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Chloramphenicol

January 2004

Authors:

Fernando Ramos

University of Coimbra

Stability of chloramphenicol in non-sterile soil

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Growth rate of chickens under different feeding conditions

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. Production of chloramphenicol in sterilized soil infested with S. venezuelae

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Plasma kinetics of parent chloramphenicol in pigs given a single subcutaneous dose at 30 mg/kg bw

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Effect of elimination half-life on steady-state concentrations

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Figures - uploaded by Fernando Ramos

Content may be subject to copyright.

Content uploaded by Fernando Ramos

Content may be subject to copyright.

CHLORAMPHENICOL

First draft prepared by

J. Wongtavatchai1,J.G. McLean2,F. Ramos3and D. Arnold4

1Faculty of Veterinary Medicine, Chulalongkorn University,

Bangkok, Thailand;

2Camberwell, Victoria, Australia;

3Laboratório de Bromatologia, Nutrição e Hidrologia, Faculdade de

Farmácia, Universidade de Coimbra, Portugal; and

4Berlin, Germany

Explanation............................................................................... 8

Biological data .......................................................................... 9

Biochemical aspects .......................................................... 9

Toxicological studies .......................................................... 11

Cytotoxicity and genotoxicity ....................................... 11

Haematoxicity .............................................................. 13

Observations in humans .................................................... 15

Problems in trade caused by residues in foods ...................... 18

Analytical methods ................................................................... 22

Preparation of the primary extract of the sample.............. 22

Puriﬁcation of the primary extract...................................... 23

Detection and quantiﬁcation of residues of

chloramphenicol........................................................... 23

Production, occurrence and fate of chloramphenicol

in the environment ....................................................... 26

Discovery............................................................................ 26

Biosynthesis ....................................................................... 26

Production and stability in soil ........................................... 26

Environmental fate of chloramphenicol.............................. 28

Results of environmental monitoring for

chloramphenicol........................................................... 31

Microbial resistance to chloramphenoicol in the

environment—is it an argument for the

presence of the drug? ................................................. 32

Feed and soil intakes and growth of some terrestrial

farm animals ................................................................ 33

Pigs ............................................................................... 33

Chicken .............................................................................. 35

Cattle ............................................................................... 35

Hypothetical intake of chloramphenicol from the

environment by soil ingestion ...................................... 35

Soil ingestion by farm animals........................................... 35

Intake of chloramphenicol and resulting residues

in tissues ............................................................... 36

Pigs .............................................................................. 36

Chickens ...................................................................... 41

Hypothetical exposure to persisting environmental residues... 43

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39834 FOOD TEXT 26/5/05 9:52 am Page 7

Examples of integrated farming......................................... 45

Processing and use of manure.......................................... 45

Production of excreta by farm animals.............................. 46

Residues in manure resulting from therapeutic

treatment of farm animals............................................ 47

Stability of chloramphenicol in manure.............................. 48

Uptake by farm animals of residues from manure ............ 49

Human intakes resulting from the consumption of

contaminated ﬁsh and shellﬁsh.......................................... 51

Comparison of dietary intakes with low-level exposure

from ophthalmic formulations ...................................... 52

Systemic effects following ocular administration of

drugs as evidence for systemic bioavailability ............ 53

Pharmacokinetic evidence of systemic absorption

of ophthalmic drugs ..................................................... 53

Results obtained with ophthalmic formulations of

chloramphenicol........................................................... 54

Quantitative considerations................................................ 54

Comments ............................................................................... 55

Evaluation ............................................................................... 61

References ............................................................................... 62

Appendix I ............................................................................... 77

Appendix II ............................................................................... 82

1. EXPLANATION

Chloramphenicol is a broad-spectrum antibiotic with historical veterinary uses

in all major food-producing animals and with current uses in humans and com-

panion animals. Chloramphenicol was evaluated previously by the Committee at

its twelfth, thirty-second and forty-second meetings (Annex 1, references

and

110

). A number of other agencies have also reviewed chloramphenicol (e.g.

International Agency for Research on Cancer (IARC), 1990; European Committee

for Veterinary Medicinal Products, 1994; United States Food and Drug Adminis-

tration, 1985). Concerns have been expressed about the genotoxicity of chloram-

phenicol and its metabolites, its embryo- and fetotoxicity, its carcinogenic potential

in humans and the lack of a dose–response relationship for aplastic anaemia

caused by treatment with chloramphenicol in humans. Deﬁciencies identiﬁed in

data on the toxicity of chloramphenicol include information necessary for the

assessment of carcinogenicity and effects on reproduction. An acceptable daily

intake (ADI) has never been allocated and consequently a maximum residue limit

(MRL) has not been assigned. This has resulted in the restriction of the use of

chloramphenicol in veterinary medicine to non-food use.

Chloramphenicol was originally isolated from the soil organism

Streptomyces

venezuelae

in 1947, but is now produced synthetically.Three common forms are

used for systemic therapy, depending on the route of administration; a free base

form of chloramphenicol, chloramphenicol palmitate and chloramphenicol succi-

nate. Other formulations are also available for topical use. Chloramphenicol usually

acts as a bacteriostatic, but at higher concentrations or against some very sus-

ceptible organisms it can be bactericidal. It is used in the treatment of human infec-

tion with

Salmonella typhi

(typhoid) and other forms of salmonellosis, and other

CHLORAMPHENICOL

39834 FOOD TEXT 26/5/05 9:52 am Page 8

life-threatening infections of the central nervous system and respiratory tract

(Parﬁtt, 1999). In veterinary medicine, chloramphenicol is used for the treatment

of a variety of infections in animals, particularly those caused by anaerobic bac-

teria or those that are resistant to other antimicrobial agents. Chloramphenicol in

animals is well absorbed by both oral and parenteral routes (Plumb, 2002).

There is good evidence for a haemotoxic effect of chloramphenicol in humans,

with two forms of toxicity being described. The ﬁrst is a commonly occurring, dose-

related reversible bone-marrow depression, which develops during treatment and

is reversible following the withdrawal of the drug. The second is a severe aplastic

anaemia, which is non-dose-related and often irreversible.

This monograph summarizes the recently published literature and submitted

unpublished information on the toxicity of chloramphenicol.

2. BIOLOGICAL DATA

2.1 Biochemical aspects

The usual therapeutic range for chloramphenicol in serum in most animal

species is 5–15 mg/ml. After dosing, chloramphenicol is widely distributed through-

out the body. The volume of distribution of chloramphenicol reported in compan-

ion animals is 1.8 l/kg in the dog, 2.4 l/kg in the cat and 1.41 l/kg in the horse.

Hepatic metabolism by a glucuronidative mechanism is the principle pathway foy

whichr chloramphenicol undergoes biotransformation to an inactive metabolite,

chloramphenicol glucuronide. Only about 5–15% of the drug is excreted

unchanged in the urine. Dogs excrete only about 6% of unchanged drug into the

urine. Cats have a limited ability to form glucuronide conjugates with drugs and

therefore excrete chloramphenicol more slowly than other animals, with 25%

or more of the administered dose excreted unchanged in the urine. The elimina-

tion half-life of chloramphenicol is 1.1–5.0 hin dog, <1hin foals and ponies, and

4–8 h in cats (Adams, 1995; Plumb, 2002).

The pharmacokinetic properties of orally administered chloramphenicol in

broiler chickens indicate that chloramphenicol is rapidly absorbed. At a dose of 30

or 50 mg/kg bw, the drug reached the maximum plasma concentration at 0.72 h or

0.60 h, it was eliminated with a mean half-life (t1/2 b)of 6.87 or 7.41hand had a

bioavailability of 29% or 38% respectively. A concentration of chloramphenicol of

>5mg/ml was achieved in plasma at 15min, and persisted up to 2 or 4 hafter admin-

istration of chloramphenicol at a dose of 30 or 50 mg/kg bw. When chickens

received an oral dose of chloramphenicol at 50 mg/kg bw once daily for 4 days,

three metabolites, dehydrochloramphenicol; nitrophenylaminopropanedione-

chloramphenicol (NPAP-chloramphenicol); and nitrosochloramphenicol were

found in kidney, liver and muscle. The study found a slow clearance of residues,

particularly of the NPAP and nitrosochloramphenicol residues, which were

detected in tissues at 12 days after dosing (Anadon et al., 1994).

Plasma concentrations of chloramphenicol were determined in four calves

given four oral doses of chloramphenicol palmitate, each corresponding to a dose

of chloramphenicol of 25 mg/kg bw, at 12 h intervals. After the fourth dose, the

CHLORAMPHENICOL

39834 FOOD TEXT 26/5/05 9:52 am Page 9

plasma concentration of chloramphenicol reached a steady state of 5–6mg/ml. The

half-life of elimination was 4.5 h. Dehydrochloramphenicol at a concentration of

3–7 mg/ml was also detected in the plasma. The authors suggested that dehy-

drochloramphenicol, which is a metabolite produced by intestinal bacteria and sug-

gested to be associated with fatal aplastic anaemia in human, may occur in edible

tissues of animals treated with chloramphenicol (Gassner & Wuethrich, 1994).

Several metabolites of chloramphenicol were identiﬁed in urine samples

obtained from male Wistar rats and from a human volunteer given tritiated

chloramphenicol at a dose of 10 mg/kg bw by mouth. In rats, the two most abun-

dant metabolites detected in the ﬁrst 24 h by high-performance liquid chromatog-

raphy (HPLC) and gas chromatography–mass spectrometry (GC–MS) were

chloramphenicol-base and chloramphenicol-acetylarylamine. The remaining

metabolites were unchanged chloramphenicol, chloramphenicol-oxamic acid,

chloramphenicol-alcohol, chloramphenicol-glucuronide and chloramphenicol-

oxamylethanolamine. Similar end-products were also found in the human

volunteer.The amount of chloramphenicol-oxamylethanolamine, which is an end-

product of chloramphenicol biotransformation that was previously reported in birds,

represented 0.74% and 1.37% of the ingested radioactivity found in the rat and

human urine samples. The formation of chloramphenicol-oxamylethanolamine as

an end-product of the metabolism of chloramphenicol by the liver was proven by

the release of chloramphenicol-oxamylethanolamine after incubation of tritiated

chloramphenicol with hepatocyte microsomes from rats treated with phenobarbi-

tal (Cravedi et al., 1995).

Chloramphenicol-aldehyde as a metabolic product of chloramphenicol was

identiﬁed in a study in four children with major infections treated with chloram-

phenicol at a dose of 50 mg/kg bw per day). The residues in samples of urine col-

lected during the treatment were analysed using HPLC and GC–MS. Results

indicated the existence of compounds with characteristics corresponding to the

synthesized chloramphenicol-aldehyde derivatives. The author concluded that

chloramphenicol-aldehyde, a metabolite that was toxic to bone marrow and previ-

ously observed only in rat hepatic tissue, was a new metabolite in humans (Holt,

1995).

A study performed in vitro in human bone-marrow cells from 72 donors showed

that chloramphenicol succinate was metabolized to chloramphenicol and other

metabolites. In all 72 samples, the HPLC analysis of cell-free supernatant obtained

from samples of bone marrow incubated with chloramphenicol succinate for 3 h at

37 °C revealed a substance with a retention time corresponding to that of chlo-

ramphenicol. Other metabolites, nitrosochloramphenicol and unidentiﬁed metabo-

lites, were also presented in some bone marrow samples. The study referred to

the ultimate toxic derivatives of chloramphenicol produced in the bone marrow in

situ as resulting from the metabolic biotransformation of the prodrug, thus indicat-

ing the marrow as both the site of metabolic conversion and the target of injury

(Ambekar et al., 2000).

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2.2 Toxicological studies

2.2.1 Cytotoxicity and genotoxicity

Catalan et al. (1993) reported that chloramphenicol induced sister chromatid

exchange in bovine lymphocyte cultures, an effect that is indicative of DNA damage

and repair, and also observed a delay in the cell cycle.

The cytotoxicity and genotoxicity of chloramphenicol and six metabolites were

investigated in human bone-marrow cells (RiBM cells) in vitro. The six metabolites

tested in the study were: nitrosochloramphenicol, chloramphenicol-glucuronide,

chloramphenicol base (NAPD), and an alcohol derivative (hydroxy-amphenicol,

HAP), dehydrochloramphenicol and nitrophenyl-aminopropanedione-

chloramphenicol (NPAP-chloramphenicol). The cytotoxic effect was demonstrated

by inhibition of incorporation of tritiated thymidine into DNA. The genotoxic

effect was evaluated by the induction of DNA single-strand breaks. Cytotoxic

effects were found with three metabolites, nitrosochloramphenicol, dehydrochlo-

ramphenicol and NPAP-chloramphenicol, at concentrations ranging from 2 ¥10-5

to 2 ¥10-4mol/l. Nitrosochloramphenicol appeared to be the most potent cytotoxic

compound tested, while chloramphenicol-glucuronide and HAP were not cytotoxic

in RiBM cells. A similar cytotoxic response was reported earlier in human peri-

pheral blood lymphocytes, but dehydrochloramphenicol was the most inhibitory

compound. Genotoxic potential was observed with nitrosochloramphenicol

and dehydrochloramphenicol at a concentration of 1–2 ¥10-4mol/l, with a

dose–response pattern; chloramphenicol and other metabolites were devoid of

genotoxic effect at concentrations up to 4 ¥10-3mol/l. On the basis of the response

found in RiBM cells compared with the previous investigation using peripheral

blood lymphocytes, the authors concluded that RiBM cells were much less sus-

ceptible to the genotoxic effect of chloramphenicol metabolites than were human

lymphocytes (Lafarge-Frayssinet et al., 1994; Robbana-Barnat et al., 1997).

An increase in apoptosis in marrow progenitor cells was reported in patients

with aplastic anaemia (Philpott et al., 1995). The ﬁrst evaluation of apoptosis in

toxicity caused by chloramphenicol was assessed in vitro in a study using a

monkey kidney-derived cell line and human haematopoietic progenitor cells from

human neonatal cord blood. At a concentration of 2–5mmol/l, chloramphenicol

caused apoptosis in dividing cells of both systems. In a subsequent study of myelo-

toxicity in vivo, morphological evidence of apoptosis was seen in erythroid and

myeloid precursors in femoral marrow of B6C3F1mice given chloramphenicol at

adose of 200 mg/kg bw. The authors suggested that effect of chloramphenicol is

at the differentiation stage of the committed marrow progenitor cells, rather than

the replication stage of the stem cells, and therefore this response appears to be

paralleling the reversible bone-marrow depression seen in the treated patient (Holt

et al., 1997, 1998).

The observation that chloramphenicol induces apoptosis in haemopoietic

stem cells was conﬁrmed by additional studies using models in vitro and in vivo.

Phenotypic analyses using ﬂow cytometry (with a ﬂuorescence-activated cell

sorter, FACS) have demonstrated the induction of apoptosis in puriﬁed human

bone-marrow CD34+cells treated with chloramphenicol (Kong et al., 1999). Alink

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39834 FOOD TEXT 26/5/05 9:52 am Page 11

between this cytotoxicity and chloramphenicol-induced apoptosis was conﬁrmed

in vivo in BALB/c mice treated orally with a single high dose of chloramphenicol

at 4000 mg/kg bw or with thiamphenicol. Apoptosis in femoral mononuclear cells

sampled at 36 h after dosing, as indicated by morphological evidence for increased

numbers of apoptotic nucleated marrow cells, was only induced by chloram-

phenicol, while thiamphenicol gave a negative result. The authors suggested

that the induction of apoptosis in marrow progenitor cells might account for

chloramphenicol-induced toxicity associated with aplastic anaemia in humans

(Turton et al., 2002b).

Inhibition of protein synthesis in the mitochondria of bone-marrow cells has

been considered as a mechanism by which bone-marrow depression is induced

by chloramphenicol. The underlying cytotoxicity may be caused by the similarity

between mitochondrial ribosomes and bacterial ribosomes, both of which are 70S.

Thus chloramphenicol can also inhibit mitochondrial protein synthesis in mam-

malian cells, particularly in erythropoietic cells, which appear to be sensitive to the

drug (Sande & Mandell, 1993; Kucers et al., 1997). It was reasoned that the inhi-

bition of mitochondrial protein synthesis suppressed the division of mitochondria

and resulted in the formation of megamitochondria. Investigation of the toxicity

caused by chloramphenicol in mouse hepatic cells in vivo, however, showed that

antioxidants prevented the formation of megamitochondria (Matsuhashi et al.,

1996). The role of antioxidants in reducing the cytotoxic effects of chlorampheni-

col was also reported to occur in vitro in a study using a monkey kidney-derived

cell line and haematopoietic progenitor cells from human neonatal cord blood. Also,

in cells in culture, the cytotoxic effects of chloramphenicol on apoptosis and sup-

pression of progenitor cell growth were not pronounced when cells were co-

cultured with antioxidants such as mercaptoethylamine or vitamin C (Holt et al.,

1997). Both studies suggested that toxicity caused by chloramphenicol relates inti-

mately to oxidative stress, with a possible link between a metabolic event—the

production of free radicals—and bone marrow suppression.

The cytotoxic potential of chloramphenicol with regard to cell membrane func-

tion was examined in a study investigating inhibition of protozoan motility.The

effect of chloramphenicol on the locomotion of the protozoan

Tetrahymena pyri-

formis

,amodel widely used for the evaluation of toxicity in excitable tissue, was

tested. Chloramphenicol appeared to depress the motility of the test organism

more effectively than did chloramphenicol succinate, the hydrophilic form of chlo-

ramphenicol. Results suggested that chloramphenicol, with its hydrophobic free

form, has the ability to partition into the lipid bilayer of the cell membrane and thus

the potential to cause membrane-mediated toxic effects. The authors postulated

that such effects might explain the acute toxicity of chloramphenicol in excitable

tissues, such as myocardium, and are a possible mechanism for chloramphenicol-

induced cardiovascular collapse in neonates, or “grey baby syndrome” (Wu et al.,

1996).

In contrast to the membrane-mediated toxic effects observed in

Tetrahymena

spp., chloramphenicol had no adverse effects on the morphologic characteristics

and migration of canine corneal epithelial cells in vitro. A monolayer of cultured

cells from the canine corneal epithelium was treated with a number of different

CHLORAMPHENICOL

39834 FOOD TEXT 26/5/05 9:52 am Page 12

antibiotics after a defect had been made on the monolayer. The toxicity of the

antibiotics was determined by the morphologic characteristics and the migration

of treated cells. Pure antibiotics were used at a concentration similar to that in

tears, obtained with topical use of the commercially available antibiotic products

in humans. Comparison between control cells and cells treated with antibiotics indi-

cated that chloramphenicol had no cytopathologic effects on the monolayer and

cellular morphologic characteristics, and migration of the treated cells was similar

to that of control cells (Hendrix et al., 2001).

2.2.2 Haematoxicity

Two types of chloramphenicol-induced toxicity in humans have been widely

discussed. The ﬁrst is a frequently occurring, dose-related, bone-marrow depres-

sion that develops during treatment with chloramphenicol. The condition is seen

as a mild anaemia, with decreased haemoglobin concentrations and reticulocy-

topenia, with the bone marrow showing reduced erythroid precursors, increased

myeloid : erythroid cell ratio and vacuolation of erythroid cells. The patient returns

to normal after drug withdrawal. Inhibition of protein synthesis in bone-marrow cells

has been proposed as the mechanism of these effects (Kucers et al., 1997). The

second is a severe, non-dose-related aplastic anaemia, which is irreversible.

Aplastic anaemia is evident as severe pancytopenia in peripheral blood, with an

acellular or hypocellular bone marrow. This might also result in leukaemia in

humans (Dollery,1999; Turton et al., 2002a).

Severe bone-marrow failure induced by chloramphenicol in man is relatively

infrequent. Susceptibility to chloramphenicol-induced aplastic anaemia and

leukaemia in man is considered to involve a genetic element. It has been sug-

gested that chloramphenicol-induced aplastic anaemia and leukaemia are related

to the DNA damage caused by nitrosochloramphenicol, which is a product of

the reduction of the para-nitro group of chloramphenicol. The ability to reduce the

para-nitro group to the nitroso derivative is genetically determined, and thus gives

rise to an individual metabolic predisposition to such drug-induced conditions.

The assumption is supported by investigations in vitro and in vivo demonstrating

that the haematological response to chloramphenicol in mice is partly strain-

dependent (Festing et al., 2001). However,the exact biochemical mechanism

responsible for aplastic anaemia in man has not yet been elucidated.

A battery of toxicological studies was performed in an attempt to develop a

rodent model for chloramphenicol-induced aplastic anaemia in humans. However,

recent studies have conﬁrmed several previous reports that no suitable or reliable

laboratory animal model of aplastic anaemia exists, although administration of

chloramphenicol succinate in rodent models induces haematological changes

comparable to the chloramphenicol-induced reversible, dose-dependent bone-

marrow depression seen in humans (Young & Maciejewski, 1997; Holt et al., 1998;

Yallop et al., 1998; Turton et al., 1999).

Haematoxicity induced by chloramphenicol was investigated in a study in CD-

1weanling mice. Animals were given chloramphenicol at a dose of 1400mg/kg bw

by gavage daily for 10 days, and blood samples were taken at 1, 4 and 15 days

CHLORAMPHENICOL

39834 FOOD TEXT 26/5/05 9:52 am Page 13

after the last dose. Haematological data at day 1 after dosing showed a signiﬁcant

reduction in erythrocytes, erythrocyte volume fraction and haemoglobin values,

which returned to normal by day 4 or 15. The investigator suggested that the

reversible, dose-dependent anaemia seen in man could develop in CD-1 mice

given chloramphenicol succinate (Turton et al., 1999).

In a study of the potential of chloramphenicol succinate and thiamphenicol to

induce aplastic anaemia, female BALB/c mice were given chloramphenicol succi-

nate at 2000 mg/kg bw per day or thiamphenicol at 850mg/kg bw per day by gavage

for 17 days. On days 1, 13, 22, 41, 98 and 179 after the ﬁnal dose, blood and

marrow samples were collected for haematological examination and assays for

haematopoietic stem cells. Chloramphenicol succinate and thiamphenicol were

found to have similar effects. Signiﬁcant reductions in values for peripheral blood

parameters (erythrocyte count, erythrocyte volume fraction and haemoglobin

concentration) and bone marrow parameters (erythroid colony forming units and

granulocyte-macrophage colony forming units) were found in samples at day 1

after dosing. At the later sampling times, values for all the observed parameters

gradually returned to normal, and there was no evidence of marrow suppression

by the end of the experiment. On the basis of this observation, the authors deter-

mined that chloramphenicol succinate and thiamphenicol induced reversible

anaemia in BALB/c mice; however, aplastic anaemia does not appear in the

BALB/c mouse (Turton et al., 2000).

The induction of haematoxicity by administration of chloramphenicol was

attempted in another rodent species, after the induction in mice was unsuccess-

ful. Guinea-pigs were examined for susceptibility to bone-marrow depression

induced by chloramphenicol succinate. In a dose range-ﬁnding study,chloram-

phenicol succinate administered at a dose of 825 mg/kg bw for 16 days induced

changes comparable to the reversible bone-marrow depression seen in humans,

but there was no evidence of late-stage marrow depression, as would be seen in

marrow aplasia. The authors concluded that rodents are not susceptible to myelo-

toxicity induced by chloramphenicol. The guinea-pig, like the mouse and rat, serves

as a model for early events, but is not a good model for aplastic anaemia induced

by chloramphenicol in man (Turton et al., 2002a).

Despite the well-recognized potential toxicity of chloramphenicol in humans,

the drug is considered by most experts to be of low toxicity in adult companion

animals when they are appropriately dosed. The development of aplastic anaemia

as seen in humans does not appear to be a signiﬁcant problem in animals.

However, a dose-related reversible bone-marrow suppression is seen in all

species, primarily after long-term therapy. Early signs of bone-marrow toxicity can

include vacuolation of many of the early cells of the myeloid and erythroid series,

lymphocytopenia, and neutropenia. Other adverse effects that may be noted in

animals treated with chloramphenicol include anorexia, vomiting, diarrhoea and

depression. Cats tend to be more sensitive to developing adverse reactions to

chloramphenicol than are dogs; cats given chloramphenicol at a dose of 50mg/kg

bw every 12 h for 2–3 weeks do develop in high incidence of adverse effects

(Plumb, 2002).

A bone marrow disorder was reported in a study of toxicity in a dog that was

dosed orally with chloramphenicol at 300 mg/kg bw per day for 14 days. The results

CHLORAMPHENICOL

39834 FOOD TEXT 26/5/05 9:52 am Page 14

showed a decrease in the number of total erythroid cells, with proportional increase

in myeloid cells, yielding a signiﬁcantly increased myeloid to erythroid cell ratio

(Baig et al., 1994).

Breeder turkey hens given drinking-water containing chloramphenicol at a con-

centration of 500 mg/l for 4 days showed a decrease in egg production. The toxic

effects of chloramphenicol were mortality and cessation of egg production, which

were more severe when a combination of chloramphenicol and monensin was

given (Friedman et al., 1998).

Overall analysis of toxicity with chloramphenicol has suggested that the most

serious toxic effect, aplastic anaemia reported in humans, is not seen in animals.

However,reversible, dose-related bone-marrow suppression can be observed in

all species given chloramphenicol in excessive doses or for prolonged periods.

Other signs of toxicity caused by chloramphenicol are evident in animals in sus-

ceptible states, e.g. in neonatal animals or pregnant animals in which the hepatic

biotransformation of chloramphenicol is impaired, or where the drug causes a

decrease in protein synthesis in the fetus. However, owing to the potential toxic-

ity of chloramphenicol in humans, and because of the possibility that metabolites

of chloramphenicol might be found in the edible tissues of animals treated with

chloramphenicol, the use of chloramphenicol in veterinary practice is not permit-

ted in food-producing animals in many countries.

2.3 Observations in humans

Toxicity caused by chloramphenicol in humans has been widely discussed

because it induces bone-marrow depression. The more common dose-related

bone-marrow depression is evident when the daily dose of chloramphenicol is

>4 g in humans. Toxicity is reversible if the treatment is discontinued or the dosage

is reduced. A more serious and unpredictable reaction is aplastic anaemia, which

is not considered to be dose-related. Although the incidence of aplastic anaemia

has been shown to correlate with several risk factors, it is estimated to occur with

afrequency of 1 in 24 000–40 000 courses of treatment with chloramphenicol. Mor-

tality from aplastic anaemia occurs in >50% of cases (Greenwood, 2000; Maluf

et al., 2002).

Rappeport & Bunn (1994) suggested that aplastic anaemia in humans is an

idiosyncratic reaction to chloramphenicol, which has an immunological basis and

which is related to the nitrobenzene structure. This hypothesis is supported by clin-

ical evidence showing that 40–50% patients with aplastic anaemia have a partial

or complete response to a variety of immunosuppressive agents.

Young (2002) reviewed the pathophysiology of aplastic anaemia and reported

that most cases can be characterized by a T-cell mediated destruction of bone-

marrow haematopoietic cells.

This aberrant immune response may be a reaction to chemicals, drugs or viral

infections, but endogenous antigens may also be involved. Many drugs can cause

idiosyncratic haematopoietic failure; however, it is rare that patients, some of whom

may have only ingested small quantities of the drug, show bone-marrow failure as

acomplication. Owing to the idiosyncratic nature of the response, it is difﬁcult to

CHLORAMPHENICOL

39834 FOOD TEXT 26/5/05 9:52 am Page 15

study aplastic anaemia, and animal models do not exist (Young & Maciejewski,

1997). The therapeutic use of chloramphenicol has been followed by the devel-

opment of aplastic anaemia in humans. This was particularly notable in the period

following the introduction of chloramphenicol as a therapeutic agent in 1948, and

before its association with aplastic anaemia had been recognized.

Other indications of toxicity associated with treatment with chloramphenicol in

humans have been recognized. Circulatory collapse (“grey baby syndrome”) has

occurred in human neonates treated with chloramphenicol. This adverse reaction

may be explained by the poor hepatic biotransformation of the drug in neonates

as a result of slow glucuronidation of chloramphenicol. Toxic concentrations of

chloramphenicol in blood and tissues develop secondary to an inability to conju-

gate the drug or to excrete the conjugate efﬁciently. However, the precise reason

for the occurrence of cardiovascular collapse in grey baby syndrome is poorly

understood. It has been stated that nitro-reduction derivatives of chloramphenicol

might play a role in causing hypotension, and the hypothesis was assessed by

perfusion of chloramphenicol through the isolated lobules of human placenta. A

decrease in blood pressure was found at the time coinciding with a peak in con-

centration of nitric oxide, which is a product of the nitroreduction of chloram-

phenicol (Holt & Bajoria, 1999).

The potential for an adverse reaction induced by treatment with chloram-

phenicol is of critical importance in seriously ill or compromised patients. In patients

with pre-existing haematologic abnormalities or hepatic failure, or in neonates,

chloramphenicol is only used when no other effective antibiotics are available.

Chloramphenicol has not been determined to be safe for use during pregnancy.

The drug may decrease protein synthesis in the fetus, particularly in the bone

marrow. Chloramphenicol is found in human milk at 50% of serum concentrations

in humans and therefore the drug should be given with extreme caution to nursing

mothers (Greenwood, 2000; Plumb, 2002).

The most serious adverse effect of treatment with chloramphenicol in humans

is its association with acquired aplastic anaemia. Many population-based studies

have been carried out to identify etiological factors associated with aplastic

anaemia and to determine a link between the use of chloramphenicol and the

development of marrow aplasia. Young & Alter (1994) reported that the published

estimates of incidence of aplastic anaemia are signiﬁcantly inﬂuenced by the

methods used to acquire the data, and the diagnostic exclusion criteria. The inci-

dence estimates reported in some former studies were too high owing to the inclu-

sion of cases improperly classiﬁed as aplastic anaemia; the reported incidence of

aplastic anaemia declines when rigorous diagnostic criteria have been applied. On

the basis of an extensive review, the authors concluded that the incidence rate for

aplastic anaemia is 2–6 cases per million population, with most cases of aplastic

anaemia being classiﬁed as idiopathic (Young & Alter, 1994).

There are only a few recently documented cases of aplastic anaemia in patients

that were sensitive to chloramphenicol. Possible etiologic factors associated with

aplastic anaemia were identiﬁed in 151 Turkish patients who met the diagnostic

criteria. The ﬁndings suggested that these cases of aplastic anaemia were most

often idiopathic (99 out of 151 cases). The most common identiﬁable etiologic

CHLORAMPHENICOL

39834 FOOD TEXT 26/5/05 9:52 am Page 16

factor was the use of drugs (23 out of 151 cases), which were mainly non-steroidal

anti-inﬂammatory agents, while chloramphenicol appeared to be speciﬁed in 1 out

of 23 cases of drug use associated with aplastic anaemia. Exposure to benzene

was the second most common causal agent in the studied cases (19 out of 151

cases) (Alnigenis et al., 2001).

In an investigation of potential risk factors associated with aplastic anaemia in

the state of Parana, Brazil, the statistical evaluation of 125 cases of aplastic

anaemia showed no positive association between use of chloramphenicol and

development of aplastic anaemia. Instead, the causes of aplastic anaemia in Brazil

were apparently identiﬁed as common factors related to the disease, such as expo-

sure to certain chemicals. The incidence found in this study was similar to that

reported in Thailand and Europe (Maluf et al., 2002).

Additional reports evaluating the correlations between the incidence of aplas-

tic anaemia and use of chloramphenicol were documented in cases of aplastic

anaemia in Nigeria and in Nepal. In a 5-year prospective study in Nigeria, it was

estimated that aplastic anaemia developed in 0.002% of non-obstetric patients

treated with chloramphenicol. Of 18 cases of aplastic anaemia diagnosed in Nepal,

16 were identiﬁed as being idiopathic and one was found to be associated with

toxicity caused by treatment with chloramphenicol. Both studies concluded that

chloramphenicol-induced aplastic anaemia is rare (Durosinmi & Ajayi, 1993; Sah

et al., 1999).

It has been claimed that the topical ophthalmic use of chloramphenicol causes

bone-marrow aplasia, but this issue has not been completely resolved.

Recent observations have shown that the use of chloramphenicol as a topical

eye medication is unlikely to introduce aplastic anaemia. Two extensive popula-

tion-based studies in industrialized and developing countries presented no support

for the claim that eyedrops containing chloramphenicol increase the risk of

aplastic anaemia. The investigators found that there was no history of use of eye-

drops containing chloramphenicol in more than 400 cases of aplastic anaemia

examined. On the basis of this observation, the authors disagreed with the

general recommendation stating that use of eyedrops containing chloramphenicol

should be avoided because of an increased risk of aplastic anaemia (Wiholm

et al., 1998).

Serum concentrations of chloramphenicol were monitored in a study in 40

patients treated with eyedrops containing chloramphenicol. HPLC with a minimum

limit of detection (LOD) of 1 mg/l was used to measure the serum accumulation of

chloramphenicol after topical therapy. After a course of treatment in which the

mean dose of chloramphenicol received in 1 week of treatment was 8.0 mg, and

in2weeks was 15.3 mg, serum concentrations of chloramphenicol were below the

limit of detection. The authors considered that the topical use of chloramphenicol

was not a risk factor for induction of dose-related toxicity in bone marrow, and the

suspension of use of topical chloramphenicol in ophthalmic practice was ques-

tioned (Walker et al., 1998).

Despite the failure of epidemiological studies to ﬁnd an association between

the topical use chloramphenicol and development of aplastic anaemia, the

CHLORAMPHENICOL

39834 FOOD TEXT 26/5/05 9:52 am Page 17

hypothesis of a metabolic predisposition in individuals predisposed to blood

dyscrasias cannot be disregarded. Small doses of chloramphenicol similar to those

used in topical therapy may cause this idiosyncratic reaction in certain individuals.

In 1993, 23 cases of blood dyscrasias in patients treated topically with chloram-

phenicol for ophthalmic purposes were reported to the national register of drug-

induced ocular side-effects in Oregon, USA (Fraunfelder et al., 1993).

In a critical review of the potential risk of developing aplastic anaemia attribut-

able to topical use of chloramphenicol, the authors postulated that the risk posed

by topical use of chloramphenicol may be similar to that of orally administered chlo-

ramphenicol. This is because topical administration achieves systemic effects by

absorption through the conjunctival membrane or through drainage down the

lacrimal duct followed by absorption from the gastrointestinal tract. In their view, it

is not possible to justify subjecting patients to such potential risk, and therefore

ocular chloramphenicol should be used only when there is no alternative (Doona

& Walsh, 1995).

Contact sensitivity to chloramphenicol is rare in humans. Two cases of skin

ulcer, secondary to contact dermatitis, were reported in a woman aged 48 years

and a man aged 46 years. Both patients had applied chloramphenicol in the form

of chloromycetin cream to their wounds for about 1 month and developed skin

ulcer at the application sites. Hypersensitivity to chloramphenicol was conﬁrmed

by patch tests in both patients. The ulcer healed after use of the drug was dis-

continued (Matsumoto et al., 1998).

The teratogenic risk of chloramphenicol was studied in a population-based

dataset of the Hungarian case–control surveillance of congenital abnormalities,

1980–1996. Retrospective investigation of the effects of oral treatment with chlo-

ramphenicol during pregnancy was implemented in 38 151 pregnant women who

had healthy babies (control group) and 22 865 pregnant women who had con-

genitally abnormal newborns or fetuses. The case–control pair analysis of preg-

nant women who were treated in the second month or third month of pregnancy

did not reveal any teratogenic potential of chloramphenicol in humans. The authors

concluded that treatment with chloramphenicol during early pregnancy presents

little, if any, teratogenic risk to the fetus in humans (Czeizel et al., 2000). However,

the human embryo implants at day 6–7 of gestation and organogenesis begins at

day 21; heightened susceptibility to malformations occurs during this period

(Rogers & Kavlock, 2001). Therefore, this study may have overlooked the occur-

rence of early abnormalities.

3. PROBLEMS IN TRADE CAUSED BY RESIDUES IN FOODS

Although the use of chloramphenicol in veterinary medicine has been restricted

to non-food animals, residues have been found in samples taken from domesti-

cally produced animals in national monitoring programmes and foods, and in

samples moving in international trade. For example, results of analyses carried out

in Germany in the years 2000–2002 in accordance with directive 96/23/EEC

showed that a small fraction (<0.2%) of all samples (

>17 500) taken at farms

and slaughter houses contained residues of chloramphenicol. The concentrations

CHLORAMPHENICOL

39834 FOOD TEXT 26/5/05 9:52 am Page 18

that were found in a total of 11 positive samples from fattening cattle, swine and

poultry ranged from 0.3 to 3.3 mg/kg with eight values of <1mg/kg (BVL, 2003).

Unfortunately, comparable information was not available from many countries and,

the limits of detection or quantiﬁcation (LOD or LOQ) of the analytical methods

used in some countries were—until recently—too high to allow quantiﬁcation of

traces of chloramphenicol.

The Health and Consumer Protection Directorate-General of the European

Commission communicated ranges of concentrations of chloramphenicol in some

food items. These had been reported by Member States during the years

2000–2003 (European Commission, 2004). The information included concentra-

tions for the following commodities: skimmed milk powder (range, 0.021–

1.23 mg/kg), milk products (range, 0.3–1.27 mg/kg), honey (range, 0.3–4.0 mg/kg;

one sample at 38.7 mg/kg), washed pollen (0.58 mg/kg), shrimps (range, 0.1–

7.7 mg/kg; two samples at 31.89 and 297mg/kg), crabmeat (range, 0.3–1 mg/kg),

crayﬁsh (range, 0.14–6.3 mg/kg), casings (range, 0.5–2.9 mg/kg), rabbit meat

(0.3 mg/kg), turkey breasts (0.82 mg/kg), and chicken breasts (range, 0.4–

1.2 mg/kg).

The presence of residues of chloramphenicol has caused major food scares

in the past 2 to 3 years. Shrimps, prawns, food products from other aquatic

animals, honey,royal jelly,meat and offal, casings, rabbit, poultry meat and milk

powder are among the commodities in which the drug was found. Many of the

shipments of commodities in which residues of chloramphenicol were found orig-

inated in south-east Asia.

The Food Standards Agency of the United Kingdom, for example, has pub-

lished a table with test results (Food Standards Agency, 2002/2003) obtained with

samples of honey that were on sale in the United Kingdom. Using a method with

a“reporting limit” (RL) of 0.3 mg/kg, the results ranged from “none detected above

the RL set” to a maximum of 7.2mg/kg. Several samples also contained strepto-

mycin, for which the RL was 50mg/kg. A group of 20 samples of honey that were

analysed in the Netherlands (Voedsel en Waren Autoriteit, 2004) gave an average

mass concentration of 1.9 mg/kg (range, 0.06–5.9 mg/kg). Positive ﬁndings of

residues of chloramphenicol in honey from different geographic regions have also

been reported in the scientiﬁcliterature (Verzegnassi et al., 2003). Most of the con-

taminated honey originated from China. Reybroeck (2003) analysed samples of

honey available on the Belgian market; samples were screened using an enzyme-

linked immunosorbent assay (ELISA) (LOD, 0.1–0.3mg/kg, depending on

clean-up). Positive samples were subjected to high-performance liquid chro-

matography–mass spectrometry (HPLC–MS) conﬁrmatory analysis (LOD,

0.1 mg/kg). Of the samples with known origin, only samples of Chinese origin

contained residues of chloramphenicol (31 out of 40 samples). It has been hypo-

thesized that contamination of honey with chloramphenicol could be related to

treatments against foulbrood disease (Dharmananda, 2003).

While some of the positive ﬁndings are most likely to be the result of intentional

uses of chloramphenicol rather than of environmental contamination, it has also

been argued that very low concentrations of chloramphenicol detected in certain

foods of animal origin, e.g. in poultry and in products from aquaculture, could

CHLORAMPHENICOL

39834 FOOD TEXT 26/5/05 9:52 am Page 19

perhaps be derived from environmental sources—either from chloramphenicol

produced naturally by microorganisms or from residues resulting from past uses,

which still persist in the environment.

Examples of low concentrations from information published by the Food

Standards Agency of Ireland (Food Standards Agency of Ireland, 2002/2003) for

the year 2002 are summarized in Table 1. The Food Standards Agency of Ireland

has placed on the Internet information on alert/non-alert notiﬁcations of the

Member States of the European Union concerning residues of chloramphenicol in

shrimps, prawns, ﬁsh, ﬁshery products and several other food commodities. A

review of the data shows that at least nine of the Member States had communi-

cated such notiﬁcations. In the majority of approximately 110 short summaries on

notiﬁcations, no quantitative data on residue concentrations were communicated.

However, an evaluation of a total of 47 quantitative results (33 for shrimp samples,

4for prawn samples, 1 for ﬁsh and 9 for crabmeat, crayﬁsh and surimi samples)

showed the following distribution characteristics of the concentrations found:

range, 0.1–34 mg/kg; median: 0.5 mg/kg.

Although these data are not representative and the samples were not all inde-

pendent, it cannot be ruled out that there could exist two distributions of concen-

trations of residues:

—One distribution representing very low concentrations of chloramphenicol,

which could be explained either by environmental contamination (e.g. from nat-

urally produced chloramphenicol or from residues attributable to previous uses

of the drug), or by intentional uses of the drug followed by long withholding

times before harvesting;

—A second distribution representing higher concentrations of chloramphenicol

that are more likely to result from recent intentional uses and failure to observe

long withdrawal times before harvesting.

CHLORAMPHENICOL

Table 1. Range of concentrations of

chloramphenicol found in samples of aquaculture

products

Lower class limit Upper class limit Number of results

(mg/kg) (mg/kg)

0.1 0.5 25

>0.5 1.0 7

>1.0 1.5 2

>1.5 2.0 0

>2.0 2.5 2

>2.5 3.0 0

>3.0 3.5 3

>3.5 4.0 1

>4.0 4.5 1

>4.5 5.0 1

>5.0 34.0 5

From Food Standards Agency of Ireland (2002/2003).

39834 FOOD TEXT 26/5/05 9:52 am Page 20

The countries involved were not equally represented and the above numbers

of samples are too low for any valid comparison between countries of origin.

Another set of results was made available for evaluation by the Committee: the

average value of a population of 50 samples of shrimp in which the presence of

chloramphenicol was conﬁrmed indicated an average value of 0.25 mg/kg, (range,

0.06–0.69 mg/kg), with two outlying values of 3.0 and 3.7 mg/kg (Voedsel en Waren

Autoriteit, 2004).

The Centre of Analytical Services and Experimentation of Ho Chi Minh City

conducted a validation study of GC and HPLC–MS methods for detection of chlo-

ramphenicol; a number of shrimp samples were analysed. During the ﬁrst three

quarters of 2002, a total of 44 samples were found to contain chloramphenicol

(range of concentrations, 0.4–1.4mg/kg) (Ngoc Son, 2002).

The Fourteenth Session of the FAO/WHO Codex Alimentarius Committee for

Residues of Veterinary Drugs in Foods (CCRVDF) discussed the possibility that

such traces of chloramphenicol found in food-producing animals could originate

from environmental contaminations, rather than from intentional use. The report of

the session reﬂects the discussion as follows (Joint FAO/WHO Food Standards

Programme, 2003):

114. The request from Indonesia to consider the elaboration of an MRL for chloramphenicol

in shrimp was addressed by the Joint Secretariat who discussed the possibility that this com-

pound could ﬁnd its way into animal tissues via other routes than its use as a veterinary drug.

Limited data showed that chloramphenicol may persist in the environment or even be formed

by soil microorganisms. Hypothetically,very low levels found in animal products could there-

fore not be related to the use of chloramphenicol as a veterinary drug. Several delegations

stressed that it would be premature to draw any conclusions or to discuss a possible clas-

siﬁcation as a contaminant and that illegal use of the drug was a primary concern. It was

noted that international trade had been disrupted severely during the past year by the rejec-

tion of products which had been contaminated at very low levels with chloramphenicol and

some other veterinary drugs. The Committee noted the offer of the FAO Secretariat to JECFA

to examine the potential persistence of chloramphenicol in the environment or its formation

by soil microorganisms on the basis of data to be provided by Indonesia.

An attempt to investigate these possibilities has been made in the present

monograph. After discussion of the development and current status of analytical

methods, essentially two hypotheses for a possible environmental origin for very

low concentrations of residues in foods were tested. In the ﬁrst hypothetical sce-

nario it is assumed that:

—Chloramphenicol is naturally produced in the soil;

—Farm animals (e.g. pigs, chickens) ingest certain amounts of soil in a certain

proportion of their daily intake of dry matter;

—This may result in an uptake of chloramphenicol and subsequently in residues

of chloramphenicol in tissues and products of pigs and chickens, which are not

related to veterinary uses of chloramphenicol as a drug.

The second hypothetical scenario assumes that food-producing animals may

currently still occasionally be exposed to persisting environmental residues of chlo-

ramphenicol resulting from historical veterinary uses.

CHLORAMPHENICOL

39834 FOOD TEXT 26/5/05 9:52 am Page 21

This monograph also assesses the hypothetical dietary intakes resulting from

low-level contamination of seafood with residues of chloramphenicol and com-

pares these intakes with the lowest known human therapeutic exposures.

4. ANALYTICAL METHODS

The ideal method for the analysis of residues of chloramphenicol should be

sensitive, accurate and precise, and provide unambiguous information on the iden-

tity of the analyte. Furthermore, it should be as cost-effective and robust as

possible. In practice, it is difﬁcult to develop methods that combine all these char-

acteristics. Therefore, the analytical strategies used for the control of residues of

chloramphenicol in animal tissues frequently include the initial application of a

screening method, followed by a conﬁrmatory analysis of those samples that gave

positive results with the screening method.

Typically, the three basic steps used in the majority of methods of analysis for

chloramphenicol are:

—Preparation of the primary extract of the sample;

—Puriﬁcation of the primary extract;

—Detection and quantiﬁcation of residues of chloramphenicol.

4.1 Preparation of the primary extract of the sample

This step usually includes homogenization and extraction of the tissue with suit-

able organic solvents, the separation of liquids and solids and the removal of lipids

from the crude extract. Temperature control during storage and extraction of the

sample is important in order to avoid metabolism in vitro, particularly in samples

of liver and kidney, respectively, owing to the action of metabolizing enzymes

(Parker & Shaw,1988; Sanders et al., 1991). Analysis of liver and kidney requires

enzymatic hydrolysis of conjugates of chloramphenicol; this step can apparently

be omitted when working with trout tissues (Baradat et al., 1993; Mottier et al.,

2003).

Use of the following organic solvents has been described for the extraction of

chloramphenicol:

—Ethyl acetate (van Ginkel et al., 1990; Van der Heeft et al., 1991; Sanders

et al., 1991; Nagata & Saeki, 1992; Keukens et al., 1992; Kijak, 1994; Epstein,

1994; Munns et al., 1994; Li et al., 2001; Neuhaus et al., 2002; Gantverg

et al., 2003; Impens et al., 2003; Stuart et al., 2003);

—Acetonitrile (Borner et al., 1995; Pfenning et al., 1998);

—A mixture of acetonitrile and acetate buffer pH 5.0 (Posyniak et al., 2003);

—A mixture of ethyl acetate and acetonitrile (Pfenning et al., 2002a);

—A mixture of chloroform and acetone (Perez et al., 2002).

CHLORAMPHENICOL

39834 FOOD TEXT 26/5/05 9:52 am Page 22

The lipids were removed through solvent partition, using:

—

-Hexane (Nagata & Saeki, 1992; Kijak, 1994; Epstein, 1994; Chevalier et al.,

1995; Gude et al., 1995; Li et al., 2001; Perez et al., 2002; Pfenning et al.,

2002a; Turnipseed et al., 2002; Storey et al., 2003; Gantverg et al., 2003;

Pfenning et al., 2003);

—A mixture of

-hexane and chloroform (1 : 1) (Sanders et al., 1991);

—

-Heptane (Rupp et al., 2003; Stuart et al., 2003).

4.2 Puriﬁcation of the primary extract

There are numerous procedures used to purify the primary extract in order to

remove substances interfering with the detection and quantiﬁcation step. Solid-

phase extraction is the most widely used technique for puriﬁcation in the analysis

of residues of chloramphenicol in food matrices (Chevalier et al., 1995; Neuhaus

et al., 2002; Turnipseed et al., 2002; Storey et al., 2003; Gantverg et al., 2003;

Pfenning et al., 2003; Posyniak et al., 2003; Mottier et al., 2003; Impens et al.,

2003).

There are, however,other puriﬁcation techniques, for example, immunoafﬁnity

chromatography (van de Water et al., 1989; Gude et al., 1995), which takes advan-

tage of highly selective hapten–antibody interactions.

4.3 Detection and quantiﬁcation of residues of chloramphenicol

In past decades, several analytical methods have been developed and

reviewed for the detection and quantiﬁcation of chloramphenicol in foods and

biological ﬂuids.

Gas chromatography

:the presence of polar functional groups in the chloram-

phenicol molecule requires a derivatization step, usually through a sylilation reac-

tion, before gas chromatography analysis. The attachment of particular functional

groups onto the chloramphenicol molecule may also lower the LOD of the elec-

tron capture detector (ECD) and MS. The silylation reaction is usually catalysed

by acids or bases. Frequently used silylation reagents include:

—A mixture of hexamethyldisilazane (HMDS), trimethylchlorosilane (TMCS) and

pyridine (Berry, 1987; Gude et al., 1995);

—

-bis(trimethylsilyl)-triﬂuoroacetamide (BSTFA) (Bories et al., 1983; Costa

et al., 1993);

—A mixture of BSTFA and TMCS (van Ginkel et al., 1990; Keukens et al., 1992;

Gantverg et al., 2003);

—

-methyl-

(trimethylsilyl)-triﬂuoroacetamide (MSTFA) (Impens et al., 2003).

ECD has been widely used for the analysis of chloramphenicol. The GC–ECD

method for analysis of chloramphenicol in muscle of prawns, described by Munns

et al. (1994) for example, reached a LOD of 1mg/kg.

CHLORAMPHENICOL

39834 FOOD TEXT 26/5/05 9:52 am Page 23

When GC is coupled with MS, the most frequently applied ionization techniques

are chemical ionization (CI) and electron impact (EI).

Negative chemical ionization (NCI) presents a limited number of fragments;

however, the molecular ion is always part of the spectrum. Owing to the presence

of two chlorine atoms in the chloramphenicol molecule, GC–MS in NCI mode is

one of the most reliable techniques, and it is the most commonly used method for

the conﬁrmation of residues of chloramphenicol. The LOD can be <0.1 mg/kg in

muscular tissue (van Ginkel et al., 1990; Epstein, 1994; Borner et al., 1995).

GC–MS in EI mode is less sensitive; however, it produces spectra of fragments,

which are reproducible with different instruments, and can therefore be stored in

electronic databases for reference purposes.

HPLC

:The development of atmospheric pressure ionization (API) mass detec-

tors, coupled HPLC, has become one of the most reliable and widespread tech-

niques in the analysis of residues of chloramphenicol. This combination of liquid

chromatography and mass spectrometry (LC–MS) enables the detection and

quantiﬁcation, without derivatization, of polar non-volatile analytes, such as

chloramphenicol.

Neuhaus et al. (2002) have described an LC–MS/MS method with a LOD of

0.08 mg/kg and an LOQ of 0.3 mg/kg in prawns. Mottier et al. (2003) have devel-

oped a method for meat (chicken, turkey,pork and beef) and aquatic products

(crab, prawn and ﬁsh), using liquid chromatography (electrospray ionization)

tandem mass spectrometry (LC(ESI)–MS/MS), with isotopic dilution, reaching LOD

and LOQ values of 0.003 mg/kg and 0.01 mg/kg, respectively.

Impens et al. (2003) have described an analytical strategy for the screening

and conﬁrmation of residues of chloramphenicol in prawn tissue, using ELISA for

screening and GC–MS/MS and LC–MS/MS for conﬁrmation; both selective tech-

niques reached LODs of 0.1 mg/kg.

Gantverg et al. (2003) have developed a very sensitive method for the detec-

tion and conﬁrmation of chloramphenicol in pork and beef muscle and in urine.

After extraction, chloramphenicol was determined through LC–MS/MS in CI mode

at negative atmospheric pressure, and by GC–MS in EI mode.

In summary,GC–MS and LC–MS/MS are, nowadays, the most reliable tech-

niques for the determination of residues of chloramphenicol in edible animal

tissues.

Selected examples of the development of analytical methods between 1990

and 2003 are listed in Table 2.

The European Union has recently deﬁned minimum required performance

limits (MPRLs) for analytical methods used for the determination of substances for

which no permitted limit has been established, and in particular for those sub-

stances, like chloramphenicol, whose use is not authorized or speciﬁcally prohib-

ited by Community legislation. For chloramphenicol, a MRPL of 0.3mg/kg was

established (European Commission, 2003).

CHLORAMPHENICOL

39834 FOOD TEXT 26/5/05 9:52 am Page 24

CHLORAMPHENICOL

Table 2. Overview of the development of analytical methods for residues of chloramphenicol in foods of animal origin

Analytical Food matrix LOD LOQ Levels reported Reference

method (mg/kg) (mg/kg) (mg/kg)

GC(NICI)–MS Muscle/egg 0.1 >0.1 —van Ginkel et al. (1990)

LC/UV Calf muscle 1 — — Sanders et al. (1991)

LC–MS Some aquatic species 0.1 0.3 — Van de Riet et al. (1992)

GC(NICI)–MS Bovine muscle 0.6 — — Epstein (1994)

GC(NICI)–MS Cows’ milk 0.5a— — Kijak (1994)

GC–ECD Shrimp 1 — — Munns (1994)

GC(NICI)–HRMS Egg 0.3 0.5 —Borner et al. (1995)

GC–ECD Egg 0.3 0.5 —

HPLC (RP) Foie gras 2.5 — — Chevalier et al.

LC/UV Pasteurized milk 50a— — Perez et al. (2002)

LC–MS/MS ESI(-)Shrimp <0.5 — — Pfenning et al. (2002b)

LC–MS/MS Shrimp 0.08 0.3 —Neuhaus et al. (2002)

GC–ECD Shrimp 0.05 —0.65–0.72 Ngoc Son (2002)

GC(NCI)–MS Shrimp 0.3 — —

LC–MS/MS APCI(-)Shrimp 0.1 — —

LC–MS/MS APCI Equine, porcine and bovine muscle 0.02 — — Gantverg et al. (2003)

GC–MS/MS Shrimp 0.1 — — Impens et al. (2003)

LC–MS/MS Shrimp 0.1 — —

LC(ESI)–MS/MS Chicken meat 0.003 0.01 —Mottier et al. (2003)

LC–MS/MS (ESI) Shrimp, crab 0.1 — — Storey et al. (2003)

GC–ECD Bovine muscle —0.25 —United States Department of

GC(NICI)–MS Bovine muscle —0.25 —Agriculture (2003)

APCI, atmospheric pressure chemical ionization; ECD, electron capture detection; ESI, ion electrospray; ESI(-), negative ion electrospray; GC,

gas chromatography; HRMS, high-resolution mass spectrometry; LC, liquid chromatography; LOD, limit of detection; LOQ, limit of quantiﬁcation;

MS, mass spectrometry; NICI, negative ion chemical ionization; RP, reverse phase; UV, ultraviolet.

a(mg/l).

39834 FOOD TEXT 26/5/05 9:52 am Page 25

5. PRODUCTION, OCCURRENCE AND FATE OF CHLORAMPHENICOL

IN THE ENVIRONMENT

5.1 Discovery

Chloramphenicol was ﬁrst described as a new antibiotic produced by cultures

of an actinomycete isolated from soil by Ehrlich et al. (1947). The soil samples

from which the strains were isolated were collected from a mulched ﬁeld near

Caracas, Venezuela (strain ATCC 10712) and from a compost soil on the horti-

cultural farm of the Illinois Agricultural Experiment Station at Urbana (strain ATCC

10595), respectively. It was demonstrated by Ehrlich et al. (1948) that this actino-

mycete was a new species. The dynamics of the synthesis of chloramphenicol

were studied under laboratory conditions by Legator & Gottlieb (1953), who

showed that the peak concentration of chloramphenicol in the culture medium was

reached hours after the growth peak of the microorganisms. The antibiotic was not

accumulated intracellularly.Addition of chloramphenicol to the culture medium

inhibited the synthesis of the antibiotic.

Chloramphenicol was also isolated from the soil actinomycete

Streptospo-

rangium viridogriseum

var.

kofuense

by Tamura et al. (1971) and from the marine

snail

Lunatia heros

(moon snail) by Price et al. (1981).

5.2 Biosynthesis

The biosynthetic route of chloramphenicol starts with the general shikimate

pathway for assembling aromatic structures. It then branches at chorismic acid to

generate

-amino-phenylalanine, which serves as an advanced precursor of the

-nitrophenylserinol moiety of chloramphenicol (He et al., 2001; Lewis et al., 2003).

3¢-

-Acetyl-chloramphenicol, which is commonly formed from chloramphenicol by

many resistant bacteria, has also been isolated from the antibiotic-producing

organism. It has been suggested that it is a protected intermediate in chloram-

phenicol biosynthesis, implicating acetylation as a self-resistance mechanism in

the producing organism (Gross et al., 2002). 3¢-

-Acetyl-chloramphenicol esterase

activity was detected in cell-free extracts of strains of

Streptomyces venezuela

other

Streptomyces

spp. and

Streptosporangium viridogriseum

var.

kofuense

which produced chloramphenicol (Nakano et al., 1977).

5.3 Production and stability in soil

Gottlieb & Siminoff(1952) studied the adsorption, stability,and rate of produc-

tion of chloramphenicol in soil under different laboratory conditions. Chloram-

phenicol was poorly adsorbed to soil. When the drug was added to sterilized soil

at a concentration of 50 mg/kg, approximately 80% could be recovered over the

whole observation period of 14 days. When the same experiment was carried out

using non-sterile soil, the antibiotic was slowly degraded. When sterilized soil was

infested with

S. venezuelae

and was incubated for long periods, the authors were

able to show the presence in soil of chloramphenicol formed by the microorgan-

ism following a lag phase of several weeks. The highest concentration measured

was 1.12 mg/kg. Table 3 summarizes some of the results obtained in experiments

CHLORAMPHENICOL

39834 FOOD TEXT 26/5/05 9:52 am Page 26

CHLORAMPHENICOL

Table 3. Production of chloramphenicol in sterilized soil infested with

S. venezuelae

Time Concentration of Number pH Concentration of Number pH Concentration of Number pH

(days) chloramphenicol of cells chloramphenicol of cells chloramphenicol of cells

(mg/g) (105/g) (mg/g) (105/g) (mg/g) (105/g)

Experiment 1 Experiment 2 Experiment 3

0 — 2.00 5.90 —0.39 5.90 0.0 38.7 5.45

70.00 23.30 5.57 0.00 35.50 5.05 0.0 12.0 5.05

20 0.00 28.50 5.10 1.12 48.20 5.60 0.0 37.0 5.63

36 0.58 18.20 5.45 — — — — — —

65 — — — 0.56 62.50 5.63 0.5 30.0 5.45

93 0.50 168.00 5.75 — — — — — —

100 — — — 0.82 945.00 5.85 0.79 1230.0 5.78

39834 FOOD TEXT 26/5/05 9:52 am Page 27

designed to study the interaction of

S.venezuelae

and chloramphenicol-sensitive

Bacillus subtilis

.The table gives the results of the control experiments in which

S. venezuelae

was the only microorganism added.

When organic substrates were added to the soil before sterilization, the pro-

duction of chloramphenicol increased after the addition of

S. venezuelae

.Under

the most favourable conditions of growth (in the presence of tryptone), chloram-

phenicol accumulated in the soil at a concentration of 25.0–27.8 mg/kg during

18–31 days of incubation. Addition of 1% of more “natural” substrates like alfalfa,

corn stover and soybean straw also increased the production of chloramphenicol.

However, only in the presence of alfalfa were signiﬁcant quantities (concentration,

1.4 mg/kg) observed.

These results should not be interpreted to mean that

S. venezuelae

produces

chloramphenicol in soil in appreciable amounts under natural conditions. Natural

soil is not usually a good substrate for production of antibiotic (Gottlieb, 1976).

Ehrlich’s group has investigated soils from 91 cultivated and grassland sites in nine

states of the USA and from 13 other countries and found that soil samples were

either infested with

Streptomyces venezuelae

or were not infested. No chloram-

phenicol was identiﬁed in extracts from either of these soils. Initially, the LOD of

chloramphenicol was 0.3 mg/kg using a test based on the antimicrobial activity of

chloramphenicol. In other experiments with LOD of 0.05mg/kg in a chemical assay

selective for the nitro group in the chloramphenicol molecule, these negative

results were conﬁrmed. If the soils were sterilized before seeding, chlorampheni-

col was found and identiﬁed in the infested soils (Ehrlich et al., 1952a, 1952b).

In their soil studies, Ehrlich et al. also investigated the recovery of smaller

amounts of chloramphenicol added to non-sterilized soil. When chloramphenicol

was incubated in sterilized soil at a concentration of 4.6mg/kg for 92 days at 23–

27 °C, recoveries were approximately 40% throughout the observation period.

When the same incubation was performed with non-sterile samples of soil, the

concentrations of chloramphenicol declined as a function of the incubation time as

shown in Table 4. A graph of the same data (see Figure 1) suggests a biphasic

curve for the degradation of chloramphenicol in soil. However, the second phase

could be the result of the closeness to the LOQ of the observed concentrations.

For the values ranging sufﬁciently above the LOQ (days 0–17 of the experiment),

ahalf-life in the order of 3–4 days was estimated.

Whether antibiotics are produced in soil in appreciable amounts by indigenous

soil organisms has remained a scientiﬁc dispute for several decades. Only recently,

it has been demonstrated that an antibiotic can be synthesized in detectable

amounts in soil. Using biosensor methods with very low LODs, Hansen et al.

(2001) have demonstrated the presence of oxytetracycline produced by

Strepto-

myces rimosus

in untreated soil. However, similar studies with chloramphenicol

were not found.

5.4 Environmental fate of chloramphenicol

The Food and Drug Administration of the USA has published an environmen-

tal assessment of chloramphenicol in the context of a proposal made in March

CHLORAMPHENICOL

39834 FOOD TEXT 26/5/05 9:52 am Page 28

CHLORAMPHENICOL

Figure 1. Stability of chloramphenicol in non-sterile soil

0.01

0.1

0 10 20 30 40 50

Time after addition (days)

Concentration (mg/kg)

Recovery LOQ, limit of quantiﬁcation

1985 to withdraw approval of new animal drug applications (NADAs) using chlo-

ramphenicoloral solution (Food and Drug Administration, 1985). Further informa-

tion was obtained from the Hazardous Substances Databank, a database of the

National Library of Medicine’s TOXNET system (Hazardous Substances Databank,

2003). The information available from these sources (most of the original literature

cited was not available for this review) is summarized as follows. The solubility of

chloramphenicol in water at 25°C is 2.5 g/l over a wide range of pH. Chloram-

phenicol is not adsorbed to clay or soil to any signiﬁcant degree and therefore has

Table 4. Recovery of chloramphenicol from non-

sterile soil after the addition of chloramphenicol at

4.6mg/kg

Time (days) Recovery of chloramphenicol from soil

(mg/kg)

1/24 (1 h) 1.900 1.700 2.200

1 1.300 1.300 1.200

2 0.850 1.000 0.890

3 0.770 0.740 0.820

4 0.550 0.240 0.470

5 0.600 0.690 0.600

7 0.250 0.290 0.440

11 0.063 0.069 0.270

17 0.130 0.130 0.150

29 0.041 0.093 0.041

45 <0.027 0.036 0.033

92 <0.027 <0.027 <0.027

39834 FOOD TEXT 26/5/05 9:52 am Page 29

very high mobility in soil. Adsorption to sediment and bioconcentration in aquatic

organisms should not be important processes. Chloramphenicol is degraded by

biological, chemical, and photolytic means and undergoes oxidation, reduction and

condensation reactions upon exposure to light in aqueous solution. Photochemi-

cal decomposition of chloramphenicol in vitro by ultraviolet-A (UV-A) light leads to

the formation of

-nitrobenzaldehyde (pNB),

-nitrobenzoic acid (pNBA) and

-nitrosobenzoic acid (pNOBA); the latter comprises up to 45% by molarity of the

starting amount of chloramphenicol (de Vries et al., 1994).

The half-life of chloramphenicol in soil at 25 °C is 4.5 days; in pond water the

half-life is 10.3 days at 25°Cand pH 8, and 20.8 days at 37 °Cand pH 6.

The log Kow for chloramphenicol is 1.14. With regard to sorption coefﬁcients to

soil solids (Kd,solid), the range of values for chloramphenicol is in the same group

as, for example, olaquindox, sulfamethazine, sulfathiazole, and metronidazole,

which also appear to have little sorption afﬁnity to soil particles, as is evidenced

by their low values of Kd,solid (0.2–2 l/kg) (Tolls, 2001; Rabolle & Spliid, 2000).

Lai et al. (1995) estimated Kd,solid values of 0.4 l/kg for a freshwater sediment

(salinity, 0g/kg ; pH 7.7; sulfate, 4.8 mmol/l) from an eel pond in Taiwan, China,

and of 0.2 l/kg for a marine sediment (salinity,33g/kg; pH 8.2; sulfate, 25.6 mmol/l)

from a shrimp farm in Taiwan, China. The authors used top sediment (0–5cm).

The typical concentration of chloramphenicol was 60–70 mg/l throughout all exper-

iments. However,possible effects of the concentration of chloramphenicol were

also studied using concentrations of 100, 200, and 400mg/l. The average rates of

chloramphenicol transformation were much higher under anaerobic conditions

than under aerobic conditions. Some selected results are summarized in Table 5.

Chloramphenicol also degraded very slowly in sterilized slurries.

Although the physicochemical mechanism of sorption interactions is not known,

it is possible that chloramphenicol might form charge–transfer complexes with soil

constituents (Haderlein & Schwarzenbach, 1993).

Lai et al. (1997) added sodium chloride to brackish water top sediment

(0–5 cm) slurries (10% slurry; salinity,24g/kg; pH 7.9) of a shrimp pond to obtain

salinities of 30 and 36 g/kg and conducted sorption and transformation experiments

at ﬁnal concentrations of chloramphenicol of 60–70 mg/l and temperatures

CHLORAMPHENICOL

Table 5. Rate of degradation (mg/l per day) of chloramphenicol in slurries of

aquaculture pond soils

Condition Sediment Concentration of chloramphenicol (mg/l)

incubation 100 200 400

Aerobic Freshwater eel pond 6.4 9.2 9.7

Marine shrimp pond 1.6 1.8 1.6

Anaerobic Freshwater eel pond 20.7 21.1 20.4

Marine shrimp pond 20.3 21.3 15.6

39834 FOOD TEXT 26/5/05 9:52 am Page 30

of 20–25 °C. The experiments were carried out under either anaerobic or aerobic

conditions. While sorption to soil of chloramphenicol was not inﬂuenced by

changes in salinities and by aerobic or anaerobic conditions, the compound was

much more stable under aerobic conditions. Increasing salinities also slowed down

the degradation process under aerobic conditions but not under anaerobic condi-

tions. Selected results from this study are summarized in Table 6.

Chien et al. (1999) studied the degradation of chloramphenicol in aquaculture

pond sediment. Freshwater (salinity,0g/kg) eel pond sediment slurries (10% w/v)

were treated with sodium chloride to obtain salinities of 12, 24 and 36g/kg. There

were no signiﬁcant differences in sorption rate either between aerobic and anaer-

obic conditions or among various salinities. Degradation of chloramphenicol ﬁtted

well to an exponential curve. The degradation rates under anaerobic conditions

were signiﬁcantly greater than those under aerobic conditions. As salinity

increased, the degradation rates decreased under both aerobic and anaerobic con-

ditions in this experiment. Selected results are summarized in Table 7.

These studies demonstrate that chloramphenicol can be quite stable under

suitable aerobic and ionic conditions and at normal pH.

5.5 Results of environmental monitoring for chloramphenicol

Hirsch et al. (1999) have estimated that 20.1 million daily doses of chloram-

phenicol were prescribed in 1995 in Germany for human medical use (indications

and doses not given). They estimate that 5–10% of the doses were excreted

unchanged and 70–90% were excreted as the glucuronide. When they analysed

10 samples of sewage treatment plant efﬂuents with a LOQ of 0.02mg/l, they found

one sample containing 0.56 mg/l of chloramphenicol. Of 52 samples of surface

water,four contained chloramphenicol at concentrations greater than the LOQ,

with a maximum of 0.06 mg/l in one sample. As a general rule, concentrations of

antibiotic residues in sewage treatment plant efﬂuents were approximately one

order of magnitude higher than the concentrations found in surface water.In 59

samples of ground water, no chloramphenicol residues at concentrations greater

than the LOQ were found. Chloramphenicol residues were not found in samples

CHLORAMPHENICOL

Table 6. Inﬂuence of salinity and oxygen on sorption and stability of

chloramphenicol in slurries of pond soils (brackish water)

Salinity % Chloramphenicol -

at1/2 -

at1/2

(g/kg) adsorbed within 1 h (days) (days)

Aerobic Anaerobic Aerobic Anaerobic

24 22 25 0.067 10.0 0.573 1.2

30 18 24 0.031 23.0 0.642 1.1

36 17 18 0.012 57.8 0.578 1.2

From Lai et al. (1997).

is the ﬁrst order rate constant of the degradation of chloramphenicol. Its dimension is

1/day.

39834 FOOD TEXT 26/5/05 9:52 am Page 31

taken during a study conducted in the USA (Kolpin et al., 2002). It was also not

found in samples of groundwater,surface water and drinking-water analysed in a

recent study conducted in the Netherlands (Stolker et al., 2003) and using sensi-

tive analytical methods with a LOQ for chloramphenicol of 0.005mg/l (Versteegh

et al., 2003).

Hamscher et al. (2003) studied sedimentation dust collected between 1981 and

2000 in a pig ﬁnishing unit (350–420 animals). Each year, 10–15 samples were

collected over periods of 14–30 days. One randomly selected sample was

analysed for each year.Chloramphenicol was detected in three out of 20 samples

(representing the years of sampling 1989, 1991, and 1992) at concentrations of

1.96, 0.07, and 5.49 mg/kg, respectively.The samples had been stored for more

than 10 years before analysis.

5.6 Microbial resistance to chloramphenoicol in the environment—is it

an argument for the presence of the drug?

The mere isolation of chloramphenicol-resistant microorganisms from the envi-

ronment, including soil, can probably not be used as an argument for the pres-

ence of the drug. The phenomenon of resistance is too complex and its occurrence

does not need to be related to any history of the use of chloramphenicol itself. For

example, Kardavy et al. (2000) isolated chloramphenicol-resistant species of

Prov-

idencia rettgeri

from the gut of larvae of the oil ﬂy inhabiting the 40 000-year-old

asphalt seeps of Rancho La Brea in California. They found a correlation between

antibiotic resistance and organic solvent tolerance, which could be explained by

the presence of an active efﬂux pump maintained by the constant selective pres-

sure of the solvent-rich environment. These efﬂux pumps expel a broad range of

comparatively hydrophobic antibiotics (chloramphenicol, erythromycin, nitrofuran-

toin, novobiocin, rifampin, spectinomycin, and vancomycin), most of which contain

aromatic ring systems.

Providencia

spp. are also known as agents of nosocomial

infections. Efﬂux pumps play also an important role in resistant strains of other

bacterial species (Malléa et al., 2003).

CHLORAMPHENICOL

Table 7. Inﬂuence of salinity and oxygen on sorption and stability of

chloramphenicol in slurries of pond soils (eel pond)

Salinity % Chloramphenicol -

at1/2 -

at1/2

(g/kg) adsorbed within 1 h (days) (days)

Aerobic Anaerobic Aerobic Anaerobic

0 28 23 0.297 2.4 1.915 0.4

12 25 21 0.155 4.5 0.957 0.7

24 27 25 0.080 8.9 0.495 1.4

36 28 23 0.039 18.4 0.286 2.4

From Chien et al. (1999).

is the ﬁrst order rate constant of the degradation of chloramphenicol. Its dimension is

1/day.

39834 FOOD TEXT 26/5/05 9:52 am Page 32

Resistance to chloramphenicol in

Salmonella enterica

serovar

typhimurium

iso-

lated from cattle in the USA has drastically increased over the years (Davis et al.,

1999) owing to sharply increased occurrence of isolates displaying DT104-linked

resistance. Although the use of chloramphenicol is prohibited in the USA, it was

never authorized for use in food-producing animals and monitoring results do not

suggest widespread illegal use. A gene conferring cross-resistance to ﬂorfenicol

and chloramphenicol has been isolated from

Salmonella enterica

serovar

typhimurium

(

S. typhimurium

)DT104 (Bolton et al., 1999). A conjugative plasmid,

pOLA52, conferring resistance to the antibiotic growth promoter olaquindox has

been isolated from

Escherichia coli

from swine manure. It also confers resistance

to ampicillin and chloramphenicol and has a high frequency of transfer between

strains of

E. coli

(Sørensen et al., 2003).

Petersen et al. (2002) have studied the development of antibiotic resistance in

integrated ﬁsh farms in Asia. The farms used antimicrobial agents and animal

manure was shed directly into ﬁsh ponds as fertilizer in these farms. Three of the

farms were using chloramphenicol in ducks and pigs. The impact of the use of

antibiotics on the development of antimicrobial resistance among the indicator

microorganisms was greatest at the beginning of a ﬁsh production cycle. However,

the most signiﬁcant increase in resistance to chloramphenicol occurred on a farm

where this antibiotic was not used (amoxicillin, enroﬂoxacin, norﬂoxacin, tylosin

were used on this farm).

For such reasons, the many reports dealing with resistance phenomena dis-

covered in the environment have not been used here as an argument for the pres-

ence of chloramphenicol in the environment.

6. FEED AND SOIL INTAKES AND GROWTH OF SOME

TERRESTRIAL FARM ANIMALS

6.1 Pigs

Table 8 provides an example and short summary of the relationship between

age, live weight, feed intake and daily live-weight gain in pigs raised in the western

hemisphere (Peer et al., 2001).

Areview of recent research papers indicates that the situation in south-east

Asia could differ signiﬁcantly from that observed in, for example, certain agricul-

tural areas of the USA. Figure 2 summarizes the results of this review.More details

are given in the Appendix I at the end of this document. From these details it can

be seen that the typical daily dry matter intake of the animals is in the same order

in the different hemispheres. However, the growth rates of the pigs used in the

studies in south-east Asia were comparatively lower.

The data for the pigs in Iowa in Figure 2 were retrieved from the Internet (The

pig site, 2003). The data for pigs in south-east Asia were taken from the studies

summarized in the tables given in Appendix I at the end of this monograph. These

data were collected from published results of research projects carried out in south-

east Asia. They may give an incomplete picture and real conditions may vary

largely from country to country, and between regions and provinces of a given

CHLORAMPHENICOL

39834 FOOD TEXT 26/5/05 9:52 am Page 33

country,and may also be subject to rapid changes. However,acomplete review

of animal feeding practices is outside the scope of this monograph. The informa-

tion provided in the Appendix I is limited to the minimum necessary to derive suit-

able relationships between live weight, live-weight gain and feed intake since this

information is needed to estimate soil ingestion as function of growth and intake

of dry matter of the animals, and to relate hypothetical environmental doses of

chloramphenicol to estimated soil ingestion.

As a result of the data discussed in this section, one can assume for simple

model calculations that pigs generally eat approximately 35 gof dry matter per kg

bw per day. Using the data in Figure 2, the average daily live-weight gain is esti-

mated as a function of body weight (within the limits of 20 g and 75 kg body weight)

according to the formula:

CHLORAMPHENICOL

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 25 50 75 100 125

Body weight (kg)

Average daily gain (kg)

Pigs in south-east Asia

Pigs in Iowa

Predicted body-weight gain (kg)

Body weight is the mean of the initial and the ﬁnal body weight of a feeding period.

Average daily gain is the average over the whole feeding period.

Figure 2. Comparison of growth rates of pigs raised in different regions of the

world

Table 8. Typical feed intakes and live-weight gain in pigs raised in the western

hemisphere

Production Live weight Average daily Dry matter intake

class (kg) gain (kg)

(kg/animal per day) (kg of dry matter/kg

average daily gain)

Starter 10–20 0.450 0.80–0.84 1.78–1.87

Grower 20–50 0.700 1.61–1.91 2.04–2.73

Finisher 50–100 0.820 2.63–3.29 2.94–4.01

From Peer et al. (2001).

39834 FOOD TEXT 26/5/05 9:52 am Page 34

Average daily live-weight gain (g) =0.164g +0.00693 (g/kg) ¥Body weight (kg)

6.2 Chickens

It is not within the scope of this monograph to describe the range of different

production systems. An example of data that have been gathered in the western

hemisphere can be found in tabular form in a publication of the National Academy

of Sciences of the USA (National Academy of Sciences, 1994). However, under

the hypothesis that soil ingestion could be the cause of chloramphenicol residues

in tissues and edible products, extensive systems—as they still exist, for example,

in south-east Asia, with free-ranging scavenging chickens receiving supplemen-

tary feeds—are of particular interest. Local breeds may play an important role, at

least regionally (Nguyen Dang Vang & Le Viet Ly, 2000). Some of these breeds

have small bodies and growth rates and feed conversion may vary largely (Tran

Thi Mai Phuong et al., 2003). A limited amount of data from south-east Asia were

available from published results of research projects. These results, which are not

necessarily representative, are summarized in more detail in Appendix I. Selected

data are presented in Figure 3. The study of Duong Duy Dong (2003) provided

useful data for model calculations.

As a result of the evaluation of the data discussed in this section, the daily live-

weight gain of chickens with a body weight of between 150 and 1000 gwas cal-

culated according to the formula:

Average daily live-weight gain (g) =3.599 g+0.01623 (g/kg) ¥Body weight (kg)

The intake of dry matter is approximately 3 g per g of average daily live-weight

gain.

6.3 Cattle

Chloramphenicol is not highly systemically bioavailable after per-oral adminis-

tration to ruminating cattle. The drug is largely degraded by microﬂora in the rumen.

Corresponding calculations have not, therefore, been performed for cattle.

7. HYPOTHETICAL INTAKE OF CHLORAMPHENICOL FROM

THE ENVIRONMENT BY SOIL INGESTION

7.1 Soil ingestion by farm animals

Soil ingestion varies seasonally and according to farm management. Using

the titanium content of faeces as a stable indicator of soil ingestion, Thornton &

Abrahams (1983) found that grazing cattle involuntarily ingest from 1% to nearly

18% of their dry matter intake as soil; sheep may ingest up to 30%. Abrahams et

al. (2003) studied rates of soil ingestion by sheep grazing on metal-enriched ﬂood-

plain soils and found very high rates of soil intake during the winter/spring season

with maximum rates during March, when soil ingestion exceeded 30% of the dry

matter intake at two of the 11 sites investigated. No detailed quantitative data on

soil ingestion of chickens and pigs were available for evaluation, although the

CHLORAMPHENICOL

39834 FOOD TEXT 26/5/05 9:52 am Page 35

phenomenon of soil ingestion in (food) animals is well recognized and frequently

described in the literature.

Fries et al. (1982) investigated soil ingestion by dairy cattle using quantitative

analysis of titanium in faecal samples and in soils to which the animals had access.

Selected results are summarized in Table 9.

7.2 Intake of chloramphenicol and resulting residues in tissues

7.2.1 Pigs

Using the information collected in section 6, hypothetical intakes of chloram-

phenicol were calculated as a function of intake of dry matter and corresponding

soil intake of the animals. It was assumed that soil represented 2% of dry matter

intake. The concentration of chloramphenicol in soil was set at one of the follow-

ing concentrations:

—£0.05 mg/kg, which corresponds to the LOD of the methods used by

researchers from Parke-Davis who were unable to detect chloramphenicol

above this limit when they analysed a large number of soil samples from dif-

ferent countries; or

—1 mg/kg, which roughly corresponds to the highest concentration produced in

soil samples to which “natural” organic matter was added, and which were then

sterilized and infested with

S. venezuelae

;or

—25 mg/kg, which roughly corresponds to the highest concentration found in

laboratory experiments with soils treated with tryptone, and under the most

favourable conditions.

CHLORAMPHENICOL

Figure 3. Growth rate of chickens under different feeding conditions

0 500 1000 1500

Average live weight during treatment (kg)

Average daily live weight gain (g)

Hong Samnang 1999

Duong Duy Dong 2003—trial 1

Duong Duy Dong 2003—trial 2

Du Thanh Hang 2003

Average of trial 1

39834 FOOD TEXT 26/5/05 9:52 am Page 36

The following steps were performed in the calculations1:

—Calculation of body weight for every day of growth from 20 kg to 75 kg, using

the formula for the average daily live-weight gain developed in section 6.1;

—Calculation of daily dry matter intake as a function of body weight;

—Calculation of soil intake as 2% of dry matter intake;

—Multiplication of the soil intake with the assumed chloramphenicol concentra-

tions in soil;

—Summing up of the individual results obtained for days 1–119 of the theoreti-

cal period of growth of the animals, from 20 to 75 kg of body weight.

The hypothetical cumulative intake of chloramphenicol by a single animal

whose live weight increases from 20 kg to 75 kg during 119 days (the average time

required to achieve this body-weight gain) varies from £183 mgto 91.3 mg under

these conditions. The results are summarized in Table 10. The cumulative intake

overestimates the amount of chloramphenicol in the body of the animal. This value

is much smaller since—even if the drug were 100% bioavailable—every daily dose

is partly eliminated before the next dose is ingested.

In order to correctly estimate the amount of chloramphenicol in the body,

bioavailability and elimination rate must be known. Unfortunately,the pharmaco-

kinetic behaviour of low doses of chloramphenicol in pigs is not known. The elim-

ination half-life for high doses is not well understood because quantitative

determinations of chloramphenicol in plasma have usually not been extended to

CHLORAMPHENICOL

1Adetailed description of the model used for the calculations is given in Appendix II

Table 9. Soil ingestion by dairy cattle

Description of the group of animals Range of mean soil ingestion (% of dry

matter intake)

Lower bound Upper bound

Mean Standard Mean Standard

error error

Lactating cows

Conﬁned to concrete 0.14 0.02 0.53 0.05

Housed in freestall barns with soil bedding 0.35 0.06 0.64 0.18

Access to unpaved lots with no vegetation 0.60 0.07 0.96 0.22

Yearling heifers and dry cows

Conﬁned to concrete 0.52 0.11 0.81 0.19

Access to unpaved lots with no vegetation 0.25 0.04 2.41 0.26

Access to unpaved lots with sparse vegetation 1.56 0.21 3.77 1.50

On pasture but receiving supplemental feed 1.38 0.33 2.43 0.50

From Fries et al. (1982).

39834 FOOD TEXT 26/5/05 9:52 am Page 37

sufﬁciently long time periods after the administration of the dose. Boertz (1984)

and Boertz et al. (1985) have carried out a residue study using 24 pigs each with

abody weight of approximately 100 kg. The animals were given a single subcuta-

neous injection of chloramphenicol of 30 mg/kg bw. Two animals were slaughtered

at each of 12 time-points between 4 h and 30 days after dosing. The kinetics in

plasma and in all tissues examined suggest that there are at least two elimination

phases, the ﬁrst one characterized by a half-life of 6–10 h and a second one with

ahalf-life of up to 100 h. There was excellent agreement between the results

obtained with radioimmunoassay and with GC–ECD. However,the number of data

points covering the terminal phase was too small to estimate the parameters of

this phase with sufﬁcient accuracy.A study using the same dose was carried out

later in the same laboratory with the aim of producing reference material (Balizs

&Arnold, 1989). Blood samples from ﬁve pigs were taken to predict the appropri-

ate time of slaughter in order to obtain a muscle sample with a concentration of

chloramphenicol of approximately 10 mg/kg. The seven time-points used covered

the period between 1 h and 107 h. The following half lives were found under these

conditions in the ﬁve animals: 10.6, 7.4, 6.5, 10.9 and 14.2 h, respectively. The

data obtained in these two studies are summarized in Figure 4.

The following assumptions were made and corresponding calculations were

performed in order to obtain a crude estimate of the hypothetical amounts in the

bodies of the animals corresponding to the cumulative intake shown in Table 10:

—Oral bioavailability was assumed to be 100%.

—The conditions deﬁned in section 6, which are summarized in Appendix I, result

in an oral dose of £0.035 or 0.7 or 17.5 mg/kg bw per day in pigs, depending

on the hypothetical concentration of chloramphenicol in the soil.

—Two different models concerning the elimination half-life were then tested. In

the ﬁrst model, a single exponential term was used represented by half-lives

of 2, 4, 6, 8, 10, or 100 h. In the second model, it was assumed that 95% of a

daily dose is eliminated with a half-life of 10h, and 5% is eliminated with a half-

life of 100 h.

The results are summarized in Table 11.

CHLORAMPHENICOL

Table 10. Hypothetical cumulative intake through ingestion of soil containing

chloramphenicol, in pigs

Average Dry matter Soil Day Body Concentration of Cumulative intake of

daily gain intake intake weight chloramphenicol chloramphenicol

(g) (g/kg bw) (kg) in soil (mg/g) (mg)

120 £0.05 £0.7

2% of 114

0.164 +dry 25 350

0.00693 ¥35 matter

bw intake 119 75 £0.05 £183

1 3 651

25 91 267

39834 FOOD TEXT 26/5/05 9:52 am Page 38

CHLORAMPHENICOL

Figure 4. Plasma kinetics of parent chloramphenicol in pigs given a single

subcutaneous dose at 30 mg/kg bw

0.01

0.1

100

1 000

10 000

100 000

0 200 400 600 800

Time after dosing (h)

Concentration of chloramphenicol (mg/l)

Balizs_a1 Balizs_a2 Balizs_a3

Balizs_a4 Balizs_a5 Boertz

From Boertz et al. (1984) and Balizs & Arnold (1989)

Table 11. Estimated amounts of chloramphenicol in the body resulting from

soil ingestion, in pigs

Concentration of £0.05 1 25

chloramphenicol in soil (mg/g)

Cumulative intake of £183 3651 91 267

chloramphenicol (mg/animal)

Daily dose (mg/kg bw) 0.035 0.7 17.5

Half-life I (h) Half-life II (h) Estimated amount of chloramphenicol in the bodies

of the pigs after 119 days (mg)

2—£0.0006–£2.62 0.013–52.5 0.32–1312

4—£0.042–£2.66 0.84–53.3 21–1333

6— £0.17–£2.80 3.5–55.9 87–1399

8— £0.37–£2.99 7.4–59.9 186–1498

10 — £0.61–£3.23 12.1–64.6 303–1615

10 100 £1.26–£3.88 25.2–77.7 630–1942

100 — £12.8–£16.3 274–326 6838–8150

aResults are given as the range between the minimum amounts (calculated for the time-

point immediately before the last dose) and the maximum amounts (calculated for the

time-point immediately after the last dose).

39834 FOOD TEXT 26/5/05 9:52 am Page 39

The apparent volume of distribution for chloramphenicol in pigs is given as

1.4 l/kg (Kroker, 1994). The studies of Boertz et al. (1984) have shown that con-

centrations of chloramphenicol in muscle were directly proportional to the con-

centrations found in plasma. Assuming 100% bioavailability of the ingested

chloramphenicol, a crude estimate of maximum residue concentrations expected

in pig muscle would result in the values presented in Table 12.

For a given dose and all other conditions remaining constant the values of the

minima and maxima and the differences between the extreme values depend only

on the half-lives of elimination. This is illustrated in Figure 5 for constant multiple

doses of 17.5 mg/kg bw per day and hypothetical elimination half-lives of 4, 10 and

100 h, respectively.

The uncertainties of the calculations are:

—The values used for development of body weight and intake of dry matter are

likely to have a narrow range of variability.

—The amount of soil ingested by pigs is not known, but the estimate used here

(2% of dry matter intake) is probably realistic or somewhat too low for free-

ranging animals.

—The most inﬂuential factor is the production and resulting hypothetical (steady-

state) concentration of chloramphenicol in soil. There is no evidence to assume

that it is greater than 0.05 mg/kg in non-amended soil, and the possibility that

it is much lower cannot be excluded. However,the effect of organic matter

(straw, manure, compost, etc.) reaching the soil is totally unpredictable.

CHLORAMPHENICOL

Table 12. Estimated muscle concentrations of chloramphenicol derived from

soil ingestion, in pigs

Concentration of £0.05 1 25

chloramphenicol in soil (mg/g)

Cumulative intake of £183 3651 91 267

chloramphenicol (mg/animal)

Half-life I (h) Half-life II (h) Estimated concentration of chloramphenicol in

muscle (mg/kg)

2 — <0.000–£0.025 0.000–0.5 0.003–12.5

4— <0.000–£0.025 0.008–0.51 0.2–12.7

6— £0.002–£0.027 0.033–0.53 0.83–13.3

8— £0.004–£0.029 0.070–0.57 1.77–14.3

10 — £0.006–£0.031 0.115–0.62 2.89–15.4

10 100 £0.012–£0.037 0.24–0.74 6–18.5

100 — £0.12–£0.16 2.6–3.1 60.8–77.6

From Boertz et al. (1984).

aResults are given as the range between the minimum amounts (calculated for the time-

point immediately before the last dose) and the maximum amounts (calculated for the

time-point immediately after the last dose).

39834 FOOD TEXT 26/5/05 9:52 am Page 40

—The pharmacokinetics of low oral doses of chloramphenicol in pigs are not

known. Should it be justiﬁed to use parameters derived from the kinetics of

high doses—as has been done here—then the maximum hypothetical con-

centration in muscle tissue could be in the order of 0.04mg/kg for chloram-

phenicol concentrations of £0.05 mg/kg in soil (see Table 12).

—Only assuming approximately 100% bioavailability and an extremely long elim-

ination half-life of very small, ingested amounts of chloramphenicol, could

residues exceed concentrations of 0.1 mg/kg for chloramphenicol concentra-

tions of £0.05 mg/kg in soil (see Table 12).

—It is not known whether the addition of organic material (straw, bedding mate-

rial, manure, slurry,etc. could enhance production of chloramphenicol by soil

bacteria. If this were the case, as has been shown under laboratory conditions,

residues in the above calculations could reach or even exceed concentrations

of 1 mg/kg in muscle tissue according to Table 12.

7.2.2 Chickens

The calculations performed for chickens are similar to those described in

section 6.1 for pigs. Therefore the description of the individual steps of the calcu-

lations already explained is not repeated here. The formulae describing live-weight

gain and dry matter intake were developed in section 4.2. The basic assumptions

CHLORAMPHENICOL

Figure 5. Effect of elimination half-life on steady-state concentrations

0.1

100

0 20 40 60 80 100 120

Time (days)

Concentration (mg/l)

max

(half-life = 4 h)

max

(half-life = 10 h)

max

(half-life = 100 h)

min

(half-life = 4 h)

min

(half-life = 10 h)

min

(half-life = 100 h)

39834 FOOD TEXT 26/5/05 9:52 am Page 41

CHLORAMPHENICOL

Table 13. Hypothetical muscle concentrations of chloramphenicol derived from ingestion of soil, in chickens

Average Dry Soil Concentration of Body weight Cumulative intake Amount in body Concentration in

daily matter intake chloramphenicol (kg) of chloramphenicol (mg) muscle (mg/kg)

gain (g) intake in soil (mg/g) (mg)

Day 1 Day 75 Day 1 Day 75

3.599 +3¥2% of £0.05 150 1002 £0.018 £2.6 —£0.02 —£0.02

0.01623 ¥average dry 0.02

bw daily matter 1150 1002 0.362 52 —0.45 —0.30

gain intake 25 150 1002 9.050 1308 — 11.30 —8.10

Using a half-life of 7 h and a bioavailability of 35%.

39834 FOOD TEXT 26/5/05 9:52 am Page 42

and the results are summarized in Table 12. Only the maximum amounts and con-

centrations calculated for the time-point immediately after the last dose are given

in Table 13.

A half-life of 7h and a bioavailability of 35% were used for the above calcula-

tions on the basis of data published by Anadon et al. (1994). These authors

have studied bioavailability, pharmacokinetics and residues of chloramphenicol in

chickens. The pharmacokinetic properties (on the basis of a two-compartment

open model) of chloramphenicol were determined in broiler chickens after intra-

venous and after oral administration. After oral administration at a dose of 30 or

50 mg/kg bw, chloramphenicol was absorbed rapidly (time to maximal concentra-

tion, 0.72 or 0.60 h, respectively) and eliminated with a mean half-life (t1/2 beta) of

6.87 or 7.41 h, respectively. The bioavailability was 29% at a concentration of chlo-

ramphenicol of 30 mg/kg bw and 38% at 50 mg/kg bw.

The uncertainties of the calculations are similar to those described for the

model in pigs:

—Again, the major uncertainty is associated with the estimates of likely concen-

trations of chloramphenicol in the soil.

—The well-established pharmacokinetic parameters given in the publication of

Anadon et al. might not adequately describe the behaviour of chlorampheni-

col at very low doses. As has been noted for pigs, the kinetics of chloram-

phenicol at low doses is not known.

8. HYPOTHETICAL EXPOSURE TOPERSISTING

ENVIRONMENTAL RESIDUES

This section discusses the hypothetical possibility that residues resulting from

past treatments of (humans and) animals could persist in the environment. Chlo-

ramphenicol was widely used in food producing animals in nearly all regions of the

world until, within about the past 10 years, many countries and regions, including

the European Union, imposed a complete ban on all uses in such animals in order

to protect the health of consumers. A wide range of formulations, dosages and

routes of administration, which cannot be reviewed here, were in use in terrestrial

animals. Table 14 provides some additional examples of historical uses in

aquaculture in Asia (Arthur et al., 2000). In Asia, chloramphenicol was widely

used as a veterinarian antibiotic in aquaculture, according to a contemporary view

(Somjetlertcharoen, 2002).

Environmental residues of chloramphenicol in farm animals are most likely to

originate from excreta of treated animals and a signiﬁcant risk of secondary con-

tamination could be assumed for farming systems in which manure is used as

fertilizer.

Integrated farming of ﬁsh or shrimp and livestock, for example, combines aqua-

culture with production of pigs, ducks, chicken, and/or other livestock animals.

Farming of such animals produces manure that may be used as soil fertilizer;

however, it is also possible to make use of the nutrients contained in this manure

CHLORAMPHENICOL

39834 FOOD TEXT 26/5/05 9:52 am Page 43

CHLORAMPHENICOL

Table 14. Some historical uses of chloramphenicol in aquaculture in Asia

Country Type of culture or product Examples of uses for disease control Reference

Bangladesh Coastal shrimp culture Occasional use, in a small percentage of—mainly Phillips (2000)

semi-intensive—farms; no further details given

China Valuable broodstock, larval shrimp Bath treatment, 1–1.8 mg/kg Jiang (2000)

Other species of high value, such Injection at 20mg/kg bw; oral treatment at

as eel and soft-shelled turtle 25–100 mg/kg bw

India Extensive and semi-intensive No details given Pathak et al. (2000)

carp farms and semi-intensive

shrimp farms

Indonesia Shrimp culture Immersion therapy at 5 mg/kg Supriyadi & Rukyani (2000)

Malaysia and Aquaculture; no further details “It is usually administered through feed at 3–5g/kg.” Shariff et al. (2000)

Singapore given

Philippines

Penaeus monodon

hatcheries Every other day from Z1to harvest, long bath, at Cruz-Lacierda et al. (2000)

1mg/kg; or 3 days, long bath, at 2–4 mg/kg

Penaeus monodon

grow-out ponds Medicated feed, 2–2.5g/kg of feed; ﬁve times daily

for 3 days

Spotted scat (

Scatophagus argus

)Bath for 10 h, 50 mg/kg or oral administration,

500–750 mg/kg of feed given at 3–10% body

weight for 5–7 days, or by injection at 15 mg/kg

of ﬁsh

Sri Lanka Larvae and post-larvae in shrimp 10–15 mg/kg; no further details given Wijegoonawardena &

hatcheries Siriwardena (2000)

Eel Daily for 21 days by oral administration at 100 mg/kg bw Liao et al. (2000)

Taiwan, China Shrimp For bath, at 1–10 mg/kg

From Arthur et al. (2000).

39834 FOOD TEXT 26/5/05 9:52 am Page 44

inﬁsh or shrimp ponds. These nutrients are taken up by bacteria and microalgae,

which themselves are food for ﬁltrating organisms, mostly zooplankton. Some of

these organisms are then consumed by ﬁsh or shrimp.

8.1 Examples of integrated farming

According to the literature (Vincke, 1991), the number of pigs required per

10 000 m2of pond area varies from 40 to 300. Typically, about 100 weaned piglets

(aged 2 months; average body weight, 12–15 kg) may be used per 10 000 m2

(ponds are frequently much smaller than 10 000 m2). The pigs reach a body weight

of 70–85 kg after 6–7 months. The pigsty may be constructed near the ﬁsh pond

in traditional systems. Manure is then washed down to the pond.

If production of duck and ﬁsh is combined, ponds provide living and foraging

space for both. A variety of strains of duck are used, each having a different fat-

tening period. About 750–4000 ducklings may be stocked per 10000 m2, these

animals reaching slaughter weight within 7–9 weeks. Both ducks and chickens

(broilers or layers, 1000–6000 animals per 10 000 m2)are traditionally reared in

pens beside or over the ponds.

If, in such systems, the terrestrial animals were to be treated with chloram-

phenicol, drug residues contained in their excreta would constantly be added to

and diluted by the pond water, and would disappear according to the rate of degra-

dation discussed in previous sections (half-life, 10–20 days in pond water; see

section 5.4), and also according to the extent to which the water of the pond is

exchanged.

However, it is also possible that fresh manure may be either directly applied to

soil or that it remains in the housing or in a speciﬁcstorage area before further

use. In cases where manure is stored or processed, potential residues of chlo-

ramphenicol would be subject to degradation processes; however, published infor-

mation about the kinetics of degradation of chloramphenicol under various

conditions of storage and processing of animal excreta is extremely limited (see

section 8.5). De Liguoro et al. (2003) demonstrated that processes that occur

between the production of faeces and the application of manure to the soil are

very effective in reducing the load of tylosin and oxytetracycline in the environ-

ment. Tylosin degraded rapidly in manure of treated Simmental calves, and was

no longer detected 45 days after the last treatment. However, the half-life of oxy-

tetracycline in manure was 30 days, and after 5 months maturation, oxytetracy-

cline at a concentration of 820mg/kg of manure was still detected.

In outside storage areas in particular,chloramphenicol would also be washed

out and diluted by rainfall and possibly end up in basins retaining urine and manure

ﬂuids, where the conditions of stability of this compound are unknown and might

be different from those prevailing in solid manure.

8.2 Processing and use of manure

There are many ways of processing manure before its use as fertilizer. In some

regions, farmyard manure is the traditional manure. It is a decomposed mixture of

CHLORAMPHENICOL

39834 FOOD TEXT 26/5/05 9:52 am Page 45

cattle dung and urine, together with straw and litter used as bedding material and

feed not consumed by the animals. The waste material is collected regularly and

placed in trenches ﬁnally plastered over with cow dung or earth slurry. It becomes

ready for application as fertilizer after 3–4 months (Indiaagronet, 2003).

Frequently, the processing of livestock manure by anaerobic digestion in “bio-

digesters” is a key component in integrated aquaculture systems. The main prod-

ucts from the biodigester are biogas and efﬂuent, which is a potential fertilizer

because the anaerobic digestion process results in conversion of organic nitrogen

of the manure to ionized ammonia (NH4+)(San Thy & Preston, 2003). Efﬂuent may

be superior to raw manure in supporting a higher yield of biomass in agricultural

crops (Le Ha Chau, 1998a, 1998b). Fish may also grow faster when ponds are

fertilized with biodigester efﬂuent instead of unprocessed manure (Pich Sophin &

Preston, 2001). Chloramphenicol is not likely to be stable under the described con-

ditions of processing of manure.

The literature offers a wide range of numbers characterizing the quantity of

manure produced by different species of animal under different conditions of hus-

bandry, according to the way of processing the manure (e.g. capacity, initial

loading, daily volumes added, water addition and retention times in biodigesters),

and the amounts added to soil and/or ponds either in terms of dry matter or nitro-

gen per 10 000 m2.The amount of manure or biodigester solids or efﬂuents added

also largely depends on the aquatic species and its density in the pond. It may be

added directly or suspended in special manure bags. Fertilization is a basic part

of pond preparation since plankton has to be available in adequate quantities

before stocking with ﬁsh or shrimp.

8.3 Production of excreta by farm animals

The values given for production of excreta by terrestrial farm animals in the

western hemisphere in Table 15 have been transformed into metric units from the

non-metric units used in the original literature (Barker et al., 2003). Other sources

give different values that are up to 25% higher or lower.Unfortunately, the term

“manure” is rarely deﬁned.

Manure output by domestic animals is affected by many factors, which could

differ widely from region to region in the world. In the tropics, a common method

of comparing farm livestock is the tropical livestock unit (TLU) (LEAD Virtual

Center, 2004). The TLU is based on the cow with a body weight of 250 kg and

other animals are compared with it using (weight)3/4 for scaling. The TLUs for

selected tropical livestock are given in Table 16.

The annual production of manure by one TLU (250kg live weight) in an exten-

sive system is roughly estimated to be about 1000 kg of dry matter. This estimate,

which is not greatly different from the values given in Table 15, is based on the

assumptions of a daily feed intake varying from 2 to 2.5% of body weight and a

digestibility of feed varying from 40–60% (LEAD Virtual Center, 2004b).

CHLORAMPHENICOL

39834 FOOD TEXT 26/5/05 9:52 am Page 46

8.4 Residues in manure after therapeutic treatment of farm animals

In order to avoid over-sophisticated calculations performed using a purely

hypothetical background, the following simplifying assumptions may be made.

When pigs or cattle are treated with chloramphenicol they receive a dose of

50 mg/kg bw (per-oral, intramuscular or subcutaneous administration in pigs, and

intramuscular or subcutaneous administration in cattle, except non-ruminating

calves in which the per-oral route is useful) daily over 7 days. Chicken may be

treated orally with drinking-water containing chloramphenicol (250 mg/l) over 7

days. Water intake (Guyer, 1996) by chickens is approximately 0.3 l/day per animal.

The resulting total doses would be approximately 87.5 g for a cow with a body

weight of 250 kg, 26.25 g for a pig with a body weight of 75 kg, and 0.525 g per

chicken. Assuming that a maximum of 50% of the dose is excreted as parent drug

or hydrolysable conjugate of the parent drug in the combined mixed excreta within

the treatment period plus an additional 3 days, these amounts would be contained

CHLORAMPHENICOL

Table 15. Production of faeces and urine by farm animals

Species or Live Faeces and urine productionaTotal solids

production weight (tonnes/year per

class (kg) (kg/head (tonnes/ (tonnes/year tonne of liveweight)

per day) head per tonne of

per year) liveweight

Dairy cows 635.00 55.50 20.20 31.9 4.43

Beef cattle 362.90 22.00 7.53 20.8 3.05

Pigs 61.20 5.03 1.72 28.1 2.90

Laying hens 1.81 0.12 0.04 23.5 5.85

Broiler chicken 0.91 0.07 0.02 24.0 6.14

Duck 1.36 0.15 0.05 33.3 9.00

From Barker et al. (2003).

aAs voided.

Table 16. TLU equivalents of some farm animals

Animal TLU

Cow 1.00

Bull 1.20

Heifer 0.70

Calf 0.50

Asian Buffalo 1.20

Goat 0.15

Sheep 0.15

Sow 0.20

100 Chickens 0.60

TLU, tropical livestock unit.

39834 FOOD TEXT 26/5/05 9:52 am Page 47

inapproximately 152 kg of beef cattle excreta, 62 kg of pig excreta and 0.7–1.5 kg

of chicken excreta.

The expected concentrations of chloramphenicol equivalents, therefore, would

range in the order of 0.29 g equivalents (chloramphenicol plus conjugates) per kg

of bovine excreta, 0.21 g per kg of pig excreta and 0.18–0.38 g per kg of chicken

excreta. Therefore one could expect to ﬁnd chloramphenicol equivalents at a con-

centration of roughly 0.3 g per kg of combined liquid and solid excreta from some

major farm animals.

8.5 Stability of chloramphenicol in manure

The degradation of chloramphenicol in manure has not been studied system-

atically.Since the molecule has a number of functional groups (e.g. the aromatic

nitro group, the dichloro-acetyl side-chain) that could be attacked by known biotic

processes, the behaviour of the compound cannot be predicted. As with other

aromatic nitro compounds, the nitro group is expected to be particularly sensitive

under anaerobic conditions (Hallas & Alexander, 1983). Chloramphenicol—like

several other antibiotics (Johnson, 1994; Sanz et al., 1996; Lallai et al., 2002)—

is, on the other hand, itself a powerful inhibitor of many anaerobic degradation

processes. There are a few examples in the literature of situations in which the

concentrations and persistence of other antibiotics and feed additives have been

determined in various kinds of manure.

Runsey et al. (1977) conducted experiments in which chlortetracycline was

used in a combination of feed additive for feedlot beef cattle. Fresh manure, stored

manure, runoffwater,manure weathered on pasture, and soil from pasture fertil-

ized with manure were analysed for the additives. Seventeen percent of the

chlorotetracycline fed to cattle appeared in fresh manure and 11% appeared in

manure stored for 12 weeks. Aga et al. (2003) used an ELISA method to screen

manure samples collected from hog lagoons and cattle feedlots for the presence

of tetracycline residues. The concentrations measured varied from below the LOD

(0.5 ng/kg) to 200 mg/kg. The degradation of tetracyclines in soil-applied manure

was also followed using ELISA.; detectable concentrations were found for up to

28 days. Analysis of selected manure extracts by liquid chromatography with mass

spectrometry (LC–MS) showed lower concentrations of total tetracyclines com-

pared with the values obtained by ELISA, indicating the presence of other struc-

turally related compounds or transformation products of tetracyclines. De Liguoro

et al. (2003) followed the levels of oxytetracycline and tylosin over time in faeces,

bedding and manure, and then in the soil of a manured ﬁeld and surrounding

drainage courses, after oral treatment of calves. Fifty Simmental calves were

treated for 5 days with oxytetracycline at 60 mg/kg bw per day. After 15 days, the

animals were treated for 5 days with tylosin at 20 mg/kg bw per day. Tylosin

degraded rapidly and was no longer detected in manure 45 days after cessation

of treatment, and no trace of the compound was detected in soil or surrounding

water (LOD, 10 mg/l). The half-life of oxytetracycline in manure was 30 days, and

the compound was still detectable in this matrix (concentration, 820mg/kg) after 5

months maturation. Hamscher et al. (2002) investigated the distribution and per-

sistence of tetracyclines and tylosin in a ﬁeld fertilized twice with liquid manure.

CHLORAMPHENICOL

39834 FOOD TEXT 26/5/05 9:52 am Page 48

Onthe ﬁrst fertilization, the manure contained tetracycline at 4.0 mg/kg and chlor-

tetracycline at 0.1 mg/kg. Similar concentrations were applied again 1 year later.

Soil sampling was performed 1 and 7 months after the ﬁrst application and 1 month

after the second application. At the third sampling time, the highest average con-

centrations of tetracycline of 86.2 (soil sublayer, 0–10cm), 198.7 (10–20 cm), and

171.7 mg/kg (20–30 cm) and chlortetracycline at 4.6–7.3 mg/kg (all three sublayers)

were found, indicating that tetracylines persisted and accumulated in the soil.

Oxytetracycline and tylosin could not be detected in any sample analysed.

Campagnolo et al. (2002) analysed samples from swine waste storage lagoons

and surface water and groundwater obtained from sites proximal to large-scale

swine and poultry operations for multiple classes of antimicrobial compounds, and

found multiple antimicrobial residues (commonly at concentrations of >100 mg/l) in

storage lagoons. Schlusener et al. (2003) developed sensitive methods for the

analysis of macrolides and ionophores and tiamulin in manure. The maximum con-

centrations found in manure samples were tiamulin at 43 mg/kg and salinomycin

at 11mg/kg. Ingerslev & Halling-Sorensen (2001) studied the biodegradability of

olaquindox, metronidazole, and tylosin, in soil–manure slurries with 50 g of soil per

litre. None of these substances persisted in the biodegradation experiments.

Degradation half-lives for the primary degradation were: tylosin, 3.3–8.1 days;

olaquindox, 5.8–8.8 days; and metronidazole, 13.1–26.9 days. Loke et al. (2000)

studied the stability of tylosin A in manure under methanogenic conditions. Tylosin

A is the major component (usually about 90% and not less than 80%) of tylosin.

The half-life was less than two days. The authors could not determine whether the

decrease in the concentration of tylosin A under aerobic and anaerobic consitions

is caused by sorption, or by abiotic or biotic chemical degradation.

Haller et al. (2002) analysed six grab samples taken in Switzerland from

manure pits on farms where medicinal feed had been applied and found total sul-

fonamide concentrations of up to 20 mg/kg of liquid manure.

The feed additive roxarsone is excreted unchanged by poultry.In experiments

conducted by Garbarino et al. (2003) it was also found to be stable in fresh dried

litter. However, when water was added to litter at about 50% w/w and the mixture

was allowed to compost at 40 °C, roxarsone disappeared and arsenate was formed

within about 30 days. Increasing the amount of water and the incubation temper-

ature increased the rate of degradation. The degradation process was most likely

to be biotic.

8.6 Uptake by farm animals of residues from manure

According to the calculations performed in section 7, a concentration of

0.3 g/kg of manure as estimated in section 8.4 would be high enough to cause

signiﬁcant residues in tissues of, for example, chickens scavenging on

unprocessed manure within approximately ten half-life periods of the chloram-

phenicol residues. In practice, however,the excreta of treated animals would prob-

ably be diluted with excreta from untreated animals.

Assuming half-lives of <1day to several days (depending on conditions such

as temperature, water content, content of bedding material and particles), manure

CHLORAMPHENICOL

39834 FOOD TEXT 26/5/05 9:52 am Page 49

should no longer play a role as a potential source of contamination several weeks

after discontinuation of the use of chloramphenicol in terrestrial animals. Since

chloramphenicol is relatively unstable under anaerobic conditions, it would most

likely not survive the process of farmyard manure production described above and

the biodigester process. However, the compound could probably survive for

several weeks or longer if administered to soil or grassland shortly after excretion

by treated animals. Quantiﬁable amounts of residues could be taken up by free-

ranging animals for a certain period of time. As mentioned in section 5.5, chlo-

ramphenicol could persist for many years in dry dusts formed during the mixing of

chloramphenicol with dry feeds. The high concentrations of chloramphenicol

present in small quantities of dust could occasionally cause residues in some

animals, even a long time after the last use of chloramphenicol on a farm.

If unprocessed manure were to be directly added to aquatic systems, two dif-

ferent “worst-case” scenarios could be discussed:

Hypothetical “worst case” scenario 1

:the daily excreta of 10 pigs with a body

weight of 75 kg are washed directly into a pond with an area of 1000 m2and a depth

of 1 m. After treatment of all animals as assumed in section 5.4, 0.5 ¥26.25 ¥10

=0.13 kg of chloramphenicol equivalents (parent drug plus conjugates) would be

added by this means to the pond during the days following treatment, resulting in

amaximum concentration of £0.13 kg/1000 m3or £130 mg/l. The half-life would be

£10–20 days. Water exchange rates need also be considered in this scenario.

There are no data enabling an estimate to be made of the quantities that could be

taken up, e.g. through the gills, by aquatic species under these conditions.

However, one cannot exclude the formation of traces of detectable residues in

tissues. The problem would no longer exist several months after discontinuation

of treatment of terrestrial animals.

Hypothetical “worst case” scenario 2

:unprocessed undiluted manure of treated

animals is used in pond preparation. In practice the amounts would vary widely

depending on, for example, the nitrogen requirements of the particular system.

Assuming, for example, that in the preparation phase of a pond with an area of

10 000 m2amaximum primary dose of 3000 kg of unprocessed manure contami-

nated with chloramphenicol is applied when the water depth is 10 cm, followed by

two maximum secondary doses of 400 kg each applied when the water depth is

30 cm and 100 cm, respectively (e.g. Pathak et al., 2000), the maximum total dose

of chloramphenicol would be £0.3 ¥3800, that is, £1140g, depending on the

degree of decomposition of active chloramphenicol residues. This amount would

probably be quickly dissolved in the water and result in an initial concentration of

£1.14 kg/10 000 m3or £114 mg/l, which would decrease owing to degradation and

water exchange. In this example, the true concentrations of chloramphenicol could

be much less than 114mg/l since the excreta would have been collected over a

certain period of time, which would favour decomposition of chloramphenicol.

Again, one could not exclude the possibility that the remaining chloramphenicol

could lead to concentrations of residues in aquatic species at greater than the

LOQ. However, such problems would rapidly disappear after discontinuation of the

therapeutic use of chloramphenicol in terrestrial animals.

Another source of environmental contamination is the direct therapeutic use of

chloramphenicol in aquatic species. In particular,its use in medicated feed had

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the potential to leave residues in the pond environment. Addition of medicated feed

containing chloramphenicol at 2.5 g/kg of feed, ﬁve times per day for 3 days could

mean, in practice, the introduction of up to 30 kg of feed/day per 10 000m2(e.g.

Cruz-Lacierda et al., 2000) depending on the density of the aquatic species. With

this amount of feed, 22.5 g of chloramphenicol would be introduced into a pond

with an area of 1000 m2,for example; at least 15% of this dose would not be con-

sumed (Primavera, 1994) by the aquatic species. Depending on the stability of the

feed particles in water (Obaldo, 2001), and of the coating in particular, chloram-

phenicol could leach out at a rate of <0.5–5% per h. Assuming more stable

particles and good quality coating, all the chloramphenicol contained in the feed

could also fall down to the mud on the pond bottom and persist there with half-

lives of up to several weeks, depending on conditions, such as oxygen content

and salinity.

In summary,all the hypothetical considerations performed in this section

suggest that after the implementation of a complete ban on the use of chloram-

phenicol in farm animals, the problems discussed in this section would vanish

within several months.

The problems of potential signiﬁcant carry-over in, for example, feed mills are

not discussed in this document.

9. HUMAN INTAKES RESULTING FROM THE CONSUMPTION OF

CONTAMINATED FISH AND SHELLFISH

Assuming occasional contamination of ﬁsh or shellﬁsh containing chloram-

phenicol at a concentration of 0.5 mg/kg, the additional chloramphenicol burden for

average and preferential eaters of ﬁsh and shellﬁsh could be estimated using data

on consumption habits. Such data are not available at the international level.

However, some data have been published for certain regions and for certain

populations with very high consumption of ﬁsh and shellﬁsh.

Asurvey of 212 people living in Singapore was conducted by Burger et al.

(2002) to examine the relative importance of ﬁsh, shellﬁsh, and other meat in the

diet. From the authors’ discussion of their ﬁndings in the context of international

surveys on ﬁsh consumption, it appears that people in the Far East eat signiﬁ-

cantly more ﬁsh than do most people whose families have lived for generations in

industrialized societies. In the study by Burger et al., it was found that people ate,

on average, ﬁsh for about ten meals per week, chicken for eight meals per week,

and shrimp and pork for about six meals each per week. While only 8% of people

never ate ﬁsh, 18% ate ﬁsh at all 21 meals per week, and >20% ate shellﬁsh for

all 21 meals. Therefore, it seems to be appropriate to base an estimate of high

seafood intake by preferential eaters on data obtained in surveys of people of Asian

origin.

Sechena et al. (2003) described and quantiﬁed rates of seafood consumption,

and acquisition and preparation habits of 202 ﬁrst- and second-generation

Asian–American and Paciﬁc Islanders from 10 ethnic groups (Cambodian,

Chinese, Phillippine, Hmong, Japanese, Korean, Lao, Mien, Samoan, and

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Vietnamese) in King County, Washington in 1997. Participants in the study were

all consumers of seafood; only one person did not eat shellﬁsh, which was other-

wise the predominant seafood consumed by these people (45.9% of all seafood

consumed). Rates of ﬁsh consumption were skewed considerably for all ﬁsh

groups, indicating that a few respondents had a larger consumption rate than other

respondents. The 90th percentile of all consumption rates for “all seafood” con-

sumption was 3.928 g/kg bw per day.

Using this conservative estimate of seafood intake and multiplying it by the

median concentration (0.5 mg/kg) of chloramphenicol found in the published alerts

from the Food Standards Agency of Ireland, mentioned above, would result in

an estimated daily intake of chloramphenicol of approximately 2 ¥10-9g/kgbw, or

0.12 mgfor a person with a body weight of 60 kg. This estimate of intake could be

slightly low since other products of animal origin could also occasionally contain

traces of chloramphenicol.

10. COMPARISON OF DIETARY INTAKES WITH LOW-LEVEL

EXPOSURE FROM OPHTHALMIC FORMULATIONS

Systemic drug absorption after ocular administration is well known and has

been reported in hundreds of research papers for a large number of substances

by numerous research groups. For a better understanding of these variable ﬁnd-

ings, it is important to consider the anatomy and physiology of the eye.

After topical ocular administration of drugs, the highly vascularized conjuncti-

val and nasal mucosa are the major sites of systemic absorption. Drug absorption

by this route bypasses the ﬁrst-pass effects in the gastrointestinal tract and in the

liver.However,local ophthalmic bioavailability can be very low,since the tear ﬂuid

is rapidly drained from the lower conjunctival sac through the puncta and the

lachrymal duct into the lachrymal sac, from where it passes through the naso-

lachrymal duct into the inferior nasal meatus. From here, the ﬂuid moves to the

nasopharynx where it is swallowed into the gastrointestinal tract. It has been

reported that drainage of the administered dose via the nasolacrimal system into

the nasopharynx and the gastrointestinal tract takes place when the volume of ﬂuid

in the eye exceeds the normal lacrimal volume of 7–10ml. Thus, the portion of the

dose that is not eliminated by spillage is drained quickly and the time that the dose

is in contact with the surfaces of the cornea and sclera is reduced to approximately

2 min (Saettone, 2002). According to Saettone (2003), the rate of loss of drug from

the eye can be such that only 1–5% or less of the drug applied topically as a solu-

tion reaches the inner eye. For example, using timolol as a probe, Alvan et al.

(1980) found that 12–88% of the dose administered was lost when 16 volunteers

received two drops of 0.5% timolol ophthalmic solution in each eye, twice daily for

2weeks. These anatomical and physiological properties of the eye explain the

short pre-corneal residence time and the poor bioavailability of many eyedrop solu-

tions that do not contain a viscosity agent in their formulation to prolong residence

time (Sirbat et al., 2000).

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Insummary, there are three possible systemic absorption sites for drugs admin-

istered topically to the eye—conjunctival, inferior nasal, and gastrointestinal

mucosa—and total bioavailability, therefore, should be assessed by estimating

drug concentration in plasma or urine.

10.1 Systemic effects after ocular administration of drugs as

evidence for systemic bioavailability

Part of the current knowledge of systemic bioavailability of drugs administered

topically to the eye has accumulated from observations of systemic adverse

effects after the use of topical ophthalmic preparations (Gerber et al., 1990; Flach,

1994; Jones et al., 1996; Shiuey & Eisenberg, 1996; Diamond, 1997). Urtti &

Salminen (1993) emphasize the need to take into account the problem of systemic

drug absorption in designing ocular drug and dosage forms to minimize systemic

absorption and increase the oculospeciﬁcity of drugs, for example, reducing

volume and increasing viscosity of eyedrops, controlling drug release from

depot preparations, prodrug-derivatization, and addition of vasoconstrictive

agents.

10.2 Pharmacokinetic evidence of systemic absorption of

ophthalmic drugs

In many other studies in humans and animals, the systemic availability of oph-

thalmic drugs has been convincingly demonstrated by pharmacokinetic research.

A few examples of hundreds of papers may be given here. Anderson (1980) deter-

mined that systemic absorption of epinephrine and dipivefrin hydrochloride was

55–65% of the ocularly applied dose. Chiang et al. (1983) gave d9-tetrahydro-

cannabinol to rabbits by ophthalmic administration, measured plasma concentra-

tions, and compared with intravenous data to establish bioavailability. Kumar et al.

(1985) studied the systemic absorption, plasma concentrations (maximum plasma

concentrations are achieved within 10–20 min after topical instillation) and cardio-

vascular effects of ophthalmic solutions of phenylephrine hydrochloride. Salminen

(1990) reviewed literature on human plasma concentrations after instillation of

ocular timolol, levobunolol, atropine, cyclopentolate, scopolamine, phenylephrine,

betamethasone and technetium-99. Kaila et al. (1999) determined the mean

bioavailability of ocularly administered atropine to be 63.5% (

=6; range,

19–95%). Sasaki et al. (2000) using tilisolol as a model substance and car-

boxymethylcellulose sodium salt as viscous polymer developed an in vivo phar-

macokinetic model that accounts for corneal diffusion in albino rabbits and predicts

the concentration of beta-blockers in the anterior segments, and characterizes the

systemic absorption of instilled drug with ophthalmic viscous vehicle. Vainio-Jylha

et al. (2001) studied the concentration of betaxolol in plasma after its topical ocular

use. The drug showed a biphasic concentration–time curve in plasma, the ﬁrst

peak occurring already after several minutes, and concentrations were detectable

even at 12 h after dosing. Korte et al. (2002) estimated the systemic bioavailabil-

ity of timolol in eyedrops containing 0.5% timolol, and compared the cardiopul-

monary effects of intravenous and ophthalmic timolol. The peak concentration of

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39834 FOOD TEXT 26/5/05 9:52 am Page 53

ophthalmic timolol in plasma was measured in most subjects within 15 min after

drug administration. The systemic bioavailability was 78.0% (

=8). Ophthalmic

timolol resembled intravenous timolol in terms of systemic bioavailability, plasma

kinetics, and cardiopulmonary effects. A recent article reviews the principles of

systemic absorption of insulin applied topically to the eye. The physiological and

pharmaceutical considerations for formulation development and the strategy of

improving the systemic absorption and bioavailability of insulin are also discussed

(Lee et al., 2002).

10.3 Results obtained with ophthalmic formulations of chloramphenicol

The above examples were given since with chloramphenicol alone, a sub-

stance that typically easily passes through all biological membranes and barriers,

the systemic bioavailability after topical application is a controversial subject of dis-

cussion. However, this discussion has taken place in the absence of adequate

studies using sensitive analytical methods.

In an early study by Trope et al. (1979), ﬁve children aged <9years received

eyedrops containing chloramphenicol, which were administered every 2hto each

eye for 5–7 days. Systemic absorption was not detected with the available assay

methods. Walker et al. (1998) used HPLC with a limit of detection of 1mg/l to inves-

tigate whether serum accumulation of chloramphenicol occurred after topical

therapy in 40 patients. The mean dose of chloramphenicol received from eyedrops

after 1 week of treatment was 8.0 mg, and after 2 weeks, 15.3 mg. As the authors

had expected, chloramphenicol failed to accumulate to detectable levels. Contrary

to these ﬁndings, chloramphenicol applied topically to the eye in ointment produced

bacteriostatic concentrations of chloramphenicol in the aqueous humor, which

lasted for several hours (George & Hanna, 1977). Hanna et al. (1978) reported

that repeated drops of solutions containing 0.5% chloramphenicol for several hours

were required to produce a concentration of chloramphenicol of 1mg/ml in the

aqueous humor.Yamada & Hiraki (1995) studied the ocular pharmacokinetics in

rabbits of a combination formulation containing 0.25% chloramphenicol (CP) under

various experimental conditions. When they sealed the cornea with cyanoacrylate

glue to block transcorneal absorption, the absorbed fraction of chloramphenicol

was <10% of that in the control, indicating that most of the chloramphenicol in the

aqueous humor was derived from the transcorneal route. Ismail & Morton (1995)

evaluated the bioavailability of three commercial chloramphenicol ophthalmic prod-

ucts supplemented with [14C]chloramphenicol. Samples of 50mlwere applied to the

corneas of isolated bovine eyes. Their results indicate a greater bioavailability of

chloramphenicol in ophthalmic ointments than in a liquid preparation, which gave

extremely low levels of chloramphenicol in the aqueous humour and corneal.

10.4 Quantitative considerations

Typical concentrations of chloramphenicol in eyedrops and ointments are

5mg/ml(0.5%) and 10 mg/ml(1%), respectively. The recommended frequency of

application varies. A dosage regimen that has been recommended on the basis of

clinical trial suggests four daily treatments. Under these conditions, acute bacter-

ial conjunctivitis was 88% healed within 9 days of treatment (Laerum et al., 1994).

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39834 FOOD TEXT 26/5/05 9:52 am Page 54

The locally absorbed fraction of the drug dose enters the anterior chamber by

penetration through the three layers and two membranes of the cornea. When

adrop (50–75 ml) is applied to the eye it becomes diluted owing to reﬂex tearing,

and the volume that is in excess of the normal lacrimal volume is drained from the

eye. The average maximum capacity of the human conjunctival sac is 25–30ml

(Peterson, lecture notes). In consequence, it can be estimated that 66–75% of the

administered dose is lost immediately, and that a maximum of 0.33 ¥5¥50 =

82.5 mgper application would be available for possible absorption through the

cornea. Ointments have prolonged contact with the cornea, which improves

absorption. Chloramphenicol apparently exhibits good penetration if the residence

time is sufﬁciently long. Beasley et al. (1975) gave topical applications of an oph-

thalmic solution containing 0.5% chloramphenicol to patients at various times

before cataract surgery. Aqueous humor obtained at the time of surgery contained

intact chloramphenicol at a concentration of 3.5–6.7mg/ml 1–2 hafter topical

administration. Aqueous humor is the watery ﬂuid produced by the ciliary body that

ﬁlls the space between the lens and cornea of the eye, which serves as a nutri-

ent delivery system for the avascular cells in the cornea and eventually drains into

avein or lymphatic vessel. The normal human aqueous humor production during

daytime is 2.75 ±0.65 ml/min (mean ±standard deviation (SD)), while at night pro-

duction is approximately half that amount (Larsson, 1998). The volume of the ante-

rior chamber is dependent on age. In normal healthy subjects in two age groups,

the volume of the anterior chamber was 247 ±39 mlin the younger group (aged

20–30 years,

=51) and 160 ±39 mlin the older group (aged ≥60 years,

=53)

(Toris et al., 1999).

Using these values, the fraction of the bioavailable dose that is absorbed

through the cornea per application of one drop (0.6–1.6mgor 2.4–6.4 mgper day)

during a treatment period can be calculated. This value probably represents a low

estimate of systemic absorption, as it does not include absorption occurring at

other possible sites described above. Nevertheless, this low estimate of systemic

absorption of chloramphenicol from eyedrops is up to 50 times higher than the

intake estimate calculated in section 7 and is of about the same order of magni-

tude as the intake corresponding to the highest contaminated seafood sample

found in 2002.

11. COMMENTS

No toxicological data on chloramphenicol were submitted to the Committee at

its present meeting. The current evaluation was made on the basis of an exten-

sive review of the scientiﬁcliterature, particularly that published since the forty-

second meeting, and with a focus on the toxicological data in humans.

Reports on uptake and metabolism in humans and animals showed that chlo-

ramphenicol is rapidly absorbed when administered orally and that it is extensively

metabolized. Astudy with human bone marrow in vitro also showed evidence of

metabolism in this tissue.

A number of studies with chloramphenicol and several metabolites of chlo-

ramphenicol have shown that they are cytotoxic to bone marrow in vitro.

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39834 FOOD TEXT 26/5/05 9:52 am Page 55

Inorder to assess the genotoxicity of chloramphenicol, the Committee

reassessed the results of tests reported at its thirty-second and forty-second meet-

ings, and also considered new studies available in the published literature. Chlo-

ramphenicol was shown to cause DNA damage in a human ﬁbroblast cell line and

in primary cultures of rat hepatocytes, but not in human bone-marrow cells in vitro.

The results of tests for reverse mutation in bacteria were mostly negative. In mam-

malian cells in vitro, chloramphenicol consistently gave positive results in tests for

chromosomal aberrations, but results of tests for gene mutations and for sister

chromatid exchange were inconsistent. Overall, these results indicated that chlo-

ramphenicol was genotoxic in vitro.

In tests for genotoxicity in vivo, chloramphenicol caused chromosomal aberra-

tions in the bone marrow of mice, but gave negative results in tests for micronu-

cleus formation in the bone marrow of mice and rats. It is not clear why contrasting

results were obtained in these two assays, but the Committee considered that it

was prudent to regard chloramphenicol as a mutagen in somatic cells in vivo.

According to data on heritable mutation reviewed by the Committee at previ-

ous meetings, chloramphenicol gave negative results in tests for dominant lethal

mutation in mice and in

Drosophila melanogaster

At its present meeting, the Committee reviewed studies on genotoxicity with

chloramphenicol and its metabolites in human bone marrow cells or peripheral

blood lymphocytes in vitro. Only nitrosochloramphenicol and dehydrochloram-

phenicol induced DNA strand breaks, while chloramphenicol and other metabo-

lites were without effects. This conﬁrmed the results of previous studies that

showed that some of the metabolites of chloramphenicol are genotoxic.

No adequate studies were available to evaluate the carcinogenicity of chlo-

ramphenicol in experimental animals. Chloramphenicol has been classiﬁed as

“probably carcinogenic in humans” by IARC (IARC, 1990).

Anumber of toxicological studies in rodents were conducted in an effort to

develop a model for chloramphenicol-induced aplastic anaemia in humans. While

bone-marrow depression was conﬁrmed in these studies, it did not progress to the

characteristic aplastic anaemia of humans and was therefore not considered to be

asuitable model for the disease in humans. The reversible bone-marrow depres-

sion that is seen in animals and humans receiving chloramphenicol can be attrib-

uted to its cytotoxicity.

A number of reports of epidemiological studies on the oral and injectable use

of chloramphenicol in humans were reviewed; they conﬁrmed that chlorampheni-

col was toxic to the bone marrow. In many cases, the toxic effects could be

reversed by reducing or discontinuing treatment with chloramphenicol. There are,

however, cases of aplastic anaemia that appear to be unrelated to dose and that

are associated with a high mortality rate. In humans, the aplastic anaemia that is

attributable to treatment with chloramphenicol is often fatal and is an idiosyncratic

reaction that may have an immunological component. There is also evidence that

some of the survivors of aplastic anaemia induced by chloramphenicol subse-

quently develop leukaemia.

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39834 FOOD TEXT 26/5/05 9:52 am Page 56

The ophthalmic use of chloramphenicol represents the lowest therapeutic dose

of the compound. Epidemiological data relating to the ophthalmic use of chloram-

phenicol in humans suggest that this form of administration is unlikely to be asso-

ciated with aplastic anaemia. While any occurrence of aplastic anaemia associated

with this form of administration is extremely rare, it is not possible to quantify the

absolute risk of the ophthalmic use of chloramphenicol in humans because of the

low background occurrence of idiopathic aplastic anaemia.

No adequate studies were available to fully assess potential reproductive

toxicity with chloramphenicol. However, chloramphenicol has been shown to be

embryotoxic and fetotoxic in a number of laboratory animal species.

In a case–control study in humans, the authors concluded that oral treatment

with chloramphenicol in the second and third months of pregnancy presents little,

if any,teratogenic risk. However, it is difﬁcult to determine if any effects might occur

during the ﬁrst month of pregnancy.

Residue data

Most countries in the world do not permit the use of chloramphenicol in

food-producing animals, in order to protect the health of consumers. Despite

such restrictions, chloramphenicol has been detected in food samples collected

in national monitoring programmes during the past 2 years and these residues

have caused safety concerns. Shrimps, prawns, food products from aquatic

animals, honey, royal jelly, meat and offal, sausage casings, rabbit and poultry

meat and milk powder were among the commodities in which the drug was

detected.

Chloramphenicol—an environmental contaminant?

While the results of monitoring clearly indicate intentional uses of chloram-

phenicol in some cases, it has also been argued that very low levels of chloram-

phenicol, such as those found in poultry and in products from aquaculture, could

result from environmental contamination. The possibility that chloramphenicol

could persist in the environment, or even be formed by soil microorganisms was

discussed by the Fourteenth Session of CCRVDF, held in 2003 (Joint FAO/WHO

Food Standards Programme, 2003).

Chloramphenicol was ﬁrst described as an antibiotic produced by cultures of

an actinomycete isolated from soil by Ehrlich et al. (1947). The soil samples were

collected from a mulched ﬁeld near Caracas, Venezuela, and from a compost soil

on the horticultural farm of the Illinois Agricultural Experiment Station at Urbana.

In studies conducted in 1952, the adsorption, stability, and rate of production of

chloramphenicol in soil under different laboratory conditions were determined.

When sterilized soil was inoculated with

Streptomyces venezuelae

and was incu-

bated for long periods, the presence in soil of chloramphenicol formed by the

microorganism after a lag phase of several weeks was demonstrated. The highest

concentration measured was 1.12 mg/kg. However, when workers of the same

group and in the same year analysed samples of normal soils collected from 91

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39834 FOOD TEXT 26/5/05 9:52 am Page 57

cultivated and grassland sites from nine states of the USA and from 13 other coun-

tries, no chloramphenicol was identiﬁed in extracts from these soils. In this initial

study (1952), the limit of detection of chloramphenicol was 0.3 mg/kg (turbidimet-

ric assay using

Shigella sonnei

). In other experiments with an improved LOD

of 0.05 mg/kg, and including the 91 samples of the previous study, these results

were conﬁrmed. Chloramphenicol was found and identiﬁed in soils only when

organic material was added before sterilization and seeding with

Streptomyces

venezuela.

Whether antibiotics are produced in soil in detectable amounts by indigenous

soil organisms has remained a subject of scientiﬁc dispute for several decades. It

was only recently demonstrated that an antibiotic could be synthesized in

detectable amounts in soil. Using biosensor methods with very low LODs,

Strep-

tomyces rimosus

was found to produce oxytetracycline in untreated soil. However,

similar studies have not been carried out for chloramphenicol.

With this background information and on the basis of an extensive search of

the current literature, the Committee at its present meeting examined the follow-

ing two hypothetical scenarios to explain potential environmental contamination of

foods of animal origin with chloramphenicol:

Scenario 1

Assuming that:

—Chloramphenicol is naturally produced in the soil;

—Farm animals (e.g. pigs, chickens) ingest certain amounts of soil in their daily

intake of dry matter;

—This may result in an uptake of chloramphenicol and subsequently in residues

of chloramphenicol in tissues and products of those animals that are not asso-

ciated with uses of chloramphenicol as a veterinary drug.

The Committee conducted a number of simple model calculations to estimate

hypothetical intakes of chloramphenicol as a function of soil intake of animals. On

the basis of data in the literature, it was assumed that 2% of the dry matter intake

of pigs and chickens was soil. The concentration of chloramphenicol in soil was

set at one of the following values:

—£0.05 mg/kg, corresponding to the LOD of the methods used by the authors of

the historical studies, who were unable to detect chloramphenicol above this

limit when they analysed a large number of soil samples from different coun-

tries; or

—1 mg/kg, roughly corresponding to the highest concentration of chlorampheni-

col produced by

S. venezuelae

in inoculated sterilized soil samples supple-

mented with organic matter; or

—25 mg/kg, roughly corresponding to the highest concentrations of chloram-

phenicol produced under experimental conditions with soils enhanced with

tryptone and under the most favourable conditions.

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39834 FOOD TEXT 26/5/05 9:52 am Page 58

The following steps were performed in the calculations:

—Calculation of average daily live-weight gain;

—Calculation of daily dry matter intake as function of body weight/live-weight

gain;

—Calculation of soil intake as a ﬁxed fraction of dry matter intake;

—Calculation of chloramphenicol intake using the above assumed chloram-

phenicol concentrations in soil;

—Estimation of the resulting tissue concentrations on the basis of the known

pharmacokinetic behaviour of chloramphenicol.

The Committee concluded that concentrations of chloramphenicol in soil, as

they were found under laboratory conditions in the presence of organic material,

would sufﬁce to explain occasional traces of chloramphenicol in tissues and prod-

ucts of free-ranging and/or scavenging livestock animals. With the LOD achieved

in the 1950s, however, it was not possible to demonstrate the production of

detectable amounts of chloramphenicol in soil. No further empirical data have been

obtained since 1952. The possibility that chloramphenicol, produced naturally by

soil microorganisms, could lead to the residues found in food-producing animals

cannot be ruled out, but remains an unexplored hypothesis that is currently not

supported by experimental data.

Scenario 2

Assuming that:

—Residues observed are caused by the exposure of some food-producing

animals to chloramphenicol that persists in the environment. Such environ-

mental sources result from historical uses as veterinary drug.

—Any persisting residues of chloramphenicol in farm environments are most

likely to originate from excreta of treated animals, and a possible source of

contamination could be farming systems in which manure is used as fertilizer,

e.g. in integrated farming. Integrated farming of ﬁsh/shrimp and livestock com-

bines aquaculture with production of pigs, ducks, chickens, and/or other live-

stock animals. Manure produced by livestock may be used as soil fertilizer.

Additionally, it is possible that manure may be used as a nutrient in ﬁsh/shrimp

ponds. The nutrients contained in manure are taken up by bacteria and micro-

algae, which themselves feed ﬁltrating organisms, mostly zooplankton. Some

of these organisms are then consumed by ﬁsh or shrimp.

The Committee reviewed published literature in order to investigate the condi-

tions of integrated farming as a potential cause of chloramphenicol residues in food

of animal origin. Chloramphenicol was in use in farm animals as a veterinary drug

before authorizations were withdrawn in many countries and regions. Before these

restrictions, signiﬁcant amounts of manure, probably containing intact chloram-

phenicol, had been used as fertilizers. Concentrations of chloramphenicol in fresh

manure and certain patterns of its use in integrated farming of aquatic species

could, in principle, also explain low concentrations of residue in certain farm

CHLORAMPHENICOL

39834 FOOD TEXT 26/5/05 9:52 am Page 59

animals, such as scavenging chickens, free-ranging pigs and in aquatic animals.

However, when reviewing the available information on the half-life of chloram-

phenicol under different environmental conditions, no evidence was found to show

that chloramphenicol could persist in the environment for periods longer than

several months, except in dry dusts. Therefore, if there was any risk of food con-

tamination resulting from historical use in the farm environment these problems

should disappear within several months of cessation of the use of chlorampheni-

col. Similar considerations apply to the persistence of chloramphenicol in aqua-

culture after past uses of the drug as medicated feed that had been directly applied

in ponds.

Analytical methods

In the past decade, several methods have been developed for the screening,

quantiﬁcation and conﬁrmation of chloramphenicol in foods.

Screening for chloramphenicol could be performed with validated ELISA kits.

The majority of these kits had a LOD of <1mg/kg. However, conﬁrmatory methods

needed to be used in order to avoid false-positive results. For conﬁrmatory pur-

poses, highly sensitive methods based on GC–MS, either electron impact or

negative chemical ionization mode, have been used. More recently, LC–MS/MS

methods allow the determination and identiﬁcation of chloramphenicol in food com-

modities such as honey, meat (chicken, turkey, pork and beef), ﬁsh and shellﬁsh,

at concentrations of <1mg/kg. The LOD and LOQ are as low as 0.05mg/kg and

0.1 mg/kg, respectively.

Conclusions

The Committee concluded that:

—There was no evidence supporting the hypothesis that chloramphenicol is

synthesized naturally in detectable amounts in soil. Although this possibility

is highly unlikely,data generated with modern analytical methods would be

required for conﬁrmation.

—There was evidence that the low concentrations of chloramphenicol detected

by food monitoring programmes in the year 2002 could not originate from

residues of chloramphenicol persisting in the environment after historical vet-

erinary uses of the drug in food-producing animals. Owing to the high vari-

ability in the half-life of chloramphenicol under different environmental

conditions, however, such a mechanism might occasionally cause low-level

contamination in food.

Valid analytical methods are available to monitor low concentrations of chlo-

ramphenicol in foods; however, conﬁrmatory methods require sophisticated and

expensive equipment.

Since the advent of sensitive routine analytical methods for the quantitative

determination of residues of chloramphenicol in foods of animal origin at concen-

trations far less than 1 mg/kg, illegal use of this drug is no longer attractive. Although

CHLORAMPHENICOL

39834 FOOD TEXT 26/5/05 9:52 am Page 60

only limited kinetic residue data exist that describe the terminal elimination from

“deep compartments” (Arnold & Somogyi, 1986), these data suggest that the with-

holding times necessary to allow residues to deplete below 1mg/kg with reason-

able statistical certainty are already in the order of 15 to much longer than 30 days,

depending on species, product, dose and route of administration. Therefore, the

relatively frequent observation of residues far less than 1mg/kg in monitoring pro-

grammes has stimulated a discussion on whether traces of chloramphenicol in

foods could occasionally originate from environmental contamination.

The possible production of chloramphenicol in natural soils was investigated

50 years ago at the time of the discovery of chloramphenicol as a product of a soil

microorganism. With the methodologies of that era it was not possible to con-

vincingly demonstrate the production of appreciable amounts of chloramphenicol

in soil. The question has never again been examined using adequate analytical

methods. However,reliable laboratory experiments conducted by the group that

discovered chloramphenicol have shown that in the presence of organic material

as it may well occur on farmland chloramphenicol could be formed in concentra-

tions up to 1 mg/kg. However no further empirical data have been generated since

that time. These concentrations in soil which were found under laboratory condi-

tions would sufﬁce to explain occasional traces of chloramphenicol in tissues and

products of free ranging and/or scavenging livestock animals.

Chloramphenicol was in common use as a veterinary drug before authoriza-

tions were withdrawn in many countries and regions. Before such restrictions,

enormous amounts of manure likely to contain active chloramphenicol were used

as fertilizers. Concentrations of chloramphenicol in fresh manure and certain pat-

terns of its use could in principle explain low residue concentrations in certain farm

animals, e.g. in scavenging chickens, free-ranging pigs and in aquatic animals.

However, no convincing evidence has been found to show that chloramphenicol

could persist in the environment for long times—except in dry dusts.

The occasional daily dietary intake of chloramphenicol by preferential eaters

of ﬁsh and shellﬁsh contaminated at the levels reported in the year 2002 (median

value, 0.5 mg/kg) is more than one order of magnitude lower than the systemically

bioavailable fraction of a daily dose of a typical ophthalmic formulation used in

human medicine.

12. EVALUATION

As there is evidence that chloramphenicol is a genotoxin in vivo, it is prudent

to assume that chloramphenicol could cause some effects, such as cancer,through

agenotoxic mechanism for which there is no identiﬁable threshold dose.

Epidemiological studies in humans show that treatment with chloramphenicol

is associated with the induction of aplastic anaemia, which may be fatal. It was

not possible to establish any dose–response relationship or threshold dose for the

induction of aplastic anaemia.

The Committee noted that aplastic anaemia induced by chloramphenicol is

arare idiosyncratic response in humans, which may have an immunological

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39834 FOOD TEXT 26/5/05 9:52 am Page 61

component. In common with many other idiosyncratic immune system-mediated

adverse reactions, no animal model could be developed. As a consequence of

these considerations, and because the mechanism of chloramphenicol-induced

aplastic anaemia remains unknown, the Committee could not identify any studies

in animals or epidemiological studies that would assist the further toxicological

evaluation of chloramphenicol.

The Committee concluded that it was not appropriate to establish an ADI for

chloramphenicol.

Taking into account the present gaps in scientiﬁc knowledge, the Committee

could not completely rule out the possibility that occasionally foods are contami-

nated from environmental sources. The easiest way to rule out this possibility

would be a thorough investigation of chloramphenicol concentrations in soil using

modern analytical methods.

The Committee was unable to propose options for regulating traces of chlo-

ramphenicol in foods. The human exposure which may have resulted from con-

sumption of contaminated seafood in the years 2001–2003 was probably lower

than the systemic exposure resulting from the use of ophthalmic formulations.

However, the resulting risk could not be estimated.

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39834 FOOD TEXT 26/5/05 9:52 am Page 76

14. APPENDIX I

Summary of data from feeding experiments

Table 1. Feeding experiments with pigs in south-east Asia

Breed or production Feeding scheme Initial Final Duration of Dry matter Average Reference

class body body treatment intake daily body-

weight weight (days) (g/kg LWweight gain

(kg) (kg) per day) (kg)

F1 Large white ¥Rice bran, ensiled cassava 20 88.2 120 £31 0.568 Nguyen Thi Loc

Mong Cai roots, maize, ﬁsh meal, 19.9 93.3 (estimated 0.611& Le Khac

soya bean meal, 19.6 97.5 from the 0.649 Huy (2003)

supplemented with 0, 20 95.4 data) 0.628

0.1, 0.2 or 0.3%

DL-methionine (on the

basis of dry matter)

Different Rice bran, fresh water 14–35 150 (5 0.405 Khieu Borin

crossbreeds ﬁsh silage (FWFS), sugar months) (0.325– et al. (2000)

palm juice, and water 0.476)

hyacinth (

Eichhornia

crassipes

)

Crossbred pigs Rice bran, maize, ﬁsh 0

(local ¥large meal, groundnut 25 20.1 55.6 90 —0.395 Bui Van Chinh

white) cake, soya bean 38 20.1 56.6 90 —0.406 et al. (1993)

meal plus soya 020.2 59.4 90 —0.435

bean foliage silage 25 48.5 84.7 60 —0.601

substituted for 38 (% of total protein) 48.5 84.5 60 —0.600

0–38% of total 48.7 85.3 60 —0.610

protein in the diet

Castrated Water spinach, broken 0

crossbred male rice, dried ﬁsh plus 59.5 10 33.6 —Prak Kea et al.

piglets palm oil 10 (8–10) 31.6 —(2003)

15 (% of dry matter) 31.8 —

30.1 —

39834 FOOD TEXT 26/5/05 9:52 am Page 77

Table 1.

(contd)

Breed or production Feeding scheme Initial Final Duration of Dry matter Average Reference

class body body treatment intake daily body-

weight weight (days) (g/kg LWweight gain

(kg) (kg) per day) (kg)

Castrated male Basal diet of cassava 14.5 23.3 4¥10 35 0.22 Ly et al. (2001)

Mong Cai bran, wheat bran

and ﬁshmeal or

basal diet with 30%

of protein replaced

by leaf meal

(Basal diet only, leaf

meal: trichanthera

basal diet only,

leaf meal: mulberry)

Castrated Mong Sugar palm syrup and 012.3 36.6 0.14 Ly & Pok Samkol

Cai male pigs dry ﬁsh, substituted 50 (% of dry matter (2001)

by ensiled cassava substituted)

Large white Wheat bran (progressively 13.6 26.4 42 51.0 0.315 Tean et al.

castrated male substituted by 0, 10, 10.6 27.3 (6 weeks) 61.7 0.341 (2002a)

and female pigs 20 and 30% ground 10.1 24.3 61.0 0.364

whole RSM), dried ﬁsh 8.5 20.6 64.0 0.310

Large white Broken rice, water 0

castrated male spinach, 0.25 29.2 47.8 56 32.5 0.332 Ly et al. (2002)

and female supplemented with 0.50 26.7 46.8 37.6 0.360

pigs (1 :1) DL-methionine 0.75 (% of dry matter) 28.0 53.8 32.3 0.442

25.1 50.9 36.3 0.459

Castrated Mong Sugar palm syrup, 0.5

Cai male pigs dried fresh water ﬁsh 1.0 (% of dry matter) 19.2 Approximately 0.143 ±Ly (2002)

and ensiled cassava 22.5 30 0.025

leaves (73.5% of dry

matter) supplemented

with DL-methionine

39834 FOOD TEXT 26/5/05 9:52 am Page 78

Landrace ¥large Broken rice, rice bran,

white soybean, extracted,

soybean, dehulled, plus

1. Cassava root meal, 57.8 92.4 26.5–30.7 0.685 Le Thi Men et al.

2. Cassava root waste, 57.3 85.5 (estimated 0.598 (2003)

3. Cassava root waste, 57.6 94.0 from the 0.715

catﬁsh oil 5% data)

4. Cassava root waste, 57.3 89.3 0.634

catﬁsh oil 10%

Mong Cai and Cassava bran, rice 022.9 Mong Cai, Nguyen Thi Thuy

Large white bran, dried fresh 27.3 (% dry matter 0.273; & Ly (2002)

female pigs water ﬁsh and whole replaced) large

rubber seed white,

replacing part of the 0.533;

dry matter control,

0.377;

rubber

seed,

0.429

Mong Cai piglets Rice bran, plus ensiled Ensiled cassava roots 6.38 22.6 0.200 Nguyen Van Lai

and large white cassava roots or duck Duck weed 11.3 20.7 0.087 (1998)

pigs weed (at libitum)

Castrate male pigs, Fresh, chopped water 030 15.2 Tean et al.

either Mong spinach plus partially 20 17.9 (2002b)

Cai or large defatted rubber 30 20.5

white seeds (rubber seed 40 (% replaced) 23.9

cake) replacing part

of the daily feed

intake . ..

RSM, rubber seeed meal; FWFS, fresh water ﬁsh silage; LW, live weight.

39834 FOOD TEXT 26/5/05 9:52 am Page 79

Table 2. Feeding experiments with chickens in south-east Asia

Breed or Feeding scheme Initial age Initial Final body Duration of Daily feed Average daily Reference

production class of the body weight (g) treatment intake body-weight

animals weight (days) (g/bird) gain (g)

(days) (g)

Native chickens Supplements p1 p2 p1 p2 70 p1ap2ap1 p2 Hong Samnang

of both sexes offered in the 607 542 1567 1424 40.5 45.65 13.7 12.6 (1999)

scavenging in evening: 50 g692 523 1814 1344 58.5 63.00 16.0 11.7

two different broken rice, 551 499 1255 241 38.7 43.2 10.1 10.6

places, p1 50 g duckweed

and p2 50g broken rice,

50 g ground

soya beans

50 g of broken

rice

Egg type layers Starter ration 42 27.6 Farooq et al.

(109 ﬂocks) Grower ration 84 49.3 (2002)

Layer ration 241 131.5

Growing and Diets containing Group (% 133.8 128 10.8** Duong Duy

scavenging RSM. The (RSM) 34.0 10.6 Dong (2003)

coloured highest two RS0 (0) 34.4 9.7

feather doses of RS10 (10) 34.4 11.9

chickens rubber seed RS20 (20) 33.8 11.6

(RS) had RS30 (30) 34.1 9.3

negative RS40 (40)

effects on RS50 (50)

survival rates

39834 FOOD TEXT 26/5/05 9:52 am Page 80

Tam Hoang Diets of RSM 0

chickens 5 1 550 1700 1–28 0–70 2.91b3.55b17.9 27.5

10 (% RSM) 570 1829 2.87 3.56 18.7 28.2

554 1747 3.00 3.66 18.1 27.4

Luong Phuong Basal diet based 30 1326c30 70.1d19.2eDu Thanh

chickens on maize and 1380 81.6 23.9 Hang (2003)

concentrate 1395 92.9 29.56

(30 :70 ratio; 1456 99.9 31.96

16% crude 1452 106.5 33.64

protein in the

dry matter)

offered ad libitum

or restricted to 60,

70, 80 or 90% of

the ad libitum

intake. On all the

diets the birds

had free access

to fresh duck weed.

RSM, rubber seeed meal.

aEstimated from the data assuming 90% dry matter for broken rice and soya beans and 5% for duckweed.

bFeed conversion rate (kg of feed/kg gain).

cCarcass weight.

dDry matter intake.

eThe authors report the following relationship between dry matter intake (

) and live-weight gain (

=0.415

-9.75.

39834 FOOD TEXT 26/5/05 9:52 am Page 81

15. APPENDIX II

Model for the calculation of the concentrations in muscle of chloram-

phenicol ingested with soil.

—Chloramphenicol is administered orally (ingestion with soil). The cumulative

intake is calculated as follows:

• Intake of soil is 2% of the intake of dry matter.

• Intake of dry matter is related to body weight (for pigs) or average daily body-

weight gain (for chickens). The choice of body weight (pigs) and average daily

body-weight gain (chickens) was dictated by the nature of available empirical

data. From the available empirical data, linear relationships were derived that

were valid for a certain period of growth of the animals.

—The site of “measurement” of chloramphenicol within the body is the plasma.

—Concentrations of chloramphenicol in plasma and in muscle are assumed to

be equal (this is strongly supported by experimental data).

—Absorption is the process by which unchanged drug proceeds from the site of

administration to the site of “measurement”.

—The combined processes of distribution and elimination are called disposition,

whereby elimination means the loss of the drug from the site of “measurement”.

—The rate of change of drug in the body is equal to the sum of the rates of

absorption and the rate of elimination.

—For simpliﬁcation of the model, the following assumptions were made:

The drug is administered as a single daily dose (the amount contained in the

daily dose increases as a function of average daily body weight gain and

increased intake of feed).

The rates of absorption and distribution are short compared with the rate of

elimination.

All drug in the body is expressed as equivalents of parent chloramphenicol.

—Calculations are performed for a range of assumed elimination half-lives (t1/2).

—The amount

remaining in the body at time

after ingestion of the amount

is calculated using a mono-exponential term:

Only in one example is the sum of two exponential terms used.

The relationship between the half-life and the elimination rate constant

is:

=0 693

AA e

=¥

CHLORAMPHENICOL

39834 FOOD TEXT 26/5/05 9:52 am Page 82

—The amount remaining in the body just after each of three successive inges-

tions is calculated according to the following scheme:

CHLORAMPHENICOL

Time Amount remaining:

from 1st from 2nd from 3rd

ingestion ingestion ingestion

0,1

0,1 ¥

0,2

0,1 ¥

0,2 ¥

kt A

0,3

—The daily ingested amount is not constant, but increases as a function

of increasing intake of feed during the growth phase. It is assumed that pigs

grow from 20 to 75 kg of body weight within 119 days; and it is assumed that

chickens grow from 150 to 1000 g of body weight within 75 days. If the ingested

dose is expressed as ingested amount divided by body weight, then it is con-

stant in the model for pigs, since feed intake is related to body weight in that

model. However, in the model for chickens, feed intake is related to growth

rate. Therefore, the dose decreases steadily.

—The amount of the drug remaining in the body at the end of the growth phase

is used to calculate the concentration of chloramphenicol in the plasma accord-

ing to the formula:

where

is the concentration,

is the amount remaining in the body and

the volume of distribution. Values for

are taken from the literature.

Alternatives:

The average plasma concentration at steady state after multiple doses can be

calculated from (Rowland & Tozer, 1995):

where

is the fraction absorbed (dimensionless),

is the daily dose

(mg/kg bw), and

is the body clearance .

Values for

have to be taken from the literature.

Use of this model has been proposed by Sanders & Laurentie (2004).

Like the present monograph, this alternative model assumes:

Áˆ

min

Dose

ss av,

=¥

39834 FOOD TEXT 26/5/05 9:52 am Page 83

—The linearity of pharmacokinetics as function of the dose;

—A fraction of dose absorbed by oral route of 100% which is the worst-case

scenario;

—A muscle/plasma ratio of chloramphenicol concentrations equal to 1.

The model gives the following results:

Using the selected model, it can be shown that the conditions described for

pigs in Table 1, while producing an average steady-state concentration of chlo-

ramphenicol of 1 mg/kg, would at the same time cause a ﬂuctuation at steady state

of between approximately 0.06 mg/kg (minimum, shortly before the next dose) and

4.4 mg/kg (maximum, shortly after the last dose).

Clearance, volume of distribution and elimination half-life are linked through

the equation:

Using, for example in the model for pigs, a clearance value of 4.16 (ml/min ¥

kg) and a volume of distribution of 1400 ml would correspond to a half-life of

233 min or 3.89 h.

Cl V

=¥0 693

CHLORAMPHENICOL

Table 1. Daily oral dose necessary to maintain an average steady-state

concentration of chloramphenicol of 1

g/kg in muscle

Species Clearance Daily doseaDaily intake of Concentration

(ml/min ¥kg) (mg/kg) feed (g) or water (mg/g or mg/ml)

(ml) per kg bw

Feed Water Feed Water

Pigs 4.16 6

40 70 0.15 0.08

Poultry 3.84 5.5 240 150 0.02 0.04

6.62 9.5 0.04 0.06

aIt is assumed that either feed or water is the only source of contamination.

39834 FOOD TEXT 26/5/05 9:52 am Page 84

Analytical strategies for the detection and quantification of antibiotic residues in Aquaculturefishes: A review

Article

Apr 2016
TRENDS FOOD SCI TECH

Background: Aquaculture is one of the worldwide strategic development fields, and its importance is evident in its significant worldwide growth in the last decades. This growth is associated with the implementation of intensive and semi-intensive production methods, with the use of antibiotics in order to prevent the emergence and spread of infectious diseases in fish. Fluoroquinolones, tetracyclines, and sulfonamides, among others, are widely used for this purpose. This practice constitutes a real public health concern, not only due to the presence of antimicrobial residues in edible tissues, which can cause allergic reactions in hypersensitive individuals, but also due to the emergence of bacterial resistance. Consequently, the European Union's Regulatory Agencies have established maximum residue limits and specific requirements regarding the performance of analytical methods. Scope and approach: This article reviews the most recent analytical methodologies concerning antimicrobial residues in fish, reported in the literature, given emphasis on sample procedures, extraction/purification methods, chromatographic conditions and validation techniques according to legislation. Key findings and conclusions: Liquid chromatography coupled to mass spectrometric detection is used as preferential tool in the analysis of antimicrobial residues in fish. The current analytical strategy is shifting towards multi-residue and multiclass methods, which save time, and surely represent the future trend in this field. The extraction process still represents the limiting factor of any multi-residual method, since it should provide acceptable recovery of all analytes with a broad range of physicochemical properties, and therefore this is probably the step that requires more in-depth research.

Investigation on the Uptake of Ciprofloxacin, Chloramphenicol and Praziquantel by Button Mushrooms

Article

May 2021
FOOD CHEM

Button mushrooms are widely produced edible basidiomycetes. Commercially, they are cultivated on substrates containing fermented horse manure and chicken feces. Since pharmacologically active substances (PAS) might be introduced into the food chain via animal treatment, their residues may be present in manure used for mushroom growth. Previous studies in plants have demonstrated an uptake of PAS from the agricultural environment. The present study was performed to investigate the presence of PAS in button mushrooms. For analysis, a multi-analyte method for the detection of 21 selected PAS using liquid chromatography tandem-mass spectrometry was developed, successfully validated and applied to commercially available button mushrooms. Traces of chloramphenicol were detected in two of 20 samples. Additionally, in a mushroom cultivation experiment an uptake of ciprofloxacin, chloramphenicol and praziquantel was conducted. Throughout the whole experiment, praziquantel was present in quantifiable amounts in mushrooms and in high quantities in soil.

Determination of total florfenicol residues as florfenicol amine in bovine tissues and eel by liquid chromatography–tandem mass spectrometry using external calibration

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Mar 2019
J CHROMATOGR B

A reliable liquid chromatography-tandem mass spectrometry method was developed to determine total florfenicol residues in bovine tissues and eel. Florfenicol and its metabolites (florfenicol amine, monochloroflorfenicol, florfenicol oxamic acid, and florfenicol alcohol) were analyzed as the marker residue, florfenicol amine, as defined by several regulatory agencies. After hydrolysis with hydrochloric acid, samples were defatted and subjected to solid-supported liquid extraction and Oasis MCX-cartridge cleanup before analysis. The method was validated for florfenicol and its metabolites at two levels in eel and bovine muscle, fat, and liver. Excellent recoveries were obtained (93–104%), with relative standard deviations of <6% for all compounds. Negligible matrix effects and minimal analyte loss during sample preparation enabled accurate quantification by external calibration using solvent standards. No interfering peaks were observed around the retention time of florfenicol amine, indicating the high selectivity of the method. Retention times in the spiked samples corresponding to that of the calibration standard in solvent did not exceed ±0.1 min. Ion ratios from the spiked sample were within ±10% (relative) of the calibration standards. Calibration curves were linear in the range of 0.5 to 100 ng/mL, with coefficients of determination higher than 0.998. The limits of quantification and limits of detection of the proposed method were estimated to be 0.01 mg/kg and 0.0005 mg/kg, respectively, in all food samples. Thus, the developed method is considered reliable and suitable for regulatory use.

Single‐Residue Quantitative and Confirmatory Methods

Chapter

Nov 2011

IntroductionCarbadox and OlaquindoxCeftiofur and DesfuroylceftiofurChloramphenicolNitrofuransNitroimidazoles and Their MetabolitesSulfonamides and Their N4-Acetyl MetabolitesTetracyclines and Their 4-EpimersMiscellaneousSummaryAbbreviationsReferences

Distribution, fate and risk assessment of antibiotics in sewage treatment plants in Hong Kong, South China

Article

Mar 2011

Occurrence, removal, consumption and environmental risks of sixteen antibiotics were investigated in several sewage treatment plants (STPs) featuring different treatment levels in Hong Kong, China. Cefalexin, ofloxacin and erythromycin-H(2)O were predominant with concentrations of 1020-5640, 142-7900 and 243-4740 ng/L in influent, respectively; their mass loads were comparable to levels reported in urban regions in China and were at the high end of the range reported for western countries. The target antibiotics behaved differently depending on the treatment level employed at the STPs and relatively higher removal efficiencies (>70%) were observed for cefalexin, cefotaxime, amoxicillin, sulfamethoxazole and chloramphenicol during secondary treatment. ß-lactams were especially susceptible to removal via the activated sludge process while macrolides were recalcitrant (<20%) in the dissolved phase. Two fluoroquinolones, ofloxacin (4%) and norfloxacin (52%), differed greatly in their removal efficiencies, probably because of disparities in their pK(a) values which resulted in different sorption behaviour in sludge. Overall antibiotic consumption in Hong Kong was back-calculated based on influent mass flows and compared with available prescription and usage data. This model was verified by a good approximation of 82% and 141% to the predicted consumption of total ofloxacin, but a less accurate estimate was obtained for erythromycin usage. Risk assessment indicated that algae are susceptible to the environmental concentrations of amoxicillin as well as the mixture of the nine detected antibiotics in receiving surface waters.

Detection of Multidrug-Resistant Salmonella entericaSerotype typhimurium DT104 Based on a Gene Which Confers Cross-Resistance to Florfenicol and Chloramphenicol

Article

Full-text available

May 1999

Salmonella enterica serotype typhimurium ( S. typhimurium ) DT104 (DT104) first emerged as a major pathogen in Europe and is characterized by its pentadrug-resistant pattern. It has also been associated with outbreaks in the United States. The organism typically carries resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline. The mechanism of chloramphenicol resistance in DT104 was determined by producing antibiotic-resistant Escherichia coli host strain clones from DT104 DNA. DNA from chloramphenicol-resistant clones was sequenced, and probes specific for the genes flo S. typhimurium ( flo St ), int , invA , and spvC were produced for colony blot hybridizations. One hundred nine Salmonella isolates, including 44 multidrug-resistant DT104 isolates, were tested to evaluate the specificities of the probes. The gene flo St , reported in this study, confers chloramphenicol and florfenicol resistance on S. typhimurium DT104. Florfenicol resistance is unique to S. typhimurium DT104 and multidrug-resistant S. typhimurium isolates with the same drug resistance profile among all isolates evaluated. Of 44 DT104 isolates tested, 98% were detected based on phenotypic florfenicol resistance and 100% had the flo St -positive genotype. Resistances to florfenicol and chloramphenicol are conferred by the gene flo St , described in this paper. Presumptive identification of S. typhimurium DT104 can be made rapidly based on the presence of the flo St gene or its resulting phenotype.

Analytical Strategy for the Regulatory Control of Residues of Chloramphenicol in Meat: Preliminary Studies in Milk

Article

Mar 1992

An analytical strategy Is described for the regulatory control of residues of the veterinary drug chloramphenicol (CAP) In meat. Screening is performed directly in meat by a simple immunochemical card test with a limit of detection of about 2 μg/kg. Statistical evaluation of a collaborative study involving 13 laboratories showed that at CAP concentrations exceeding 8 μg/kg, no false negatives are found (N = 554). In positive samples, CAP Is quantltated with a routinely applicable, collaboratively tested column liquid chromatographic method with a limit of detection of about 1 μg/kg. At concentrations exceeding 10 μg/kg, the Identity of CAP Is established by Its UV spectrum obtained by using diode-array UV/VIS detection. A further confirmation can be obtained by the combination of gas chromatography/ mass selective detection In the electron Impact mode. Using 2 diagnostic Ions (m/z 225 and 208), the limit of identification Is about 5 μg/kg. The combination of the different analytical principles ensures reliable quantitation and Identification of CAP in positive samples, as established experimentally in Incurred samples and spiked samples (n > 100), and theoretically by the estimation of the uncertainty factor. The proposed set-up makes a regulatory program possible in which screening can be performed In a simple laboratory environment, followed by quantitation and Identification under more sophisticated conditions. Preliminary experiments Indicate that the analytical strategy Is also applicable to the control of CAP in milk. Application of mass spectrometry with negative chemical ionization permits the confirmation of CAP concentrations as low as 0.2 μg/L.

Confirmation of Chloramphenicol Residues in Bovine Milk by Gas Chromatography/Mass Spectrometry

Article

Jan 1994

Philip James Kijak

A gas chromatographic/mass spectrometric method was developed to confirm the presence of chloramphenicol (CAP) at 0.5 ng/mL in bovine milk. meta-Nitrochloramphenicol (20 ng) is added to 10 mL milk as a surrogate standard. The milk is mixed with ethyl acetate and then loaded onto a diatomaceous earth-filled, solid-phase extraction (SPE) column; CAP is eluted with additional ethyl acetate. A solution of 4% NaCI in water is added to the eluant, and ethyl acetate is removed by evaporation under a flow of N2. The sample is defatted with hexane and then loaded onto a C18-SPE column. The column is washed with water, and CAP is eluted with methanol. The methanol is evaporated under a flow of N2, and the trimethylsilyl derivative is prepared with Sylon HTP (hexamethyldisilazane–trimethylchlorosilane–pyridine, 3 + 1 + 9). The excess reagent is evaporated under N2, and the residue is taken up in cyclohexane-hexane (60 + 40). CAP is separated on a 30 m × 0.25 mm id methylsilicone column having a film thickness of 0.25 pm and analyzed by methane negative chemical ionization with selected ion monitoring. The ions with m/z 304, 322, 358, 360, 466, and 468 are monitored. Confirmation is based on the presence of 5 ions with relative ion abundances within 10% of that obtained by using standards.

The production and role of antibiotics in soil. II. Chyloromycetin

Article

Jan 1952
PHYTOPATHOLOGY

Fresh water fish silage as protein source for growing-fattening pigs fed sugar palm juice

Article

Jan 2000

Eighteen families who owned at least 10 sugar palm trees and were interested to rear pigs participated in this study which was carried out in Kandal village, Ang Snoul district in Kandal province, 40 km west of Phnom Penh city. The juice from 157 sugar palm trees of both sexes (119 males and 38 females) was used as the principal energy source for fattening pigs. The average daily juice yield varied among families (P=0.001) in the range of 3.00 to 4.92 kg/day with a median value of 3.89 kg/day. The corresponding values for the Brix (total sugars) content were a range of 11.6 to 12.8% (P=0.001) and a median value of 12.3%. Seventy two pigs (23 males and 49 females) of different crossbreeds were reared during 5 months on a diet of ad libitum sugar palm juice, 500 g/day of rice bran and 400 g/day of fresh water fish silage which was made by mixing the partly eviscerated fish with sugar palm syrup and rice bran (Fresh weight ratios were: Fish, rice bran, sugar palm syrup; 50:10:40) and storing anaerobically in sealed plastic bags. The average daily intake of sugar palm juice was 8 kg. The protein intake was estimated to be 130 g/pig/day. The growth rates of the pigs varied among families in the range of 325 to 476 g/day with an average of 405±47 g/day.

Biodigester effluent versus manure from pigs or cattle as fertilizer for production of cassava foliage (Manihot esculenta)

Article

Dec 1998

H.C. Le

A field experiment was conducted during five months from 1 December 1997 to 30 April 1998) in the University of Tropical Agriculture on the College of Agriculture and Forestry Campus, Thu Duc, Ho Chi Minh City, Vietnam. The experimental soil was grey podzolic. The trial was laid out in a randomized block design, replicated three times with a plot size of 4 x 2.5m and four treatments of organic fertilizer application to cassava grown for forage. The treatments arranged in a 2*2 factorial were: • Animal species (cows vs pigs) • Biodigestion (fresh manure vs effluent derived from the same manure after being fermented in a biodigester) The fresh manure was applied before planting and after the first harvest (at three months). The effluent was applied every three days. Thus the effect of biodigestion was confounded wiith the method of application. Quantities of each source were estimated to provide 200 kg nitrogen/ha/year. The cassava was harvested for forage (cutting height 70 cm above ground level) after 3 months and again 2 months later. The foliage was separated into stem and leaf + petiole and analyzed for dry matter and nitrogen. Soil analysis was done before planting and again 5 months later. Effluent was significantly better than raw manure in supporting a higher biomass yield and protein content of the foliage. The source of the manure did not affect these parameters. Yields of fresh leaf plus petiole were 6.45 and 5.16 tonnes/ha/harvest for effluent and manure, respectively (SE±0.15; P=0.001). Protein percentages in dry matter of leaf and petiole were: 27.6 and 24.2 (SE±0.165; P=0.001). There were marked improvements in soil fertility parameters as a result of applying either manure or effluent.

Biodigester effluent versus manure, from pigs or cattle, as fertilizer for duckweed (Lemna spp.)

Article

Dec 1998

H.C. Le

The research had the following objectives: to document the observed improvements in nutritive value (protein content) and yield of duckweed when biodigester effluent, rather than original manure, is used to fertilize duckweed ponds; to determine optimum fertilizing level of manure and effluent for duckweed; to determine if there are differences between manure and effluent from cows versus pigs as fertilizer for duckweed ponds. The treatments were: • Source of manure / effluent: From cows (C) or pigs (P) • Processing: Fresh manure (M) or effluent (F) • Concentration of nitrogen in the medium (10, 20 and 30 mg N/litre) The design was a factorial arrangement (2*2*3) and there were 4 replications in a completely randomized layout. The experiment was conducted for 24 days in the rainy season (August 1998). Plastic baskets (n=48) lined with plastic film were used as the experimental ponds. The surface of the water in each basket had an area of 0.145 m2 and with 10 cm depth the volume was 14.5 litres. Each container was inoculated with 40g (250g/m2) of duckweed (Lemna spp.). The yield of duckweed was calculated by subtracting the inoculum from the total biomass production measured every 24 hours and was expressed as fresh duckweed (DW) yield g/m 2/d. Manure and effluent were taken from two plug-flow tubular plastic biodigesters: one charged with cow manure the other with pig manure. Manure and effluent were added daily in quantities calculated to maintain pond nitrogen levels at approximately10, 20 and 30 mg/litre. The results of the experiment showed that with the same input of nitrogen, plants nutrients derived from biodigester effluent supported higher concentrations of crude protein in duckweed, than nutrients from raw manure. Manure and effluent from pigs tended to support higher concentrations of crude protein in duckweed than when cows were the source of these inputs. The optimum level of nitrogen in the pond water was in the range of 20 to 30 mg/litre. Root length of duckweed was inversely related with protein content. Higher pH of the pond water in the range pH 6.4 to 7.2 was associated with duckweed of higher protein content.

Two cases of skin ulcer due to chloromycetin® cream

Article

Jan 1998

Two cases of skin ulcer due to Chloromycetin® cream are reported. Case 1 is a 48-year-old woman and Case 2 is a 46-year-old man. Both patients had applied Chloromycetin® cream to their wounds for about 1 month and developed skin ulcers on the applied sites. These ulcers had healed after discontinuance of the treatment with Chloromycetin® cream. Patch tests on each patient showed strongly positive reactions to Chloromycetin® cream (as is) and 2% chloroamphenicol (aq.), and 2% chloramphenicol (pet.) with 1% sodium lauryl sulfate, but negative reactions to 2% chloramphenicol (pet.). From these results, we diagnosed both cases as skin ulcer based on contact dermatitis due to chloramphenicol.

Goodman and Gilmans The Pharmacological Basis of Therapeutics

Article

Jan 1990

Utilization of catfish oil in diets based on dried cassava root waste for crossbred fattening pigs in the Mekong delta of Vietnam

Article

Apr 2003

An experiment was carried out at Song Hau State farm from April to June in 2002 with 48 crossbred growing pigs (Landrace * Large White) with an average initial live weight of 57 kg and final live weight of 90 kg. The trial included 4 diets: Cassava root meal, catfish oil 0 (Control, CRM-OIL0); Cassava root waste, catfish oil 0 (CRW-OIL0); Cassava root waste, catfish oil 5% (CRW-OIL5) and Cassava root waste, catfish oil 10% (CRW-OIL10) and 4 blocks, corresponding to the initial live weights of the groups, randomly allocated to 16 pens. The mean dietary crude protein (CP) content was 15.3 % in dry matter and the daily DM feed intake equal to 3 % of the mean pen live weight. Mean daily gains were significantly different (P<0.05) among treatments, and were 685 g, 598 g, 715 g and 634 g for CRM-OIL0, CRW-OIL0, CRW-OIL5 and CRW-OIL10 diets, respectively. The FCRs were 2.9, 3.1, 2.9 and 3.0 for these diets, respectively. The carcass quality parameters of back fat thickness (2.2, 2.2, 1.9 and 2.4 cm, respectively) and loin eye area (46.7, 45.2, 47.5 and 45.6 cm2, respectively) were not significantly different between diets. Similarly the content of crude protein in loin muscle was not affected by treatment (P>0.05). The Iodine No of back fat was higher (P<0.01) in the 10 % catfish oil diet. The highest economic benefit was for the CRW-OIL5 diet.

Chloramphenicol

Figures

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