Indian Journal of Experimental Biology Vol. 4 1, November 2003, pp. 1306- 13 10

Oxygen uptake and filtration rate as animal health biomarker in Lamellidens marginalis (Lamarck)

Shami k Das & B B Jana*

Aquaculture and Applied L imnology Research Uni t, Departmenl of Zoology . Uni versity of Kalyani , Kalyan i 74 1 235. India

Received 28 j {/ J/uorv 200J; revised 22 May 2003

The freshwater bivalve. L. lIIargiJ/olis was experimentally exposed to 10 and 30 ppm concentrations of CdCl2 to exami ne fill rat ion rate, oxygen uptake and glycogen level of li ver and gill s for hea lth assessment ror their reuse in the reclamalion of cadmium intox icated environments. 111 situ experiment was also performed for better apprai sal of the fi ltration rate in the lake. Oxygen uplake in the treated group exceeded that of control by 15-22% during the early 24 hI' after cadmium exposure, bUI fo llowed an essential dec line (23-30%) thereafter. T he reduction of II llration rate ranged fro m 12-62% in laboratory to 83-85% in field tria ls. At the ti ssue level. glycogen content was reduced by 6 1-72% in li ver and 52-63% in gi ll. In both tissues . glycogen content was inversely proporti onal to the cadmi um contents or the animal. Crit ical appraisal of data suggests that the threshold va lues of cadmium in gill and l iver were 50-80 }J.g/g dw for oxygen uptake and 50-60 Ilg/g for fi l tration rate because of marked reduction of these parameters beyond the values of cadmium. It is concluded that filtra tion rate, oxygen uptake of the freshwater bi valve. L. lIIarg illolis can be used as biomarker for ani mal heal th assessment and for possible reuse of the stock animals.

K eywords: A nimal health, Fil trati on rate, Lalllell idell .l' lIIargillalis, Oxygen uptake

Cadmium, whi ch has long been recogni zed as a tox ic metal, has become a common aquatic pollutant in recent years due to its widespread use in industries like zinc smelters, electroplating, all oy and pi gment fac tories, etc. Cadmium has become potentiall y hazardous causing metaboli c, physiological, structural and functi onal di sorders of aquatic animals l

. The short term effects of cadmium tox icity are due to its binding with thiol (-SH ) groups and protein denaturation, while long term effects are attributed to DNA damage, when it accumul ates in the body of animals2

. At the ti ssue level, heavy metal causes hypox ia or anox ia leading to profound alterations in respiration and carbohydrate metabo li sm}·5 and fi ltration rate in some molluscs l

.

Filter feeding bi valve molluscs are often se lected and can be used fo r rec lamation of intoxicated aquatic environments due to their immense biofilter potenti aI6-

11. Nevertheless, the freshwater bivalve

Lamellidens marginalis undergo modi fications in their fu ncti onal response as well as biochemical characteri sti cs of ti ssue in response to ambient

*Correspondent author Phone: 033 -25826323 (R), 2580-92 12 (0) Fax : 033-2582 8282 E-mail : bbj @caI2.vsnl.nel.in:bbj icee @rediffmail.com

cadmium level. There are marked changes in the rate of filtration and oxygen co nsumption of the animal when cadmium accumul ation increases in the ti ssues over time. These ac ti vities can be used as biomarker fo r assess ing ani mal health before re-employ ing them in bioremediation of cadmium-pollu ted aquatic environments. The present study foc ussed on the changes in filtration rate, oxygen consumption and ti ssue glycogen of the freshwater bi valve mollusc, Lamellidens marginaiis for use as biomarker for monitoring environmenta l qu ality.

Materials and Methods

Oxygen uptake - Oxygen uptake and fi ltration rate in re lati on to ti ssue glycogen were examined in three parallel experiments in the Lamellidens ex posed to cadmium. Aquaria (36) fill ed with 3 liter ground water (PH 7.2-7.5) were treated with CdCI2 (analytical grade; 55.83% cadmium) at the rate of 0, 10 and 30 mg/l using 12 aquaria per batch. Each batch has essenti ally 6 aquari a with Lamellidens and 6 without. Adult Lamellidens (5 1 ± 2.5 g; 7.2 ± 1.5 cm), collected from a local pollution-free fi sh pond, were acclimatised for a week under laboratory conditi ons and 2 animals were placed in to each of 6 aquaria used fo r Lamellidens of each treatment dose (0, 10 and 30 mg/l ),

;

DAS & JA A: O2 UPTA KE & FILTRATION AS ANIMAL HEALTH BIOMARK ER IN £AMELL/DENS 1307

while the rem,lInmg 6 aquaria of each cadmium concentration without Lamellidens served as control.

The concentration of di ssolved oxygen of water and the routine water quality parameters were determined for each aquarium using specific DO probe (Jenway Model 9070) prior to, and at regular intervals after introducti on of Lamellidens into it. The oxygen conce ntration (mg/l ; weight ) was converted into volume (mill ) by dividing the fonner with 1.43 (ref. 12). Di ffe rences of DO (mill ) of aquari a water with and wi thout introducti on of Lal1l ellidens gave the oxygen uptake (VO") by Lall/ ellidens at that hour9.

FilTratioll raTe-Aquari a (3 liter capacity, 108) were fill ed with eut rophic water predominated by Euglena sp. and grouped into 3 batches for treatments with 3 doses of cadmium (0, 10 and 30 mg/l ). Chl orophyll-a content of phytoplankton in the aquaria water was determined to monitor filtrati on rate of Lalllellidell.,·. A pilot study indicated that applicati on dose of cadmium did not influence the chl orophyll -a content of phytoplankton in the aquari a.

Two acc limati sed Lamellidens (51 ± 2 .5 g; 7.2 ± I .) cm) were pl aced into each aquarium all otted to diffe rent dose treatments and held for 24 hr. Aquarium without cadmium treatment served as control. Eighteen aquaria, each representing both treated and control groups, were sacri ficed at different time intervals for determin ation of chlorophyll-a content of phytoplankton.

Chlorophyll-a content of phytoplankton was ex tracted in chilled 80% acetone for 24 hr from 100 ml water after filt ering through Whatman GFIC filter paper. The absorbance was measured at 665 and 650 nm and chlorophyll-a content was calculated following the method described by Holden 13.

An experiment in situ was performed to check the va lidity of laboratory data. Glass jars (3-1, 234) fill ed with lake water were suspended in a lake projecting sli ghtly above the surface water. They were grouped into three batches of 78 each and treated with cadmium in 0, 10 and 30 mgll concentration. Two Lamellidens (51 ± 2.5 g; 7.2 ± 1.5 cm) were introduced into each of the 39 jars representing all the doses of cadmium and held for 84 hr, the remaining 39 jars without introducti on of Lamellidens served as control. Arter spec ifi c intervals, three jars from each of the treatment and control group, were removed and used for es timation of chlorophyll-a content of phytoplankton. The chl orophyll-a content of water was then used to determine the filtration rate (FR) according to the formul a given by Coughl an 14 _

V FR = - x r(ln Co - In Ct) - (In Co - In Ct )l

n.t.

where, FR = filtrati on rate; V = volume of container; n = the number of mussels; t = time span; Co and Ct = the concentration of chlorophyll -a at time 0 and t respecti vely; and Clo and Cit = same for the bl ank .

Lall/ellidens used for determin ation of filtration rate were sacrificed for examining cadmium content in gill and liver following the method described by Walsh IS.

Cadmium and glycogen conrenT of g ill and li ver ­Out of 33 replicates for each treatment dose, 3 sets were sacrificed every sampling hour for estimation of glycogen and cadmium contents of gill and li ver. Gl ycogen content was determined foll owing the method of Montgomery' 6 using anthrone reagent in a spectrophotometer, and cadmium content by Walsh IS.

Data were statistica ll y evaluated using analys is of vari ance (A NOV A) and LSD test. The level of signifi cance was accepted at P < 0 .05 .

Results Oxygen uptake- Ox ygen uptake in Lamellidens

was hi ghly dose-dependent. The values tended to ri se graduall y till 24 hI' of exposure followed by decline. [n the treated groups, the rate of decline was maximum (23-30 %) between 144-168 hr. The oxygen uptake of Lamellidens held in capti vity without cadmium treatment was always significantly lower than the treated groups (Fig. I).

Filt ration raTe - Filtration rate of Lamellidens did not differ in control and treated groups (Fi g. 2) during

3.5 ~o ppm .....e- IO ppm .....- 30 ppm

0.5

12 18 24 }6 48 96 144 192 240 288 336

H o ur s

Fig. I - Pallern of changes in oxygen uptake by Lall/ellidellS arti f iciall y exposed to different concentrations of cadmium. Each point represent mean (±SE) for three repli cates w ith two animals per aquarium. Note the increased response up to 2 1 hr then de­cline gradually

1308 I DIAN J EXP BIOL, NOVEMBER 2003

the initi al 9 hr of cadmium ex posure, but the fi ltration rate in the treated group reduced 12-62% (in laboratory) and 83-85 % (in lake) more th an that of their respective control s. The filtration rate in the 30 ppm ex posed group was significantl y lower than that of 10 ppm till 50 hr. Differences were, however, not signi ficant during the rest of the peri od.

CadllliulII upraJ.:.e - The cadmium contents varied from 10-11.5 to 8.4-9.5 J1 g/g dw in li ve r and gill s, respecti ve ly of Lalllellidens held in aquaria without cadmium treatment. A t 30 ppm, cadmium cOlltent of gill (60 Il g/g dw.) was signifi cantl y higher than that of li ver (4 1 Il g/g dw.) during 24 hI' of ex posure, but the situation reversed thereafter (Fig. 3).

Challges 111 gIycogell COlllelll - The initial glycogen contents of li ver and gill (Fig. 4) were 4.38±0.85 and 12.75 ± 1.23 mg/g wet weight , respectively, but it differed between treated and contro l groups during the period of study. There was gradual decline in the values of glycogen with time; the per cent decrease over control was 6 1 % in li ver and 52% in gill at 10 ppm and 72% in liver and 63% in gill at 30 ppm. The values in control group remained almost unchanged up to 96 hr, but reduced considei'ab ly during the next 144 hours followed by gradual increase during the remaining hours of the study.

35

" 15 .~ 1:: ii:

] ::> E

~ S ~ :! c

. ~

~ Ii:

100

80

60

40

20

_____ 0 ppm

___ 10 ppm (A) --.- 10 ppm

9 12 16 24

-.- 0 ppm (B) -o- lOppm

-.- 10 ppm

12 10 48 84

H 0 u r s

Fig. 2 - Pallern of changes of cadmium induced f iltration rate of Lall/ellidens exposed to different concentrations of cadmium in fi eld experiment (A) and in laboratory experiment (B)

Discussion Signifi cant rise in oxygen uptake by the test an imal

Lalllellidens during the earl y phase of cadmium exposure was due to its high energy requi rement for increased metabo lic act i vity when there was physio logical adj ustment of the animal requiring high level o f oxygen to compensate cadmium stress of th e ce ll s. It becomes apparent from the data (Fig. 5) that the cr iti ca l level of ti ssue cadmium wa ' 50-80 Ilg/g dw. beyond which a decrease of oxygen uptake was inev itab le regardl ess of the dose of cadmium. Marked ri se in oxygen consumption due to cadmium tox icity was also evident in Ju venile Bay sca llop A rgopeclell

100 -- 30ppm. gill -- tOpprn. gill -- JOppl1l,li ", l'r -- IOppm.l l\ cr

~ 80 ." CD

Ob 70 ~

5 60 E 0 50 <.>

E ::> 40 E ."

30 '" :.;

20

10

12 16 24 30 36 42 48 60 72 84

H 0 U r S

Fig. 3 - Pattern of changes of cadmium accu mulation by LlIllleI­/idc l/ s in gill and li ver artifi ciall y exposed to di fferent concentra­ti ons of cadmium. Each point represent mean (±SE) for thrce replica tcs with two anima ls per aquarium. Note the increased response of cadmiulll over time in both thc ti ssue

16

4.5

~ 00 OIl} .§. c ~ o <.>

.?:-1.5 o

Liver

16 24 48 96 144 192 240 288 }}6

___ Oppn -e- . 10 ppn -.- l Oppm Gill

16 24 48 96 144 192 240 288 336

H 0 u r S

Fig. 4 - Pattern of changes of ti ssue glycogcn of Lame//idells exposed to different concentrations of cadmium

DAS & JANA: O2 UPTA KE & FILTRATION AS AN IMAL HEALTH BIOMARKER IN LAMELL/DENS 1309

4

y = 3E-06x' - 0.00 I x' + 0.0989x - 0.7036

~ 3 1;:, '" x 0

E ';;'2 "'" .. a " c 1;:, '" I Y-o

0

0 50 100 150 200 250

Tissue cadmium contenl (.(Jglg dw)

Fig. 5 - Rel atio nship betwee n the tissue cadmiulll induced ox y­gen uptake of cad mium ex posed Lallll' lIide/l .\·

16

14 y = 0.0005x' - 0.1345x + 13.919

20 40 60 80 100 120 140 160 180

y = 0.000 I x' - 0.048x + 5.0657

4.5

_ 4 i • ).5 ~

Gill !. ) ~ S 2.5

6' 2

1.5

o 20 60 80 100 1M I ~ 160 IW

Cadmium content «-gig dw)

Fig. 6- Relationship be tween the tissue cadm ium and tissue g lycogen content of LalllelLiden.\· showi ng an inverse relationship

irradians l 7, mud snail Nassorius obsoletus4 within the

concentration range of 500-940 ppb and gastropod Nassorius obroletus over an ex posure range of 0_5-4_0 mg/14

. Prolonged exposure produced similar effect even at a much lower dose (500- 1000 ppb for 30 days, 50 ppb for 60 day s4_

Glycogen is the immediate source of energy during stress I8

-20

, with the liver acting as a sink for metal ions and glycogen playing a significant role in glucose turnover and the mobilisation of liver glycogen _ The maximal reduction of liver glycogen of the animal

)0 y = O.OOOh' - 0.0307x' + 1.844 Ix - 14 . I~ S

2M

~ 26 tt f !'l 24

E

'" S 22

~ 20 t

~ IX c

.2 16

~ !!

w: 14

12

10

10 )0 50 70 t 10

TisS UI! cadmium conlenl (.u glg dw )

Fig 7 - Relatio nship between the ti ssue cadmium cOlllent and cad mium induced filtration rate of Lalllellidl'lI.\·

may be the result of cadmium st ress- induced hypoxia reflecting the elevation of glutamate dehydrogenase (G DH) activ ity in liver for mobilisation of glucose from the ti ssue glycogen to meet the hi gh energy demand_ As a result, ti ssue glycogen and cadmium contents of the animal were inversely related (Fig_ 6) , Glycogen reduction due to heavy metal toxicity has been observed in some other an imals2 1

-n .

The results further indicated that cadmium stress was responsible for reducti on in filtration rate of the animal as evident from the in verse relationship between these two parameters supported by other workerss.23

. The filtration rate of the animal was adversely affected when the tissue cadmium exceeded the critical level of 50-60 J-tg/g dw_ (Fig. 7).

The freshwater bivalve L marginalis has been used as a potential specimen for restorati on of cadmium intox icated aq uatic environment. It appears that the rate of the filtration and respiration of the anima l exposed to cadmium polluted habitats can be used as biomarker for assessment of animal health to determine the actual state of health for further use in reclamation _

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