Bull. Environ. Contam. Toxicol. (1988)41:352-359 �9 1988 Springer-Verlag New York Inc.

E n v i r o n m e n t a l C o n t a m i n a t i o n

~and Tox ico logy

Effect of an Anionic Detergent on the Lipid Moieties of Various Cell Types in the Opercular Epidermis of Rita rita

Debasish Roy

Department of Zoology, Banaras Hindu University, Varanasi-221 005, India

With the fise of industrial civilization, the use of synthetic dstergents as cleansing agent in India has become so wide spread that if has aven replaced the traditlonal soap. This extensive use has created havoo by pollutin9 the uhole of the aquatic eco- system and th~eatenin 9 the Government for its immediate vemediee. Various reports conce~ning the effects of synthetic detergents on different organ systems of freshwater and marine fiches have been reviewed by Abel ( 1974)o But very less attention has besn given on the histopatholo9y of epidermis (8ardach et al. 1955; Pohla- Gubo and Adam 1982; Roy 1987) especially the change in the cyto- logy and cytochemistry influenced by the detergent. Zaccone et al. ( 1985)~howeverp studied the pattern of change of activity of feu enzymes of the epidermis of Heteropneustes fossilis under the Infl- uence of an anionic detergent~ sodium alkyl benzene sulphonate (LAS)

Biological membranes are mainly composed of lipids and proteins to which carbohydrates may be covalently bound and itis probable that the detergent may come in contact with the membrane and exert its influence. Therefore~ the aim of thie investigation has been to exa- mine the histochemical change caused by an anionic detergent, do- decylbenzene sodium sulfonate in the various lipid moieties of different cell types in the opercular epidermis at different rime intervals of exposure.

MATERIALS AND METHODS

Fish, Rit__~a rit__~a (14~ 2 cms in length) were collected from rivers- ide of Ganges at Varanasi,U.P. durin 9 January and kept for 30 days in optimum laboratory conditions for acclimatization. Static bio- assay test was performed to determine g6-hr LCSO value of an an�8 chic detergent, dodecylbenzene sod�8 sulfonate (C19-LA$) obtain- ad from Koch Light Laboretories~ Colnbrook~ England~ to the fish following the methods of APHA,AWWA~WPCF (i5th sdn.). 80 fish were divided into 8 batches having tan fish in each batch and were kept in aquaria of size (60cms x 30 cms x 45 cms) containing 30 litres of tap water to avoid crowding. The tap wa~er used was having pH 7.1 ~ 0.3, hardness 158 mgs/litre (as CACO5) , dissolved oxy9en

Send reprint requests to D.Roy~464,Saheednagar,Bhubaneswar, lndia.

352

5.5 to 8.4 ppm and temperature 21 ~ 0.8~ The first batch expos- ad to normal tap water only wae treated as control and ai1 others as experimental. In all the experimental aquaria, 207 mgs of det- argent per aquarium i.e. for 30 litres of water at the rate of 5.9 mgs/litre (g6-hr LC50 of the detergent) were dissolved in a small amount of water end properly mixed in the aquaria. Care was taken to avoid froathing. Dead fish,as soon as detected were st once discarded. Control as well as experimental fish were sacrif- iced st frequent intervals (15minp 30minp 45minp I hourp 2h~ 3hp 6h~ 12h t 1 dayp 2d, 3d~ 4d™ 5d~ 6d~ 7d, 8d ) . Opercule of the fish (right side only) were diseectad out and fixed in 10% neutr- al formalin and formel calcium for 6 hours. Frozen eections were cut st 10-159um using cryostat (AO modal 849C) st-30 ~ C and wsre mcunted on clean g�8 slides and dried for 10-15 minutes st room temperature (20-25 ~ C) before subjecting them to histoch8mical techniques for the damonstration of various lipid moieties. For controlsp sections were elso subjected to different extration procedures following Bayliss High (1982) in order to confirm the staining reactions.

RE5ULTS ANO DISCUSSION

With 5udan black B with and without prior treatment of various extraction techniques, the epithelial cells throughout the operc- ular epidermis indicate the presence of moderate amount of lipids. The intansity of Sudan black B colour enhances greatly after bro- mine watsr treatment. This colour intensity markedly gets reduced with prior acetone treatment. Prior chloroform methanol treatment completely extracts the lipid contente of these cells which also give moderate reaetion for neutral lipids with Oil red O. Prior acetone treatment both st room temperature and st 4~ completely inhibits the Oil red© staining of these cells. Relatively a weak resction for phospholipids is observed with acid haematein techn- ique which toc is extracted by chloroform-meth8nol treatment with and without I% HCI and 4% H20. With nile blue sulphate, the eplt- helial cells stain bluish pOrple which becomes light blus if tre- ated with acetone prior to treatment with stain. Prior chloroform methanol treatments with or without 1% HCI or 4% HgO donot cause fading of this colour reaction with this technique~ These ce�9239 also contain small amount of fatty acids t cholesterol and choies- terol estsrs as evidenced by moderate reaction with various tech- niques (Table I).

Transferring the fish into the detergent medium for threa or more days causes disappearance of most of the lipid moieties from the epithelial celle as evidenced by negative reactions with most of tha lipid staining techniques. Such condition prevails througho- ut the period of the experiment. However t onl~ neutral lipids co- uld be Ioc~ted in these cells upto 5 days of detergent treatment t aftar which the reaction for neutral lipids is lost also~euggest- ing complete disappearance of all the types of lipid moieties from the epithellal cells of the ope~cular epidermis.

The secretory products of the goblet mucous cells~distributed ma- inly in the outer layer of the opercular epidermis remaln unstain -~

353

L~

L~

Table I. A

summary of ths hietochemical

changes in the lipld constituante

of the epithelial in the

opercular epidsrmis of Rit.~a rira st va~ious intervals of detergent

exposure.

Chemical

Histochemical

techniques

Colour

Constitu

ents

Epithelial celle (after days of exposure)

Ge

ne

ral

lipids

symbol

Outer layer

Middle layer

Basal layer

5udar black

B

Blb

Acetone/Sudsnblack

B

Bro

min

e/$

udan bla

ck B

B

C

hZoro

form

-meth

anol

uit

h &

w

ithout

I%

HC

I or

4~ H20 / Sudan black 8

C

1 2

3 4

5 5

t08

C

1

2 3

4 5

6to

8

C

1 2

3 4

5 6

toB

§

+ +

§

- §

§ §

24

7

- +

+ +

+§

-

++

+

§

+ +

+ --

++

+

+

++

+

+ +

--

m

++

+

+

++

+

+ --

--

Ne

utr

al

Oil

re

d

0 R

+

+ +

+ +

+ -

+ +

+ +

+ +

- +

+ +

+ +

+ -

lipids

Acetone/Oil Red 0

-

Dif

fere

nti

a-

Nil

e

blu

s

su

lph

ats

P

ur

+ +

+ +

++

-

+ +

+ +

+ +_

. -

+ +

+ +

+ +

- tion of neu-

Acstons/Nile

blue

BI

+ +

+ t~al and acl- sulphate

dic lipids

Chloroform-methanol

with

BI

+ +

+

Phospho-

lipids

&

wit

ho

ut

1%

HC

Z o

r 4%

H

20

/Ntl

e

blu

e

su

lph

ats

Bro

min

e/a

ce

ton

e/

Blb

+

+ +

Sudan black B

Chloroform-methanol

with

- &

without 1% HCI or

H^O/Bromine/acstons/

S~dan black B

Acid haematein

BI

+ +

+ +

- -

- Chloroform-msthanol

with

- &

without I% HCI or 4%

H20/acid hsematein

+ +

+§

--

+ +

§

+ §

+ §

-- --

--

+ §

§

--

Table 1

Continued

A summary of

th

e histochemical changes in

thelipid constituante of

the epithelial in the opercular

epidermis of Rira rira ai various intervals of detergent exposuCe.

L~

Chsmical

Histochemical techniques

Colour

Constituents

symbol

Free fatty

Copper ~ubeanic acid

G acids

Acatone/Copper

- rubeanic acid

Cholesterol

Perchloric acid naph-

P &

cholesterol thoquinone(PAN)

esters

Cold acetone/PAN

-

Free

Digitonin/PAN

P cholesterol

Digitonin/acetone~PAN

-

Triglyce~ides Calcium-lipase

Acetone/

calcium-lipase

Epithslial cells (after days of exposure)

Du

rer

lay

er

Mid

dle

la

ye

r B

as

al

lay

er

C 1

2 3

4 5

6to

8 C

1 2

3 4

5 6t

o8

C 1

2 3

4 5

6to

8 +

+ +

§ +

+ +

+ ....

+ +

+

+ +

+

Symbole: Blpblue; BLbpbluish black; G,green; P~

pink

; R~red; Pur~purple;-~negative; ~~

ueak;

+tmoderate; ++~strong resction. C~ control conditions

ad w�8 most lipid stains except with nile blue sulphata which et- aine the contente purple which le acetone lebile.The peripheral areas of these cells stain bluish black and deep black uith 5udan black B and bromination/$udan black B techniques and appear red with Oil red 0 which gets extracted by prier acetone and chlorof- orm-methanol treatment with and uithout I% HCI er 4% H_O t~eatme- nts. The peripheral areae of the goblet mucous celle a~so stain blus uith nile blue sulphate and/uith copper rubeanic acid for free fatty acid m bluish black with acid haemateln for phospholip- ids and pink with PAN for cholesterol estere uhich are extracted by various extraction procedures applied in this study. The secr- etory contsntsp however~ remain unstained for tr�8

3 days of detergent treatment induces a decrease in most of ths lipid moieties in tha peripheral regions of the goblet mucous celle. However~ with acid haematein test a weak reaction for pho- epholipids persiste upto 5 days after which no reaction for eny of the lipid moieties could be observed till the end of the expe- riment. The secretory contente t like those of the control condit- ion~ remain unstained throughout.

The perinuclear area and the boundaries of the club cells,distri- buted throughtout in the opercular epidermis stain positively with 5udan black B that is intensified by prier brominationp and stain purple with nile blue sulphate. When stained with these methods after prier extraction (Table 2), these cells show negative reac- tion. These areas give weak reactions with copper rubeanic acid technique for free fatty acids~ with acid haematein test for pho- spholipids and with PAN test for choleeterol and cholesterol est- ets. The contente of ths club celle remain mostly unstained with various histochemical techniques applied in this study.

The intensity of resction for general lipids uith Sudan black B and Bromine/$udan black B, for acidic and nautral lipids with nile blue sulphate™ for free fatty acide uith coppsr rubsanic acid gets weaker and only e weak reaction is observsd upto 2 days after the fimh are transferred te the detergent medium. After this period, the �8 of reaction further decrssses and after 5 days,no reaction for these lipid moieties could be observed.

This study experiences a decreaees in various lipid moieties of the epithelial cellsp goblet mucous celle and club celle under the influence of the detergent treatment. The effect of the dete- rgent treatment on the mucous membrane is instant as evidenced by immediate profuse secretion by the celle of the epidermis (Roy 1987). The mucous coat effets a quick and first barrier te prevent the penetration of the toxicity of the detergent. This coat is™ howeverp net sufficient ner is a permanent ons but synthesis and secretion of mucus is a continuous process.

The toxici™ of a datergent is a function of both its surface activity and chemical toxicity (Abel 1974). Ha further explained that reduced surface tension might modify the esquence of evente during acute poisoning but definitely net the only cause of

356

Tabl

e 2,

A s.ummary of

the

hiat

oche

mica

l ch

ange

s in

th

e li

pid

constituante of

ths gl

and

cell

s in

th

s

L~

~J

opercular epidermls af

Ri

te ri

te

et

vari

ous

intervals of

d

ete

rge

nt

expo

sure

.

Chemical

Histochemical te

chni

ques

Constituante

General

Suda

n black B

lipi

da

Acetone/Sudan bl

ack

B Bromine/Sudan bl

ack

B Chloroform-methanol with

&

wit

ho

ut

I%

HC

1 o

r 4%

H

20

/Su

da

n

bla

ck

B

Neutral

Oil re

d 0

R +b

lipi

da

Acetcne/Oil re

d 0

- -

Diffe~entia-

Nile

bl

ue sulphate

Pur

+a

2b

tion

of

n

eu

- 81

tral and acid-

ic li

pids

Acetone/Nile

BI

+b

blue

eulphate

Chloroform-methanol wi

th

BI

+b

& wi

thou

t I%

HC

I or

4%

H2

0/Ni

le blue su

lpha

ts

Phospho-

Bromine/acetone/Sudan

Blb

+b

lipi

de

black B

Chloroform-methanol with

- &

with

out

1% HC

I or

4%

H~O/Bromine/acetone/

SOda

n black 8

Colour

symbo�8 C

Blb

+b

; ~2

b

Gland cells( after da

ys of de

terg

ent

expo

sure

)

Gobl

et mu

cous

ce

lle

Club

ce

lls

1 2

3 4

5 6

to8

C

1

2 ..

.. 3

4

, 5

,, 6

to8

§

.b

.b~ ~

b _"

m

§247

171

..

..

s

~b ~

b ~b

~b

~ s

: :

+b

+b

m

+a

+a

~~

b

+b

-

+b

-

m

m

!

m

m

o

m

m I m

+c

~d

+c

,d+

c~

d .

..

.

+a

+a

+a

..

..

m

~ m

~

m

+c,d+c,d .

..

..

OO

Ta

ble

2

Continued

A eummary of

the hietochemical changes in

the lipid constituante of the gland cella in the

operoular epidermie of

Rita rita at varioua intervale of detergent exposure.

Chemical

Constituents

Free fatty

acids

Cholesterol

& cholesterol

esters

Free Chole-

sterol

Triglyce-

rides

5ymbols:

Histochemical techniques

Colour

symbol

Gland oeils ( after days of

detergent expoeure)

Goblet mucoue cells

Club celle

C i

2 3

4 5

6toB

C I

2 3

4 5

6to8

Acid haematein

Blb

+b

Chloroform-methanol with

- &

without I%

HCI or

4~

H20/acid haematein

Copper rubeanic acid

G +b

Acetone/Coppe~

- -

rubeanic acid

Perchloric acid naphth-

P +b

oquinone(PAN)

Cold acetone/PAN

- -

Digitonin/PAN

p +b

Digitonin/

- acetone/PAN

Calcium-i~pase

- -

Acetone/calcium lipase

- -

+b

+b

+b

+b

+b

- +c,d+c,d+c,d .

..

.

+b

-

m m

m m

m

m m

m

m

m m

I m I

m u

+c,d+ctd+c,d .

..

.

+c~d .

..

..

.

m m

m m

m m

m y

m m

m ~

m

Blpblue; Blbtbluish black; Gjgreen; P,plnk; Rtred; Pur,purple;-~negative; ~~weak;

+~moderate; ++~strong;reactions

a,secretory contente; bpperiphery; c~perinuclear

area

; dtcellboundary. Ctcontrol condition

disruption.Biological membranes constituts extremely complex mix- ture of lipide,protein ions etc.. In spire of greater complexiti- es of membranes, if se~ms clear fo us that ths general principlss of detergent action apply to ail as well. There will be modifying factors dus to speciel structurel features of biological msmbranss. Itis believed that the lytic process in haemoglobin as described by Helsnius & Simon (1975) may also be opsrating in this case bain 9 a resultant of tha interaction betueen the surfactant and the li- pids of the membzane.

Although, ths question of skin permaability fo water is complex (Isaia 1984;Rankin & Bolis 1984) and the structure of the epider- mis is probebly mors important from this point of view than the chemistry of the secretions t the lipid in the skin surface sacre- tions may provide a barrisr betwean the internal and ths sxternal environment of the fish, acting as a water repellsnt and limiting the entry of water into the body of these spsclss.

Acknowledgements. Financial assistance by U.G.C.& I.C.A.R. t New Oelhi and the concerned Principal Investigator~ in the form of Research Fellow is gratefully acknowled9ed.

REFERENCES

Abel PO (1974) Tox�8 of synthetic detergents te fish and aquatic invertebrates. 3 Fish Biol 6:279-298

APHA~AWWApWPCF ( 15th edition) Standard methods for the examination of water and wasts water, Washington

Bardach 3Ep Fujiys M, Holl A (1955) Detsrgents: sffects on the chemical senses of ths fish Ictalurus na™ (le sueur) Science NY 148:1605-1607

Bayl iss High OB (1982) L ip ids. In:Bancrof t JD and Stevens A (ad) Theo~y and Pract ice of H is to log ica l Techniques Churchi l l L iv ingstone, Edinburg~ London~ Melbournep New York

Hslenius A, 8imon K (1975) 5o lub i l i za t i on of membranes by dstsrgents.Biochimica et Biophysic ia Acte 415:29-?9

Isa ia J (1984) Water and nonelectrolyte permeation. In:Hoar WS and Randall D3 (ad) Fish Physiology Vol X Academic Press, p 1-38

Pohla-Gubo G~Adam H (1982) �9 of the anionic active dete~gent Na-alkyl-benzenesulphonate (LAS) on the head epidermis of Juvenile rainbow trout (Salmo 9airdneKi Richardson) Zool Anz (Jena) 209:97-110

Rankin 3C, Bolis L (1984) Hormonal control of water movemant across the gills. In: Hoar W8 and Randall DJ (ed) Fish Physiology vol X.Academic Pr© t p 177-202

Zaccone,G~ Locascio Pi Fasulo Sp Licata A (1985) The effect of an anionic datergent on complex carbohydrates and snzymes activities in ths spidermis of the catfish Heteropneustes fossilioe (Bloch) Histochem 3 17:453-466

Received November 30, 1987; accepted April 2, 1988.

359