Indian Journal of Experimental Biology Vol. 42, November 2004, pp. 1107-1111

Pathology of benzalkonium chloride toxicity and its effect on body weight gain in broiler birds* .

P K Mandai , M K Guptat & K K Singh

Department of Veterinary Pathology Ranchi Veterinary College, Birsa Agricultural University, Kanke, Ranchi 834 006, India

Received 26 November 2002; revised 16 July 2004

Four groups comprising 16 broiler birds each were given benzalkonium chloride (BC) at 100, 300, 500 and 700 ppm in drinking water for 40 days and one group of 16 birds (control) was kept on plain water. Clinical signs in higher dose groups were respiratory di stress, drooling of saliva, difficulty in deglutition, inappetence, apathy, lethargy and loss of body weight. Better body weight gain was recorded in 100 ppm dose rate. At 300 ppm, no significant body weight variation was recorded, whereas, at 500 and 700 ppm dose rates, significantly poor body weight gain was recorded. Major pathological changes were seen in 500 and 700 ppm groups, which exhibited formation of yellow diphtheritic plaques in the buccal cavity, swollen and pale commissures of beak and shortening of tongue. Minute necrotic and ulcerative foci were seen in oesophagus and crop. Hyperplastic and hypertrophic alterations were seen in mucosa of the upper digestive tract. Crop of 300 ppm group revealed formation of well developed epithelial nest with pseudoepitheliomatous hyperplasia at the margin of the lesion . Serum alanine transaminase, urea and uric acid in 500 and 700 ppm groups were elevated whereas no significant variations were observed in the 100 and 300 ppm groups. BC could enhance performance of broiler birds at 100 ppm dose rate. It should not be used beyond 300 ppm.

Keywords: Benzalkonium chloride toxicity, Broiler birds, Body weight.

There is a growing concern over the problem of antibiotic resistance due to its indiscriminate use in poultry farming as water and feed additive for growth promotion. A ban on the use of certain antibiotics has been imposed by the European Communit/. It has, thus, become imperative to explore other resources available to counter the menace of harmful microorganism, which causes mortality, growth impediments· resulting in economic loss to the poultry farmers.

Water sanitizers such as quaternary ammonium compounds (QACs) have gained popularity . The recommended dose for use of QAC as water sanitizer is 100 ppm2

.3. The QACs are cationic surfactants, which act by affecting permeability of the plasma membrane of the organism4

. Amongst QACs, benzalkonium chloride (BC) is hailed as one of the most effective biocidal agents. Although effective sanitizer and potent bactericidal, QACs can be detrimental to the health of the birds when used indiscriminate! y. The difference between effective dose and toxic dose of QAC is reported to be less5

.

*Part of research for MVSc degree of PKM. +Correspondent author E-mail: myapex2001 @yahoo.com Phone: 0651 -2450912 (0)

Keeping in view the popular use of BC as water sanitizer and paucity of literature regarding its deleteriou? effects, the present study has been taken up to determine the safety level of the drug and to know its pathological impact and effect on the body weight gain.

Materials and Methods Experimental design-Apparently healthy, broiler

chicks (day-old Hub chick; 80) obtained from Eastern Hatchery, Bhubaneshwar were kept in a disinfected room under proper management ___illld hygienic condition for 12 days prior to start of the experiment. All the birds were vaccinated against Ranikhet disease using Lasota strain on the 6th day and infectious bursal disease using intermediate strain on the 15th day of age. Birds were divided into 5 groups of 16 birds each. Pure benzalkonium chloride (BC) (Qualigens 50% w/v aqueous solution) was used. Drinking water containing 100, 300, 500 and 700 ppm of BC was given to groups TI> T2, T3 and T4, respectively, for 40 days, whereas, control birds (C) were kept on plain water. The selection of dose was done on the basis of previous reports on QAC toxicity2

•3

• Treated water and feed were given ad libitum to all groups. An incubation period-of 15 min

1108 INDIAN J EXP BIOL, NOVEMBER 2004

was allowed for disinfection of the drinking water. Prior to use, water samples were subjected to bacteriological examination on MacConkey agar and nutrient agar. No antibiotic was added to the water during experimental period .

The birds were observed for clinical signs of toxicity . Body weight of the birds was taken on the 12th day of age and then at weekly interval. It was canied out in the morning hours , before giving food. Four birds from each group were sacrificed by cervical dislocation on the lOth, 20th, 30th and 40th days of experiment to study gross and histopathological alterations of the tissues.

Necropsy and histopathology-A detailed post­mortem examination of all the sacrificed birds was carried out and different organs were examined for the presence of gross lesions. Pieces of liver, kidneys, heart, lungs, trachea, tongue, oesophagus, crop, proventriculus, duodenum, ileum, caecum, spleen and brain were collected and fixed in 10% formal saline solution. Tissues were routinely processed, sectioned (4-5 11m thick) and stained with haematoxylin and eosin (HE) stain6.

Biochemical studies-Blood was collected from the jugular vein of four birds of each group on the 110th, 20th, 30th and 40th days of the experiment before sacrifice. Serum was separated and used for the estimation of alanine amino transferase (ALT) 6, urea8 and uric acid9 using commercial kits from Span Diagnostic Pvt. Ltd., Surat, India. Data collected during the experiment were subjected to analysis of variance to study the effect of period and treatment on biochemical parameters and body weight gain. Critical difference test was done to differentiate two means.

Results and Discussion Bacteriological examination-BC-treated water

was found to be sterile, whereas water of control group contained 4-8 coliform colonies per 100 mi.

Clinical sign-The clinical signs of toxicity developed only in T3 and T4 groups which included respiratory distress characterized by gasping and open-mouthed breathing, drooling of slimy fluid from beak, difficulty in deglutition, sneezing and shaking of head. Birds also showed huddling tendency, apathy, lethargy and inappetance. Gurgling sound was produced by some birds. No such clinical signs were observed in T 1 and T2 groups.

No death due to chronic BC toxicity was recorded in any of the experimental groups. Clinical signs such

as repeated swallowing, anorexia and ataxia in turkey poult due to QAC toxicity have been reported2

. These clinical signs suggest that at higher dose rate BC is irritant , and conosi ve in nature due to increased alkalinity of water.

Gross pathology-Major gross pathological changes were observed in upper digestive tract of the birds kept on 500 and 700 ppm BC. The lesions were formation of yellowish diphtheritic patches in the buccal cavity, mainly in the inner surface of cheek, under surface of tongue and soft palate which later developed into plaques of different sizes. Commissures of beak were swollen and pale. Considerable shortening in the length of tongue was observed due to necrosis. Oesophagus and crop also revealed presence of minute necrotic foci on the mucosal surfaces.

At 100 ppm, no significant gross lesion was seen. At 300 ppm dose minute necrotic foci were observed in buccal cavity in some cases. These changes show that BC at higher dose rate is capable of causing necrotic and ulcerative lesion due to its irritant and conosive properties, which were in accordance with those

d . . I d . al ? 3 10 reporte previOus 111 pou ts an amm s-· · . Histopathology-Adaptive hypertrophic and

hyperplastic changes along with necrosis and ulceration in the mucosal surface of upper digestive tract in response to higher concentration of BC were observed. Tongue revealed marked hyperkeratosis at the necrosed blunt end. Buccal mucosa revealed ulcerative and proliferative changes (Fig. 1). In the

Fig. !-Hyperkeratosis (H) at the necrosed blunt end of tongue on 40th day of chronic toxicity with 700 ppm BC. HEx 400

MANDAL eta!.: PATHOLOGY OF BENZALKONIUM CHLORIDE TOXICITY IN BROILER BIRDS 1109

oesophagus, there was hyperkeratosis of the mucosal lining with hypertrophic oesophageal glands distended with retained mucous.

Crop was one organ which showed distinct histopathological alteration because of greater sojourn of the BC in it. Crop harbours feed and water for maximum petiod 11

• The most remarkable histopathological alteration of crop was observed in the birds of T2, since this group was given relatively higher dose rate (300 ppm) of BC and no significant reduction in water consumption was observed, therefore consistently getting exposed to BC, hence inducing histopathological alteration which have hitherto not been mentioned in the earlier literature dealing with BC or QAC toxicity. On the other hand, birds receiving 500 and 700 ppm BC, showed marked reduction in water consumption, hence suffered far lesser local toxic effect in the digestive tract during later phase of the experiment.

In the earlier stages, hyperkeratosis of mucosa of crop was prominent in T3 and T4. After 30th day, birds of T2 showed marked keratinization of the mucosa of crop alongwith pseudoepitheliomatous hyperplastic changes resulting in the formation of distinct hyalinized epithelial nest on 40th day. Moderate degree of dysplastic changes were seen in the margin of invaginating hyperplastic reaction (Figs 2 and 3). At this stage, birds of T3 and T4 also revealed disorganized growth of cells in squamous layer with hyalinization and tendency towards formation of epithelial nest.

Development of dysplastic changes in the crop points towards the possible mutagenic property of BC on long-term exposure. Influences inducing the dysplastic atypicalities when sufficiently prolonged or severe, ultimately serves as promoters in the carcinogenic process12

• Considering its wide use for different purposes, it becomes mandatory to explore this aspect of BC toxicity in future.

Other observations in the digestive tract included degenerative changes in the cells of tubular and glandular proventriculus, fusion of zymogen cells at the central area of the lobules of glandular proventriculus alongwith infiltration of inflammatory cells. Degenerative changes in the lining epithelium of the villi and goblet cell hyperplasia were commonly seen in the intestine. These changes were most prominent in T3 and T4. Except for mild hyperplastic change, no significant histopathological change was seen in the digestive tract of the birds ofT 1•

Lungs showed congestion, alveolar emphysema and brochiectasis in the birds of T3 and T4 groups. Degenerative changes in the lining epithelium alongwith hyperplasia of mucous secreting cells were observed in the trachea. In the later phase of

Fig. 2-Hyperkeratosis (H), dysplasia (D) and tendency of epithelial nest formation (N) alongwith lipping of its margin (L) in wall of crop on 30th day of chronic toxicity with 300 ppm BC. HE x400

Fig. 3--Formation of epithelial nest (N) due to pseudoepitheliomatous hyperplasia in wall of the crop on the 40th day of chronic toxicity with 300 ppm BC. HEx 400

1110 INDIAN J EXP BIOL, NOVEMBER 2004

experiment, keratinization of the tracheal epithelium along with squamous metaplasia and loss of cilia were evident. Excessive secretion of mucous by the goblet cells was mainly responsible for accumulation of mucous in trachea, which resulted into gurgling sound and respiratory distress during the first phase of the experiment. Death du~ to respiratory paralysis induced by curare-like effect of QAC has been previously reported2

. Lung . pathology due to QAC toxicity reported earlier include bronchoconstriction and bronchopneumonia 13 .

Liver, kidney and heart revealed congestion and · .1cuolar degeneration of the parenchymal cells in the

. ~<ds o( T3 and T4 groups (Figs 4 and 5). ; 'ypofunction of these organs lead to disturbanc.e in .netabolism and reduced excretion of the metabolic .vaste products. Hepatic toxicity and cardiac toxicity Jue to QAC has been earlier reported14.

Biochemical studies-Results of biochemical estimations of different groups at different time intervals are given in Table 1. At low dose exposure of birds with BC, no significant variation was observed in the level of serum ALT, urea and uric acid.

Birds given higher dose levels of BC in group T3 and T 4 revealed moderate yet significant elevation in the levels of serum ALT, urea and uric acid (Table 1). This finding is substantiated by the fact that there was vacuolar degeneration of hepatocytes in these groups,

resulting into elevated level of serum ALT. Uric acid and urea constitute major nitrogenous excretory products, which mostly originate as an end-product of protein catabolism. Uric acid constitutes 60-80% of total nitrogen in the urine of the birds 13

.

Hyperuricaemia and hyperuraernia observed in T 3 and T4 are suggestive of renal malfunction, which was also observed in the histopathological study of kidney in these groups in the form of nephrosis. Hyperuraemia due to chronic QAC toxicity in birds has been previously reported14.

Body weight gain-At higher dose rates of 500 and 700 ppm, BC caused reduction in body weight possibly because of ulceratives lesions in buccal cavity with subsequent inappetance due to painful and difficult deglutition and the degenerative changes in intestine and liver affecting absorption and metabolic function of the body. Further, alkaline nature of drinking water at higher dose rate may possibly have altered the optimal acidic environment of proventriculus, thereby affecting the proteolytic activity, resulting in protein malnutrition. On the other hand, a better body weight gain (g/g of initial weight) was achieved by the birds of T1 as compared to control at the maturity age. At 300 ppm, no significant variation in body weight as compared to control group was observed (Table 2) .

Thus a dose rate of 300 ppm could be considered as threshold value for BC toxicity. BC can be safely

Table !-Effect of different dose levels of BC on serum AL T, uric acid and urea in broiler birds at different time intervals (n=4 in each group)

[Values are mean± SE from 4 animals in each group]

Days c Tt T2 T3 T4

Serum ALT (Units/ml)

10 35.75±0.62b 35.50±0.95b 36.00±1.82b 43 .50±0.64" 37.75±0.85b 20 46.50±1.71 b 46.00±2.16b 45 .50±1.70b 45.50±1.70b 53.00±1.29"

30 46.00±2.16bc 44.25±1.65c 46.002.16bc 51.50±1.5ab 53.00±1.91" 40 47.00±2.38b 45 .50±1.55b 45.50±1 .55b 63.25±1.253 64.00±1.77"

Serum uric acid (mg/dl)

10 5.39±0.383 5.24±0.46" 5.59±0.23" 6.47±0.403 5.75±0.363

20 5.15±0.35b ' 5.50±0.34b 5.70±0.31b,m 6.17±0.2b 7.55±0.443

30 5.83±0.33b 5.78±0.05b 6.31±0.10b 9.05±0.35" 9.18±0.35" 40 6.27±0.33c 6.34±0.26c 7.14±0.31c 8.53±0.45b 10.06±0.30"

Serum urea (mg/dl)

10 1.26±0.06c 1.30±0.09c 2.90±0.28b 3.17±0.17b 3.96±0.07" 20 1.93±0.06d 2.11±0.12d 2.48±0.11c 2.82±0.09b 3.41±0.09" 30 2.23±0.07c 2.38±0.09c 2.41±0.14bc 2.97±0.15b 3.83±0.343

40 3.06±0.23c 3.12±0.12c 4.44±0.24b 5.11±0.28b . 6.28±0.343

Values bearing same superscripts (a-d) in a row did not differ significantly. C =Control Tl = 100 ppm BC; T2 = 300 ppm BC; T3 = 500 ppm BC; T4 = 700 ppm BC.

MANDAL et al.: PATHOLOGY OF BENZALKONIUM CHLORIDE TOXICITY IN BROILER BIRDS 1111

Table 2-Effect of different dose levels of BC on body weight gain (g/g of initial weight) during its chronic intoxication in broiler birds at different ranges of age

[Values are mean± SE from 4 animals in each group]

Period (days) c T1 12-18 0.97±0.05" 1.02±0.02" 18-25 0.65±0.04"b 0.71±0.05" 25-33 0.43±0.03" 0.44±0.02" 33-39 0.35±0.03" 0.36±0.02" 39-46 0.25±0.02bc 0.34±0.03"b 12-33 3.15±0.19b 3.99±0.17" 12-39 4.63±0.29b 5.79±0.32"

Fig. 4.-Congestion of sinusoids (C) and vacuolar degeneration (V) of hypatocytes in liver on 40th day of chronic toxicity with 700 ppm BC. HE x 400

Fig. 5--{:ongestion (C) and vacuolar degeneration (V) of the tubular epithelium in the kidney on 40th day of chronic toxicity with 700 ppm BC. HEx 400

Treatment

T z T3 T4 0.86±0.06"b 0.74±0.06b 0.54±0.llc 0.62±0.01 ab 0.54±0.06b 0.20±0.05c 0.37±0.02"b o.35±0.o5"b 0.22±0.06c 0.32±0.06" 0.29±0.03" 0.09±0.04b 0.28±0.05bc 0.19±0.05c 0.10±0.06d 3.12±0.09bc 2.63±0.24c 1.26±0.10d 4.48±0.33bc 3.72±0.39c 1.45±0.08d

used at 100 ppm as a water disinfectant and sanitizer for more hygienic and economical poultry farming .

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