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Formaldehyde content of milk in goats fed formaldehyde‐treated soybean oil‐meal a
J. L. Barry & D. Tomé
a b
a
Laboratoire de Technologie Appliquée à la Nutrition, Rue de la Géraudière , Institut National de la Recherche Agronomique , BP 527, Nantes Cedex 03, 44026, France b
Inserm U 290, Hôpital Saint‐Lazare, 107, rue de Faubourg Saint‐Denis, Paris, 75010, France Published online: 10 Jan 2009.
To cite this article: J. L. Barry & D. Tomé (1991) Formaldehyde content of milk in goats fed formaldehyde‐treated soybean oil‐meal, Food Additives & Contaminants, 8:5, 633-640, DOI: 10.1080/02652039109374017 To link to this article: http://dx.doi.org/10.1080/02652039109374017
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sublicensing, systematic supply, or distribution in any form to anyone is expressly
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FOOD ADDITIVES AND CONTAMINANTS, 1991, VOL. 8, NO. 5, 6 3 3 - 6 4 0
Formaldehyde content of milk in goats fed formaldehyde-treated soybean oil-meal J. L. BARRY and D. TOMÉ † Institut National de la Recherche Agronomique, Laboratoire de Technologie Appliquée à la Nutrition, Rue de la Géraudière, BP 527, 44026 Nantes Cedex 03, France
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(Received 24 September 1990; accepted 1 March 1991) Formaldehyde is used in ruminant feeding for different purposes including the protection of dietary proteins from ruminai degradation. The formaldehyde content of milk of goats fed various levels of formaldehyde-treated soybean oil-meal has been determined by using a sensitive HPLC method. Results showed a significant linear correlation between ingested formaldehyde and formaldehyde concentration in milk. About 0.02% of ingested formaldehyde was excreted in milk, as free formaldehyde. Keywords: ruminants, treated protein, milk, formaldehyde
Introduction
Formaldehyde is used in ruminant feeding for different purposes. Its bacteriostatic properties make it useful as a preservative in silage (Kreula and Rauramaa 1977, Rauramaa and Kreula 1977, Arnould et al. 1978) and in liquid whey (Buckley and Fisher 1984, Buckley et al. 1988). Formaldehyde can be used as a complex with urea to lower the rate of urea degradation in the rumen (Lall et al. 1982, Setälä and Syrjälä-Qvist 1982a, b). Formaldehyde can also be used to protect dietary protein from ruminai degradation (Ferguson et al. 1967, Zelter et al. 1970). This process, applied to soybean oil-meal, increases by 50-90% the flow of dietary amino acids in the intestine of adult ruminants (Vérité et al. 1977) and is commercially developed in Europe (Delort-Laval 1985). Formaldehyde is a toxin (Beall and Ulsamer 1984, Ulsamer et al. 1984), whose presence in animal products is undesirable. Metabolism of formaldehyde ingested by ruminant is well known (Mills et al. 1972). 14C-formaldehyde is transformed in the rumen mainly to 14CC>2 and 14CH4, about 20% of the radioactivity is excreted in the faeces and a small fraction is absorbed (Mills et al. 1972). Absorbed formaldehyde enters three metabolic pathways. It is metabolized mainly to CO2: rats metabolize more than 80% of intraperitoneally injected formaldehyde (Neely 1964, Mashford and Jones 1982). Some absorbed formaldehyde is transformed into various metabolites found in tissues (Mills et al. 1972), milk (Mills et al. 1972, Kreula and Rauramaa 1977) and urine (Neely 1964, Mills et al. 1972). At least small amounts of formaldehyde are found in milk of ruminant fed formaldehydecontaining silages (Beck and Gross 1973, Kreula and Rauramaa 1976, Arnould † Present address: Inserm U 290, Hôpital Saint-Lazare, 107, rue de Faubourg Saint-Denis, 75010 Paris, France. 0265-203X/91 $3.00 © 1991 Taylor & Francis Ltd.
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et al. 1978, Syrjälä-Qvist and Setälä 1982b), or formaldehyde-protected proteins (Barry and Tome 1982, Syrjälä-Qvist and Setälä 1982a). Many difficulties occur in formaldehyde determination in milk. Quantitative discrepancies have been pointed out (Syrjälä-Qvist and Setälä 1982b, Buckley et al, 1988). Some of them could be due in part to the form in which formaldehyde is ingested (Buckley et al. 1988) but, as noticed by Syrjälä-Qvist and Setälä (1982b), the exactness of methods of determining formaldehyde contents of milk explains in part these discrepancies. That agrees with the fact that these discrepancies are observed even when no formaldehyde is ingested. Concentrations usually found are very low and colorimetric or fluorimetric techniques used formerly for formaldehyde determination are not sensitive and specific enough for a correct quantification of formaldehyde in milk. Our work aims to determine precisely formaldehyde concentrations in milk from goats fed various amounts of formaldehyde-treated soybean oil-meal by using a more sensitive method than those used previously. This method employs the extraction of formaldehyde by steamdistillation (Tome etal. 1979) and the formation of a 2,4-dinitrophenylhydrazone (DNPF) which is separated by HPLC (Selim 1977, Van Schalm 1983).
Experimental section
Animals, diets and experimental design Five adult lactating Alpine goats, weighing a mean of 45 kg (42-47 kg), were fitted, several months previously, with rumen cannulae. They were housed in individual boxes. The experiment was performed during their 4th and 5th month of lactation. Animals were milked twice each day. Animals received two daily meals consisting of a basal diet of 750 g of medium quality grass hay and 600 g of a maize-based concentrate (maize: 90%, molasses: 5%, mineral complement: 5%). The basal died was completed with an experimental feed constituted of 600 g of soybean oil-meal. Soybean oil-meal was either untreated or industrially treated with 0-3% (w/w) of formaldehyde. Treatment of soybean oil-meal was performed by spraying a 31% formaldehyde aqueous solution on the oil-meal. Formaldehyde content in soybean oil-meal was quantified as extractable formaldehyde according to the procedure described by Tome et al. (1979): formaldehyde released by phosphoric acid was steam extracted and colorimetrically quantified according to Nash (1953). Soybean oil-meal was given to animals in three forms: (i) untreated soybean oil-meal, (ii) a mixture 50:50 of treated and untreated soybean oil-meal, (iii) treated soybean oil-meal. The experimental feed was given at the beginning of each meal. So that soybean oil-meal consumption was rigorously controlled, refused soybean oil-meal was eventually introduced into the rumen via a cannula. During a three week pre-experimental period, animals were adapted to the basal ration and untreated soybean oil-meal and to the feeding procedure. The experiment consisted of three periods of seven days: during each period, each animal was randomly assigned to one of the three experimental diets. On the whole, each animal received each diet once. Daily milk production was determined for each animal. Milk samples, pooled for morning and evening milking, were taken for each goat during the five last days of each experimental period. Samples were frozen at -20°C until analysis.
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Determination of free formaldehyde in milk The method used for the determination of free formaldehyde in milk was derived from the technique of Van Schalm (1983). Fifty ml of milk or of formaldehyde standard solution and 1 ml of phosphoric acid were introduced in a flask of 250 ml connected with a steam-distillation apparatus (Tome et al. 1979). Two hundred ml were steam-distilled and 50 ml of the distillate were transferred into a beaker with 10 ml of a solution of 2,4-dinitrophenylhydrazine 2-5 g DNPH in HCI(6N) and 10 ml of chloroform. This mixture was shaken for 15 min at room temperature and transferred to a separatory funnel. The two phases were separated and the aqueous phase was extracted twice with 25 ml of HC1(2N), three times with 25 ml of H2O and concentrated to dryness under reduced pressure at 25°C. The residue was dissolved in 2 ml of acetonitrile. A volume of 20jnl was injected in a Waters HPLC system equipped with a RP C-18 ^m Bondapack column (350x4-6 mm) and a UV detector at 350 nm. Residual components were eluted with a mobile phase of water/acetonitrile 50:40 (v/v) at a flow rate of 1 -5 ml/min. The position of the DNPF derivative was determined by referring to a standard of DNPF and the concentration of formaldehyde by the peak area in reference to a calibration curve prepared with standard solutions of formaldehyde treated in the same way as milk samples. With this methodology, a small contaminating peak, eluting at the same retention time as DNPF, was observed in HPLC chromatograms with blank solution. Buckley et al. (1986), quantifying DNPF by GLC, have also reported the presence of this peak due to the contamination of derivatizing reagent. Detection limit was determined as the upper limit of this background signal calculated as: y = j o + 3ffy
where yo and ay were respectively the mean and standard deviation of the background of the signal (Buckley et al. 1986). In our methodology, yo was 0-0086 mg/kg and ay was 0-0012 mg/kg: the detection limit of our methodology was calculated to be 0-012 mg/kg. Results Animal health and feeding To make goats ingest high quantities of formaldehyde we fed them with an excessive amount of soybean oil-meal. Though the animals were consequently fed hypernitrogenous diets, they remained in good health with no incidence of mastitis. Some of the soybean oil-meal was sometimes not ingested by animals and was then introduced into the rumen. As this fraction was very small (less than 10%) and as it has been previously shown (Barry et al. 1979) that feeding concentrate via rumen cannula does not change its digestive fate, no distinction has been made between ingested or rumen cannula-introduced soybean oil-meal: in the following results, ingested soybean oil-meal refers to all the oil-meal consumed by the animals. Formaldehyde in soybean oil-meal Formaldehyde-treated soybean oil-meal contained 2 • 40 g of extractable formaldehyde per kg of dry matter. The incomplete recovery of formaldehyde is in agreement with the findings of Tome et al. (1979) who showed that some
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formaldehyde is irreversibly bound to proteins and that extractable formaldehyde represented about 80% of the formaldehyde used during the treatment of various oil-meals. With untreated soybean oil-meal, a mixture (50:50) of untreated and treated soybean oil-meal, and treated soybean oil-meal, animals ingested 0, 0-63 and 1 • 26 g of extractable formaldehyde per day, respectively. Milk and formaldehyde in milk During the whole experiment, mean daily milk production was 2-00 ±0'031 kg per goat, with no statistical influence of either time or formaldehyde ingestion. Typical HPLC chromatograms for formaldehyde determination in standard and milk samples are given in figure 1. Milk formaldehyde concentrations are shown in table 1. When 0, 0*63 or 1-26 g of formaldehyde were ingested daily, mean formaldehyde concentrations in milk (mean ± standard error) were 0-033 ± 0-007, 0-083 ± 0-007 and 0-153 ± 0-011 mg/kg respectively. As shown in table 2, these figures differed significantly from each other (p < 0-01). Experimental points could be fitted by a linear regression with a very high correlation coefficient (y = 0-02947 + 0-0952*; r- 0-938; p < 0-01; where y is milk formaldehyde concentration expressed in mg/kg and x daily formaldehyde ingested expressed in g/day). The relation between the level of milk production (z, expressed in kg/day; and milk formaldehyde concentration (w,
DNPF
Blank
Standard 0.2 mg/l
Milk sample
10
20
Figure 1. HPLC separation of the formaldehyde-2,4-dinitrophenylhydrazone (DNPF). The blank, the standard solution of formaldehyde (0-2 rag/1) and the milk sample have been extracted and derived as described in the experimental section.
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Table 1. Milk formaldehyde concentration (mg/kg) in goats fed various levels of formaldehyde (mg/day).
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Daily ingested formaldehyde Animals
0-000
0-630
1-260
Goat Goat Goat Goat Goat
0-054* 0-046 0-017 0-023 0-024
0-072 0-112 0-086 0-061 0-083
0-152 0-198 0-110 0-150 0-154
O-033a 0-007
0-083" 0-009
O-153c 0-014
1 2 3 4 5
Mean** Standard error
*Each figure is the mean of five consecutive days. ** Figures with a different superscript are significantly different ( p < 0 - 0 1 ) .
Table 2. Statistical analysis of formaldehyde concentrations in milk.
Ingested formaldehyde Animal Interaction Residual error
Sum of square deviation
Freedom degrees
Mean squares
0-172 0-022 0-011 0-033
2 4 8 60
0-086 0-011 0-001 0-001
F
Level of statistical significance
158-56 10-21 2-57 —
0-01 0-01 0-05 —
expressed in mg/kg) was: w= 1-033 10~3 + 1-564 10" 2 z (r = 0-507; p > 0 - 0 5 ) w = 5-839 10" 2 + 1-965 10" 2 z (r = 0-522; p> 0-05) w = 6-567 1(T 2 + 4-291 1(T 2 z (r = 0-876; p>0-05) respectively when 0, 0-63 and 1-26 g of formaldehyde were ingested daily and showed no significant influence of level of milk production on milk formaldehyde concentration. Discussion A main problem was first to determine whether formaldehyde was present in the milk of animals fed diets containing no formaldehyde. Formaldehyde is a normal product of intermediary metabolism in mammals (Mashford and Jones 1982, Buckley and Fisher 1984). It has been detected in various animal tissues. Florence and Milner (1981) found 19, 19 and 23 mg/kg of formaldehyde respectively in liver, kidney and muscle of pigs fed diets containing no formaldehyde. More recently, Buckley et al. (1988) found lower but significant amounts of endogenous formaldehyde in calf tissues (3-6, 1-4 and 0-18 mg/kg
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respectively in liver, kidney and muscle). Whether or not this endogenous formaldehyde can enter the udder and be excreted in milk is not well established. Using a colorimetric determination of formaldehyde in milk (chromotropic acid), Beck and Gross (1973) found no endogenous formaldehyde in cow's milk; using the same methodology, Syrjälä-Qvist and Setälä (1982a) determined levels of formaldehyde up to 0-2 mg/kg in milk of cows fed diets containing no formaldehyde. Using the colorimetric determination of Czech (1973), Barry and Tome (1982) found 0-017 mg/kg of endogenous formaldehyde in goats' milk. The main reason for these discrepancies could be the inaccuracy of colorimetric methods to quantify trace levels of formaldehyde in milk. With the specific and sensitive HPLC method we developed, endogenous formaldehyde level in milk was found in the present experiment to be 0-033 mg/kg. Using a GLC determination of formaldehyde in milk, Buckley etal. (1986, 1988) found 0-017 mg/kg of formaldehyde in milk of cows fed diets containing no formaldehyde. As this level was lower than the detection limit of their technique (0-026 mg/kg), they could not conclude as to the actual presence of endogenous formaldehyde in milk. Our figures obtained with a more sensitive method indicate that there are low but significant levels of formaldehyde in milk of animals fed diets containing no formaldehyde. The other question is the transfer of ingested formaldehyde in milk. With goats fed I4C-formaldehyde-treated casein, Mills et al. (1972) detected small amounts of radioactivity in milk; but this was not present as formaldehyde and they concluded that ruminants effectively metabolize formaldehyde and that there is no accumulation of this compound in milk. In their experiment, they used a colorimetric determination of formaldehyde (chromotropic acid) and found very high levels of formaldehyde in milk of control animals (1 mg/kg): their methodology, as mentioned above, might not have been sensitive enough to determine any increase in formaldehyde concentration in milk. The results obtained in the present experiment showed a significant increase of formaldehyde concentration in milk of animals fed formaldehyde-treated soybean meal. These results are in good agreement with those previously published in goats (Barry and Tome 1982) and in cows (Syrjälä-Qvist and Setälä 1982a) which indicate that a fraction of dietary formaldehyde used to protect dietary protein from ruminai degradation was recovered as free formaldehyde in milk. Such a transfer of formaldehyde from the diet to the milk has also been observed with formaldehyde used as silage preservative (Beck and Gross 1974, Kreula and Rauramaa 1976, Arnould et al. 1978, Syrjälä-Qvist and Setälä 1982b), whey preserver (Buckley and Fisher 1984, Buckley etal. 1988) or as a protective agent of fats from rumen degradation (Wrenn et al. 1975). In conclusion, when introduced in ruminant feeds either as a preservative agent or as a reagent used for the protection of dietary components from ruminai degradation, formaldehyde is probably always recovered at low levels in ruminant milk. For comparative purposes, one can estimate that the daily ingestion of 1 -26 g of formaldehyde in goats weighing 45 kg corresponds to a daily ingestion of 16-8 g in a cow weighing 600 kg. With such levels of dietary formaldehyde, very conflicting results have been obtained in cows. With daily formaldehyde intakes of 14-6, 18-7 and 18-2g, Syrjälä-Qvist and Setälä (1982a), Kreula and Rauramaa (1986) and Beck and Gross (1973) reported milk formaldehyde concentrations of 1-7, 1-2 and 2-5 mg/kg respectively. In contrast, with daily ingestion of formaldehyde of 27-7 and 23-9 g, Buckley and Fisher (1984) and Wrenn etal.
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(1975) measured milk formaldehyde concentrations of 0*095 and 0-225 mg/kg respectively. These latter values are relatively near those we measured in goats. They indicate that there are no systematic differences between formaldehyde concentrations of milk of goats vs. cows and that former concentrations were overestimated. Milk formaldehyde concentration was not influenced by the level of milk production: the transfer of formaldehyde from body to milk seems to be a passive phenomenon. Milk formaldehyde concentration probably reflects formaldehyde concentration in the body which is a result of ingested formaldehyde and body size. The close linear relationship observed between milk formaldehyde concentration and level of ingested formaldehyde agrees with this explanation. Calculation of the quantity of formaldehyde excreted in milk shows that about 0*02% of ingested formaldehyde was excreted in milk. This result is in good agreement with our previous findings (Barry and Tome 1982). However, because milk formaldehyde concentrations do not depend on milk production, milk formaldehyde excretion for a level of ingested formaldehyde is linked to the level of secreted milk: this could explain why the correlation between formaldehyde ingestion and formaldehyde excretion is relatively low (r= 0-758). This also may explain why formaldehyde excretion varies between experiments: the rate of transfer of formaldehyde from feed to milk was reported to be between 0-008% and 0-023% according to Wrenn et al. (1975), about 0-07% according to Syrjälä-Qvist and Setälä (1982a) and could be as high as 0-1% according to Beck and Gross (1973). In fact, milk formaldehyde concentration being independent of the level of milk production, the rate of transfer of formaldehyde from feed to milk probably increases with the level of milk production. Acknowledgements
We wish to express our best thanks to S. Gueneau and C. Llamas for their excellent technical help during this study. References ARNOULD, R.,
VANBELLE, M.,
JOASSARD, J. M. MOREELS, A. and VAN HOLM, L.,
1978,
Le
formaldéhyde utilisé comme conservateur d'ensilage. I—Comparaision de son effet avec celui de l'acide formique pour la conservation du fourrage dans le silo Revue de l'Agriculture (Bruxelles), 31, 79-88. BARRY, J.-L., and TOMÉ, D., 1982, Effet de la protection par let traitement au formol des protéines alimentaires sur la teneur en formaldéhyde du lait chez la chèvre laitière. Science des Aliments, 2, 537-543. BARRY, J.-L., DEBRAS, E., CHAMPREDON, C , and PION, R., 1979, Quantitative estimation of ammonia
absorption by goats fed high starch diets with urea supplement. Annales de Recherches Vétérinaires, 10, 297-299. BEALL, J. R., and ULSAMER, A. G., 1984, Formaldehyde and hepatotoxicity: a review. Journal of Toxicology and Environmental Health, 14, 1-21. BECK, T., and GROSS, F., 1973, Zur frage der Rückstande bei der Verwendung Formaldehydhaltiger Zusatzmitell bei der Gärfutterbeireitung. Wirtschafteigene Futter, 19, 282-289. BUCKLEY, K. E., and FISHER, L. J., 1984, Formaldehyde residues in the tissues and milk of dairy cattle consuming formalin-treated liquid whey. Review of Research., Research Station, Agassiz B.C., March-August, 14-15 BUCKLEY, K. E., FISHER, L. J., and MACKAY, V. G., 1986, Electron capture gas chromatographic
determination of traces of formaldehyde in milk as the 2,4-dinitrophenylhydrazone. Journal of the Association of Official Analytical Chemists, 69, 655-657. BUCKLEY, K. E., FISHER, L. J., and MACKAY, V. G., 1988. Levels of formaldehyde in milk, blood and
tissues of dairy cows and calves consuming formalin-treated whey. Journal of Agricultural and Food Chemistry, 36, 1146-1150.
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CZECH, F.P., 1973, Simplex optimized acetylacetone method for formaldehyde, Journal of the Association of Official Analytical Chemists, 35, 1496-1502. DELORT-LAVAL, J., 1985, Chemical treatments of oil meals. Abstract of Papers. Work Conference on Emerging Technologies in Fats and Oils Industry. (Cannes: AOCS), edited by A. R. Baldwin, pp. 218-220. FERGUSON, K. A., KEMSLEY, J. A., and REIS, P. J., 1967, The effect of protecting dietary protein from microbial degradation in the rumen. Australian Journal of Agriculture Research, 30, 215-217. FLORENCE, E., and MILNER, D. F., 1981, Determination of free and loosely protein-bound formaldehyde in the tissues of pigs fed formalin-treated skim milk as a protein supplement. Journal of the Science of Food and Agriculture, 32, 288-292. KREULA, M., and RAURAMAA, A., 1976, Transfer of formaldehyde from feed to milk during the feeding of fresh cut grass treated with formaldehyde containing preservative. Journal of the Scientific Agricultural Society of Finland, 48 154-157. KREULA, M., and RAURAMAA, A., 1977, Formaldehyde and HCOOH in the ensiling process and the metabolism of dairy cows. Agrochimica, 21, 341-355. LALL, D., LOLAN, O. P., and NEGI, S. S., 1982, Partial replacement of protein by urea formaldehyde complexes in the concentrate mixtures fed to cattle. Indian Journal of Animal Science, 52, 142-146. MASHFORD P. M., and JONES, A.R., 1982, Formaldehyde metabolism by the rat: a re-appraisal. Xenobiotica, 12, 119-124. MILLS, S.C., SHARRY, L. F., COOK, L. J., and SCOTT, T. N., 1972, Metabolism of
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when fed to ruminants as an aldehyde-casein-oil complex. Australian Journal of Biological Science, 25, 807-816. NASH, T., 1953, The colorimetric estimation of formaldehyde by means of Hantzsch reaction. Biochemical Journal, 55, 416-421. NEELY, W. B., 1964, The metabolic fate of formaldehyde-14C intraperitoneally administered to the rat. Biochemical Journal, 13, 1137-1142. RAURAMAA, A., and KREULA, M., 1977, On the formaldehyde content of the silages prepared with formaldehyde-containing preservative. Journal of the Scientific Agricultural Society of Finland, 49, 199-202. SELIM, S., 1977. Separation and quantitative determination of traces of carbonyl compounds as their 2,4-dinitrophenylhydrazone by high-pressure liquid chromatography. Journal of Chromatography, 136, 271-277. SETÄLÄ, J., and SYRJÄLÄ-QVIST, L., 1982a, Untreated and formaldehyde treated urea as nitrogen sources for lactating dairy cows. Journal of the Scientific Agricultural Society of Finland, 54, 43-52. SETÄLÄ, J., and SYRJÄLÄ-QVIST, L., 1982b, Untreated and formaldehyde treated urea as nitrogen for young growing' bulls. Journal of the Scientific Agricultural Society of Finland, 54, 53-62. SYRJÄLÄ-QVIST, L., and SETÄLÄ, J., 1982a, Formaldehyde content of milk. 1—Cows fed on protein concentrates treated with different amounts of formaldehyde, Journal of the Scientific Agricultural Society of Finland, 54, 63-67. SYRJÄLÄ-QVIST, L., and SETÄLÄ, J., 1982b, Formaldehyde content of milk. 2—Cows fed on grass silage preserved with formaldehyde containing additive and on formaldehyde treated urea. Journal of the Scientific Agricultural Society of Finland, 54, 69-76. TOMÉ, D., BERTRAND, D., VIROBEN, G., and DELORT-LAVAL, J., 1979, Tannage par le formol des
aliments protéiques du ruminant. I. Etude préliminaire du mode d'action du formol sur un substrate protéique modèle Annales de Technologie Agricole (Paris), 28, 299-318. ULSAMER, A. G., BEALL, J. R., KANG, H. K., and FRAZIER, J. A., 1984, Overviews of health effects
of formaldehyde. Hazard Assessment of Chemicals: Current Developments, Vol. 3, edited by J. Saxena (New York: Academic Press), pp. 337-399. VAN SCHALM, K. J., 1983, Determination of traces of formaldehyde in milk as the 2,4dinitrophenylhydrazone by HPLC. Netherlands Milk Dairy Journal, 37, 59-64. VÉRITÉ, R., PONCET, C , CHABI, S., and PION, R., 1977, Utilisation des tourteaux traités au formol par
les vaches laitières. Annales de Zootechnie, 26, 167-181. WRENN, T. R., WEYANT, J. R., WOOD, D. L., BITMAN, J., RAWLINGS R. M., and LYONS, K. E., 1975,
Increasing polymerisation of milk fats by feeding formaldehyde treated protected sunflowersoybean supplement. Journal of Dairy Science, 59, 627-635. ZELTER, S. Z., LEROY, F., and TISSIER, J. P., 1970, Protection des protéines alimentaires contre la
désamination bactérienne dans le rumen. I—Etude in vitro du comportement en milieu de rumen de quelques protéines tannées avec du tannin de châtaignier ou certains aldéhydes (formaldéhyde, glutaraldéhyde, glyoxal). Annales de Biologie Animale, Biochimie et Biophysique, 10, 111-122.
Food Additives & Contaminants Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac19
Formaldehyde content of milk in goats fed formaldehyde‐treated soybean oil‐meal a
J. L. Barry & D. Tomé
a b
a
Laboratoire de Technologie Appliquée à la Nutrition, Rue de la Géraudière , Institut National de la Recherche Agronomique , BP 527, Nantes Cedex 03, 44026, France b
Inserm U 290, Hôpital Saint‐Lazare, 107, rue de Faubourg Saint‐Denis, Paris, 75010, France Published online: 10 Jan 2009.
To cite this article: J. L. Barry & D. Tomé (1991) Formaldehyde content of milk in goats fed formaldehyde‐treated soybean oil‐meal, Food Additives & Contaminants, 8:5, 633-640, DOI: 10.1080/02652039109374017 To link to this article: http://dx.doi.org/10.1080/02652039109374017
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sublicensing, systematic supply, or distribution in any form to anyone is expressly
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FOOD ADDITIVES AND CONTAMINANTS, 1991, VOL. 8, NO. 5, 6 3 3 - 6 4 0
Formaldehyde content of milk in goats fed formaldehyde-treated soybean oil-meal J. L. BARRY and D. TOMÉ † Institut National de la Recherche Agronomique, Laboratoire de Technologie Appliquée à la Nutrition, Rue de la Géraudière, BP 527, 44026 Nantes Cedex 03, France
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(Received 24 September 1990; accepted 1 March 1991) Formaldehyde is used in ruminant feeding for different purposes including the protection of dietary proteins from ruminai degradation. The formaldehyde content of milk of goats fed various levels of formaldehyde-treated soybean oil-meal has been determined by using a sensitive HPLC method. Results showed a significant linear correlation between ingested formaldehyde and formaldehyde concentration in milk. About 0.02% of ingested formaldehyde was excreted in milk, as free formaldehyde. Keywords: ruminants, treated protein, milk, formaldehyde
Introduction
Formaldehyde is used in ruminant feeding for different purposes. Its bacteriostatic properties make it useful as a preservative in silage (Kreula and Rauramaa 1977, Rauramaa and Kreula 1977, Arnould et al. 1978) and in liquid whey (Buckley and Fisher 1984, Buckley et al. 1988). Formaldehyde can be used as a complex with urea to lower the rate of urea degradation in the rumen (Lall et al. 1982, Setälä and Syrjälä-Qvist 1982a, b). Formaldehyde can also be used to protect dietary protein from ruminai degradation (Ferguson et al. 1967, Zelter et al. 1970). This process, applied to soybean oil-meal, increases by 50-90% the flow of dietary amino acids in the intestine of adult ruminants (Vérité et al. 1977) and is commercially developed in Europe (Delort-Laval 1985). Formaldehyde is a toxin (Beall and Ulsamer 1984, Ulsamer et al. 1984), whose presence in animal products is undesirable. Metabolism of formaldehyde ingested by ruminant is well known (Mills et al. 1972). 14C-formaldehyde is transformed in the rumen mainly to 14CC>2 and 14CH4, about 20% of the radioactivity is excreted in the faeces and a small fraction is absorbed (Mills et al. 1972). Absorbed formaldehyde enters three metabolic pathways. It is metabolized mainly to CO2: rats metabolize more than 80% of intraperitoneally injected formaldehyde (Neely 1964, Mashford and Jones 1982). Some absorbed formaldehyde is transformed into various metabolites found in tissues (Mills et al. 1972), milk (Mills et al. 1972, Kreula and Rauramaa 1977) and urine (Neely 1964, Mills et al. 1972). At least small amounts of formaldehyde are found in milk of ruminant fed formaldehydecontaining silages (Beck and Gross 1973, Kreula and Rauramaa 1976, Arnould † Present address: Inserm U 290, Hôpital Saint-Lazare, 107, rue de Faubourg Saint-Denis, 75010 Paris, France. 0265-203X/91 $3.00 © 1991 Taylor & Francis Ltd.
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et al. 1978, Syrjälä-Qvist and Setälä 1982b), or formaldehyde-protected proteins (Barry and Tome 1982, Syrjälä-Qvist and Setälä 1982a). Many difficulties occur in formaldehyde determination in milk. Quantitative discrepancies have been pointed out (Syrjälä-Qvist and Setälä 1982b, Buckley et al, 1988). Some of them could be due in part to the form in which formaldehyde is ingested (Buckley et al. 1988) but, as noticed by Syrjälä-Qvist and Setälä (1982b), the exactness of methods of determining formaldehyde contents of milk explains in part these discrepancies. That agrees with the fact that these discrepancies are observed even when no formaldehyde is ingested. Concentrations usually found are very low and colorimetric or fluorimetric techniques used formerly for formaldehyde determination are not sensitive and specific enough for a correct quantification of formaldehyde in milk. Our work aims to determine precisely formaldehyde concentrations in milk from goats fed various amounts of formaldehyde-treated soybean oil-meal by using a more sensitive method than those used previously. This method employs the extraction of formaldehyde by steamdistillation (Tome etal. 1979) and the formation of a 2,4-dinitrophenylhydrazone (DNPF) which is separated by HPLC (Selim 1977, Van Schalm 1983).
Experimental section
Animals, diets and experimental design Five adult lactating Alpine goats, weighing a mean of 45 kg (42-47 kg), were fitted, several months previously, with rumen cannulae. They were housed in individual boxes. The experiment was performed during their 4th and 5th month of lactation. Animals were milked twice each day. Animals received two daily meals consisting of a basal diet of 750 g of medium quality grass hay and 600 g of a maize-based concentrate (maize: 90%, molasses: 5%, mineral complement: 5%). The basal died was completed with an experimental feed constituted of 600 g of soybean oil-meal. Soybean oil-meal was either untreated or industrially treated with 0-3% (w/w) of formaldehyde. Treatment of soybean oil-meal was performed by spraying a 31% formaldehyde aqueous solution on the oil-meal. Formaldehyde content in soybean oil-meal was quantified as extractable formaldehyde according to the procedure described by Tome et al. (1979): formaldehyde released by phosphoric acid was steam extracted and colorimetrically quantified according to Nash (1953). Soybean oil-meal was given to animals in three forms: (i) untreated soybean oil-meal, (ii) a mixture 50:50 of treated and untreated soybean oil-meal, (iii) treated soybean oil-meal. The experimental feed was given at the beginning of each meal. So that soybean oil-meal consumption was rigorously controlled, refused soybean oil-meal was eventually introduced into the rumen via a cannula. During a three week pre-experimental period, animals were adapted to the basal ration and untreated soybean oil-meal and to the feeding procedure. The experiment consisted of three periods of seven days: during each period, each animal was randomly assigned to one of the three experimental diets. On the whole, each animal received each diet once. Daily milk production was determined for each animal. Milk samples, pooled for morning and evening milking, were taken for each goat during the five last days of each experimental period. Samples were frozen at -20°C until analysis.
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Determination of free formaldehyde in milk The method used for the determination of free formaldehyde in milk was derived from the technique of Van Schalm (1983). Fifty ml of milk or of formaldehyde standard solution and 1 ml of phosphoric acid were introduced in a flask of 250 ml connected with a steam-distillation apparatus (Tome et al. 1979). Two hundred ml were steam-distilled and 50 ml of the distillate were transferred into a beaker with 10 ml of a solution of 2,4-dinitrophenylhydrazine 2-5 g DNPH in HCI(6N) and 10 ml of chloroform. This mixture was shaken for 15 min at room temperature and transferred to a separatory funnel. The two phases were separated and the aqueous phase was extracted twice with 25 ml of HC1(2N), three times with 25 ml of H2O and concentrated to dryness under reduced pressure at 25°C. The residue was dissolved in 2 ml of acetonitrile. A volume of 20jnl was injected in a Waters HPLC system equipped with a RP C-18 ^m Bondapack column (350x4-6 mm) and a UV detector at 350 nm. Residual components were eluted with a mobile phase of water/acetonitrile 50:40 (v/v) at a flow rate of 1 -5 ml/min. The position of the DNPF derivative was determined by referring to a standard of DNPF and the concentration of formaldehyde by the peak area in reference to a calibration curve prepared with standard solutions of formaldehyde treated in the same way as milk samples. With this methodology, a small contaminating peak, eluting at the same retention time as DNPF, was observed in HPLC chromatograms with blank solution. Buckley et al. (1986), quantifying DNPF by GLC, have also reported the presence of this peak due to the contamination of derivatizing reagent. Detection limit was determined as the upper limit of this background signal calculated as: y = j o + 3ffy
where yo and ay were respectively the mean and standard deviation of the background of the signal (Buckley et al. 1986). In our methodology, yo was 0-0086 mg/kg and ay was 0-0012 mg/kg: the detection limit of our methodology was calculated to be 0-012 mg/kg. Results Animal health and feeding To make goats ingest high quantities of formaldehyde we fed them with an excessive amount of soybean oil-meal. Though the animals were consequently fed hypernitrogenous diets, they remained in good health with no incidence of mastitis. Some of the soybean oil-meal was sometimes not ingested by animals and was then introduced into the rumen. As this fraction was very small (less than 10%) and as it has been previously shown (Barry et al. 1979) that feeding concentrate via rumen cannula does not change its digestive fate, no distinction has been made between ingested or rumen cannula-introduced soybean oil-meal: in the following results, ingested soybean oil-meal refers to all the oil-meal consumed by the animals. Formaldehyde in soybean oil-meal Formaldehyde-treated soybean oil-meal contained 2 • 40 g of extractable formaldehyde per kg of dry matter. The incomplete recovery of formaldehyde is in agreement with the findings of Tome et al. (1979) who showed that some
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formaldehyde is irreversibly bound to proteins and that extractable formaldehyde represented about 80% of the formaldehyde used during the treatment of various oil-meals. With untreated soybean oil-meal, a mixture (50:50) of untreated and treated soybean oil-meal, and treated soybean oil-meal, animals ingested 0, 0-63 and 1 • 26 g of extractable formaldehyde per day, respectively. Milk and formaldehyde in milk During the whole experiment, mean daily milk production was 2-00 ±0'031 kg per goat, with no statistical influence of either time or formaldehyde ingestion. Typical HPLC chromatograms for formaldehyde determination in standard and milk samples are given in figure 1. Milk formaldehyde concentrations are shown in table 1. When 0, 0*63 or 1-26 g of formaldehyde were ingested daily, mean formaldehyde concentrations in milk (mean ± standard error) were 0-033 ± 0-007, 0-083 ± 0-007 and 0-153 ± 0-011 mg/kg respectively. As shown in table 2, these figures differed significantly from each other (p < 0-01). Experimental points could be fitted by a linear regression with a very high correlation coefficient (y = 0-02947 + 0-0952*; r- 0-938; p < 0-01; where y is milk formaldehyde concentration expressed in mg/kg and x daily formaldehyde ingested expressed in g/day). The relation between the level of milk production (z, expressed in kg/day; and milk formaldehyde concentration (w,
DNPF
Blank
Standard 0.2 mg/l
Milk sample
10
20
Figure 1. HPLC separation of the formaldehyde-2,4-dinitrophenylhydrazone (DNPF). The blank, the standard solution of formaldehyde (0-2 rag/1) and the milk sample have been extracted and derived as described in the experimental section.
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Table 1. Milk formaldehyde concentration (mg/kg) in goats fed various levels of formaldehyde (mg/day).
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Daily ingested formaldehyde Animals
0-000
0-630
1-260
Goat Goat Goat Goat Goat
0-054* 0-046 0-017 0-023 0-024
0-072 0-112 0-086 0-061 0-083
0-152 0-198 0-110 0-150 0-154
O-033a 0-007
0-083" 0-009
O-153c 0-014
1 2 3 4 5
Mean** Standard error
*Each figure is the mean of five consecutive days. ** Figures with a different superscript are significantly different ( p < 0 - 0 1 ) .
Table 2. Statistical analysis of formaldehyde concentrations in milk.
Ingested formaldehyde Animal Interaction Residual error
Sum of square deviation
Freedom degrees
Mean squares
0-172 0-022 0-011 0-033
2 4 8 60
0-086 0-011 0-001 0-001
F
Level of statistical significance
158-56 10-21 2-57 —
0-01 0-01 0-05 —
expressed in mg/kg) was: w= 1-033 10~3 + 1-564 10" 2 z (r = 0-507; p > 0 - 0 5 ) w = 5-839 10" 2 + 1-965 10" 2 z (r = 0-522; p> 0-05) w = 6-567 1(T 2 + 4-291 1(T 2 z (r = 0-876; p>0-05) respectively when 0, 0-63 and 1-26 g of formaldehyde were ingested daily and showed no significant influence of level of milk production on milk formaldehyde concentration. Discussion A main problem was first to determine whether formaldehyde was present in the milk of animals fed diets containing no formaldehyde. Formaldehyde is a normal product of intermediary metabolism in mammals (Mashford and Jones 1982, Buckley and Fisher 1984). It has been detected in various animal tissues. Florence and Milner (1981) found 19, 19 and 23 mg/kg of formaldehyde respectively in liver, kidney and muscle of pigs fed diets containing no formaldehyde. More recently, Buckley et al. (1988) found lower but significant amounts of endogenous formaldehyde in calf tissues (3-6, 1-4 and 0-18 mg/kg
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respectively in liver, kidney and muscle). Whether or not this endogenous formaldehyde can enter the udder and be excreted in milk is not well established. Using a colorimetric determination of formaldehyde in milk (chromotropic acid), Beck and Gross (1973) found no endogenous formaldehyde in cow's milk; using the same methodology, Syrjälä-Qvist and Setälä (1982a) determined levels of formaldehyde up to 0-2 mg/kg in milk of cows fed diets containing no formaldehyde. Using the colorimetric determination of Czech (1973), Barry and Tome (1982) found 0-017 mg/kg of endogenous formaldehyde in goats' milk. The main reason for these discrepancies could be the inaccuracy of colorimetric methods to quantify trace levels of formaldehyde in milk. With the specific and sensitive HPLC method we developed, endogenous formaldehyde level in milk was found in the present experiment to be 0-033 mg/kg. Using a GLC determination of formaldehyde in milk, Buckley etal. (1986, 1988) found 0-017 mg/kg of formaldehyde in milk of cows fed diets containing no formaldehyde. As this level was lower than the detection limit of their technique (0-026 mg/kg), they could not conclude as to the actual presence of endogenous formaldehyde in milk. Our figures obtained with a more sensitive method indicate that there are low but significant levels of formaldehyde in milk of animals fed diets containing no formaldehyde. The other question is the transfer of ingested formaldehyde in milk. With goats fed I4C-formaldehyde-treated casein, Mills et al. (1972) detected small amounts of radioactivity in milk; but this was not present as formaldehyde and they concluded that ruminants effectively metabolize formaldehyde and that there is no accumulation of this compound in milk. In their experiment, they used a colorimetric determination of formaldehyde (chromotropic acid) and found very high levels of formaldehyde in milk of control animals (1 mg/kg): their methodology, as mentioned above, might not have been sensitive enough to determine any increase in formaldehyde concentration in milk. The results obtained in the present experiment showed a significant increase of formaldehyde concentration in milk of animals fed formaldehyde-treated soybean meal. These results are in good agreement with those previously published in goats (Barry and Tome 1982) and in cows (Syrjälä-Qvist and Setälä 1982a) which indicate that a fraction of dietary formaldehyde used to protect dietary protein from ruminai degradation was recovered as free formaldehyde in milk. Such a transfer of formaldehyde from the diet to the milk has also been observed with formaldehyde used as silage preservative (Beck and Gross 1974, Kreula and Rauramaa 1976, Arnould et al. 1978, Syrjälä-Qvist and Setälä 1982b), whey preserver (Buckley and Fisher 1984, Buckley etal. 1988) or as a protective agent of fats from rumen degradation (Wrenn et al. 1975). In conclusion, when introduced in ruminant feeds either as a preservative agent or as a reagent used for the protection of dietary components from ruminai degradation, formaldehyde is probably always recovered at low levels in ruminant milk. For comparative purposes, one can estimate that the daily ingestion of 1 -26 g of formaldehyde in goats weighing 45 kg corresponds to a daily ingestion of 16-8 g in a cow weighing 600 kg. With such levels of dietary formaldehyde, very conflicting results have been obtained in cows. With daily formaldehyde intakes of 14-6, 18-7 and 18-2g, Syrjälä-Qvist and Setälä (1982a), Kreula and Rauramaa (1986) and Beck and Gross (1973) reported milk formaldehyde concentrations of 1-7, 1-2 and 2-5 mg/kg respectively. In contrast, with daily ingestion of formaldehyde of 27-7 and 23-9 g, Buckley and Fisher (1984) and Wrenn etal.
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(1975) measured milk formaldehyde concentrations of 0*095 and 0-225 mg/kg respectively. These latter values are relatively near those we measured in goats. They indicate that there are no systematic differences between formaldehyde concentrations of milk of goats vs. cows and that former concentrations were overestimated. Milk formaldehyde concentration was not influenced by the level of milk production: the transfer of formaldehyde from body to milk seems to be a passive phenomenon. Milk formaldehyde concentration probably reflects formaldehyde concentration in the body which is a result of ingested formaldehyde and body size. The close linear relationship observed between milk formaldehyde concentration and level of ingested formaldehyde agrees with this explanation. Calculation of the quantity of formaldehyde excreted in milk shows that about 0*02% of ingested formaldehyde was excreted in milk. This result is in good agreement with our previous findings (Barry and Tome 1982). However, because milk formaldehyde concentrations do not depend on milk production, milk formaldehyde excretion for a level of ingested formaldehyde is linked to the level of secreted milk: this could explain why the correlation between formaldehyde ingestion and formaldehyde excretion is relatively low (r= 0-758). This also may explain why formaldehyde excretion varies between experiments: the rate of transfer of formaldehyde from feed to milk was reported to be between 0-008% and 0-023% according to Wrenn et al. (1975), about 0-07% according to Syrjälä-Qvist and Setälä (1982a) and could be as high as 0-1% according to Beck and Gross (1973). In fact, milk formaldehyde concentration being independent of the level of milk production, the rate of transfer of formaldehyde from feed to milk probably increases with the level of milk production. Acknowledgements
We wish to express our best thanks to S. Gueneau and C. Llamas for their excellent technical help during this study. References ARNOULD, R.,
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