloxacin were found in feather meal products
tively, for fish fed the highest level of dietary enrofloxacin.
(68 lg kg
1) followed by poultry high ash meal
For fish fed the lowest dietary level of enrofloxacin,
(53 lg kg
1) and pork low ash meal (13 lg kg
1).
0.58  0.03 lg kg
1 was the highest observed level in fish
of enrofloxacin, and levels of ciprofloxacin were non-
detected in this treatment group. The muscle plus skin
enrofloxacin uptake rates were 11  3.1 and 9.3 
No mortality was observed in the enrofloxacin exposed fish
3.3% day
1 for fish fed the highest and lowest dietary
groups, the SGR during the accumulation phase was
levels, respectively, and no significant differences were
0.94  0.08 and 0.89  0.05% day
1 for high and low die-
observed. The ciprofloxacin formation rate in fish fed the
tary enrofloxacin groups, respectively. The FI for high and
highest enrofloxacin level was 0.25  0.015% day
1. The
low dietary was 0.64  0.04 and 0.60  0.05%
muscle plus skin elimination was biphasic with a rapid ini-
body weight day
1, respectively. During the depuration
tial elimination followed by a slower terminal phase. The
period, the SGRs were 0.91  0.02 and 0.89  0.03%
terminal half-lives did not differ significantly between fish
fed the different dietary enrofloxacin levels and were
Table 1 Levels (lg kg
1) of enrofloxacin and ciprofloxacin in ani- 17  0.4 and 18  0.7 days for high and low dietary enro-
mal by-products (ABPs) floxacin fed fish, respectively. The elimination rate of cipro-
ABP description Ciprofloxacin Enrofloxacin floxacin was monophasic with a half-life of 2.8  0.2 days,
which was significantly (P < 0.001) lower than the enroflox-
Poultry low ash meal-1 0.71  0.11 2.35  0.32
acin half-life (Table 2).
Poultry low ash meal-2 1.31  0.32 12.9  0.22
Poultry high ash meal-1 >0.03 0.58  0.01
Poultry high ash meal-2 4.21  0.23 52.6  0.91
Feather meal-1 1.21  0.14 5.22  0.34
Feather meal-2 5.31  0.23 60.9  0.64
Feather meal-3 4.81  0.24 67.5  0.51
Poultry fat >0.03 1.08  0.32
Pork blood meal nd >0.03 The present study demonstrated that ABPs can be a source
Pork low ash meal-1 0.12  0.15 1.94  1.16
Pork low ash meal-2 1.41  0.32 13.3  0.34
of low background levels of enrofloxacin and ciprofloxacin
Pork high ash meal-1 nd 0.36  0.11 when these ABPs are used in aquafeeds. In the present
Pork high ash meal-2 nd >0.03 study, the analytical method had low detection levels
Pork greaves meal-1 nd >0.03
(<0.03 lg kg
1) which is much lower than the EU-MRL
Pork greaves meal-2 nd >0.03
(100 lg kg
1) set in finfish muscle plus skin samples (EC
Mean  SD of triplicate chemical analyses, ‘>0.03’ indicates posi-
2009a). All of the analysed ABPs had detectable levels of
tively detection (>0.03 lg kg
1) of enrofloxacin and ciprofloxacin,
and ‘nd’ indicates non-detected levels (<0.03 lg kg
1) of cipro- enrofloxacin while 11 of 15 ABPs also had detectable levels
floxacin. of ciprofloxacin. Enrofloxacin was the most dominant form
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Aquaculture Nutrition, 20; 712–721 ª 2014 John Wiley & Sons Ltd

Figure 1 (a–c) Muscle plus skin enro-
floxacin and ciprofloxacin levels
(lg kg
1wet weight, n = 9 per sampling
point) in Atlantic salmon fed diets
enriched with enrofloxacin for 41 days
followed by a 90 days depuration period
(mean  SD). (a) enrofloxacin muscle +
skin levels in fish fed 0.1 mg kg
1, this
group had no detectable ciprofloxacin
levels (<0.03 lg kg
1), (b) enrofloxacin
muscle + skin levels in fish fed
4 mg kg
1 enrofloxacin and (c) cipro-
floxacin muscle + skin levels in fish fed
4 mg kg
1 enrofloxacin.
Table 2 Enrofloxacin and ciprofloxacin uptake or formation rates
rate (a), terminal elimination (kel) constants and half-life (t½),
in Atlantic salmon (Salmo salar L.) fed two levels (0.1 and
4 mg kg
1) of dietary enrofloxacin
Uptake Terminal
formation elimination
rate (% day
1) rates (day
1)
Enrofloxacin-low 9.3  3.3a 0.041  0.005a
Enrofloxacin-high 11  3.1a 0.038  0.008a
Ciprofloxacin1 0.25  0.015b 0.24  0.051b
Values (mean  SD) in the same column with the same super-
script letters are not significantly different (P < 0.05).
1
Formation rate of ciprofloxacin was calculated as the net rate
constant (day
1) of ciprofloxacin formation during the acclimati-
zation period of fish fed high (4 mg kg
1) levels of enrofloxacin,
calculated from the slope of concentration over time.
with highest levels found in feather meal products
(12–67 lg kg
1). Studies with intramuscularly treated poul-
try showed that enrofloxacin was more persistent in feather
than muscle (Martin et al. 2007). This could explain why
high residue levels were found in the feather products in
the present study. A recent study on pharmaceuticals and
personal care products in five poultry feather products on
the US market also showed the presence of both enro- and
ciprofloxacin (Love et al. 2012). Particularly high levels,
ranging from 279 to 1050 lg kg
1 for enrofloxacin and
37–76 lg kg
1 for ciprofloxacin, were reported for two
feather products imported from China. One US feather
product contained enrofloxacin at a level of 48 lg kg
1
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Aquaculture Nutrition, 20; 712–721 ª 2014 John Wiley & Sons Ltd
(a) (b)
(c)
and no detectable ciprofloxacin (Love et al. 2012). In this
study, background levels of enrofloxacin were also
observed in porcine products indicating that enrofloxacin
ABP residues are not limited to feather products.
Terminal
half-lives
(days)
17  0.4a
18  0.7a
2.9  0.2b
The present trial studied the toxicokinetics of enrofloxacin
and the metabolite ciprofloxacin in Atlantic salmon fed a
continuously non-therapeutical low level of enrofloxacin
and ciprofloxacin. Several pharmacokinetic studies have
been published for a variety of fish species that are treated
with clinical enrofloxacin doses (5–50 mg kg
1 BW day
1)
for a relatively short period (either single oral dose or max-
imum 10 days). These studies mainly describe bioavailabil-
ity and maximum concentration (Cmax) in relation to the
pathogen minimum inhibitory concentration (Bowser et al.
1992; della Rocca et al. 2004; Samuelsen 2006; Xu et al.
2006; Koc et al. 2009; Fang et al. 2012; Liang et al. 2012).
The pharmacokinetic studies show a more rapid enrofloxa-
cin elimination from muscle compared with cartilaginous
tissue such as skin or bones (Stoffregen et al. 1993, 1997;
Lucchetti et al. 2004). Accordingly, skin was found to be
the most important deposition reservoir for enrofloxacin-
treated Atlantic salmon (Steffenak et al. 1991). EU legisla-
tion on residues of pharmacologically active substances in
finfish is based on the examination of muscle plus skin in
natural proportions (EC 2009a). In the present study,
terminal half-lifes of enrofloxacin from muscle plus skin (a)
were 17–18 days, which is in the same range as earlier
reported terminal half-lives for clinically treated Atlantic
salmon (Stoffregen et al. 1993). The present study had a
two-phase elimination with a rapid initial phase a slow ter-
minal phase. The slow terminal phase is of importance in
the accumulation potential of enrofloxacin in muscle plus
skin and is one of the kinetic parameters used in the accu-
mulation model. Only the highest dietary enrofloxacin
exposure gave detectable muscle plus skin levels of cipro-
floxacin and estimated daily ciprofloxacin bioformation
(b)
and muscle plus skin accumulation was only 0.25% of the
enrofloxacin dose. Sea bass (Dicentrarchus labrax) treated
with a single oral clinical dose (5 mg kg
1 BW) only had
significant but low ciprofloxacin levels in liver but not mus-
cle (Intorre et al. 2000). Seabream (Sparus aurata L.)
receiving a similar enrofloxacin treatment with a higher
dose (10 mg kg
1 BW) had no detectable levels of cipro-
floxacin in plasma or muscle (della Rocca et al. 2004). Tur-
bot (Scophtalmus maximus) fed a similar dose over a longer
period (7 days) showed low but detectable ciprofloxacin
Figure 2 (a, b) Model predictions of maximum accumulated mus-
levels in muscle (Liang et al. 2012). Low ciprofloxacin and cle plus skin enrofloxacin + ciprofloxacin levels at steady state con-
enrofloxacin plasma area under the curve ratios confirmed ditions over a period of 5 months in Atlantic salmon fed
a low transformation of dietary enrofloxacin into ciproflox- enrofloxacin enriched feeds at 0.1 mg kg
1(a) or 4 mg kg
1 (b).
acin in fish (Liang et al. 2012). In the present study, the Closed lines are the model predictions and open lines are the
observed values. The vertical dotted line indicates the end of the
terminal half-life of ciprofloxacin was approximately sixfold
experimental exposure period after 41 days.
higher than for enrofloxacin, which further contributes to a
relative low ciprofloxacin muscle plus skin accumulation.
floxacin accumulation in fish fed the low and high dietary
exposure groups over an extended period. The predictions
model includes dietary enrofloxacin accumulation as well
The uptake, biotransformation and terminal elimination as ciprofloxacin bioformation and accumulation while
kinetics were implemented in a simple toxico-kinetic accu- using the experimental conditions (feed enrofloxacin level,
mulation model. This model also includes aquaculture FI and growth rates) as input values. The model estimated
parameters such as growth and FI to simulate the potential steady state maximum muscle plus skin enrofloxacin + cip-
feed-to-muscle carry-over of residue levels when Atlantic rofloxacin levels are, respectively, 1.26  0.2 and
salmon are reared under different aquacultural conditions. 50.2  2.3 lg kg
1 for low and high exposed fish. The
As no apparent steady state in enro- or ciprofloxacin accu- model was further used to estimate muscle plus skin enro-
mulation was observed during the 1.5 month exposure per- floxacin + ciprofloxacin levels in Atlantic salmon reared
iod of the present trial, the model was used estimate with future ABP-based feeds. To simulate realistic salmon
enrofloxacin plus ciprofloxacin accumulation beyond the farming conditions, growth and FI data (FI of
current experimental period. Steady state conditions are 0.51% BW day
1 and growth rate of 0.53 g day
1) were
likely to be reached at five time half-lives, which would give taken from a large-scale study with consumer-sized fish
maximum estimated muscle plus skin levels after at least (approximately 5 kg) that had a similar temperature regime
3 months of continuous dietary enrofloxacin exposure. In as the present study (7–10 °C) (Lock et al. 2011). To simu-
contrast, ciprofloxacin has a far shorter half-live than enro- late potential enro- and ciprofloxacin background levels in
floxacin with expected maximum steady state accumulation ABP-based salmon feeds, the highest enro- and ciprofloxa-
after approximately 0.5 months of exposure. Figure 2 cin levels in feather products analysed from the present
shows the modulated and observed enrofloxacin plus cipro- study (68 lg kg
1 for enrofloxacin and 5.3 lg kg
1 for
..............................................................................................

Aquaculture Nutrition, 20; 712–721 ª 2014 John Wiley & Sons Ltd
 ciprofloxacin) and in the study of Love et al. (2012)
(1050 lg kg
1 for enrofloxacin and 76 lg kg
1 for cipro-
floxacin) were used. With an assumed dietary ABP inclu-
sion level of 15% (Bransden et al. 2001; Poppi et al. 2011),
the theoretical lowest and highest ABP salmon feeds levels
were 10 and 158 lg kg
1 for enrofloxacin and 0.8 and
11 lg kg
1 for ciprofloxacin. The model predicts minimum
and maximum muscle plus skin enrofloxacin + ciprofloxa-
cin values of, respectively, 0.12 and 1.8 lg kg
1, which is
below the EU limit of 100 lg kg
1 for finfish food prod-
ucts. The current model does not compensate for possible
temperature effect on enro-and ciprofloxacin muscle plus
skin accumulation. Temperature can have an effect on anti-
biotic metabolism For example, a temperature increase of
1 °C corresponded to a 10% increase in metabolic and
elimination rates of oxytetracycline in sea bass (Rigos et al.
2002a), and a similar effect on elimination rates was
observed for oxolinic acid (Rigos et al. 2002b). Liang et al.
(2012) reported a decreased elimination at lower tempera-
tures in turbot exposed to oral enrofloxacin. In contrast,
Bowser et al. (1992) found no significant effect of tempera-
ture on elimination rates in oral-dosed fingerling rainbow
trout. Further enrofloxacin trials for Atlantic salmon are
needed to assess the temperature-related toxicokinetics for
dietary enrofloxacin. In the present study, the enrofloxacin
was dissolved in fish oil and might be more bioavailable
compared with enrofloxacin present in feather meal because
cartilaginous tissue readily retains enrofloxacin.
In the European Union (EU), a MRL for enrofloxacin
including ciprofloxacin residues in finfish muscle and skin
has been set at 100 lg kg
1 (EC 2009a). In the current
study (also that of Love et al. 2012), levels of enro- and cip-
rofloxacin were detected when analysing different commer-
cial ABPs. Using an estimate of dietary inclusion of ABPs
in salmon feeds, the highest possible ABP-feed levels would
give total enro- and ciprofloxacin levels in salmon muscle
plus skin of 0.12–1.8 lg kg
1. This range is well below the
current MRL for enrofloxacin and ciprofloxacin in finfish
food products. However, this level of enro- and ciprofloxa-
cin in muscle plus skin of salmon could be detected in sur-
veillance programmes, depending on the LOD or
quantification (LOQ) used in the analytical method. LODs
and LOQs can vary between analytical laboratories. For
example, in a wide screening LC-MS/MS method that
included 59 different substances by Love et al. (2012), the
detection value and method detection limit ranged from 3
..............................................................................................
Aquaculture Nutrition, 20; 712–721 ª 2014 John Wiley & Sons Ltd
to 6 lg kg
1 for ciprofloxacin and 2–175 lg kg
1 for enro-
floxacin. In the method used in the present study, which
determined only two compounds, a lower LOD of
0.03 lg kg
1 was obtained, while in comparison other stud-
ies (a single enrofloxacin and ciprofloxacin HPLC-MS) used
a detection limit of 1.2 lg kg
1 (Martin et al. 2007). There-
fore, depending on the sensitivity of the analytical method
used, there is a potential that enrofloxacin and ciprofloxacin
in muscle plus skin would be detected if salmon were fed
diets with residual levels of enrofloxacin and ciprofloxacin
via dietary inclusion of ABPs. This finding is of particular
relevance for markets with zero tolerance for enrofloxacin
in food production. Furthermore, detection of enrofloxacin
in farmed Atlantic salmon from a production site that has
no legal report on the therapeutic use of this antibiotic
could lead to investigation and legal actions.
The authors wish to thank Elisabeth Eie, EWOS Innova-
tion research station Dirdal, Norway for excellent assis-
tance in the feeding trial. The authors would further like to
thank Joar Breivik and Rita Hannisdal, NIFES, Bergen,
Norway for the analyses on enrofloxacin and ciprofloxacin.
The project was funded by the Norwegian Research Coun-
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