J Sci Food Agric 1997, 74, 229È236

Eþects of Roxarsone on Performance, Toxicity,Tissue Accumulation and Residue of Eggs andExcreta in Laying HensPeter Wen-Shyg Chiou,*1 Kuo-Lung Chen2 and Bi Yu11 Department of Animal Science, National Chung-Hsing University, Taichung, Taiwan2 Department of Animal Science, National Agricultural Institute of Chai-Yi, Taiwan

(Received 30 November 1995 ; revised version received 26 July 1996 ; 20 January 1997)

Abstract : In this study, we examined the e†ects of a high dosage of roxarsone inthe diet on the performance, liver function, and its residue in liver, eggs andexcreta of laying hens. Seventy-Ðve 32-week-old layers were selected and ran-domly allocated into Ðve dietary treatments with three replications for eachtreatment. Feeding periods were 4 weeks with an additional week for withdrawal.The experimental diets included 0, 11, 22, 44 or 88 mg kg~1 arsenic from roxar-sone, respectively. Dietary arsenic above 44 mg kg~1 signiÐcantly decreased theegg production and feed intake of the layer (P\ 0É05). Layers ceased to produceeggs after two weeks of feeding the 88 mg kg~1 arsenic supplement diet. Wherethe enzyme activities in the serum, aspartate aminotransferase (AST), lactatedehydrogenase (LDH), creatine kinase (CK) signiÐcantly increased. Also, theliver weight did not only signiÐcantly decrease (P\ 0É05), but was also damagedon histological examination. Moreover, arsenic residues in the liver, eggs and theexcreta signiÐcantly increased as dietary arsenic level was increased (P\ 0É05).The serum enzyme activities of AST, LDH, CK returns to normal after a week ofthe drug withdrawal. Arsenic residues in liver, egg and excreta also signiÐcantlydeclined in the withdrawal period (P\ 0É05). Furthermore, the hepatic cells werevacuolised from layers treated with 88 mg kg~1 of arsenic.

Key words : arsenic, toxicity, layer, performance, liver function

INTRODUCTION

Roxarsone (3-nitro-4-hydroxyphenylarsonic acid) is anorganic arsenic having been used for more than fourdecades as a poultry feed additive not only to promotegrowth and egg production, but also to enhance feedefficiency and pigmentation. Arsenicals as feed additivesare allowed in the USA and some other countries buthave not been allowed within the European Union formany years. For this usage, the FDA (USA) approvedlevels of 25È50 mg kg~1 of the diet.

The maximum tolerable level of arsenic for poultrywas set as 100 mg kg~1 for organic and 50 mg kg~1 forinorganic arsenic according to the National Research

* To whom correspondence should be addressed.

Council (1980). In a study involving tissue clearance ofRofenaid in chickens and turkeys with arsenicals, Felliget al (1971) indicated that arsenic was detected only inthe liver and kidneys and their levels were both below1É3 mg kg~1. They also found that arsenic levels wereuna†ected by the simultaneous feeding with Rofenaid.Sullivan and Al-Timimi (1972) fed a practical cornÈsoyaration with 25È400 mg kg~1 roxarsone to youngturkeys for 4 weeks, Ðnding that the maximum safedosage ranged between 50 and 100 mg kg~1. They alsodemonstrated that the level of roxarsone for theLD5028 days of age ranged between 300 and 400 mg kg~1.Wise and Hartley (1974), however, found that allturkeys receiving 200 or 400 mg kg~1 of 3-nitro startingat 1 day of age died within 15 days. Turkey poults maybe considerably more sensitive to 3-nitro toxicity than

229J Sci Food Agric 0022-5142/97/$17.50 1997 SCI. Printed in Great Britain(

230 P W -S Chiou, K-L Chen, B Y u

fowl. Adding upto 200 mg kg~1 of roxarsone in the dietdecreased the weight gain and feed conversion of thebroilers (Czarnecki and Baker 1982, 1984 ; Czarnecki etal 1984). Czarnecki and Baker (1982) fed a practicalcornÈsoya diet supplement with roxarsone to hybridbroilers from 8 to 21 days of age. According to theirresults, the arsenic content in kidney increased with anincreasing level of roxarsone in the diet. The mortalityreached 13É3% as dietary roxarsone increased to1000 mg kg~1, whereas the mortality reached 80% atthe level of 2000 mg kg~1 of inclusion. Stute and Vogt(1968) examined the inÑuence of roxarsone on egg pro-duction and food conversion of laying hens and residueof muscles, organs and egg. Their results did not indi-cate residues in eggs above the FDA tolerance of500 mg kg~1.

Although arsenic is no longer approved for use inlaying hens in the USA, it is approved for use in broilers(Muirhead 1992). The feasibility of an unintentionalcross-contamination during feed mixing or mislabellingcould lead to feed laying hens arsenic intended forbroilers. In this study, we examined the toxicity and theaccumulation in liver, egg and excreta of arsenic inlaying hens fed conventional diets. Such an investiga-tion originates from an environmental protection andconsumer safety perspectives.

MATERIALS AND METHODS

Seventy-Ðve 32-week-old White Leghorn layers wereselected from a commercial Ñock and were housed inindividual cages with Ðve cages in an unit sharing acommon feed trough. Cages were placed in three blocksof 1, 2 or 3 tiers of Ðve units each. They were randomlyallocated into Ðve dietary treatments with three blocksas replicate groups for each treatment. Birds were main-tained on a lighting programme of 17 h light and 7 hdarkness. Feeding period was 4 weeks with an addi-tional week of drug withdrawal period. Table 1 presentsthe experimental basal diet. Diets were added 0, 11, 22,44 or 88 mg kg~1 of arsenic from 0, 38, 78, 156 or310 mg kg~1 of Roxarsone, respectively, for the dietarytreatment.

During the experimental period, egg weight and eggproduction were daily recorded. Feed and water wereprovided ad libitum and the feed was recorded weekly.The blood sample, egg and excreta were collected everyWednesday afternoon while feed and water were with-drawn that morning. The bodyweight of layer hens wasmeasured at the beginning, 4th week and at the end ofthe experimental period. At the end of the trial, twolayer hens from each replicate group were slaughteredand their livers were removed. The livers were weightedand the sample was preserved for pathological exami-nation. The samples of liver, egg and excreta were sub-jected to chemical analysis of arsenic content, while the

TABLE 1Composition of the basal diet

Feed ingredients g kg~1

Yellow corn meal 603É7Soya bean meal (44%) 240É0Fish meal 30É0Soya bean oil 29É8Dicalcium phosphate 13É0Limestone (pulverised) 77É2Iodised salt 3É0DL-Methionine 0É8Vitamin premixa 0É5Mineral premixb 2É0

Total 1000É0

Calculated valueCrude protein 178É0Metabolisable energy (MJ kg~1) 13É67Calcium 34É0Available phosphorus 4É7

a Vitamin premix supplied the following per kilogram of diet :vitamin A, 25 000 IU; vitamin 3125 ICU; vitamin E,D3 ,37É5 IU; vitamin 6É25 mg; vitamin 3É75 mg; vitaminK3 , B1,

12É5 mg; vitamin 10É0 mg; Ca pantothenate,B2 , B6 ;18É8 mg; niacin, 50 mg; biotin, 0É06 mg; folic acid, 1É25 mg;vitamin 0É05 mg.B12 ,b Mineral premix supplied the following per kilogram of diet :Cu 25É45% Cu), 6 mg; Fe(CuSO4 . 5H2O, (FeSO4 . 7H2O,20É09% Fe), 50 mg; Mn 32É49% Mn), 40 mg;(MnSO4 . H2O,Zn (ZnO, 80É35% Zn), 60 mg; Se 45É56% Se),(NaSeO3 ,0É075 mg.

serum enzymes, aspartate aminotransferase (AST),lactate dehydrogenase (LDH) and creatine kinase (CK)activity were measured.

The water of feed and excreta was analysed accordingto the method of AOAC (1980). The arsenic content wasanalysed by atomic absorption spectrophotometer witha Perkin-Elmer electrodeless discharge lamp (Perkin-Elmer 2380 and FIAS 400) at the wavelength of197É7 nm with a slit width of 0É7 nm. The sampleÏsarsenic level ranged from 10 to 40 kg kg~1. Bloodserum was analysed for AST (EC 2.6.1.1), LDH (EC1.1.1.27) and CK (EC 2.7.3.2). Those enzyme activitieswere determined by kinetic methods as recommendedby the German Society for Clinical Chemistry (1972),using Roche testing kits (Roche COBAS MIRA) bymeasuring NADH at the wavelength of 340 nm.Enzyme activities are expressed in international units(U) per liter of serum (Bergmeyer 1983). The liver wasÐxed in 10% bu†ered neutral formalin, processed bystandard methods and embedded in paraffin wax. Sec-tions cut at 5 km were stained by haematoxylin andeosin.

A completely randomised block design was applied toÐnd the e†ects of the level of roxarsone inclusion and

Safety and toxicity of roxarsone in layer hen 231

the feeding period (weeks). Variance analysis resultswere calculated with the general linear model procedureof the SAS (1984). Moreover, DuncanÏs New MultipleRange Test was used to compare the treatment meansaccording to Steel and Torrie (1960).

RESULTS AND DISCUSSION

E†ects on performance

Table 2 presents di†erent levels of arsenic in the diet onlaying Leghorn hens performances. A trend was foundin better egg production and egg weight in the diet with11 mg kg~1 arsenic inclusions (38 mg kg~1 roxarsone).It did not reach the signiÐcant level (P[ 0É05). Thisresult di†ered from most investigations that a low levelof roxarsone (25È50 mg kg~1) increased performance oflayers (Hung 1986). The contradictory result may beowing to the shorter feeding period and the cage feedingof laying hens in this trial. Layers under cage feedingthat block the life cycle of the coccidia reduced the posi-tive response of roxarsone. Such a reduction occurredsince roxarsone is an e†ective drug for the coccidiosisdisease (Izquierdo et al 1987 ; Bafundo et al 1989). Thearsenic in the diet did not signiÐcantly inÑuence the feedintake, egg production and egg weight upon the22 mg kg~1 of inclusion. The adverse e†ect became sig-niÐcant over the 44 mg kg~1 supplement (P\ 0É05).The feed intake, egg production and egg weight at44 mg kg~1 inclusion were 76%, 83% and 98% of thecontrol, respectively. Whereas at the 88 mg kg~1 sup-plement were 50%, 17% and 94% of the control, respec-tively. However, the laying hens in the 88 ppm arsenicgroup ceased to produce eggs after 2 weeks of feeding.Above data suggest that the adverse e†ects of roxarsoneon the performance, ie body weight gain, egg pro-duction, and egg weight were not signiÐcant at22 mg kg~1 (78 mg kg~1 roxarsone) ; it became signi-Ðcant over 44 mg kg~1 (156 mg kg~1 roxarsone) level

of inclusion in the diet. This Ðnding implies that themaximum tolerance level could be found between 78and 156 mg kg~1 of roxarsone. This Ðnding resemblesthose results of Frost et al (1953), in which themaximum tolerable level of roxarsone was 100 mg kg~1on the chickens with body weight between 1130 and1360 g. Czarnecki and Baker (1982, 1984) also indicatedan adverse e†ect of 200 mg kg~1 roxarsone on gainsand feed conversion of broilers.

E†ects on body weight and liver weight

Table 3 presents di†erent arsenic levels in the diet onthe body weight, liver weight and relative liver weight oflaying Leghorn hens. The e†ects of arsenic inclusion upto the 44 mg kg~1 in the diet on the body weight gainwere insigniÐcant (P[ 0É05). However, the arsenic levelover 88 mg kg~1 signiÐcantly depressed 23% of bodyweight after 28 days of feeding (P\ 0É05) as comparedto the initial body weight of the trial. This level resem-bles that of Hermayer et al (1977) whom derived a sig-niÐcant depression on body weight of laying hens thatfed the diet added 100 mg kg~1 of (75 mg kg~1As2O3arsenic). The e†ect of depressed body weight gain wasprobably owing to the reduction of the feed intake.

When the diet included 11 or 22 mg kg~1 arsenic,liver weight signiÐcantly decreased after 4 weeks offeeding (P[ 0É05) as compared to the control.However, the relative liver to the body weight did notexert any signiÐcant inÑuence by the arsenic supple-ment. After inclusion, 88 mg kg~1 arsenic acid wasadded into the diet for 4 weeks, the liver weight and therelative liver weight decreased signiÐcantly (P\ 0É05) ; itdecreased 42% in the liver and 25% in the relative liverweight. The liver weight signiÐcantly increased in the88 mg kg~1 group, ie 1É24 times, after 1 week of arsenicwithdrawal as compared to before. The liver revealedobviously vacuolised hepatic cells as fatty liver from thehistologies graph (see Fig 4 below).

TABLE 2The e†ects of di†erent levels of roxarsone inclusion in the diet on the feed intake, egg production and egg

weight of laying Leghorn hens (mean^ SE)a

Items L evel of arsenic (mg kg~1)

0 11 22 44 88

Arsenic (mg kg~1 1É1 9É1 21É2 43É0 85É1analysed value)

Feed intake 115a^ 0É4 113a^ 0É4 114a ^ 0É4 87b^ 0É4 58c^ 0É5(g per bird day~1)

Egg production 87É1a ^ 1É5 87É6a ^ 1É4 84É7a ^ 2É0 72É1b^ 1É8 14É9c ^ 3É4(%)

Egg weight (g) 58É7ab^ 0É4 60É1a ^ 0É7 59É1ab^ 0É7 57É5bc^ 0É4 55É3c ^ 0É9

Mean^ standard error.a Means in the same row with the di†erent following letters are signiÐcantly di†erent (P\ 0É05).

232 P W -S Chiou, K-L Chen, B Y u

TABLE 3The e†ects of di†erent levels of roxarsone inclusion in the diet on the body weight, liver weight and

relative liver weight of laying Leghorn hens (mean^ SE)a

Experimentperiod

L evel of arsenic (mg kg~1)

0 11 22 44 88

Body weight (g)0 week 1509^ 46 1532^ 31 1484^ 41 1524 ^ 37 1478x ^ 274th week 1496a^ 35 1520a^ 21 1490a^ 36 1439b^ 26 1132by ^ 16

Liver weight (g)4th week 39É5a ^ 2É2 33É7b^ 1É2 34É6b^ 0É5 36É2ab^ 1É1 22É2cy ^ 1É75th week 38É1b^ 1É3 38É0b^ 0É9 36É3b^ 1É0 37É6b^ 2É4 49É8ax ^ 4É3

Relative liver weight (%)4th week 2É60a ^ 0É14 2É30a ^ 0É08 2É42a ^ 0É07 2É41a ^ 0É09 1É93by ^ 0É155th week 2É58b^ 0É09 2É50b^ 0É04 2É37b^ 0É04 2É48b^ 0É08 3É79ax ^ 0É24

a Means of the same row with the di†erent following letters (aÈc) are signiÐcantly di†erent (P\ 0É05).Means of the same column with the di†erent following letters (x, y) are signiÐcantly di†erent (P\ 0É05).

E†ects on arsenic accumulation and residue

Table 4 presents the e†ects of arsenic inclusion in thediet on the arsenic contents of liver, egg and excreta oflayers. The arsenic content in liver signiÐcantlyincreased as an increased level of arsenic in the diet(P\ 0É05). Since liver is the major metabolism site inthe body, minerals could also be accumulated in theliver. Amount of arsenic that accumulated in liver was22É8 times in 88 mg kg~1 arsenic inclusions in the dietfor 4 weeks as compared to the control. This Ðndingcorresponded to the results of Frost (1953) and Proud-foot et al (1991). When laying hens were fed the dietwith any arsenic supplement for 4 weeks and followed aweek of withdrawal period, the liverÏs arsenic contentswere signiÐcantly higher than the control (P\ 0É05).Ferslew and Edds (1979) fed a diet supplement with

arsenilic acid to swine, indicating that the liver returnsto a normal arsenic content after 1 week of drug with-drawal as compared to the control. Proudfoot et al(1991) fed the growing broiler diet with 99 mg kg~1 ofarsenilic acid supplemented diet. They also found thatthe arsenic content in the liver return to a normal levelafter 1 week of the drug withdrawal. Such a contradic-tory result from ours may be due to the di†erent chemi-cal structures of the arsenic used which subsequentlyproduced a di†erent rate of excretion (Underwood1977). The arsenic residue in liver was 2 mg kg~1 fromthe 44 mg kg~1 supplement group, suggesting that theresidue exceeds the Canadian national standard(Proudfoot et al 1991). The liver residue dropped belowthe standard after 1 week of withdrawal.

The arsenic content in egg was signiÐcantly inÑu-enced by adding arsenic in the diet (P\ 0É05) ; It

TABLE 4The e†ects of di†erent levels of roxarsone inclusion in the diet on the arsenic content of liver, egg and excreta

of laying Leghorn hens (mean^ SE)a

Experimentperiod (weeks)

L evel of arsenic (mg kg~1)

0 11 22 44 88

As content of liver (ng g~1)4th 150e^ 4É5 1005dx ^ 109 1609cx^ 126 2177bx ^ 117 3429ax^ 605th 150e^ 5É6 338dy ^ 20 491cy^ 25 622by ^ 35 697ay^ 18

As content of egg (ng g~1)4th 26É4e^ 1É2 46É3dx ^ 2É1 75É0cx^ 5É7 145É1bx ^ 5É8 245É2ax ^ 6É85th 24É6d^ 0É3 32É6cy ^ 1É2 33É0cy^ 1É7 42É1by ^ 2É3 60É8ay ^ 2É9

As content of excreta (ng g~1)4th 926e^ 56 3288dx ^ 108 6037cx^ 100 18 200bx ^ 393 43 971ax^ 10855th 879e^ 45 2577dy ^ 138 2722cy^ 53 4585by ^ 380 5648ay^ 143

a Means of the same row with the di†erent following letters (aÈc) are signiÐcantly di†erent (P\ 0É05). Meansof the same column with the di†erent following letters (x, y) are signiÐcantly di†erent (P\ 0É05).

Safety and toxicity of roxarsone in layer hen 233

reached maximum at the 88 mg kg~1 inclusion levels, ieroughly 10 times the control level. The eggÏs arseniccontent signiÐcantly decreased from the 4th to 5th weekin the trial after 1 week of withdrawal. The arseniccontent of the liver was still signiÐcantly higher at the5th week in our trial than the control (P\ 0É05). Thisdata resemble the results of Donoghue et al (1994) thatalthough the arsenic concentrations precipitouslydropped in both yolk and albumen within one week ofdrug withdrawal, the amount of arsenic was still greaterthan that of eggs from control hens. They regarded theresidual arsenic as probably being a reÑection of thenormal pattern of yolk formation. This is since yolksreleased during the Ðrst week of drug withdrawal wereformed and incorporated arsenic during the periodwhen hens were fed the organic arsenicals. Their dataalso revealed that arsenic was not released into eggs in ahigh amount two weeks after removal of the drug fromthe feed of the laying hens. The maximum residue ofarsenic in egg was 245 kg kg~1 in this trial and still didnot exceed the 500 kg kg~1 of the Food and DrugAdministration. Therefore, including arsenic below88 mg kg~1 would not create the consumer safetyproblem of residues in eggs. The arsenic content in theexcreta of layers signiÐcantly increased as the level ofarsenic in the diet increases (P\ 0É05). Concentration ofarsenic in excreta was less than the level added in thediet. Although the residue in the excreta signiÐcantlydropped after 1 week of the drug withdrawal (P\ 0É05),it was still signiÐcantly di†erent from the control(P\ 0É05), ie roughly six times that of the control.Ferslew and Edds (1979) fed the diet supplemented100 mg kg~1 of the drug residue in the faeces ; The fecalresidue even dropped to 0É2È0É3 mg kg~1 after 1 weekof the drug withdrawal. Frost (1953) pointed outthat the organic arsenicals can easily be adsorbed andaccumulated in various tissue inside swine and poultryand can also be metabolised and excreted into faeces.Organic arsenicals seem to be readily absorbed andaccumulated into the body tissues ; the amount excretedmay be less than the amount consumed. This result issimilar to ours. The result of Frost (1953) was dif-ferent from our result. They demonstrated that the resi-dues in faeces return to normal arsenic level was com-pared to the control. Whereas the residues remainedsigniÐcantly higher than the control after 1 week of thedrug withdrawal in this trial. Such a di†erence may beowing to the chemical structure of the arsenicals ; sincethe rate of absorption, storage and excretion is di†erentamong forms of the arsenicals compounds. The Nation-al Standard of Taiwan (1989) required a minimum of2É5% nitrogen in the compost fertiliser where thearsenic content could not exceed 40 mg kg~1 each per-centage of nitrogen content. Restated, the arseniccontent in the fertiliser should not exceed 100 mg kg~1of the national standard. Maximum arsenic residues inthe excreta was only 44 mg kg~1 in this trial. Whether

the national standard was below the requirement of theenvironmental protection requires further investigation.

E†ects on the serum enzymes activity

Figures 1, 2 and 3, present the e†ects of the di†erentarsenic levels in the diet on serum AST, LDH, and CKactivities of laying Leghorn hens, respectively. At theÐrst week of feeding, serum AST activity was signiÐ-cantly inÑuenced by the inclusion of arsenic in the diet(P\ 0É05) ; The e†ect was signiÐcant over the88 mg kg~1 level of inclusion (P\ 0É05). Although itdid display a trend of AST increasing up to the level ofthe 44 mg kg~1 of As supplement, it did not reach asigniÐcant level. In the Ðrst week, the AST activity ofthe 88 mg kg~1 group signiÐcantly increased up to themaximum level (P\ 0É05), ie twice that of the control(414 vs 208 U litre~1). It then gradually declined after-ward. Later it further dropped in the serum AST activ-ity after 1 week of the drug withdrawal. However, theenzyme activity was still signiÐcantly higher, ie 1É5 timesthat of the control. This Ðnding implied that the birdhas still not recovered to normal functioning.

A trend of higher LDH activity was found in the 2ndand 3rd weeks of the 44 mg kg~1 supplement group ;however, it did not reach the statistical level. Theenzyme activity of the 88 mg kg~1 supplement groupreached maximum during the 2nd week, ie 2É3 times

Fig 1. The e†ects of di†erent arsenic levels in the diet onserum AST activity (U litre~1) of laying Leghorn hens. …,0 mg kg~1 ; 11 mg kg~1 ; 22 mg kg~1 ; 44 mg kg~1=, >, L,

and 88 mg kg~1.K,

234 P W -S Chiou, K-L Chen, B Y u

Fig 2. The e†ects of di†erent arsenic levels in the diet onserum LDH activity (U litre~1) of laying Leghorn hens

(symbols as in Fig 1).

that of the control, and then gradually declined.Although the enzyme activity dropped after 1 week ofdrug withdrawal, it was still signiÐcantly higher, ie 1É5times than the control. Also, a trend of increased CKactivity was found in the 1st, 2nd and 3rd weeks of the

Fig 3. The e†ects of di†erent arsenic levels in the diet onserum CK activity (U litre~1) of laying Leghorn hens (symbols

as in Fig 1).

44 mg kg~1 supplement group, but did not reach anystatistically signiÐcant level. The CK activity was sig-niÐcantly higher in the 88 mg kg~1 group than theother treatment groups during the experimental periods(P\ 0É05). The CK level reached maximum during the1st week, ie 2É9 times that of the control. It then grad-ually decreased. Nevertheless, the level still remainedtwice that of the control after 1 week of the drug with-drawal.

The serum AST, LDH and CK revealed a highertrend of activities in the 44 mg kg~1 arsenic inclusiongroup. This also coincided with the 44 mg kg~1 groupÏsinsufficient performance (Table 2). It was probablyowing to the fact that the toxic e†ect of arsenicdamaged the body tissues, thereby resulting in therelease of enzymes in serum. Therefore, the performanceof the laying hens declined. The levels of serum AST,LDH and CK of the 88 mg kg~1 group increasedduring the 1st and 2nd week (Figs 1, 2 and 3). ThisÐnding implied that the heart and muscle tissues weredamaged by the inclusion of arsenic into the diet.

Attempting to evaluate the damage of liver based onthe activities of serum AST, LDH and CK was imprac-tical. The AST is widely distributed in liver, kidney,heart and muscle cells. LDH is also widely distributedin liver, kidney and heart tissue. Whereas CK is the spe-ciÐc enzyme in heart and muscle tissue as suggested byWang (1992). Serum AST and LDH increasing withoutincreasing the level of CK activity implied that kidneyand liver were damaged while heart and muscle tissueremained intact. The increased serum level of enzymes,AST, LDH and CK represented damage in both heartand muscular cells. The activities of all the threeenzymes, AST, LDH and CK increased in the highdosage arsenic group in this trial. Such an occurrencecan only conÐrm the damage of muscle and heart tissue.Whether the liver was damaged cannot be conÐrmedfrom the data derived from this trial. Further pathologi-cal examination is required to conÐrm the damage ofliver.

E†ects on liver histology

Figure 4 presents the microscopic photographs of theliver of layer hens. This Ðgure reveals the damaged liverof layers that received a diet with the 88 mg kg~1arsenic supplement. The Ðgure also shows the abnormalliver that received treatment for 4 weeks (above), and 1week after withdrawing period (below). This Ðgureshowed a bile duct proliferation and lymphocyte inÐl-tration in the damaged liver (above). This Ðndingimplied that the liver lesion is caused by the highdosage of arsenic inclusion in the ration ; This liverlesion could also be conÐrmed by the As accumulation(Table 4) and the enzyme activities of serum AST andLDH (Figs 1 and 2). After 1 week of withdrawal period,

Safety and toxicity of roxarsone in layer hen 235

Fig 4. Microscopic photograph of the liver section from the laying hens treated with 88 mg kg~1 of arsenic (200], above) andafter 1 week of drug withdrawal (200], below).

the histologies graph of the liver also revealed a similarpicture as in the abnormal liver or vacuolarised hepaticcells (Fig 4). This also conÐrmed that longer than 1week is required for the liver to recover from the arseniclesion.

According to performance data, As accumulation andthe function of liver that were obtained in this trial, themaximum dietary tolerable level of arsenic from roxar-sone for laying white Loghorn hens seems to range from22 to 44 mg kg~1.

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