Tetracyclines in Food and Feedingstuffs: From Regulation to Analytical Methods, Bacterial Resistance, and Environmental and Health Implications.

TLDR: This review focuses on tetracyclines, usage, dosages, and regulatory issues that govern their food-related application, with particular attention to the prohibitions and restrictions that several countries have enforced in recent years by agencies from both the United States and the European Union.

Abstract: Antibiotics are widely used as growth promoters in animal husbandry; among them, the tetracyclines are a chemical group of relevance, due to their wide use in agriculture, surpassing in quantities applied almost every other antibiotic family. Seeing the considerable amounts of tetracyclines used worldwide, monitoring of these antibiotics is paramount. Advances must be made in the analysis of antibiotics to assess correct usage and dosage of tetracyclines in food and feedstuffs and possible residues in pertinent environmental samples. The tetracyclines are still considered a clinically relevant group of antibiotics, though dissemination of tolerance and resistance determinants have limited their use. This review focuses on four different aspects: (i) tetracyclines, usage, dosages, and regulatory issues that govern their food-related application, with particular attention to the prohibitions and restrictions that several countries have enforced in recent years by agencies from both the United States and the European Union, (ii) analytical methods for tetracyclines, determination, and residues thereof in feedstuffs and related matrices with an emphasis on the most relevant and novel techniques, including both screening and confirmatory methods, (iii) tetracycline resistance and tetracycline-resistant bacteria in feedstuff, and (iv) environmental and health risks accompanying the use of tetracyclines in animal nutrition. In the last two cases, we discuss the more relevant undesirable effects that tetracyclines exert over bacterial communities and nontarget species including unwanted effects in farmers.

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Review Article
Tetracyclines in Food and Feedingstuffs: From Regulation to
Analytical Methods, Bacterial Resistance, and Environmental
and Health Implications
Fabio Granados-Chinchilla
1
and César Rodríguez
2
1
Centro de Investigaci
´
on en Nutrici
´
on Animal (CINA), Universidad de Costa Rica, San Jos
´
e, Costa Rica
2
Centro de Investigaci
´
on en Enfermedades Tropicales (CIET) and Facultad de Micro biolog
´
ıa,
UniversidaddeCostaRica,SanJos
´
e, Costa Rica
Correspondence should be addressed to C
´
esar Rodr
´
ıguez; cesar.rodriguezsanchez@ucr.ac.cr
Received  August ; Revised  October ; Accepted  November ; Published  January 
Academic Editor: Jose Antonio Rodriguez Avila
Copyright ©  F. Granados-Chinchilla and C. Rodr
´
ıguez. is is an open access article distributed under the Creative
Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the
original work is properly cited.
Antibiotics are widely used as growth promoters in animal husbandry; among them, the tetracyclines are a chemical group of
relevance, due to their wide use in agriculture, surpassing in quantities applied almost every other ant ibiotic family. Seeing the
considerable amounts of tetracyclines used worldwide, monitoring of these antibiotics is paramount. Advances must be made in
the analysis of antibiotics to assess correct usage and dosage of tetracyclines in food and feedstus and possible residues in pertinent
environmental samples. e tetrac yclines are still considered a clinically relevant group of antibiotics, though dissemination
of tolerance and resistance determinants have limited their use. is review focuses on four dierent aspects: (i) tetracyclines,
usage, dosages, and regulatory issues that gover n their food-related applicat ion, with particular attention to the prohibitions and
restrictions that several countries have enforced in recent years by agencies from both the United States and the European Union,
(ii) analytical methods for tetracyclines, determination, and residues thereof in feedstus and related matrices with an emphasis
on the most relevant and novel techniques, including both screening and conrmatory methods, (iii) tetracycline resistance and
tetracycline-resistant bacteria in feedstu, and (iv) environmental and health risks accompanying the use of tetracyclines in animal
nutrition. In the last two cases, we discuss the more relevant undesirable eects that tetracyclines exert over bacterial communities
and nontarget species including unwanted eects in farmers.
1. Introduction
Some compounds of veterinary relevance deserve particular
attention from an econo mic and health standpoint, especially
because of their widespread use. Among these compounds,
the uoroquinolones, 𝛽-lactams, sulfonamides, and tetr acy-
clines are included [].
Feed and environmental antibiotic residues are a deter-
minant factor that is usually omitted and is not entirely
understood when public health or food and feed safety
are discussed. Epidemiological, toxicological, and chemical
information regarding tetracyclines is presented to assess
acute and chronic consequences of human and animal health
as the result of their usage []. As such, a multifaceted and
complex issue is better addressed with a one health approach
[].
Antibiotics are extensively utilized in productive animals
with therapeutic, prophylactic, metaphylactic, growth pro-
moting, and food eectiveness enhancing ends []. Antibiotic
usage for growth promotion is the mainstream applica-
tion in animals and is the subject of regulatory eorts to
decrease antibiotic consumption in livestock, poultry, and
aquaculture []. Growth promotion and nutrient ecacy are
considered to be accomplished with relatively small doses of
antibiotics mixed with the feed by the manufacturer or the
farmer []. Growth promotion has no counterpart in human
medicine, and for this particular practice, an timicrobials
do not require veterinary prescription []. However, it is
Hindawi
Journal of Analytical Methods in Chemistry
Volume 2017, Article ID 1315497, 24 pages
https://doi.org/10.1155/2017/1315497

 Journal of Analytical Methods in Chemistry
essential to pursue prudent use of antimicrobials to mini-
mize resistance and ensure t heir continued eectiveness and
availability of these products and curtail impacts on human
health []. Implementation of s ecurity measures on farms,
prop er monitoring and registration, animal identication,
more tting management practices, implementation of the
right antibiotic and dosage, and promoting the judicious and
prudent use of antibiotics may extend the life of antibiotics
despite their frequent, persistent, and increasing use during
the last decades [].
Tetracyclines are a family of compounds frequently
employedduetotheirbroadspectrumofactivityaswellas
their low cost, compared with other antibiotics. Currently,
there are over  tetracyclines available; however, tetra-
cycline, chlortetracycline, oxytetracycline, and doxycycline
are the most common ones in veterinary medicine []. In
addition to therapeutic purposes, in many other countries,
tetracyclines are oen incorporated into livestock feed at
subtherapeutic doses as growth promoters for swine and
poultry and in aquaculture. For years, the use of antibiotics
as growth promot ers has been linked with benecial aspects
(especially increase in nutrient uptake eciency and com-
mercial revenue for farmers), though there is data that sup-
ports the fact that this exercise promotes bacterial resistance,
allergic reactions in humans and animals, and changes in
environmental microora and bacterial p opulations among
other detrimental eects [, ].
is review is centered in tetracycline antibiotics consid-
ering that they are among the most frequently used and in
more quantities (dosage-wise) in livestock and poultry world-
wide []. Global antibiotic consumption in livestock was
conservatively estimated at   ± tons in  [],
accounting for nearly two-thirds of the worldwide antibiotic
production [], and is projected to rise. Specically, by ,
the estimated usage of tetracyclines was of  . and .
tons in the United States and the European Common w ealth,
respectively. ese stats indicate that the issue of overuse of
these antibiotics is of particular signicance in countries such
asUnitedStates,China,andIndia[]ratherthaninEurope,
where consumption gures are much lower due to the ban
of antibiotics as growth promoters []. Amounts represent
a total of ca.  million USD of a group of antibiotics still
categorized as critically important in human medicine [];
only  out of  classes of antibiotics are exclusively used in
animals []. A thorough outlook of antibiotic usage may be
found in a recent paper written by Van Boeckel et al. [].
2. Tetracyclines Usage, Dosages, and
Regulatory Aspects That Govern the Food-
Related Application of Tetracyclines
2.1. A pproved and Recommended Uses. Tetracyclines have
several therapeutic indications de aling with infections in
food-producing animal and pets. In food-producing species,
including horses, usually, the rst-generation tetracyclines
are used, while in pets the second-generation tetracyclines are
chosen. erapeutic indications in animals comprise respira-
tory infections, dermal and so tissue infections, peritonitis,
metritis, and other enteric infections as well as the treatment
infections in aquatic species and honeybe es. In food animals,
for easier administration, the antibiotics are administered to
groups simultaneously through the drinking water or feed to
treatorpreventdisease[].Tetracyclineshavealsobeenused
for growth promotion, but apprehension related to emerging
bacterial resistance has led to a removal of the utilization
of t hese antibiot ics in this capacity, especially in European
countries [, ]. Till this day, the use of tetracyclines as
growth promoters is still allowed in many countries [,
]. However, beginning from  January , tetracyclines
will no longer be allowed for use as growth promoters in
USA []. e use will be restricted to therapeutic use only
and subject to a veterinary feed directive (VFD) []. Tetra-
cyclines have been active against Mycoplasma, Chlamydia,
Pasteurella, Clostridium, Ornithobacterium rhinotracheale,
and some protozoa. Examples of commercially available
tetracyclines include chlortetracycline (Aureomycin and
ChlorMax) and oxytetracycline (Terramycin).
Of particular concern are some tetracyclines of second
and third generation such as doxycycline and tigecycline.
Doxycycline is a semisynthetic tetracycline derivative. As
a hyclate salt, doxycycline is presented as an injectable
solution (intramuscular and intravenous), water-soluble or
lactodispersable powders, and tablets and capsules (for pets).
Doxycycline hyclate is indicated in cattle, pigs, poultry,
turkeys, and pets for the treatment of bacterial infections,
susceptible to this antibacterial, at a dose of – mg kg
−1
body weight per day , for – days. Doxycycline is not
to be used in lactating cattle and layers. In contrast to
animal therapy, this antibiotic has a long history of use in
human medicine. Doxycycline was previously assessed by the
Committee for Medicinal Products for Veterinary Use and an
“acceptable” le vel of daily intake of  𝜇gkg
−1
body weight, that
is,  𝜇g per person, was recognized. Currently, doxycycline
is comprised in the Commission Regulation (EU) number
/ of  December  [].
On the other hand, there are currently no authorized
tigecycline-containing products for veterinary use in the
European Union. Ho wever, according to the Cascade rule
(a legislative provision that allows a veterinary surgeon to
prescribe unauthorized medicines that would not otherwise
be permitted), tigecycline could be used for pets [] espe-
cially since multidrug-resistant organisms in dogs, cats, and
horses are being found with increasing frequency. e extent
of use of tigecycline in veterinary medicine due to this rule
is unknown. However, some countries such as Finland have
barred the use of this antibiotic for animal use []. In Europe,
since,cattleandbroilerchickenindustriesvoluntarily
stop the utilization of all antibiotic growth promoters, and
in  the swine industry followed []. An EU ban on
all antibiotics as growth promoters went into eect in 
[].
Notwithstanding, in the United States, oxytetracycline
(OTC) and chlortetracycline (CTC) are licensed to promote
weight gain and improve feed eciency rates [] (Table ).
Doxycycline, by contrast, is reserved for periodontal disease
in pets [] (Table ).

Journal of Analytical Methods in Chemistry 
T : Approved applications of tetracyclines in medicated feed for dierent food-producing animal species and their respective withdrawal
time frames.
Pharmacologically active
substance
Indications for use
a
U sage level Withdrawal time (days)
Feed antibiotics for swine
Chlortetracycline
Increased average weight gain and feed
eciency
10–50 g ton
−1
Voluntary withdrawal
Reduction of jowl abscesses – g ton
−1
Voluntary withdrawal
Control of leptospirosis in sows  g ton
−1
Voluntary withdrawal
Control of proliferative enteropathies (ileitis)
Body weight dosage:
 mg lb
−1
d
−1
Voluntary withdrawal
Chlortetracycline & penicillin &
sulfathiazole
Abscess abatement; treatment of bacterial
enteritis; upkeep of weight gain in the presence
of rhinitis
100 chlortetracycline;100
sulfathiazole;50
penicillin
7
Oxytetracycline
Increased average weight gain and feed
eciency
10–50 None
Treatment of bacterial enteritis and bacterial
pneumonia
Body weight dosage:
 mg lb
−1
d
−1
,–days
None
Control of leptospirosis in sows
Body weight dosage:
 mg lb
−1
d
−1
,–days
None
Neo mycin & oxytetracycline
Increased average weight gain and feed
eciency
10–50 5
Treatment of bacterial enteritis and bacterial
pneumonia
Body weight dosage:
 mg lb
−1
d
−1
,–days

Control and treatment of leptospirosis in breeders
Body weight dosage:
 mg lb
−1
d
−1
,–days

Prevention or treatment of bacterial enteritis
and dysentery; maintenance of weight gain in
thepresenceofatrophicrhinitis
50–150 oxytetracycline;
neomycin body weight
dosage:
35–140 mg lb
−1
d
−1
10
Oxytetr acycline & carbadox
Treatment of bacterial enteritis and bacterial
pneumonia
– carbadox;
oxytetracycline body
weight dosage:
 mg lb
−1
d
−1

Tiamulin & chlortetracycline
Control of dysentery; treatment of bacterial
enteritis and b acterial pneumonia
 tiamulin +  CTC
(body weight dosage:
 mg lb
−1
d
−1
)

Feed antibiotics for cattle (up to 700 lb)
Chlortetracycline
Coact ing in the prevention of bacterial
pneumonia associated with shipping fever
complex caused by Pasteurella spp.
 mg head
−1
day
−1

Control of active infection of  of anaplasmoses
caus ed by Ana plasma marginale
 mg head
−1
day
−1
or
. mg lb
−1
of body weight
day
−1
, beef control of
active infection

Oxytetracycline
Finishing cattle: to increase rate of gain and
improve feed eciency
 mg head
−1
day
−1
None
Coacting in reducing incidence and severity of
liver abscesses
 mg head
−1
day
−1
None
Coacting in the prevention of bacterial diarrhe a
.–. mg lb
−1
of body
weight day
−1
to
Prophylaxis and treatment of the early stages of
shipping fever complex
.– mg lb
−1
of body
weight day
−1
to
Chlortetracycline &
sulfamethazine
Feed for 28 days coacting in the maintenance of
weight gain in the presence of respiratory
disease such as shipping fever
350 mg head
−1
day
−1
7

 Journal of Analytical Methods in Chemistry
T : C ont inued.
Pharmacologically active
substance
Indications for use
a
U sage level Withdrawal time (days)
Lasalocid & oxytetracycline
For improved feed eciency and increased rate
of weight gain and reduction of incidence and
severity of liver abscesses in cattle fed in
connement for slaughter
From 25 to 30 g ton
−1
None
Oxytetracycline & neomycin base Coacting in the prevention of bacterial enteritis From  to  g ton
−1
–
Feed antibiotics for calves (up to 400 lb)
Chlortetracycline
Increased rate of weight gain and improved feed
eciency
0.1 mg lb
−1
of body
weight day
−1
or 25–70 mg
head
−1
day
−1
None
Treatment of bacterial enteritis caused by
Escherichia coli
 mg lb
−1
of body weight
day
−1
None
Treatment of bacterial enteritis caused by
Escherichia coli and bacterial pneumonia caused
by P. m u l t o c i d a
 mg lb
−1
of body weight
day
−1
Var iabl e
Oxytetracycline
Increased rate of weight gain and improved feed
eciency
From 0.05 to 0.1 mg lb
−1
or 25–75 mg head
−1
day
−1
0to5
As an aid in the treatment of bacterial diarrhea
From . to . mg lb
−1
or
 to  g ton
−1
None
Feed antibiotics for poultry
Chlortetracycline &
oxytetracycline
Increased average weight gain and feed
eciency
From  to  g ton
−1
None
Control of synovitis caused by Mycoplasma
synoviae and avian cholera caused by Pasteurella
multocida
From  to  g ton
−1
None
To control chronic respiratory disease of the air
sacs caused by Mycoplasma gallisepticum and
Escherichia coli
 g ton
−1
None
To reduce mortality due to air sac infections
caus ed by Escherichia coli
 g ton
−1

Antibiotics used in pets (companionship animals)
Doxycycline
Topical, to treat periodontitis. In dogs, used to
treat bac terial infections and infections c aused by
Rickettsia, Canine ehrlichiosis (anaplasmosis),
Toxoplasma, Borrelia burgdorferi (Lyme disease),
leptospirosis, and Neorickettsia helminthoeca
(salmon poisoning).
Incats,usedtotreatbacterialinfectionsand
infections caused by some other organisms
including Bartonella, Hemoplasma, Chlamydia
felis, Ehrlichia, Anaplasma,andTox oplasma
NA NA
a
Rows in bold font refer to growth promotion approved applications. Data based on values set in [, ]. In USA, tetracyclines are no longer allowed for
growth promotion aer  January  .
2.2. Regulatory Aspects
2.2.1. Feedingstu. Becaus e of its substantial implications for
food and feed safety, public administrations promote inte-
grated “farm to fork” approaches to ensure food security and
verify law compliance regarding the occurrence of antibiotic
residues and contaminants in feed and feed ingredients [].
Within the EU, maximum residue limits (MRLs) of
autho rized veterinary drugs in foodstus are dened in the
Council Regulation. Prior to the antibiotic ban in ,
Annex  from the Council Directive number //CEE
[] stated maximum limits for antibiotics, including tetra-
cyclines. ree tetracyclines are listed. Tetracycline, oxyte-
tracycline, and chlortetracycline were approved for animals
bred for fur, calves, lambs, poultry, and swine with maxi-
mumcontentsofactiveingredientofmgkg
−1
feed. Also,
according to European legislati on, Good Veterinary Practices
(GVP) request producers to abide by established withdrawal
times for antibiotics []. Some countries have set still stricter
measures regarding the use of antibiotics in food-producing

Journal of Analytical Methods in Chemistry 
T : Requirements of a medicated feed mill license.
Categor y I
Compound(s) Type A (assay limits,%)
a
Type B maximum, g lb
−1
(x, %) Type B/C (assay limits,%)
a
Chlortetracycline – . (.) –/–
Oxytetracycline – . (.) –/–
Categor y II
Compound(s) Type A (assay limits, %) Type B maximum, g lb
−1
(x, %) Type B/C (assay limits, %)
Neomycin – . (.) –
Oxytetracycline – . (.) –
Sulfamethazine – . (.) –
Chlortetracycline – . (.) –/–
a
Percentage of labeled amount. Based on values set in [].
animals. A denite success example of antibiotic regulation
through prescription monitoring is Denmark which in 
adopted a “Yellow Card” system in which producers that
applied more amount of antibiotic per animal are issued a
warning. Failure in abiding and lowering their use would
even tually imply that they would be forced to either reduce
useorcuttheherdsize[,].eyalsolimitthepos-
sibility of veterinarians to prot from antibiotic sales and
prescription monitoring in farms [, ]. Application of this
measurement was accompanied in the years that followed by
a decrease in antibiotic usage [].
In this regard, the FDA has established control limits for
typesA,B,andCanimaldrugs,wheretypeAreferstopure
drugs and types B and C correspond to medicated feed. US
FDA medicated feed assay control limits for pure CTC and
OTC (medicated articles) (Table ). Similar limitations are
established for feeds medicated with CTC and OTC []. On
their appearance aer application/withdrawal windows, feed-
antibiotics can be classied into two categories. Category
I antibiotics may appear in feed at the lowest use level
for which no withdrawal period is required, and ca tegory
II antibiotics comprise compounds for which a withdrawal
period is required at the lowest use level and no residue or
“zero tolerance” is allowed.
In the absence of any other antibiotics, tetracyclines can
be considered as category I antibiotics. Subsequently, they
canfurtherbeviewedasatypeA,B,orC.AtypeAarticle
is a product that consists of one or more animal drugs
of consistent strength, intended exclusively for use in the
production of another type A article or either a type B or a
type C medicated feed. Typ e A medicated articles must be
registered with the FDA annually (FDA-/e). Type B is a
medicatedfeedthatcontainsatypeAmedicatedarticle,andit
is signicantly diluted with one or more nutrients to produce
a type C medicated feed which is intended to be presented as
a complete feed for the animal.
Before a f acility can manipulate a category II, type A
medicated article, it must hold an approved medicated feed
mill license. Registration as a drug establishment and FDA
approval of a feed mill license are required before a category
II, type A medicated feed article can be purchased (Table ).
Antibiotics in feeds, including tetracyclines, are regulated
by US FDA Current Good Manufacturing Practices (cGMP)
 CFR .- [] when medicated feed application
is required or  CFR .- where it is not [].
ey are also subjected to Part  of cGMP regulations
[].
When the US FDA approves new animal drugs or combi-
nation products, they obtain one of the following marketing
statuses: (i) over-the-counter, (ii) veterinary prescript ion, or
(iii) veterinary feed dire ctive (VFD) []. As tetracyclines
are considered medically relevant antibiotics (according to
US FDA Guidance  []), tetracyclines are in need of a
VFD to be incorporated in the feed. Furthermore, the US
FDA Guidance  [] dictates t hat all medically relevant
drugs administered require VFD and therefore inspection
and record keeping of prescription. At the time of writing,
this guidance is voluntary implementation, but it will become
mandatory in December  [].
While recent FDA actions have focused on production
uses,therearecallsintheUnitedStatesalsotorestrict
prophylactic uses as well [, ]. An economic analysis of
benets, costs, and perspectives of a possible ban has been
analyzed in depth recently by Teillant et al. []. Notewor thy,
jurisprudence in USA has usually been laxer than in other
count ries [].
Despite being considered a global reference point for
consumers, fo od manufacturers, and processors, food control
agencies, and the international food trade, the Codex Ali-
mentarius does not have a particular task force concerning
animalfeedissues[].eprimaryoutputoftheonceadhoc
Codex Intergovernmental Task Force on Animal Feeding was
the Codex Code of Practice on Good Animal Feeding [].
Regarding antibiotics in feed, it only states that antibiotics
shouldnotbeusedinfeedforgrowthpromotingpurposes
when public health safety assessment is lacking.
Other texts relevant to animal feeding have b een devel-
oped by other Codex Committees such as those on Food
Additives, Contaminants in Foods, Meat Hygiene, Residues
of Veterinary Drugs in Foods, Pesticide Residues, and Food
Labelling. Analogously, the FAO in partnership with the
International Feed Industry Federation (IFIF) has issued
a Manual of Good Practices for the Feed Industry. Good
Manufactory Practices demand feed and feed ingredients to
be free of pests and chemical, physical, and microbiological
contaminants during their production, management, storage,