THESIS LF’ ’ .. '33.}? I. .3. .L.-'..;6.o.’"’n-;.Q I {1‘3”}!!! erg, . m 1 O 0* _ 'a‘eo-fgplf‘v-r‘— '1 with! V‘hrau'au', This is to certify that the thesis entitled THE EFFECTS OF SODIUM HYPOCHLORITE ON THE GROWTH AND REPRODUCTION OF MINK (MUSTELA VISON) presented by Angeio Carmen Napolitano has been accepted towards fulfillment of the requirements for ___M._S‘__degree in Anima]_$.c;ierme flé/é 1/ / Q/ZW/ Jor professor Ri d J. Aulerich Date February 13, 1985 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution MSU LIBRARIES .-—_ V RETURNING MATERIALS: Place in book drop to remove this checkout from your record. ‘Eiflgg will be charged if book is returned after the date stamped below. THE EFFECTS OF SODIUM HYPOCHLORITE ON THE GROWTH AND REPRODUCTION OF MINK (MUSTELA VISON) By Angelo Carmen Napolitano A Thesis Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Animal Science 1985 \./ ” b \J C) .p) ABSTRAOP THE EFFECTS OF SODIUM HYPOCHLORITE ON THE GROWTH AND REPRODUCTION OF MINK (MUSTELA VISON) By Angelo Carmen Napolitano Feed and water consumption studies were done to ascertain the levels at which mink could tolerate sodium hypochlorite (NaOCl) in their feed or drinking waters Mink were more tolerant of NaOCl in their feed than in their drinking water. Feed consumption was significantly reduced athZOO ppm NaOCl while water consumption was reduced at 200 ppm. NaOCl was added to the drinking water of mink at 25, 50, 100, and 200 ppm and to the feed at 100 ppm to determine the effects on growth and reproduction. The addition of NaOCl to the drinking water or feed at the levels indicated did not have a significant effect (beneficial or detrimental) on the growth or reproduction of mink. Aerobic plate counts of mink feed treated with 100 ppm NaOCl indicated that this compound did not significantly reduce the rate of bacterial growth over a 24 hour period. ACKNOWLEDGMENTS I would like to extend my deepest appreciation to my major professor Richard J. Aulerich and to my other committee members, Steve Bursian, Theo Coleman, and George A. Padgett. To my wife Elizabeth, thank you for your constant love and encouragement during the writing of this thesis. To my son Carmen, thank you for the moments of joy you supplied over the course of this writing. ii TABLE OF CONTENTS Page List of Tables v Introduction 1 Literature Review 3 History 3 Usage and Effectiveness 4 Mode of Action 4 Toxicity 6 Related Compounds 8 Mink Experiments 1O Feed and Water Consumption of NaOCl 10 Purpose 10 Materials and Methods 10 Results 12 Discussion 12 Aerobic Plate Counts of Mink Feed 16 Purpose 16 Materials and Methods 16 Results 17 Discussion 17 Growth and Reproduction of Mink Administered Sodium Hypochlorite 21 Purpose 21 Materials and Methods 21 Results 24 Discussion 33 Conclusions 37 Appendices Appendix A. The concentrations of chlorine disinfectants (including NaOCl) commonly used in different industries......................................38 Appendix B. Biocidal effect of free available chlorine on various organisms...............................39 Appendix 0. Relationships between HOCl, 'OCl, and pH........4O Appendix D. Procedure for aerobic plate counts of mink feed............................................41 Appendix E. Composition of basal mink diet..................43 Bibliography..................................................44 iii Table Table Table Table Table Table Table Table Table 8. LIST OF TABLES Page Water consumption of male mink given various concentrations of NaOCl-treated drinking water forz daySCOOIOOOOOIOOOOOOOOOO0.00.0.00...0.0.0.0....13 Feed consumption of male mink given various concentrations of NaOCl-treated feed for 2 days......14 Aerobic bacterial plate counts H001 + Na+ + 0H” Hypochlorous acid (H001) has been shown to be the main compound responsible for the bactericidal action of NaOCl (Andrews and Orton, 1904; Baker, 1959). H001 exists in equilibrium with its ionized form ’001 according to its ionization constant. The dissociation of H001 H001 =_.__._.. H“ + ‘001 6.8 x 10'8 (Holst, 1940) to “001 depends primarily on the pH. As the pH increases the amount of H001 decreases and the germicidal prOperties of NaOCl decrease and vice-versa. This is illustrated by the graph in Appendix C showing pH versus H001 and '001. This again indicates that H001 is mainly responsible for the germicidal action of NaOCl and is a more powerful disinfectant than ”001. Fair gt 31. (1948) and Morris (1926) showed theoretically that the '001 ion possesses about 1/80 of the bacteriocidal action of H001. How H001 produces the bacteriocidal effect is a matter of debate. Baker (1926) suggests that chlorine binds with proteins of the bacterial cell membrane, forming N-chloro compounds, which interferes with cell metabolism and leads to eventual death|of the organism. Rudolph and Levine (1941), stated that an active germicidal ingredient penetrates the bacterial cell and forms toxic Nechloro compounds when it contacts the cell protoplasm. The enzyme trace substance theory of Green and Stumpf (1946) postulated that chlorine must inhibit some key enzymatic reactions within the cell, since it is effective at such low concentrations. This was later confirmed by Knox g 21. (1948), who showed that chlorine in bacteriocidal amounts or less inhibit various sulfhydryl enzymes and other enzymes sensitive to oxidation. Inhibition of the essential enzymes is irreversible and causes death to the cell. TOXICITY Sodium hypochlorite is generally considered safe for higher animals and man at use concentrations. Upon ingestion of excessive quantities of NaOCl, corrosion of mucous membranes, gastric perforations, laryngeal edema, methemoglobinemia, and death can occur. Inhalation of large amounts of NaOCl can cause severe bronchial irritation and pulmonary edema and prolonged skin contact may result in vesicular eruptions (Anonymous, 1976). The lethal dose of NaOCl give by intra-peritoneal injection to mice and guinea pigs has been reported to be between 100 to 150 mg/kg body weight (Taylor and Austin, 1918), although Cunningham (1980) found no signs of toxicity when NaOCl was given orally to mice in milk (gavage) at concentrations up to 200 mg of available chlorine/kg body weight/daily for 2 weeks. High concentrations of free chlorine (2,000 to 10,000 ppm), when added to .cake, flour, have been found to be toxic to rats. The most common signs of toxicity were reduced growth rate and enlarged liver, kidney and heart (Cunningham _e_t _e_t_l_., 1977; Cunningham and Lawrence, 1978). Hulan and Proudfoot (1982) also found increased mortality and decreased feed conversion, but a decrease in organ weights (liver, kidney, heart and testes) in chicks given 600 or 1200 ppm available chlorine in their drinking water. Furukawa 3£_§g: (1980) noted a tendency towards dehydration in rats administered water containing up to 4% NaOCl (4000 ppm) for 14 days but observed no clinical signs of toxicity. Also, no pathological changes were noticed in rats treated with 0.4% NaOCl (400 ppm) in their water for 92 days. Cunningham (1980) found no evidence of toxicosis in rats administered NaOCl at up to 1000 ppm of available chlorine in their drinking water. Druckery (1968) found no effects on fertility, growth, hematologic parameters, or histology of organs in rats administered water containing 100 ppm available chlorine over their life span. These studies indicate that at low concentrations (< 1000 ppm available chlorine) sodium hypochlorite is relativeLy non-toxic. Sodium hypochlorite is seldom used at concentrations above 1000 ppm available chlorine, so toxicity should not be a problem. Possible potential hazards may, however, arise when sodium hypochlorite is accidently used in conjunction with other compounds. The addition of acids to hypochlorites can lead to the production of deadly chlorine gas. NaOCl can also react with 8 formaldehyde to form the potentially dangerous lung carcinogen bis-chlormethyl ether with a threshold limit value of 1 part per billion (Gamble, 1977). NaOCl has also been shown to be a cocarcinogen when used with 4-nitroquinoline 1-oxide (Hayatsu §_1:_ _a_l_., 1971). Chloroform, another carcinogen, has been shown to be produced _i_1_l_ m after the ingestion of NaOCl (Vogt _1 21., 1979). Likewise, dichloroacetonitrile, which was shown to have mutagenic activity in the Ames biassay (Simmon e_t_a_l_., 1977), was found to be formed _i_n vivo after the ingestion of NaOCl (Mink gt 51-. 1983). RELATED COMPOUNDS Chlorine has long been used for the primary disinfection of drinking water in the United States. Recently, it has been shown that the treatment of drinking water with chlorine results in the formation of trihalomethanes, such as chloroform, which has been shown to be carcinogenic in mice and rats (Rock, 1974; Bellar _et 21., 1974; Anonymous, 1976). Chlorine dioxide has been suggested as a replacement for chlorine in water disinfection. Chlorine dioxide (0102) and the byeproducts of its use, chlorite (0102') and chlorate (0103'), are more oxidized forms of hypochlorite (001') and hence are more potent oxidizers. Studies using rats, mice, and chickens treated with 0102, 0102', and 0103' in drinking water have shown alterations of hematologic parameters in all species tested (Couri _e_t_ilg 1982). The effects were usually dose related with marked changes 9 occurring at concentrations above 100 ppm. These treated groups showed alterations in erythrocyte morphology and osmotic fragility, and a dose related decrease of blood glutathione content. Atrecent study with rats showed that 0102, 0102', and 0103' increased the turnover of cells of the gastrointestinal mucosa and inhibited DNA synthesis in several organs, including the testes, which could possibly cause reproductive failure in the males (Couri gt 21., 1982). Hypochlorites generated from the use of chlorine disinfectants have shown none of these adverse characteristics. MINK EXPERIMENTS FEED AND WATER CONSUMPTION 0F NaOCl Pugpose This trial was conducted to determine the concentrations of sodium hypochlorite that mink would tolerate in their feed or drinking water. These limits were then used to select the levels to be used during the growth and reproduction experiment. Materials and Methods Nine standard dark male mink were used on the water consumption trial and sight on the feed consumption experiment. The mink were housed indoors in wire cages that measured 61.5 cm length x 41.0 cm width x 36.0 cm height. The mink were allowed to acclimate for a period of five days so that animals on the water’consumption study could adjust to drinking water from water bottles and those on the feed study could adjust to their new feeding regime. Both experiments were conducted as a simple (complete block) crossover design, that is, each animal received each NaOCl treatment level including control for two days, followed by a day of no treatment. Treatment means for both experiments were compared used Dunnett's one-sided t-test (Gill, 1978). 10 ll Mink on the water consumption trial were given NaOCl-treated water in "laboratory-type" inverted glass bottles. These bottles were used to insure an accurate measurement of water consumption and to minimize water evaporation which could increase the concentration of sodium hypochlorite. The NaOCl-treated water was made fresh daily and water consumption measured daily. Sodium hypochlorite1 was added to the drinking water at the following concentrations (ppm): 0, 25, 50, 100, 200, and 400. Mink on the feed consumption study were given feed twice daily for a two hour period (8 to 10 a.m. and 3 to 5 p.m.). The amounts of feed consumed during each two hour period were summed to give the total feed consumption per day. This feeding regime was used because sodium hypochlorite is known to react with the organic matter in the feed, thus decreasing the concentration of NaOCl over time. If the mink did not consume the feed initially they might consume the feed later after the NaOCl concentration decreased thus yielding false feed consumption values. NaOCl- treated feed was made fresh prior to each two hour feeding period. Sodium hypochlorite was added to the mink diet at the following concentrations (ppm), 0, 200, 400, 800, 1600, and 3200. 1Laboratory grade sodium hypochlorite (4-6% NaOCl), Fisher Scientific 00., Chemical Manufacturing Division, Fairlawn, NJ 07410. Solution contains 4-6% available chlorine. 12 Results Water consumption by the mink was not affected at concentrations up to 50 ppm NaOCl. Above 50 ppm there was a dose-response relationship, as the concentration of NaOCl increased, water consumption decreased. Concentrations of NaOCl of 200 ppm and greater resulted in a significant reduction in water consumption, when compared with that of the control group (Table 1). As shown in Table 2, feed consumption of the mink given NaOCl-treated feed was not affected at concentrations up to 1600 ppm. At 3200 ppm NaOCl, feed consumption was reduced significantly when compared to that of the control group. Discussion The sodium hypochlorite solution used for these studies contained 4-6% NaOCl. This product yielded 4-6% available 2, so for those trials the concentration of NaOCl and chlorine available chlorine was equivalent. The results of this experiment indicated that mink are less tolerant of chlorine in their drinking water than are chicks or rate. Chicks tolerated up to 300 ppm chlorine in their drinking water (Hulan and Proudfood, 1982), while rats showed no decrease 2Available chlorine is a measurement of the oxidizing capacity and is expressed in the terms of the equivalent amount of elemental chlorine. 13 .eeos-e eoeae1oeo e.eeoeesq . A.o.o v av Hoaeeoo seam sasnoeehe harnessesemame .ponno cnmcompm “.mmoz. assuage assumes meme; saunas assures senses a 13:3 _eoavmssmeoo hope: : 00v 00m 00. On mm Aaouasoov o Aagmv Hoooz Hmusoaoamnsm no defipuuaeoomoo .mhmc N you have: msfixswho cousouauaoomz mo msoaummpsoomoo msouum> =o>wm Mafia mama ho sofipmasmnoo poem: .. canoe 14 .pmouup coowmnoeo m.»uocssa 1 Apo.o v my Homaooo 30mm acomoMMfiv havd¢oa~amwwmm .uopuo canvases H.emo:. assumes amuse? a.m...mém. semis. Thames. menses. o 335 .mowumasmsoo comm Q 00mm 000. com 00¢ oow Aaonueoov o Assay Hoosz Hmpeoaoanazm mo soaamnaeooeoo .mhmc N mom noon coamoupuaoomz no mmowammueooeoo macapm> so>am xsaa can: no soapmssmmoo comm .N manna 15 in water consumption at concentrations up to 1000 ppm available chlorine (Cunningham, 1980). As shown by the data presented in Tables 1 and 2, mink were more tolerant of sodium hypochlorite in their feed than in their drinking water. Consumption was reduced at 200 ppm NaOCl in the water while in the feed it was not reduced until a concentration of 3200 ppm NaOCl was fed. Perhaps, the NaOCl in the feed was masked by some of the other feed odors or flavors, making it more palatable to the mink. Another possibility is that the initial chlorine demand3 of the water is met much more quickly than that of the feed. Since the amount of impurities and organic matter in the water is relatively low, the amount of chlorine required to meet this initial demand is low, thus free available chlorine for the mink to detect becomes available at lower concentrations of NaOCl. 0n the other hand, mink diets contain a considerable amount of organic matter and the amount of chlorine required to meet the initial demand is high and free available chlorine for the mink to detect may not become available until this demand is met . 3"Chlorine demand" - the amount of initial chlorine that is used up by the water impurities and other organic materials. Chlorine is changed to inorganic chloride ions in these reactions. AEROBIC PLATE COUNTS 0F MINK FEED Pumas Mink ranchers who use fresh or frozen animal byéproducts in their feed are constantly exposing their animals to large numbers of bacteria which reside in these by-products. Many of these organisms, such as Staphylococci, Streptococci, and Salmonella, are potential disease producing organisms. Since NaOCl is effective against many of these microorganisms, it was thought that its addition to the feed would decrease the numbers of these microorganisms and decrease the rate of bacterial growth (spoilage) in the mink feed. Materials and Methods Five feed samples from the control diet and five from a diet containing 100 ppm supplemental NaOCl were placed in sterile petri dishes and left exposed to laboratory atmosphere at a temperature of 22 °C for 24 hours. Aerobic plate counts (Dunnigan, 1972; see Appendix D) were made on each sample (in duplicate) at 0, 3, 6, 12, and 24 hours to determine the amount of bacteria per gram of feed. Data were analyzed using a split- plot analysis with the feed samples serving as incomplete blocks. 16 17 Results As shown in Table 3, both the control and NaOCl-treated feed had increasing bacterial counts over time. There was, however, no marked difference in trends between the two groups, nor were there any significant differences (P < (L05) in the counts between the control feed and the NaOCl-treated feed at any of the five corresponding time periods. Sodium hypochlorite, when added to the feed at a concentration of 100 ppm did not significantly reduce the initial bacterial count of the mink feed, nor'did it significantly reduce bacterial growth in the feed over a 24 hour time period. Discussion Although 100 ppm of supplemental sodium hypochlorite failed to significantly reduce bacterial growth in the feed, higher levels of NaOCl might be required to obtain positive results. Since 100 ppm NaOCl did not decrease the initial amount of bacteria in the diet when it was added, it is quite possible that most of the NaOCl was destroyed by its interaction with the organic matter in the feed, leaving no residual chlorine for the destruction of microorganisms. Coates (1977) found that all types of organic material interact with NaOCl causing a serious loss of disinfection activity; Yasukawa (1931) reported that 500 ppm of chlorine did not sterilize oysters in water containing Escherichia coli and Eberthella typhosa. He concluded that much l8 .pouuo cmmcmmam “.mmom— m.o..n cm.em. m.o. . ee.n m.o. u..m.o 8.0. “.mm.o m.o. u.em.o m Aam\eao o_v ease as H osz Housmaoammsm Eng 00— m.o..u om.eea .m.o_.u ow.Pa m.o. H.em.. m.o_ “.me.o m.o._n..e.o m Asm\aeo o.v .eone Hoe coo em N. Amhfiomv 05TH. .oaH» uo>o coon coueomuuaoomz one Honumoo mo mausoo oumam Hmamouomn canomo< .n manna 19 of the free chlorine was used by organic matter and, thus, was not available for the destruction of bacteria in the oysters. Habeck gt_al: (1967) reported that available chlorine is rapidly depleted in the presence of poultry meat and drumsticks. A constantly flowing 20 ppm chlorine solution did not reduce the total bacterial counts in poultry drumsticks. He concluded that chlorine concentrations must be metered into the system at a rate which overcomes the absorption rate by poultry. Some chlorine treatments have been shown to be effective in the reduction of bacterial growth. After 10 days of storage at 38 OF, chicken fryers chilled for 2 hours in water containing 140 ppm chlorine contained 140,000 bacteria/gm while control fryers contained over 5,000,000 (Dawson ita_l_., 1956). Mallmanigl. (1959) found that up to 400 ppm chlorine in the chill water yielded birds with lower bacterial counts after seventeen days of storage. These studies suggest that in order for sodium hypochlorite to be effective in the reduction of bacterial growth, chlorine levels must be high enough to ensure adequate residual chlorine to destroy bacteria after NaOCl has reacted with any organic matter present. Since mink will tolerate concentrations up to 1600 ppm supplemental.NaOCl in their feed, additional studies should be conducted to determine whether concentrations higher than 100 ppm might reduce bacterial concentrations and growth in 20 conventional mink diets, and if this is the case whether these levels would have any adverse effects on growth and/or reproduction in the mink. GROWTH AND REPRODUCTION OF MINK ADMINISTERED SODIUM HYPOCHLORITE Purpose Mink farmers use chemical disinfectants to sanitize cages, nest boxes, water cups, water lines, and feed equipment. Since chlorine disinfectants are inexpensive and effective, they are widely used for this purpose. The mink are constantly being exposed to these disinfectants. Recent research has shown that sodium hypochlorite - the major component of most chlorine disinfectants - has a growth stimulatory effect on rate and guinea pigs when administered at low concentrations (Cunningham, 1980). This study was conducted to investigate any beneficial or adverse effects NaOCl might have on the growth and/or reproduction of mink. Materials and Methods The growth and reproduction study was started on July 2, 1980. iNinety standard dark mink were randomly allocated into six groups, each containing 12 females and 3 males. Littermates were placed on different treatment levels in order to avoid biasing the trial. Sodium hypochlorite was added either to the drinking water or the feed at the following concentrations. 21 22 Group I - No NaOCl added to the drinking water or the feed (control) Group II - 25 ppm NaOCl in the drinking water Group III - 50 ppm NaOCl in the drinking water Group IV - 100 ppm NaOCl in the drinking water Group V - 200 ppm NaOCl in the drinking water Group VI - 100 ppm NaOCl in the feed -untreated drinking water Each group (except Group VI) was fed a standardrbasal mink diet (see Appendix E). Group VI received the basal diet supplemented with 100 ppm NaOCl. The sodium hypochlorite was added to the mink feed daily, just prior to its being fed to the animals. Since NaOCl breaks down readily, especially in sunlight (Gelinas and Goulet, 1982), the mink water cups were emptied daily and refilled with fresh untreated or NaOClétreated water twice daily (morning and evening) The mink were housed in open-sided sheds and cared for according to standard mink ranch procedures (Travis and Schaible, 1960). From July 2, 1980 to February 2, 1981 the mink were housed in growing cages (61.5 cm length x 31.0 cm width x 38.5 cm height) with attached penthouse nest boxes (31.0 cm length x 23.0 cm width x 15.5 cm height). Thereafter the mink were placed in breeder cages (77.0 cm length x 61.5 cm width x 46.0 cm height) with attached outside nest boxes (38.5 cm length x 25.5 cm width x 25.5 cm height). At 10 weeks of age the mink were vaccinated for botulism, distemper and virus enteritis. Body weights were 23 recorded at the start of the experiment and at biweekly intervals from July 2 to September 24, and at monthly intervals thereafter, until the start of the breeding season (March 1). During the breeding season, the female mink were mated with males within their respective treatment groups whenever possible. Matings were confirmed, by examining vaginal smears taken from the females immediately after c0pulation. The smears were examined for the presence of live, motile sperm. After the initial sperm checked mating the females were given an Opportunity for a second mating either the day after the original mating or eight days later. At least two positive matings per female were obtained whenever possible. Beginning April 18, the nest boxes of female mink were checked daily for litters. The kits were counted, sexed and weighed at day 1, three weeks of age and six weeks of age. Prior to the breeding season, blood samples were taken by heart puncture from four females in each group and submitted to the Michigan State University Veterinary Clinic for analysis of *, 01‘, and total 002'. Blood serum blood levels of Na+, K samples were also collected from all the mink by toe clips for red blood cell counts, white blood cell counts, hematocrit, and hemoglobin values. Blood smear were made to determine mean differential white cell counts. At the end of the trial, four female adult mink from Group I (control), Group V (200 ppm NaOCl in drinking water) and Group VI (100 ppm NaOCl in feed) were necropsied and their organs weighed. 24 Data were analyzed using one way analysis of variance. Treatment means were compared using Dunnett's two-sided t-test (Gill, 1978). Results Sodium hypochlorite when added to the drinking water or the feed, did not have a significant effect on mink body weight gains during the post weaning growing and furring periods (July 2 to November 20, Table 4) at the levels employed in this study. Male and female mink in all groups had increasing body weight gains over the course of the growing period. The female mink reached their full growth (maximum body weight) about 16 weeks after the start of the trial (24 weeks of age), whereas the males continued to gain weight (depending on the severity of the weather) up to 28 weeks after the trial began (36 weeks of age). The NaOCl-treated water or feed had no effect on any of the hematologic parameters measured. No significant differences (P < 0.05) were detected between the control and treated groups in red blood cell counts, white blood cell counts, hematocrit or hemoglobin values (Table 5). Also, there was no effect on the differential white blood cell counts of mink in the various treatment groups (Table 6). Serum blood levels of 01", Na”, K+, and total 002' were not affected by any of the treatments administered. There was a significant increase in the serum potassium (K+) level of mink 25 III-Illl‘l' '1‘ '1 'I .11-}.1. I -l.‘ 'It‘l'UAIIill-‘llll I'll'" _smmw ouimxeun H zisza “.3“ Tau TE“ .12.. 73H .22 + 72“ 12“.. a.m.. 1:“ com men as; znc -n was “we man man nod ans m «.5: H so: u TS H 0.2 H TS H «.3 .+. 12 H ”.3 H i: H 92 H Nan H Go: 3 E: 8: mnmu mead awed ma~a mama sham man man an... DON Ono I ~> 92H TS.“ Ta“ “.3“ TR“ damn 4.2“ fiflw 12“ ion 13H e2 6% can So In 2... «.3 as 2n a: 3.. a | I .I I I «sous: «aqxcamu 9.5 H as: + 0.; + 92 + «.2 + it. + 1% + «.3 u inn H o.- H Nan H 3 sea 82 8.: 32 2.: :2 SS 23 5. 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M 0.00 .0.0 M 0. .0 00.0 M 00.0 0000 M 00.0 .. 0 to»: 0000...... 00.. + 0.00 00.. + ..00 00.0 + 00.0. 0000 + 000.0 0 x a. can 000v 0 00.0 M .100 00.0 M 0. .0 .0.0 M 00.0 00.0 M 00.00 0. 0 232. 0:35.... 00.. + 0.00 00.0 + 0.00 .0.0 + 00.0 .000 + .000. 0 z 0. ags 00.0 ..H 00. M 0.0 .0.0 M 0. .0 00.0 M 00.0 ..000 M 0000. .. .0 232. 000.5,... 00.. + 0.00 00.0 + 0.00 .e.0 + 00.0. .000 + 0000. 0 x as .00 00v :0 00.0 M 0. .0 00.0 M 0.00 .0.0 M .0.0 00.0 M 00000 0. 0 130.. 090.00.... 00.. + 0.00 00.0 + 0.00 .e.0 + .0.0 .000 + 0.00. 0 z a. s00 00v 2. 00.. M 0.00 .0.0 M 0...0 0.0 0 M 0.0 ..000 M 00000 .. 0 00.. + 0.00 00.0 + 0.00 .0. 0 + 0..0. ..000 + 0000. 0 : ..oaseoo .a00 00 H va Amuv as\oo.v Niai\ 00v a mom Aaoomz Heasoaoammsm 000000050: manoamoaom mmaoo woods 0H 00 mooan mo mowamnuqoomoov .60 32.. 0.8.5 .0000 no 0000: mmaxswuc on» :0 Hoomz mo 0:00ammumoomoo 050000> conoamfioaacs x005 mo mosam> manoamoao: 0mm .uamoopmao: .mumsoo HHoo cooHn com .mamsoo HHoo oooap 090:: .m manna Mean differential white blood cell counts of mink administered various concentrations of NaOCl in the drinking water or feed. Table 6. Group (Concentration of supplemental Cell type (i)1 Lymphocyte Monocyte Mature neutrOphil Band Basophil Eosinophil neutrOphil Sex n NaOCl) alr- v-CD +|+l <>t~ mxv- +|+| \Oln uxnl +|+| C>¢) \OM c> +|+I PKG c>.— I: F III (50 ppm in drinking water) 27 mt‘ F0 +|+I [‘0‘ U\U\ u\r~ \ocu +|+l b-F\ MKO MN +l+l mm 00 +l+l Otfi d-q- oxe- 00 +|+I (Dcv «x.- I F IV (100 ppm in drinking water) «)0- v-c> +I+| r-.— N\u\ +|+l \oie m\ni +|+I ON \0 own 00 +|+| L‘N FM ox<> +I+| (axe . . FF N\b— NO +|+| c>b~ K\K\ O\b- r-ni +|+| m0 . . Mc- fl'm +|+| F-G) PO +|+| min NV’ w-d- v-O +|+| c).- e e F'— (200 ppm in drinking water) VI (100 ppm in COP PO +|+| 0CD OIK\ +l+| \o-¢ U\OI +|+| OO‘ . . IS"- ‘d'KO and) CO +|+| two MVI' O‘d' (Dc: +|+I rxnl «x.- «\01 OO +|+I CM CO feed) 1Mean + standard error. 28 receiving 50 ppm NaOCl, but this trend was not verified in any of the other NaOCl-treated groups, which were comparable to the control group (Table7). Organ weights taken at the conclusion of the experiment from the female mink in groups I, V, and VI, showed no effect of NaOCl on any of the organs measured (Table 8). Upon gross examination, all organs appeared normal. The reproductive performance of females in the various treatment groups was comparable to that of the control group. There were no significant differences in conception rate, gestation period, or average number of live kits at birth per female whelped between the control and various NaOCl-treated groups (Table 9). Although, as shown in Table 9, there was no significant difference in the average number of kits at 3 weeks between the control group and any of the NaOCl-treated groups, greater kit mortality between birth and three weeks was noted among the higher NaOCl—treated groups. There was no kit mortality in any of the groups between three and six weeks of age. As shown in Table 10, there was no correlation between the level of NaOCl administered and kit body weights. Kit body weights at day 1, 3 weeks, and 6 weeks of age were generally comparable to those of the control group. The birth weight of kits from females in Group VI were significantly less than those of the control kits, however, at 6 weeks of age the male kits in this group weighed more than the controls. Also, the female kits 29 Table 7. Mean serum blood level values of Cl‘, Na+, I, and total 00 ' of mink given various concentrations of NaOCl in he drinking water or feed. Serum Concentration (mEq/L)1 Group (concentration of supplemental NaOCl) Cl- Na+ K+ Total 002 I (0 ppm; Control) II (25 ppm in drinking water) III (50 ppm in drinking water) IV (100 ppm in drinking water) V (200 ppm in drinking water) VI (100 ppm in feed) 109.0 1 0.89 109.5 _+_ 0.89 109.5 1 0.89 111.510.89 109.3 1 0.89 109.3 1 0.89 147.0 11.86 147.3 _+_1.86 150.8 11.86 148.3 11.86 152.5 11.86 150.3 11.86 4.03 1 0.17 25.5 _+_ 3.25 4.28 _+_ 0.17 23.7 1 2.65 4.83 1 0.17‘El 4.15 1 0.17 4.20 1 0.17 4.05 _+_ 0.17 23.7 1 2.65 23.0 1 2.65 23.0 1 2.65 2405 l 2030 1Mean‘1standard error. aSignificantly different from control (P < 0.01) by Dunnett's t-test. 30 mane .ponpo cumcnmum H.sso:n .uanox 5009 «0 R 0 mm commounums 7:. H m.0~0 3.0 .4. 3.0 0.0 u. 5.. 0.0.0 + $0 000.0 M 00.0 00.0 H 30 no.0 n. 00.0 0 030 3 H0002 man 00— P.>¢ + m..mm mn.o + mm.n op.o + mo.— No.0 + ow.o >0.o + mm.o 00.0 + no.0 no.0 + n¢.o ¢ poem: 003020 a“ doom: BAA CON t: ..1 902. 0.0.0 .+. 3.... 0.0 H No.0 8.0 H 0m.0 00.0 H 8.0 00.0 H $0 000.0 M 3.0 0 H2080 Amv azwfio: no>fiq :Hmnm ammo: mung moacfix mooanm 0 ages hoom undone 9.0»:Mwo: momma .0oom on» a“ H0002 Ema 00— no hope: 0:0 ca Hoowz Ema com Sufi: acumen» mason» 0:0 .msoum Homecoo on» scum xcaa oauaom mo magm«o: dunno .m manna 31 .honno cumcnmvm H.cmo:. 0 0.0. .00 + 0.0 .00 H 0... $0 .+. 06 0.. + 90.. «.0. .08.. :w Sam oo—V 9 0 0.0. 00.0 .+. 0.m 00.0 H 0.0 00.0 u. 06 .1. u. ..00 0.\0 20.2. 0033.... a“ 3mm ooNv > 0 0.0m .00 H 0.0 .00 H 0.0 «0.0 M 9m n. .... 0.2 «.0. 232. 0033.... :w Inn oo.v 3 0 0.5 2.0 u. 0.m 5.0 H 0.m 00.0 H 0.0 0.. ..1 90.. ..\0 232. 0300...... :a nun OmV HS 0 NA 00.0 ..1 ..m 00.0 H. ..m 090 H m6 0... u. 9.... SR. 232. 033.5,... :a nun mmv H. 0 0.0 2.0 ..1 0.m 00.0 H 0.m 00.0 H 0.0 .0. ... 0.: ..\0 38.000 3.... 0. H .02: 0 .ng: m .n30 0 .4 .n30 n .< . 880 .< Anmnev 00.88 .on ..0082 0» .mx: m on Sagan :ofipmamow \comaosa Hmaqoaoamnsm no comaons oHsEoM\mpax .oz .m>< moamaom .oz noaaeuauoosoov ARV hawamauoa wax macho .nafix agony mo haaaaps>fi>usm one coon no noun: muaxawuc 0.3 .3 .8002 no mcofiumuacoocoo 36:3. copoamacfiaum an“: onIom mo ooamsnompom 033033.33. .m 0.30.... 32 Table 10. Mean body weight at Day 1, 3 weeks; and 6 weeks of kits whelped and nursed by females administered various concentrations of NaOCl in the drinking water or feed. Group Kit body weight (g)1 (concentration of supplemental 6 wks. NaOCl) Day 1 3 wks Males Females I (0 ppm; 8.9 1 0.22 103 + 2.1 276 + 11.5 272 + 10.1 Control) II (25 ppm in drinking water) 8.6 1 0.19 102 + 1.8 286 + 9.8 248 + 7.7 III (50 ppm in drinking water) 9.2 1 0.22 9012.48 261 + 11.9 224 1 10.98 IV (100 ppm in drinking water) 8.5 1 0.23 108 1 2.3 279 1 12.6 260 1 9.9 v (200 ppm in drinking , water) 8.5 1 0.23 105 1 2.2 301 + 10.7 252 110.6 VI (100 ppm in feed) 7.9 1 0.2181 103 1 2.1 313 110.2 262 110.9 1Moan1standard error. aSignificantly less than control (P < 0.01) - Dunnett's one- Sided t'testo ' - 33 from females in Group III weighed significantly less than the control female kits at 6 weeks of age. Discussion The levels of sodium hypochlorite chosen for this study were derived from the water consumption data reported earlier. Since water consumption was reduced significantly at 200 ppm supplemental NaOCl. this concentration was chosen as the highest level of administration. This was done to avoid any effects of dehydration confounding the effects of NaOCl. Although mink can tolerate higher levels of NaOCl in the feed than the 100 ppm supplemental.NaOCl used in this study; this level was used so that comparisons could be made between the administration of NaOCl in the feed and in the drinking water. The addition of NaOCl at concentrations up to 200 ppm to mink drinking water or feed had no effect (stimulating or depressing) on body weight gains during the growing period. These results do not support the findings of Cunningham.(1980) who reported increased body weight gains in rats and guinea pigs administered supplemental NaOCl in their water at concentrations up to 80 ppm available chlorine with a maximum increase at 40 ppm. Hulan and Proudfoot (1982) reported no effect on body weight gains of broiler chicks administered up to 150 ppm available chlorine. At concentrations above 300 ppm they found poorer feed conversion (decreased weight gains) and increased 34 mortality. These adverse effects noticed at the higher concentrations of chlorine in broilers may be due to dehydration effects and not the effects of the sodium hypochlorite itself, since there was a decrease in water consumption at concentrations above 300 ppm. Since broilers consume a dry ration; any decrease in water consumption may lead to poorer feed conversion and increased mortality. None of these adverse effects were noticed with the mink. but the NaOCl levels administered to the mink were not nearly as high as those given to the chickens. Although a decrease in water consumption was. reported at the 200 ppm concentration of NaOCl used in this trial; this did not produce any adverse effects in the mink. This could be due to the fact that the mink's feed is approximately 60% water which may supply sufficient water to meet its requirement. Cunningham (1980) suggested that the means by which NaOCl might improve weight gain is that it may lessen the microbiological flora inhabiting the digestive tract and competing with the animal for food. One possible explanation why this increased weight gain was not seen in the mink is that the mink have a very short digestive tract and continually consume food to meet their energy needs. Any NaOCl that they ingest immediately interacts with the organic matter (feed) in their digestive tract and renders it ineffective for destroying the bacteria in the intestinal tract. NaOCl at concentrations up to 200 ppm produced no significant effects on any of the hematologic parameters 35 measured. Sodium hypochlorite is probably a less potent oxidant stressor to the blood than chlorite (0102'), since chlorite at concentrations of 100 ppm and above has been shown to decrease red blood cell counts. hemoglobin concentration and packed cell volume in the rat at 30 and 60 days exposure (Heffernan 33111., 1979). 'Very similar results were reported in the mouse by Moore and Calabrese (1980). Sodium hypochlorite forms chloride ions when it interacts with inorganic ions and organic material in water or feed. Since the interaction of 01'; K". Na“; and total 002' determines the pH of the blood. it is possible that excess chloride ions could exert a profound effect on the acid-base balance of the blood. The data presented in Table 7 indicates that the addition of NaOCl. in low concentrations to the water or feed of mink. had no adverse effects on this interaction. The serum blood levels of 01'. XI. Na+. and total 002' for animals treated with NaOCl were within the normal range for mink (Anonymous. 1980). The most common signs of chlorine toxicosis in rats are reduced body weight gain and enlarged liver. kidney and heart (Cunningham _eia_1_.', 1977; Cunningham and Lawrence. 1978). The amount of chlorine required to produce toxicity was 2.000 to 10,000 mg/kg in the diet. Hufran and Proudfoot (1982) also reported decreased weight gains in broiler chicks administered concentrations above 600 ppm chlorine in their water. but lower kidney. heart and testes weight. Such changes in organ weights 36 were not observed in this trial. but the levels of sodium hypochlorite administered were not as high. Chlorite (0102‘), when administered at 100 ppm in the water. has been shown to be capable of reducing the conception rate of A/J mice (Moore _e_t__a_l_., 1980). This effect was not seen with the NaOCl in this study. There was no significant differences in the reproductive performances of female mink between the control and the various treatment levels of NaOCl. Greater kit mortality was noticed between birth and three weeks of age among the higher NaOCl—treated groups. suggesting that young kits may be more susceptible to NaOCl than older mink. Examination of the data however. revealed that this higher mortality rate was accounted for by a few females in each of the groups that lost a large portion of their litters. This suggests that they were either very poor mothers or possibly did not have enough mammary development to support all the kits. Although there were a few significant differences in kit body weights at day 1. 3 weeks. and 6 weeks of age between the control and the NaOCl-treated mink. no trend between the level of NaOCl administered and kit body weights could be established. These results do not support the findings of Moore _t__al.. (1980) who reported a reduced growth rate in mouse pups whose dams were administered 100 ppm chlorite (0102') in their drinking water. These findings suggest that NaOCl is not as toxic as chlorite or possibly acts in a different manner. CONCLUSIONS Mink are less tolerant of sodium hypochlorite in their drinking water than in their feed. Water consumption was reduced at 200 ppm NaOCl in the drinking water. Feed consumption was reduced at 3200 ppm NaOCl in the food. One hundred ppm NaOCl added to the feed did not significantly reduce the amount of bacteria in the feed nor did it significantly slow bacterial growth (spoilage). Sodium hypochlorite added to the water at concentrations up to 200 ppm and to the feed at 100 ppm showed no effects on mink growth or reproduction. 37 APPENDICES 38 .mm. .muosom mm on o. sowvmsfiuoano unmagnsH mussam coon mom. .msoahsoc< oo. 0» 0m magmas»: was psoamfisdo 0o 00.00»...onm 06.80 0mm. .msoahsosd m on . :ofiuoomnfimao how nous: mo mofiumqwnoano 0959 now was a.mmm com. ..Amqmm.hoxoma m.o Hegemoo osma< pops: Hoom msaaafiam $0. rum fl. 83830.. N o. . 003808.30 .80 need... no noaumsfinoaso pops: Hoom wsassfirm oosonomom Aamnv oswpoano mansafis>m on: hnansosH mo soaasuusoosoo .moapumscsa ”2.090.“ch 5” com: hHsoaaoo Coos: msacsaosfiv musmuoomsanfic ocfihoano mo 33.393.30.30 0.3. .< 323%: 39 Appendix B. Biocidal effcit of [let available Chlorinc on VaflOUb organisms (Aiter Drychdula. Temp. Exposure pp." Organism ('C) Time Av. Cl. Biocidal Results ALGAE Chlorella variegata 7.8 22 --- 2.0 Growth controlled Gomphonema parVulum 8.2 22 --- 2.0 Growth cuntrolled Hicrocystis aeruginosa 8.2 22 --- 2.0 Crouch centralled BACTERIA Achromobacter metalcaligenes 6.0 21 15 seconds 5.0 1004 Bacillus snthracie 7.2 22 120 minutes 2.1-2.4 100. B. globigii 7.2 22 120 minutes 2 5-2.6 99.99. Clostridium botulinum toxin type A 7.0 25 30 seconds 0.5 1002 Escherichia coli 7.0 20-25 1 minute 0.055 100? E. typhosa 8.5 20-25 1 ninute O.1-O.29 1002 Hycobacterium tuberculosis 8.4 50-60 30 seconds 50 1001 Pseudomonas tluorcscens In 6.0 21 15 seconds 5.0 100: Shigella dysenteriae 7.0 20-50 3 sinutes 0 046-0. 1001 Staphylococcus auteus 7.2 25 30 seconds 0.8 1001 StreptucOCLus faecalis 7.5 20-50 2 minutes 0.5 1002 All vegetative bacteria 9.0 2 30 seconds 0.2 1002 BACTERIOPHACE S. tremoris 6.9-8 2 25 15 seconds 25 1002 phage strain 114? FISH Carassius auratus 7 9 Room 96 hours 1.0 Killed Daphnia magna 7.9 Ross 72 hours 0.5 Killed PROCS Rana pipisns 21 4 days 10 100: FUNGI Aspergillus niger 2 30-60 100 1002 Rhodotorula (lava 20 minutes 100 1002 5 minutes NEHAIODES C. quadrilebiatus 25 30 minutes 95-100 932 D. nudicspitstus 25 3O linutes 95-100 972 PLANTS Cabomba csrolinisns Room 4 days 5 1001 Elodea canadensis Room 4 days 5 1002 PROIOZOA Endamoebs histolytica cysts 25 150 minutes 0.08-0.12 99-1002 VIRUSES Purified sdenovirus 3 25 40-50 seconds 0.2 99.81 Purified Coxsnckie A» 27-29 3 minutes 0.92- 99.62 Purified Coxsackie a; 25 2 minutes 0.31-0 99.92 Purified Coxsackie 35 25—28 1 minute 0.21-0 99.92 Infectious hepatitis Room 30 minutes 3.2 Protected all 12 volunteers Purified poliovirus (Hahoney) 25-28 3 sinutes 0.21-0.30 99.91 Purified pcliovirus (Lsnsen) 19-25 10 Iinutea 1.0-0.5 Protected all 16. inoculated mice Purified poliovirus III (Sankett) 25-28 2 ninutes 0-11‘0 2 99.91 Purified Theilsr's 25-27 5 minutes 4-6 99.92 Appendix C. Relationship between HOCl, Baker, 1959). HOCanwcun 100 40 'OCl, I \1 and pH. (After 41 Appendix D. Procedure for aerobic plate counts of mink feed. 1. 6. Ten grams of mink food was weighed aseptically into a sterile jar of 200 ml capacity. 90 ml of sterile water was then added providing a 10“1 dilution. The food homogenate was blended for 2 minutes at 8000 RPM. Using separate sterile pipettes. decimal dilutions of 10'1, 10'2, 10’3; 10'4. 10'5 and 10‘6 of the food homogenate were prepared. Decimal dilutions were prepared by transferring 1 ml of the previous dilution to 9 ml of the diluent (sterile distilled water). All dilutions were shaken 25 times in a 1-foot arc within 7 seconds. Each decimal dilution was agitated to suspend any material that may have settled out and 1 ml of each dilution was then pipetted into each of appropriately marked duplicate petri dishes. The plates were filled with 12 to 15 ml of Brain Heart Infusion.Agar’(cooled at 45 °C) within 15 minutes of the time of the original dilution. The samples dilutions and agar medium were mixed immediately by rotating the plates on a flat surface. The agar was allowed to solidify. the petri dishes inverted, and incubated promptly at 35 °C for 48 hours. 42 9. Following incubation. all colonies on plates containing 30- 300 colonies were counted, using a colony counter and tally register. Results were recorded per dilution counted. 10. The counts obtained were averaged and reported as counts per gram of feed. 43 Appendix E. Composition of basal mink diet. Percentage Ingredients of diet Mink cereal1 20 Whole chicken 24 Ocean fish scraps2 15 Beef trips 8 Beef lungs 4 Beef trimmings 4 Beef liver 4 Water 21 1int-40 Mink Food. xx Mink Food. Inc. Subsidiary of United Feeds, Inc.. Theinsville, WI. 2Cod. haddock and flounder mix. G.M.F. Corp., Glouchester, MA. 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Proceedings, trace substances in environmental health, Vol. 13, University of Missouri, Columbia. MO. Wabeck. C.J.. D.V. Schwall. G.M. Evancho. J.G. Heck and A.B. Rogers. 1967. Salmonella and total count reduction in poultry treated with sodium hypochlorite solutions. Poultry Sci. 47:1090-1094. Yasukawa. 1931. Japan J. Expt. Med. 9:385-401. ”11111111111111111111111111111111”