CHAPTER 2

17

L_E'l'HAL. Il‘OXICI,'1'_Y STUDIES OF QOPPER END MERCURY

Excessive pollutants affect the type of organisms in theumter which may lead to immediate death, depending on thestrength of the poison and duration of exposure. The poisonfirst debilitates the organism through physiological stress andthis is often reflected first in the excitement and hyperactivity, followed by coughing, gasping and other distressmanifestations and finally, death. The physiological effect ofthe poison may be manifested in different ways on differentspecies. For example. the function of the vital organ of thefish may be seriously impaired with irreparable damage to theliver, kidney or the spleen. In some cases, the respiratoryprocesses may be adversely affected. This may arise as a resultof gill damage which may at first appear as mucus secretions andlatter tissue degeneration with fungus growth and other diseases.

The use of bioassays as part of a comprehensive approach tonmrine pollution assessment is widely accepted now a days.Toxicity is a biological response, which when quantified in termsof concentration of the toxicant can constitute the basis for abioassay procedure. Bioassay tests are defined here as theestimation of the amount of biologically active substances by thelevel of their effects on test organism (Chapman and Long, 1983).Information generated from various toxicity tests can be of usein the management of pollution for different purposes likeprediction of environment damage of waste, comparison of varioustoxicants, animals or test conditions and regulations of wastedischarge.

18

The acute toxicity of poisons is determined in thelaboratory on biological assays or bioassays, where the fish orcmher organisms are subjected to different concentration of thepmison under controlled salinity, temperature and dissolvedcmygen for a specified time; usually 48 or 96 hrs. Thecmncentration in which one half of the organisms are killed in 96lus., say, is referred to as the 96 hrs median lethalzxmcentration or LC 50. It is extremely important to note thesqmcies of organism tested and the conditions under which thetnoassy tests are made, i.e., temperature, salinity, dissolvedcmygen, pH and whether the tests are static (solutions changedcmly at specified intervals) or continuous flow. Usually thewater quality criteria for protection of fish life are based onthe LC 50 values (Mc Lee and wolf, 1963; U.S. FWPCA. 1968). But

it is quite clear that these can change from place to place,depending on such factors as temperature, salinity and dissolvedcmygen concentration (Alderdice, 1963).

Static acute toxicity tests have been the primary tool forevaluating short term effects of pesticides and other foreigncmmpounds on aquatic organisms (Nimmo, 1985). In general the LCM) values from the static tests are greater than those from theflow through tests. However a well designed static test can beuseful in determining acute toxicities for comparative studies oras indicator as for further acute or chronic tests (Nimmo, 1985).

Sprague (1969) has reviewed the problems of toxicity teststo fish. The incipient LC 50 (lethal concentration for SO

19

percent of individuals on long exposure) is recommended as thenmst useful criterion of toxicity. If this cannot be estimated,UEE4 day LC 50 is a useful substitutes and often its equivalent(Sprague, 1969). Information needed for protection of aquaticlife has been eloquently described by Tarzwell (1962 a, 1962 b,1966), and tests provide some of the required answers. Theperiod for which the LC 50 is determined is usually ofcmnsiderable importance, values normally being much lower after96in? (Holden, 1973). In aquatic toxicology. the acute toxicitytests for fish enables estimation of the exposure concentrationresulting in 50% mortality of test animals within 48 or 96 hr(expressed as LC 50 value). The numerical value of LC 50 hasassumed special importance as an index of toxicity, but with theincorporation of highly persistent substances with highconcerntration potentials and low water solubilities it canprovide only marginal information (Ernst, 1980).

The acute toxicity data have been used in conjunction withso called safety factors of 0.1 — 0.01 to estimate safeconcentration of chemicals for the protection of aquatic lifeduring chronic exposure. However, these factors do notadequately consider the specific action of the individualsubstance (Ernst, 1980). The concept of specific applicationfactor define the relationship between the acute and chronictoxicity of a chemical, the accurate estimate of the specificapplication factor for a chemical can be derived from maximumacceptable toxicant concentrations (MATC). Numerically the

20

application factor (AF) is Quotient of the MATC and the 96 hr LC50. Application factors for some pesticides show that thehighest concentration without any toxic effect may be more thantwo orders of magnitude lower than the 96 hr LC S0 (Hansen andParish, 1977; Nimmo et al. 1977). The method for determinationof medium of tolerance limit (TLM) was evolved long before (Hartet al. 1945; Doudoroff et al. 1957). Attempts were made toapply suitable factors to the TLM data for predicting long termsafe concentrations. Burdick, (1957); Henderson and Pickering,(1957); Kimura and Matssushima, (1969); and Muirhead — Thomson,

(1971) suggested that the factor to determine the long term safeconcentration should be derived from simple short term bioassayssince static bioassay stimulate most closely single or multipleapplications of a pesticide to a lake or pond (Burdick, 1967).

Standard bioassay studies were conducted for 96 hrs periodto obtain a measure of acute toxicity of copper and mercury onthe fish Macrones gglio.

MATERIAL AND METHODS

This part of the investigation has centered mainly to knowthe toxic effects of copper and mercury individually at differentlethal levels on the fish Macronesggulio.

There are various ways of investigating sublethal effects,and each techniques provides an insight into the physiology orbehaviour of the organism in question (waldichuk, 1979). Efforts

21

ana nwde to evaluate the lethal and sublethal effects of heavynwtals individually on the fish Macrones gulio.

Specimens were collected from an unpolluted area of theinland water system, confluent with Cochin backwaters. Themurals were collected using castnets causing minimum stress, andthen brought to the laboratary in large plastic bucketscontaining the water taken from the same site. Within half anhour they were brought to the laboratary. The environmentalparameters of the collection area noted were, salinity 15i2%¢,temperature 28ilOC, PH 7.5 i 0.5 and dissolved O 4.8 — 5.2 ml/lduring the time of collection. 2Laboratorx Erocequres

The animals brought to the laboratory were maintained inlarge tanks containing filtered unpolluted water of correspondinghydrographical parameters of the collection area. The fishesacclimated for one week and observed for mortality, diseasesymptoms or abnormal behaviour. During the acclimation periodthe fishes were fed with clam meat. The fishes were not fed 24hgmior to the test experiment and during the entire period of theexperiment. Only healthy fishes of immature stage of size range10-13 cm in length were chosen for the experiment.

Toxicants

The toxicants selected for the experiment were copper andIIIQICUIY.

22

Copper

Analar grade of copper sulphate (M.w.249.68) was the sourceof copper. The salt was dissolved in distilled water and addedto achieve the required concentration.

Mercury

Standard solution of mercury were prepared using analargrade mercuric chloride (M.W.27l.5O) in distilled water and inamber colored bottles. Since mercury solutions are not stablefor long periods, they were prepared fresh for each set ofexperiments and added to make up the required concentration.

Toxicant C0r1_Cen.tr§tion

The various concentrations of the toxicants are expressed inppm in terms of the individual heavy metal (copper and mercury)formulation. Commercial formulations were used and calculatedquantity was weighed out to give the desired concentrations inthe test medium.

Eiioasser for LC 50 determinationStatic bioassay procedure given by APHA et al. (1981) and

ward Parish (1982) was followed in the experiment. For thestudy, filtered unpolluted water of corresponding hydrographicalparameters was used (salinity l5i2%q temperature — 28 1 10 C, pH— 7.5 i 0.5 and dissolved oxygen > 90% saturation). After rangefinding tests, seven concentrations were used for each metal(Table 1).

23

Each experimental tank contained 50 litres of test solutionwith different concentrations of toxicants. Ten fishes were usedfor each test concentration of the toxicant. One tank was keptas control without metal solution. Duplicates were run for eachmetal concentration. The fishes were inspected every 12 hr formortality and the water was renewed every 24 hr. Dead fishes ifany were removed and recorded every 12 hr.

Data analzsisCumulative percentage mortality was determined for each

metal concentration of the mortality study experiment. This wasplotted later in a log probit paper and the concentration ofmetal, killing 50% of the test organisms (LC 50) during 48, 60,72, 84 and 96 hr exposure together with 95% confidence limit andslope function were calculated following Litchfield and wilcoxon(1949).

RESULTS

Behavioural I€5P9HS§ £9 metal exposureDuring the lethal toxicity study behavioural response of the

fish Macrgneg gulio to the metals (copper and mercury) were moreor less similar.

Altered behaviour of the metal—exposed fish includesprogressive exhibition of signs of tiredness, irregular erraticswimming, agitation, jerky movement, restlessness, frequentsurfacing gulping of air, revolving, convulsions, extension offins, accelarated ventilation with rapid rhythmic opercular and

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Figure 2 Toxicity curve for mercury.

24

mouth movements etc. Finally they lost their equilibrium andsettled to the bottom before death. water turned turbid andmucus oozed out of the dead fishes. The dead animals had blood

clots on the gill surfaces with widely opened mouth and stretchedgills. Air bubbles were seen trapped within the mucus.

Lethal texicitx of QOQPQI and mercury

The cumulative percentage mortality in differentconcentrations of copper and mercury are given in table 1 and LC50 values are summarised in the table 2.

The concentration tested for the lethal toxicity of copperranged from 0.04 ppm to 0.16 ppm and 96 hr LC 50 value worked out

was 0.063 ppm.

The concentration tested for the lethal toxicity of mercuryranged from 0.05 ppm to 1.00 ppm. The 96 hr LC 50 value formercury was 0.125 ppm.

Changes in LC 50 values with time are given in figures l and2. In the figure the copper was found to be having steeper slopethan mercury, which is an indication of acutely acting toxicity.

Discussion

The results of the acute toxicity studies of the presenteffort revealed that of the two metals tested, copper was moretoxic than mercury.

The observed effect of copper on Macrones gulio which wasfound to be more toxic than mercury, appears to be is contrary to

25

what was observed in Tilapia nilotica (Nile Tilapia) by Somsiri(1982). He observed a higher 96 hr LC 50 value for Cu (58.1mg/1) and lower values for Hg (3.71mg/1). A similar observationwas made by Krishnakumari et al. (1983) in Therapon jdlbua where96 hr LC 50 value for copper was 4.5 mg/1 and for mercury it is0.06 mg/1.

Acute toxicity on teleosts, have been conducted by severalworkers. The LC 50 values recorded for copper was O.2,pg/1 forFundulus heteroglitus (La Roche, 1974), 0.28 ,ng/1 forQnchoghynchus kishutgh (Ma Carter et al. 1981) and 6.0‘pg/1 forTilapia mossambiga (Balavenkatasubaiah et al. 1984). Those formercury were 0.07 ml/l (Roales and Perlmutter, 1984) inIfiQhQQ§$¥Qr trichopterus and 0.03 ml/l in lctalurug punctatus(Nriagu, 1979).

These studies established that each toxicant affecteddifferent species of fishes in variety of manner. The effect onsame fish can vary with environmental factors as observed byDorfman (1977) in Fundulus gp.

Nriagu (1979) made an effort to compare the 96 hrs LC 50values of mercury on different species of teleosts and observedthat rainbow trout (Salmo giardngri) was the most sensitive (4.7pg/1) species in relation to catfish, lctalurus punctatus (30pg/1) blue gill, Lepomis morochirus (88.7 pg/1). Lepomismicrolophus (137.2 pg/1) and the large mouth bass, Micropterussalmoides (140 pg/1). Krishnakumari et al. (1983) found that

26

among the metals, Co, Ni, Cu, As, Pb, V, Hg and Zn, mercury was

the most toxic and cobalt was the least to Th€rapon jarbua. Theexperimental LC 50 values would thus provide data on comparativeeffect of pollutants and are useful in identifying potentiallytoxic substance. In the present study the order of toxicity ofMacrons gulio was Cu > Hg.

Concerning behavioural patterns, similar observations as inthe present study have been reported by Eaton (1974), Spehar(1976), Nriagu (1979), Satchell (1984), Rai and Qayyam (1985),Lauren and-Monald (1987) and James (1990). These abnormalitieshave been attributed to nervous impairment and blockade ofnervous transmission between nervous system and various effectorsites as a result of heavy metal treatment (James 1990).