Toxicity and Bioaccumulation Studies of Heavy Metals on a Freshwater Fish

Test Animals

Fish samples (Clarias gariepinus) were collected for toxicological studies from a fish farm and transported in glass tank to the laboratory.

Bioassay Studies

After acclimatization, the fish samples were randomly assigned to bioassay containers. For the series of bioassays, 20 Clarias gariepinus were exposed per treatment in two replicates (10 fish per replicate). There was a control in all the treatments. In these bioassays after range finding preliminary trials, fish samples were exposed to series of concentrations of each heavy metal compounds.

A series of bioassays were carried out, but this time, the acclimatized animals were exposed to different concentrations of binary and multiple mixtures of heavy metal salts in pre-defined ratios. There was always 10 Clarias gariepnius per bioassay container exposed at each concentration or treatment. Each treatment was replicated 2 times, meaning that 20 Clarias gariepinus were exposed per concentration of heavy metal mixtures as specified below.

In all bioassays, mortality data were taken once every 24h over the 96h duration. Test animals were taken as dead if they failed to respond by moving away when touched gently with a glass rod.

Bioaccumulation Studies

Clarias gariepinus was exposed to sublethal concentration of single metal compounds (Pb, Cr, Cd) in semi-static bioassays. In this series of experiments, Clarias gariepinus was exposed to only sub-lethal concentrations (1/10th and 1/100thof 96-h LC50) of the selected metal salts acting singly as specified below. A total of 60 test animals were exposed per sublethal concentration in 3 replicates (20 animals per replicate). In this series of bioassay that went on for 28 days in order to investigate the rate of bioaccumulation, the semi-static bioassay procedure was always adopted to avoid drastic changes in concentration of test media via evaporation and excessive reduction in dissolved oxygen level. In this semi-static procedure, each test medium was changed into a fresh solution at exactly the same concentration of heavy metal salt once every 7 days, transferring the same exposed test animals into the freshly prepared test media over the 28-day period of the experiment. At predetermined time intervals (day 0, 7, 14, 21 and 28), One live Clarias gariepinus per replicate, making three per treatment were randomly selected, cleaned thoroughly with distilled water and placed in labeled polythene bags in which they were kept frozen awaiting digestion of the extracted whole animal tissue and analysis for test metals by AAS [3].

Bioaccumulation of heavy metals by Clarias gariepinus was also exposed to sublethal concentrations of binary and multiple heavy metals compounds in mixtures.In order to evaluate the possible influence of ionic competition for uptake sites or other modes of possible interactions on rates and amount of bioaccumulation of metals by animals exposed to mixtures of metallic elements, the following series of bioassays were carried out in the laboratory. A similar semi-static bioassay as described above was carried out, but for this present bioassays, Clarias gariepinus was exposed to sublethal concentrations of binary mixtures of Cd – Pb, Cd - Cr, Cr - Pb and multiple mixtures of Cd - Cr - Pb. The binary and multiple mixtures consisted of sublethal concentration of each constituent metal salt, the computation of which was based on pre-determined fractions (1/10th and 1/100th) of the 96-h LC50 values of the test metals obtained in acute toxicity of joint action experiments conducted in this work.

Statistical Analysis

Toxicological data based on quantal response (mortality) was analysed by probit analysis, after Finney (1971). The probit analysis, dependent on maximum likelihood interactive regression was done by a computer programme designed and implemented by Ge le Patourel, imperial College London and adopted by Don-Pedro [4]. The above analysis when it involved mortality-dosage data resulted in derivation of LC50, LC95 and LC5 with their 95% confidence limits and other relevant parameters, while when mortality time was analyzed, Lc50 values were obtained. Pooled cumulative quantal response (mortality) data (of replicates/treatment) was used in probit analysis following accepted toxicological procedures [4].

TF (Toxicity Factor): This was used to measure the relative potency ratio. RPR = LC50 of a compound X LC50 of another compound Y Where X is the metal with higher value of LC50 and Y is the metal with the lowest value of LC50Histograms were used to compare the LC50 values of the metals when acting singly.

Analysis of Data and Measurement of Joint Action Toxicity of Binary and multiple Mixtures of Test Compounds

Joint action toxicity was determined and analyzed using synergistic ratio model, according to Hewlett and Plackett (1969) as follows: S.R (Synergistic ratio) = LC50 of a chemical acting alone LC50 of chemical + additive mixture

Translation of Results

Where S. R = 1 – describes additive action S.R = < 1 – describes antagonism S.R = > 1 – describes synergism Significance /overlap in 95% confidence limit of the detected 96hLC50 values were determined using the Chi- Square technique. The limit of significance was 0.05.

Single Acute Toxicity of Cadmium (Cd) Against Clarias gariepinus

The 96-h LC50 values of Cadmium against Clarias gariepinus are shown in 1. The values were calculated from 24h – 96h of the study duration. There was no significant difference observed among the 96-h LC50 values of the fish species.

Single Acute Toxicity of Chromium (Cr) Against Clarias gariepinus

The 96-hLC50 values through 24h duration of fish species exposure are shown on Table 3. The LC50 values for the 24-96h exposure recorded no variation (P>0.05).

120

Comparative LC50

Binary Acute Toxicity of Cadmium (Cd) and Lead (Pb) Against Clarias gariepinus

The relative joint acute toxicity (24h – 96-h LC50 values) of the ratios of mixtures for cadmium and lead against the catfish are presented in Table 4.

The 24-h LC50, 48-h LC50, 72-h LC50 and 96-h LC50 values of binary combination of Cd and Cr in a specified

Binary Acute Toxicity of Chromium (Cr) and Lead (Pb) against Clarias gariepinus

The LC50 values for the exposure duration of Clarias gaeripinus to the binary mixture of Cr and Pb are shown in Table 6. Only 24-h LC50 was seen to be significant (P<0.05) with the heterogeneity factor used in the confidence limit calculation of 95%.

Table 6: Acute Toxicity of Binary Mixture of Cr and Pb against Clarias gariepinus (_mg/L). Joint Action Toxicity of Multiple Mixtures of Heavy Metal Compounds against _Clarias gariepinus The LC50 values of multiple mixtures of Cd, Pb and Cr over 24- 96-h exposure duration are shown on Table 7. All the LC50 values were statistically significant (P<0.05) except value of 24h duration (P>0.05) at 95% confidence limit.

From the above table, there was increasing concentration of the metals in fish with increase in exposure duration for both 1/10th and 1/100th respectively. Chromium (Cr) was the metal mostly accumulated and Lead (Pb) was the least accumulated metal.

Bioaccumulation of Binary Mixture of Heavy Metals by Clarias Gariepinus Exposed to Sublethal Concentrations

The concentration of binary combination of Cd: Pb, Cd: Cr and Cr: Pb is presented in Table 9.

The triple mixture of Cd: Cr: Pb concentrations are shown in Table 10.

On the basis of the result, similar trend of dose response relationship was seen dependent on the exposure duration. The test animal bio accumulated chromium more than any other metal.

Acute Toxicity Studies / Bioassay

Under the single action toxicity test carried out_, Cd was the most toxic to the test animal with the lowest 96- h LC 50 of (8.280mg/L) (Table 4.7) while lead (Pb) was the least toxic (96-h LC50 value of 70.183mg/L).The high toxicity of Cd has been previously demonstrated by several workers against fish species [5, 6]. The high toxicity of Cd could be attributed to its high electropositivity. The low toxicity of Pb has also been reported widely in the literature. For example, Paul, et al. [7] was unable to establish a 96-h LC50 value for lead acetate (90.43mg/L) against _Channa punctatus because the compound did not cause any mortality of the exposed animal in their study “Lead toxicity on non-specific immune mechanisms of freshwater fish Channa punctatus”.

Certainly, a number of interactions in joint toxic action of pollutants results in synergism in which the toxicity of a pollutant may be enhanced several folds by the presence of another pollutant against organisms. For example, Enajekpo [8] demonstrated that toxicity of spent engine oil against C_.africanus_ was increased by over 2000 folds by the presence of Nuran insecticides in joint action studies.

In the joint action toxicity evaluations with binary and multiple (triple) mixtures carried out on this study, most of the results upon classification with the synergistic ratio agreed mainly with the model of synergism where only a few cases Cd in Cd: Pb, Cd in Cd: Cr and Cd in Cd: Pb: Cr interactions remained consistent with the model of antagonism. Liu, et al. [9] reported that the mechanism responsible for antagonistic interaction between constituent metal components in a mixture can be attributed to the oxidative stress and competition for uptake/binding sites in the biological interface between the various types of metals. Antagonistic interaction in mixture of pollutants Cd: Pb, Cd: Cr and Cd: Pb: Cr in this study, where it exists could be an advantage in environmental management. This is so because antagonism implies that there is interaction between the constituents which result in the lowering of the toxicity of one or all the constituent of mixtures against the living species. Researchers outside the country who have also reported on synergistic interaction between constituent metals of a mixture include Abdullah and King [10] who documented the synergism that occurred in the interactions between Cu and Zn against freshwater isopod Asellus aquaticus. The observed synergistic interactions could also have occurred if the metals form complexes, which have greater penetrability with respect to the tissues of the exposed animal than the individual metals when acting alone On the basis of emerging results obtained from the joint action toxicity studies carried out in this work, it will certainly be more realistic and effective to take into consideration toxicity levels of mixture of pollutants that occurs together in a local ecosystem and not just levels of single action toxicity of individual pollutants in deriving safe limits/standard aimed at protecting organisms in the environment. Although most of the existing safe limit/standards worldwide depended mainly on the existing single action toxicity data, view of such standards in the developed countries consider the results of joint action toxicity measurements against several sensitive species [11].

Bioaccumulation Studies

The bioaccumulation of Cd and Cr in the series of test carried out in this work likely occurred because the experimental animals were able to absorb the metals directly across body surfaces, membranes and ingested food at a faster rate than they were able to metabolize and excrete the absorbed metals. This led to a net gain of the metals in their body tissues. This is in line with the findings of Otitoloju and Don-Pedro [12, 13] in their work on bioaccumulation of heavy metals (Zn, Pb, Cu and Cd) by Tympanotonus fuscatus exposed to sublethal concentrations of the test metal compounds in laboratory bioassays which recorded higher concentrations of metals that were about 2-6 times higher than the levels accumulated in control animals exposed to 30days period.

Another segment of this work reported here demonstrated the influence of binary and multiple mixtures of metals on the rate and pattern of bioaccumulation in the laboratory bioassays. When Clarias gariepinus were exposed to joint mixtures of Cd- Pb, Cd-Cr, Cr-Pb and Cd-Cr-Pb, in laboratory bioassays, the concentrations of the metals accumulated by the test animals over 28-day experiments were found to be lower than the concentrations of the respective metals accumulated under the single action studies. This is in accordance with works of Borgmann, et al. [14] which stated that bioaccumulation of metals in mixtures may demonstrate competitive, a, or non-competitive inhibition that may lead to lowered concentrations when compared to single metal bioaccumulation.