Chlorocholine chloride residue distribution in eggs, breast and femur meat of laying hens determined by labelled-¹⁵N estimates

 

 

Nurhayati^{I, II}; J.D. Kabasa^{I, III, #}; G. Thinggaard^I; U. ter Meulen^I

^IInstitute of Animal Physiology and Animal Nutrition, Göttingen University, Kellnerweg 6, D-37077, Germany
^IIDepartment of Animal Nutrition and Feed Science, Jambi University, Kampus Pinang Masak Mendalo Darat, Jambi 36361 Indonesia
^IIIFaculty of Veterinary Medicine, Makerere University, P.O. Box 7062, Kampala, Uganda

 

 

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ABSTRACT

The distribution of chlorocholine chloride (CCC) residue or its metabolites in the meat and eggs of laying hens was studied using the ¹⁵N delta value (5¹⁵N) and ¹⁵N atom % derived from ¹⁵N-CCC containing diets. In a completely randomised design, 20 laying hens were divided into four groups allocated four different diets namely; 0 mg ¹⁵N-CCC /kg feed a control diet (group A); 5 mg ¹⁵N-CCC /kg feed (group B), 50 mg ¹⁵N-CCC /kg (group C) and 100 mg ¹⁵N-CCC /kg (group D) for 11 days. During the seven days that followed, ¹⁵N-CCC diets were withdrawn and all hens were restored to feeding on the control diet. The 5¹⁵N excess and ¹⁵N atom % excess in meat and eggs of hens fed diets containing ¹⁵N-CCC, were higher than in the control diet after 11 days of treatment and seven days after withdrawal of ¹⁵N-CCC, except for the egg yolk values of hens fed 5 mg ¹⁵N-CCC /kg feed. The 5¹⁵N excess and ¹⁵N atom % excess of meat, egg yolk and egg albumen were dependent on dietary ¹⁵N-CCC concentrations and differed significantly between tissues for each of the three ¹⁵N-CCC concentrations examined. Femur meat 5¹⁵N excess and ¹⁵N atom % excess were similar to that of breast meat but differed significantly from that of other tissues. The results show that tissue type is a factor in CCC residue/metabolite accumulation in chicken products suggesting differences in exposure or risk of CCC on consumers.

Keywords: Chlorocholine chloride residues, poultry products, labeled-¹⁵N

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Introduction

Chlorocholine chloride (CCC), commonly known as Chlormequat, is a plant growth retardant widely used in commercial agriculture to improve the shape, size and yield of cereal crops. The CCC residues and its metabolites accumulate in cereal plants and can be detected in different plant products (Blinn, 1967; Bier & Dedek, 1970; 1972; Bohring, 1972; 1982). Although a small quantity of residue of CCC is allowed in foodstuffs, there are concerns that the residue may be harmful to both animals and humans. Consumption of excess CCC tended to increase the incidence of cancers such as leukaemia in rats (National Cancer Institute, 1979). Reports that CCC is completely metabolised into other compounds without leaving any residues in the tissues are not conclusive (Dekhuijzen & Bodlaender, 1973; Dekhuijzen & Vonk, 1974). To protect animals and humans from the intake of CCC residues in food, the European Union has set maximum CCC residue limits of 2 mg/kg for cereals, except for oats (5 mg/kg) and pears (10 mg/kg). Using radioactive-labelled CCC at different concentrations in feed, several workers demonstrated the presence of CCC residues in rats (Blinn, 1967; Romanowski, 1972; Gonzales, 1997), cows (Lampeter & Bier, 1970), oviducts of laying hens (Landazuri, 1992) and ovaries of pigs (Azem, 1996). When different concentrations (5, 50 and 250 mg) of ¹⁵N-labelled CCC/kg feed were fed to laying hens, significantly higher ¹⁵N concentrations were recorded in the egg yolk and albumen from hens fed the diets containing 250-mg/kg ¹⁵N-CCC (Songsang, 2000). The extent of CCC residue distribution in tissues may, however, differ due to differences in environmental factors, genotype, physiological and nutritional state of the individual (Ackermann et al., 1975; Sachse, 1977; Torner et al., 1999). High CCC concentrations in tissues increase the exposure risk of individuals consuming food products from such tissues. This study investigated the distribution of CCC residue and its metabolites in the eggs and meat of laying hens fed diets containing varying concentrations of CCC. For the purpose of this investigation the ¹⁵N-labelled CCC (¹⁵N-CCC) estimating method was used to trace the fate of the compound.

 

Materials and Methods

Twenty 280-day old Brown-breed laying hens, weighing between 1727 and 2269 g, were housed in individual layer cages and fed for seven days on a control diet consisting of ¹⁵N-CCC free wheat, maize, fish meal, a vitamin-mineral premix and feed lime. The diet contained 163.7 g crude protein/kg and 11.48 MJ ME/kg. Thereafter, the hens were divided randomly into four experimental groups. Each group was allocated one of the four experimental diets: a control diet containing 0 mg ¹⁵N-CCC/kg (group A) and diets containing 5 mg ¹⁵N-CCC/kg (group B), 50 mg ¹⁵N-CCC/kg (group C) and 100 mg ¹⁵N-CCC/kg (group D).

The ¹⁵N-CCC (atom ¹⁵N content, 99%) (MSD Isotopes, MERCK-FROSST Canada Inc., Montreal, Canada) was mixed in feed to concentrations of 5, 50 and 100 mg/kg in the final diet. The experimental diets were fed for 11 days, after which the diets containing ¹⁵N-CCC were withdrawn and the hens were fed the ¹⁵N-CCC free diet for seven days. Feed and water were offered ad libitum and cage temperatures were maintained at 19 - 21 °C during the experiment.

Eggs were collected daily, and the yolk and albumen portions of the eggs were separated immediately after collection using an egg separator (Labortek, Germany). The eggs from the different treatments were processed in separate rooms with separate equipment to avoid cross contamination with ¹⁵N. Meat samples were collected from the breast and femur muscles by biopsies on day 11 of the experimental period and seven days after ¹⁵N-CCC withdrawal. All samples were weighed, analysed for dry matter content and stored at -21 °C until further analyses.

The 11 days of experimental feeding were justified because yolk deposition takes place in 10 to 11 days (Etches, 1996; Hartmann, 2001). The seven days withdrawal period adopted was based on the observations of Coffman et al. (1999) that at least seven - eight days prior to slaughter are required to keep food derived from hens supplemented with antibiotics and/or drugs safe for human consumption.

The measurement of ¹⁵N was done according to the method described by Reineking et al. (1993) in the Forschungszentrum Waldökosysteme, Kompetenszentrum Stabile Isotope (KOSI) Göttingen University, Germany. The procedure involved using an elemental analyser (EA, Carlo-Erba 1500 nitrogen analyser) coupled into the Finnigan MAT Continuous Flow interface (ConFlo II^TM), which connects to Finnigan MAT Isotope Ratio Mass Spectrometry (IRMS, Finnigan MAT 251). Acetanilide (C₈H₉NO) was the standard material for checking the analytical variability. It had a composition of 71.1% (C), 10.4% (N), 6.7% (H) and 11.8% (O) and contained 0.366943 of ¹⁵N atom % and 1.76 ^o/oo of δ¹⁵Ν. The relative δ¹⁵Ν was calculated using the formula of Mariotti (1983):

The ¹⁵N atom % was calculated based on the international standard of ¹⁵N in air (Mariotti, 1983) as follows:

where:

R = absolute isotope ratios, measured for the sample and standard. The value of absolute isotope ratios of international standard in Goettingen was 0.003676496.

wst = work standard,

sa = sample,

i = current ion which i29 is ion of ¹⁵N and ¹⁴N, i28 is ion of ¹⁴N2

ist = international standard

Atom % excess (APE) and delta value excess (δ¹⁵ΝΕ) were calculated by difference between the ¹⁵N values of the treatment and that of the control. Similarly, the difference between δ value of the working standard and international standard of -13.468 was obtained.

 

Results and Discussion

The results of excess δ¹⁵ and excess atom % ¹⁵N in egg yolk, egg albumen, breast and femur meat of hens fed diets with varying levels of ¹⁵N-CCC for eleven days are shown in Table 1 and Figure 1. The 11 days of feeding diets containing ¹⁵N-CCC resulted in increased 5¹⁵NE and APE in the meats and eggs of hens (P < 0.05). The significant increases were more in hens fed higher concentrations of ¹⁵N-CCC. The 5¹⁵NE and APE values were highest in the egg yolks and decreased in the order: egg yolk > egg albumen > breast meat > femur meat.

The contribution of ¹⁵N in the meat N was lower than in the eggs at all doses examined. The results are similar to that of Wilbur (1975) who found that after 24 hours of oral application of 3 mg/kg CCC per laying hen per day, the CCC residue was higher in the yolk than in the albumen. However, residue levels in the muscle were not detectable (<0.01 mg/kg).

The pattern of the nitrogen distribution might be due to differences in tissue protein metabolism and flowing rate of nutrients during the production phase that is more in the egg than in muscle tissue. This is similar to reports by Paulson et al. (1983) that recorded the lowest pesticide residues concentration in the skeletal muscle of hens. Residue accumulation in the body is affected by several factors including post metabolism persistence of the applied compound and its rate of degradation (Paulson et al., 1983).

The results of excess δ¹⁵Ν value and that of excess atom % ¹⁵N (i.e. differences between the treatment and control values) in the eggs and meat of hens seven days after withdrawal of ¹⁵N-CCC diets are shown in Table 2 and Figure 2, respectively. Seven days after ¹⁵N-CCC diet withdrawal, δ¹⁵^ and APE concentrations in the meat of hens fed CCC treated feeds were still higher (P < 0.05) than in the control diets. For the eggs (yolk and albumen), the concentrations did not differ (P < 0.05) at any treatment level.

However, all values were lower than those observed after 11 days of feeding on treated diets. The δ¹⁵NE and APE in meat differed significantly at P < 0.05 among treatments, whereas those of egg yolk and albumen were similar during the same time period of eleven days.

Differences in tissue metabolism could explain these observations. Calsamiglia et al. (1996) observed differences in ¹⁵N levels and attributed their findings to variation in metabolism. The low mobility of ¹⁵N-CCC could also be a factor (Bohring, 1972; 1982) as it promotes tissue retention of the compound without any metabolism to the other compounds (Bier & Dedek, 1970; 1972). Other authors (Katz, et al., 1973; Beale et al., 1990; Shaikh & Chu, 2000; Furusawa et al., 2002) reported varying levels of drug residues in tissues, organs and eggs of poultry at different days after drug withdrawal. The residue levels were attributed to the body fat content that affects drug release back into the blood.

This study investigated the potential distribution of CCC residues in tissues of laying hens. Labelled ¹⁵N was used because it is a heavy isotope with a natural abundance of < 1% that is relatively constant in air. This abundance was approximately 0.366% at the time of the study. Since the CCC is a polar compound with low volatility, and the ¹⁵N was not degraded in the animal body under the experimental conditions, using ¹⁵N as a label of CCC was beneficial. The application of ¹⁵N in experimental diets enabled the quantification of the potential distribution of the dietary CCC and its metabolites in the chickens.

 

Conclusion

The results suggest that the CCC residue and/or its metabolites are distributed in chicken meat and eggs in varying concentrations. The ¹⁵N content was highest in egg yolk, followed by egg albumen and breast and lowest in femur meat. The nature of CCC metabolites in the chicken needs clarification.

 

Acknowledgements

We are grateful to the DUE-UNJA project (Development for Undergraduate Education Project at the University of Jambi), Departments of Tropical Animal Nutrition, Goettingen University, Germany; the Faculty of Animal Science, Jambi University, Indonesia and Physiological Sciences, Faculty of Veterinary Medicine, Makerere University, Uganda for invaluable support.

 

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# Corresponding author. E-mail: kabasajd@vetmed.mak.ac.ug