Indian Jou rnal of Experimental Biology Vol. 41 , June 2003, pp. 592-597

Immunotoxicological effects of dermal application of scum of waste crankcase oil in mice

Santosh Khanna & Ram K S Dogra

Immunotox ico logy Laboratory, Indust rial Toxicology Research Centre, Lucknow 226 00 I, India

Received 10 June 2002; revised 27 February 2003

The sc um of waste cra nkcase o il (SWCO) form s due to wea thering of waste crankcase o il , deposited on the surface of water bodi es. It is known to attach to the feat hers of aquatic birds and cause toxicity to the eggs of nestling birds . The water bodies contaminated with SWCO can also be a sou rce of toxicity to the human beings and animals en te ring such bodies. Since SWCO used in thc present study had an appreciable content of heavy metals like Zn, Pb, Cd, Mn, Cr and Ni, the present investigation was undertaken to study a probable effect on immune system of mice. Animals treated w ith SWCO at a dose of 0.5, 1.0, 2.0, or 4.0 g/kg body weight for 28 days, had no effecl on the we ight gain of vita l organs. A depress ing effec t was observed on the ce ll popu lat ion of spleen and thymus. The number of primary antibody ( lgM ) produc ing cells was sign ifi cant ly depressed in spleen. The IgM anlibody tiler of serum, reduction of NBT dye by peritoneal exudat cell s and mounting of delayed hypersensiti vity response were not affecled. In view of above immunotox ic effecls of SWCO, the waste crankcase oi l shou ld be carefu ll y disposed of, away from water bod ies.

Keywords: Crankcase o il , ImmunolOxicology, Mice dermalloxicily, Scu m of waste crankcase oi l

The scum of waste crankcase oil (SWCO) is the residual part left after the dissolution of water soluble component of waste crankcase oil (WCO). The later (WCO) , is the lubricating oil removed from crankcase of internal combustion engines after certain period of use. Nearly 150,000 metric tons of WCO is generated every year in India. Almost all the transport sections, such as automobi les (cars and trucks), rail ways (di esel locomotives), marine (diesel power engine), aviation (piston driven air crafts), agriculture, mining, forestry (tractors, harvesters, locomoti ves) generate it 1.2. The crank case oil is made by mixing of mineral base oil and additives. The final product contains organics like monocyclic and polycyclic aromatic hydrocarbons (linear and branched aliphatic alkanes, toluene, benzene, xylene) and inorgani cs like lead, copper, zinc, nickel, manganese , cadmium, aluminium, barium, arsenic, magnesium and calcium . The used crankcase oil becomes enriched with metals like lead, cadmium, zinc, arsenic and chromium from the wear and tear of engine lini ngs3.The toxicity of WCO has primarily been attributed to these metals4

. In rural areas of northern India, fanners smear the horns and body of buffaloes with WCO, to get a sheen and glow of heal th y animalS. With increasing au tomation of agricultural operations in Indi a, such application may also increase and prove toxic to animals. In some developed countries, WCO is di sposed of on land

(backyards, alleys, pits, fields and drainage ways), land fills and even sprayed on roads to suppress dud'. After di sposal on land it can percolate laterall y as well as downwards and find access to water bodies like ponds, lakes and streams. WCO also contaminates the soil and water surface by acc idental sp il lage from tankers, during transport on land and sea. The aquatic pollution by WCO has great significance because of its volume and effects on biological life. The aquat ic birds, human beings and livestock may come in co ntact with the scum, when they enter such water bodies for bathing or quenching thirst. As no toxicological studies of SWCO on the skin or organs of animals or human beings have been li sted in literature, the present study was undertaken to investigate the immunotoxicolog ical effects of SWCO painted on the shaven skin of mice.

Materials and Methods Preparation of scum of waste crank case oil

(SWCO)-It was prepared from waste crankcase oil (WCO) obtained from petrol run cars at the time of first service. The SWCO was prepared as per standard method7

. Brietly, WCO heated at 90°C for 4 hr was mixed with extraction fluid and pl aced on thermal reg ulated magnetic stirrer for 18 hr. The sample was then transferred to a separating funnel. The water portion was drained and the upper layer of scum coll ected .

KH ANNA & DOGRA : IM M UNOTOXICOLOGICAL EFFECT OF SLUM OF WASTE CRAN KCASE OIL S93

Chemical analysis of SWCO - One gram of SWCO was wet diges ted by boiling with 10 ml mixture of HN0 3, H2S04, HCl04 (6: 1: 1). The extracted meta ls were d issol ved in 10 ml of HNO} (0.1 N)8 and analysed by inducti vely coupl ed pl as ma atomic emi ss io n spectrophotomete r (IC P-AES ), as pe r the standard method'!.

Acute (14-days) dermal toxic dose determination study - Acute dermal toxicity of scum (SWCO) was carri ed out as per OECD guidelines 10 . Young Swiss male mice (20 ± 2g body weight) obta ined from the Animal B reeding Fac ility of ITRC , Lucknow, were used fo r the s tudy. They were maintained on pe lle t diet (Hind Lever) and wate r ad libitum. T wenty four animals were randomly divided into group I to IV of 6 mice each. Abo ut 1 inch2 of the inte r scapul ar space was shaved for apply ing SWCO. Each animal of group I, II , III or IV recei ved one applicatio n of 0.012, 0.02S, O.SO or 0 .100 g of SWCO , dissolved in 0.2ml of ether by oce! usio n patch tes t method II. These doses corresponded to O.S, 1.0, 2.0 or 4 .0 g/kg body weight. The animals were watched fo r changes in behav iour, food and water intake. Afte r 24 hr, the patch was removed and any skin changes like redness , oedema o r eschar were observed . Animals were furt her watched till 14-days, for above changes and any morbidity/mortality .

At 14 days, the animals were sacrificed by decapitatio n under anaesthes ia. Free fl ow ing blood was co llected in heparini zed conta iners fo r haemato logical assays. Autopsy was performed and organs like li ver, kid ney, adrenal, brain , and lympho id organs li ke, spleen, thymus, peripheral lymph nodes (ax ial and inguina l) and mesente ri c lymph nodes were collected and weight recorded . The tox ic dose was deri ved on the bas is o f changes in mo rbidity , food and water intake body weight/organ we ight ratio , haemato logy and gross patholog/o as per OECD guidelines .

Sub acute (28 days) dermal toxicity study ­Animals obtained as above were di vided into groups I to IV of 6 animals each. Multiple groups were set up as per parameter. A nimals of each gro up were treated dermally fo r 28 days with the fo llow ing dose of SWCO di ssolved in anaestheti c ether, by occlusio n patch test method II. Gr. 1-0.2 ml of ethe r o nl y Gr. II - 0.2 ml of ether conta ining 12 .S mg SWCO (O.S g/kg body wt) Gr. 1lI - 0 .2 ml of ether containing 2S.0 mg of SWCO (l .0 g/kg body wt)

Gr. IV - 0.2ml of ether conta ining SO.O mg of SWCO (2 .0 g/kg body wt)

Pathotoxicological studies-The animals were watched fo r changes in behav io ur, food and water intake throughout the treatment peri od. Fo rty e ight hours afte r the last dose at 28 days, 6 mice fro m each group were weighed and sac ri f iced by decapitation under anaesthes ia. The blood was collected fo r vari ous haematological paramete rs (total RB C count , Hb, ESR, PCV , TLC, DLC). Autopsy was performed and organs like live r, kidney, testes, brain , adrenal g land , spleen, thymus, mesenteri c, and periphera l (ax ia l and inguina l) lympho id nodes were weighed. The lympho id organs were immed iately pl aced in cold (4°C) RPMI-1 640 fo r assess ing the immunotox ic effec ts as described elsewhere I2-IS . Brie fly , fo r assess ing the effect o n humoral immunity , the treated and contro l mice were immunised intraperitoneally

with SRBC (S x 108 ce ll s) . On fo urth, day, the number of primary antibody (JgM) forming cells was assayed in spleenI6-18

. The anti-SRBC primary antibody (lgM ) ti ter of serum was assayed by titratio n against SRBC in 96 we ll titer plates. The hi ghest dilution of the serum that prod uced agg lutination of SRBC was noted and expressed as log2 titer l 8

.

The effect of SWCO on the cellul ar immunity was studied by assess ing the del ayed type hypersensiti vity (DTH) response to SRBC, as described earli er l

,}.2 )

Briefl y , 48 hr afte r the last application, the mi ce were

sens iti zed with 1 x 106 SRBC, injected sub cutaneous ly in the inte r scapular region . They were

cha llenged S day later with 1 x 106 SRBC injected subcutaneously in the left hind foo t-pad. The right hind foo t-pad was injected with sterile physiological saline and served as contro l. The increase in foot pad thickness was measured 24 hr after challenge, using a di al ca liper (Mitutoyo, Japan). Results were expressed as pe rcent increase in thickness of the left hind foot pad co mpared to the non-challenged ri ght foot pad .

The effect of treatment on the indirect immuni ty was studied by enumerating the phagocytic acti vity and reduction of nitroblue tetrazo lium (NBT) dye by perito neal ex udate cell s (PECs) . T wenty-four hours afte r las t dose, the PECs were aseptically co llected in Hank 's ba lanced sa lt so lution containing heparin and feta l calf serum I9.21. Afte r assess ing viability of PECs,

popul ati on was adjusted to S x 106/ml. The assay system fo r the reductio n of NBT dye by PECs was essenti a ll y the same as described earli e rl s.19.21. Bri efly,

S x 106 PECs were incubated in a medium conta ini ng

594 INDIAN J EX P BIOl, JU NE 2003

NET dye, for one hour. Th e PECs were then spinned down, washed with phosphate buffered saline and lysed by NaO H, to re lease inges ted dye. It was reacted with pyridine and the amo unt of fonnazan produced was measured at '5 J 5 nm and ex pressed as optical density.

Stalistical Clnalysis- The results were expressed as mean ± SE. Comparisons were made with appropriate control, employing a two-way students' t­test. Difference were considered significant at P<O.OOI to 0.05 .

Results Metal content of SWCO - The meta l content of

SWCO was apprec iably above the permissible level when compared with safe levels in water. Almost all the important metal like Cd, Cr, Fe, Pb and Zn were above permissible level. Only Cu, Mn and Ni were below the permissible level (Table 1).

Acute (j 4 day) toxic dose determination study -The animals showed no changes in behaviour, food and water intake after dermal app lication of SWCO. No mortality was observed. The animals treated with 2.0 and 4 .0 g of SWCO showed a decrease in body weight ga in . Vital organs like liver, kidney and adrenal showed no significant increase in the weight with lower doses but the weight gain was quite appreciab le with higher doses (2.0 and 4.0 g). The lymphoid organs like spleen and thymus showed an appreciab le increase in weight with all the doses. The peripheral lymph nodes showed appreciable reduction

in wei ght with 1.0 and 2.0 g doses and an increase with hi g hest dose, when compared to 0.5 g dose. The mesenteric lymph node also showed non-significant increase in weight with a ll the doses (Table 2). The total e rythrocy te count and TLC of animals showed a non-significant increase with all the doses, and corresponding increase in the haemoglobin content. The ESR and PCV values showed minor non­sig nifi cant variations with all the doses when compared to lowest dose. The treatment showed a dose-dependent increase in WEC count (Tab le 3). Since the mice treated even with a dose of 2.0 g/kg showed a decrease in the body weight gain in 14 day tox icity study, this dose and its lower mul tiples (1.0, 0.5 g/kg body weight) were used for 28 day tox icity study. Thi s dose leve ls is also recommended by OECD lo

.

Table I - Metal content (ppm) of SWCO

Metal Content in Desired leve ls SWCO in water*

Cd 0.040 0.0 10

Cr 0.173 0.050

Cu 00413 0.972

Fe 3.650 0.300

Mn 0.315 0.980

Ni 0.048 0.962

Pb 3.040 0.955

Zn 60480 0.994

*Reference No. 35

Table 2 - Percent body weight/organ weight rat io of mice treated dermally with SWCO at 14-days

[Values are ± SE of six animals and not s ignifica nt in all the observatiuon]

Dose Bod~ weight liver Kidney Spleen Thymus Pl Ml (g/kg) Initi al Terminal

0.5 28.86 ± 1.76 30.83 ± 1.05 5.30±0.69 1.35 ±0.05 o 73±0.025 0.22 ±0.02 0.35±0. 18 0041 ±0.03

1.0 28. 14± 1.05 32.00 ± 1.77 5.78±0.60 1.28 ±0.05 0.92±0.11 0.24±0.0 1 0.21 ±0.04 0.70±0.18

2.0 30. 14 ± 1044 21.00 ± 1.03 6. 14 ± 1.1 3 J .88 ±0.09 1.14±0.09 0.25 ±0.03 0.26±0.06 0.57 ± 0.O8

4.0 2857 ± 1.2 1 22.00 ± 1.67 6AO± IA3 1.66 ±0.30 1. 13 ±0.26 0.23 ±0.01 0.62 ±0.17 0.63±0. 11

Pl = Peripheral lymph nodes; Ml = Mesenteric lymph nodes

Dose (g/kg)

0.5

1.0

2.0

4.0

Table 3 - Haematological picture of mi ce treated dermally with SWCO at 14 days

[Values are ± SE of six animals and not significant in all the observations]

TEC Hb ESR PCV TlC (x 106

) (g%) (I Hr) (mm) (x JOJ)

7.00±0.93 9043 ± 1.14 0.67 ±0.33 55 .83 ±3.66 3.JO±0.54

9.50±0.76 11.26 ±0.9 1 0.50 ±0.22 45 .00 ±4.56 3.50 ±0.34

9.00 ±0.89 10.95 ±O.96 0.33 ±0.2 1 40.83 ±4.60 4.60±0.67

9.20 ±I A5 9.94 ± 1.03 0.60±0.24 50.80±3.89 8.60 ± 1.96

Adrenal

0.020 ± 0.0 I 0

0.020 ± 0.004

0.030 ± 0.0 I 0

0.030 ± 0.002

KHANNA & DOGRA: IMMUNOTOXICOLOGICAL EFFECT OF SLUM OF WASTE C RANKCASE OIL 595

Sub acute (28-days) immul1otoxicity study-The animals treated with the hig hest dose revealed an apprec iab le decrease in body weight ga in as compared to contro l animals. The animals treated with middle and highest doses had no appreciable effect on weight of liver, but treatment with lowest dose resulted in appreciable increase (Table 4). Treatment with middle dose resulted in a non-sig ni fica nt decrease in weight of kidney , and an increase with lowes t and highest dose. Similarly treatment with middle and hi ghest doses resu lted in reduction in weight of spleen. The higher doses a lso caused an increase in the weight of thymus, wh ile the lowest dose had no effect. However the middle dose caused a sli ght increase in weight of peripheral lymph nodes while the hi ghest dose caused a decrease. The treatment with lower dose resulted in highly signi fica nt increase in the cell popul ation of spleen, whi le the middle and hi ghest dose caused a reduction , which was signifi cant with highest dose.

All the doses caused an increase in the ce ll popu latio n of thymus but on ly middle dose showed a significant increase. The lowest and middl e doses caused non-sig ni ficant in crease in the ce ll populati on of peripheral lymph nodes and a reductio n with highest dose. All the doses caused a non significant increase in cell popu lation of mesenteric lymph node but the changes were more marked with lower doses (Table 5).

Treatment with lowest dose caused a non­sig nifi cant increase in RBC count wh ile the middle and hi ghest dose caused in s lig ht decrease. All the doses caused a decrease in haemoglobi n content but it was sig ni ficant only with middle and highest doses. The ESR and PCV counts showed a non-significant decrease with midd le and hi ghest doses. The lower doses resulted in significant increase in WBC count, but the increase with the highest dose was not significant (Table 6).

Table 4 - Percent body we ight/organ weight ratio (g) at 28-days de rmal ex posure

[Values are ± SE of six animals and not signi fican t in a ll the observat ions]

Dose Body Li ver Kidncy Adrenal Splcen Thymus PL (g/kg) weight

0.0 27.S0±0.6S 5.64 ± 0.57 1.27±0.27 0.030 ± 0 .00 I 0.96±0.16 0. 16±0.02 0 .33 ±0.03

0.5 28.33 ±0.33 7.09±0.2S IA7 ±0.08 0 .020 ± 0.002 1.36±0.17 0 .1 6±0.01 03 1 ±0.03

1.0 27.20±OAO SAL ±0.23 1.22±0. 10 0.020 ± 0.002 0.87 ±0.07 0.20±0.20 OA7 ±0.09

2.0 24.75 ± 1.3 1 S.21±0. 14 IAS±O.IO 0.020 ± 0.002 0.80±0.13 0.19±0.02 0.23 ±0.03

PL - Peripheral lymph node; ML - Mesen teri c lymph node

Table 5 - Effect of SWCO on cell count (x 106) and viability* of lymphoid organs at 28 days of

dermal ex posure

[Values are ± SE o r six animals]

Dose (g/kg) Spleen Thymus PL ML

0.0 289.23± 17.37 23.33 ±SA7 32.60±2.67 3S.33 ±7.73 0.5 552.33 ± 3 1 .89+" 4 1.00±S.69 48.83 ±4.83 SO.80±4.17

1.0 2S7 .24±40.17 48.00 ± 2.86+b 37.S0±2.37 4S.S0±6.S I 2.0 208 .80± 13.S lb 46AO±9.01 23.84 ± 2.06 36.67±S. 13

*Thc cell viabi lity was more than 95%; "P<O.OOI, bp <O.O I

Table 6 - Haemato logical piclUre of mice treated dermal ly with SWCO fo r 28 days

[Values are ± SE or six animals]

Dose RBC Hb ES R PCV WBC (g/kg) (x 106

) (g%) ( I hr) (mm) (x 10J)

0.00 7.98± 0.29 13.02 ±0.86 1.82±OA8 40.60±3.S7 7A4±0.S7 0.5 9AS±0.74 10.80±0.S6 1.40 ±0.24 44. 17 ±0.9S 12A3±0.92"+

1.0 7.69±0.2 1 8.50 ± 0.25''' 1.25 ±0.2S 30.17±3A9 11 .57 ± 0 .9S+b

2.0 7.60±0. 14 9.88±OA I-b 1.25 ±0.2S 37 .60±4.09 8. 18 ± 0.3 1

"P<O.OO I, hp<O.O I

ML

0.5 1 ±0.06

OA4±0.02

0.38 ±O.OS

OA6±0.OS

596 INDIA N J EXP BIOl, JUNE 2003

All the doses had a significant dose dependent depress in g effect on the primary antibody (IgM) forming cells of spleen. However the total antibody titer of serum was only slight affected by all the doses. The lower and middle doses had a stimulatory effect on delayed type hypersensitivity but the highest dose had a depressing effect. The reduction of NBT by PECs was depressed by all the dose levels but it was significant with highest dose only (Table 7).

Discussion Pol lution of aquatic surface due to accidental

spillage of petroleum products is well known. Such a pollution also affects the aquatic life2

.3

.6 .The severity

of toxicity however varies with concentration of oil per square meter of water surface and the type of oil. Contamination of water surface with 100 ml of oil per square meter is more toxic than 5 ml per square meter22 .The problem of pollution by SWCO has not received much attention and there is hardly any literature available under this heading. The authors of this paper opine that, the literature on aquatic pollution and toxicity of WCO, especially to aquatic birds, also includes effects of SWCO. It is known that, weathering process causes a number of physical and chemical changes in WCO suspended on the surface of water23-26. Organic compounds with higher vapour pressure (toluene, benzene) get volati lised to atmosphere, while semi volati le compounds (benzo(a)pyrene, nephthalene) and metals, partition to the water or sediment phase27

-3o. Access of WCO to

water bodies, thus results in the formation of two phases, water insoluble fraction , the scum (SWCO), and the water soluble fraction (WSF) containing organic and inorganic fract ion of WCO. The SWCO being lighter than water, remains floating on the surface and can attach to the skin of aquatic birds and produce toxicit/ I

-33 . Similarly, the scum may affect

the animals and human beings entering such water bodies. Thus, there is a need to understand the toxicity of such a product.

The 14 day treatment of animals with higher doses (2 .0 and 4.0 g/kg) of SWCO had a highly significant depressing effect on body weight gain. On the other hand there was an inconsistent effect on the weight gain of the vital organs. The hi gher doses resulted in disproportionate increase in the weight of vital organs. The higher doses were therefore considered toxic to the animals. The dose of 2.0 g/kg was used for further 28 day study as it produced minimal changes.

The 28 day treatment with all the doses resulted in norma l body weight gain, suggesting that there was no drastic systemic effect. The vital organs also showed no significant effects on their weight gain. The peripheral and mesenteric lymph nodes showed a slight depressing effect in their weight gain. The changes were more marked in peripheral lymph nodes. These nodes were therefore primary target of toxic effects of SWCO, as they drain thi s interscapu lar region of skin34. The toxic effect was also evident from the decrease in cell population of these nodes. The middle and the highest doses . had a non significant depressing effect on the total RBC count, ESR and PCY but the total WBC count was not affected. The treatment significantly suppressed the antibody forming cell population of spleen but had no effect on the all over antibody levels of serum showing a bidirectional effect on the cellular immunity.

The SWCO used in ' the present study had a high content of metals like Cd, Cr, Fe, Pb and Zn as compared to desired levels in water35. These metals may have been derived from the wear and tear of engine lining4. These metals, especially the lead, which has been implicated for toxicity of WCO to duck eggs, may have been responsible for the depression in the number of antibody forming cells36. The present studies have therefore shown that, SWCO enriched with metals can exerts an adverse effect on immunological competence of animals. Therefore, the disposal of WCO and its impact on aquatic life, warrants special attention.

Table 7 - Effect of SWCO of immunological parameters at 28 days

Dose (g/kg)

0.0

0.5

1.0

2.0

"P<O.OOI

[Values are ± SE of six animals)

PFq 10D/ml) HA(log2) DTH(%) NBT(OD)

219.75±3.76 7.65± 1.77 9.49±2.8 0.256 ± 0.0 13 16 1.34± 7.59'01 7.99±0.33 14.42± 1.02 0.235 ± 0.030

54.5 1 ±4.78-a 6.82 ± 1.19 10.25 ±2.32 0.198± 0.053

34.63 ± 1.7]'01 8.32 ±0.41 6.44 ± 1.90 0.152 ± 0.033

KHANNA & DOGRA : IMM UNOTOX ICOLOGICAL EFFECT OF SLUM OF WASTE CRAN KCASE OIL 597

Acknowledgement Th anks are due to Director, Indu strial Toxicology

Research Centre, Lucknow for hi s keen inte rest in the work. Thanks are a lso due to Sri Lalji Shukla, Sri S.P. Dhruva and Sri R.S . Verma for technical help.

References I CCME (Canadian Council of Mini ster of the Enviro nment),

'Used Oil Management in Canada: Existing Pract ices and Alternatives " Prepared by Monenco Consultants Ltd ., for the Indust ri al Programs Branch, Conservati on and Protection, Environment-Canada, Ottawa, Ontario, CCME-TS/WM­TRE007, 1989.

2 CH2M Hill Engi neering Ltd., Environmental Risk (~l Waste Crankcase Oil , Prepared for the office of Waste Management, Conservation and Protection , Environment, Canada, Ottawa, Ont.1992.

3 Environment Canada, Personal communication wi th F. Colin Duerden, Manager, Toxic Chemical and Waste Management Branch, Atlanti c Region , Dartmouth , N.S. 1993.

4 Headtake S F & Puglosi F A. Effect of waste oil on the survival and reproduction of the American nag fi sh, lordanell a noridae, Can J Fish Aquat Sci, 37 (1980) 757. '

5 Personal communic'!tion, Prof. Chauhan , Deptartmental of Pathology , GB Pant University o f Agriculture and Technology, Pantnagar, India 2001.

6 Canadian EPA, Waste crankcase oils: Priority substance list supporting document, Canadian Environmental Protection Act. Govt. of Canada, 1994.

7 EPA, Test Methods for Evaluating Solid Waste. Vol. IC, Laboratory Manual on Physical/Chemical methods (1990) 1311.

8 Schroeder H A & Nason A P, Clill Chem, 17 (1971) 461. 9 AOAC, Official methods of AOAC International, edited by

Patricia Cunniff, 16th edition (AOAC international Gaithersbury , Maryland, USA) 1998.

10 OECD, Guidelines, for testillg of chemicals, No. 402 and 410, Organisation for economic cooperation and development, OECD Publication Service, Cedex 16, France 1993.

II Drai ze J H, Woodward G & Col very H 0, Methods for the study of irritation and toxicity of substances applied topicall y to skin and mucus membranes. J Pharmacal Exp Ther, 83 (1944) 377.

12 Cunningham A J & Szenberg A, Further improvement in the plaque technique for detecting single antibody forming cells, IlIIlIIu/l.ology, I (1968) 599.

13 Koller L D, Exon J H & Brauner J A, Methyl mercury: Decreased an tibody formation in mice, Proc Soc Exp Biol Med, 155 (1977) 602.

14 Luster M L, Dean J H & Moore J A, Eva luat ion of immune functi ons in tox icology , Methods ill Toxicology, edited by W. Hayes (Raven Press, New York ) 1982,56 1.

15 Dogra R K S, Khanna S, Srivas tava S N, Shukla L J, Chandra K, Saxena G & Shanker R, Immunomodulation due to exposure to styrene and di octyl phthalate in mice, llI1mullopharmacollllllllunotoxicol , 15 ( 1993) 491.

16 Koller L D, Exon J H & Roan J G, Humoral antibody response in mice, after single dose exposure to lead or cadmium, Proc Soc Exp Biol Med , 151 ( 1976) 339.

17 Vos J G, Van Logton M J , Krae fenberrg J G & Krui ziga W, Hexachlorobenzene induced stimulation of the humoral immune response, New York Acad Sci, 320 ( 1979) 535.

18 Hudson J & Hay F C. Practical 1lIlIllUIlUlogy, 3rt! cd.

(B lackwell Scientific, Oxford) 1988. 19 Thong Y H & Ferrante A, Effect of tetracycline treatment on

imm unologic response in mice, Clin Exp Immun ol, 9 ( 1980) 720.

20 Ross G D, Detcction of complement receptors and Fc receptors on macrophages, in Manual of Macrophage Methodology: Co/lection characterisatioll & functioll , edited by Herscowitz H S , Holden H T, Bellante J.A. & Ghaffar A. (Marce ll Dekker, New York) 198 1.

2 1 Vos J G, Kreeftenbrerg J G, Engal H W B, Minderhoud A & Van Noorle Jansen , L M, Studies on 3,7,8-tetrachlorobernzo­p-dioxin induced immune suppress ion and decreased resistance to infection. Endotox in hypersensitivity, se rum zinc concentration and effec t of thymosin treatment, Toxicology, 9 (1978) 75.

22 O'Brien P Y, Dixon P S, The effect of oil and oil components on algae: A rev iew, Br Phyto J, I (1976) 11 5.

23 Nocaodem D E, Fernandes M C Z, Guedes C B, Correa R l , Photochemical process and environmental impact of petroleum spills, Biochemistry, 39 (1997) 121.

24 Carls M G, Rice S D & Hose J E, Sensitivity of fish Embryo to weathered Crude Oil. Part I. Low level exposure during incubation causes malformations, ge netic damage and mortality in larval pacific herring (C lupea pallasi) , Env Toxicol Chem, 18 (1999) 481.

25 Hansen H P, Photochemical degradation of petroleum hydrocarbon surface film on sea water, Marine Chelll, 3 (1975) 183 . "

26 Payne J R & Phillips C R, Photochemistry of 'petrOleum in water, Environ Sci Tee/mol, 19 (1985) 569.

27 Ray mond R L, Hudson J 0 & Jami son V W, Oil degradation in soi l, Appl Environ Microbial, 31 (1976) 522 ..

28 Supernant N F, Battye W H, Fennelly PF &BrinknW.n D W, The fate of hazardous and nOll hazardous waste in used oil disposal and recycling, Prepared for the United State Department of Energy , Washington , 1983.

29 Literathy P, Haider S, Samhan 0 & Morel G, Experimental studies on biological and chemical oxidation of disposed oil in Sea water, Water Sci Technol, 21 (1989) 845 .

30 Bobra M, Photolysis of petroleum , Prepared by Consul tech for Environmental Protection Directorate, En vironment Canada, Ottawa, Ontario, 1992.

31 Vazquez-Duhalt R . Environmental Impact of used Motor Oil, Tile science of total ellvironment, 79 (1989) I.

32 Albres P H, Transfer of crude oil from contaminated water bird eggs, En viron Res, 22 (1980) 307.

33 Hoffman D J, Eastin Wc. Jr & Gay M L, Embryotoxic and bioc hemical effects of waste crankcase oil on bird eggs, Toxico/ Appl Pilannacol, 63 (1982) 230.

34 Kuper C F, Schuurman H J & Vos J G, Pathology in illlrlwn%xicology, in Methods in imlllul1()/oxicology, Vol. I, edi ted by Burleson G R, Dean J H & Munson A E (Willy Li ss, New York) 1995, 403.

35 Indian Standard Drinking Water-Specifications. Second. Reprint. (Bureau of Indian Standards, New Delhi), 1995 .

36 Koll er L D, Immunotox ico logy of heavy metals, Ill tem J llIllllulloplwrl7lacol, 2 (1980) 269.

37 Hoffman DJ, Embryotoxicity and teratogenicity of environmental contaminants to bird eggs , Rev Enviroll COlltam Toxicol, 15 (1990) 39.