A range finding test (trial test) was carried out to determine the actual concentration of the toxicant (aminoforce containing 720 g/l of 2,4 - Dimethylamine salt) to be used for the main experimental run. The trial test rabbits were grouped into three groups. Each group which contain triplicate rabbit was exposed to 1.00 mgl^-1, 2.0 mgl^-1 and 3.00 mgl^-1 of the toxicant. The trial test lasted for 14 days. The test solution in each compartment were renewed daily.

A sub-lethal dose of the toxicant needed for the main experiment was determined based on the result of the range finding test. Three different concentrations viz: 0.04 mls, 0.08 mls and 0.12 mls were exposed to the probe organism from the original concentration of 720 g/l to make 2.0 mgl^-1, 4.0 mgl^-1 and 6.0 mgl^-1 i.e. concentration of the toxicant used. In addition, a control was set up viz: 0.0 mgl^-1. Each of the experimental group contain triplicate New Zealand rabbit. The main experiment lasted for 21 days. The test solution in each compartment were renewed daily akin to trial test period.

Blood samples were collected from the ear veins before the probe organisms (New Zealand rabbit) were killed and dissected and target organs were obtained (liver and muscle). About 0.5 g of each of the organs were macerated with a ceramic mortar. 5 ml of physiological saline was used stabilize each sample for enzyme analysis, while deionized water was used stabilize samples meant for electrolyte analyses. All samples were centrifuged at the rate of 3000 rpm for 15 minutes. The supernatant was then removed and stored in plain bottles at -20 ⁰C for analysis. Sodium and potassium ions were assayed via APHA 18 method. Chloride ion assay was carried out using Malmstadt and Winefordner 19 method, while Mg²⁺ assay was carried out via Macintyre and Davidsson 20 method. In the liver and blood samples, aspartate amino transferase and alanine amino transferase were analysed using the colorimetric technique previously described by Zimmerman 21, while ACP analysis was carried out by the method described by Andersch and Szcypinski 22.

SPSS software version 16 was employed to unveil the analysis of the data. The data were expressed as mean ± standard error and one-way analysis of variance was carried out at α=0.05 and Duncan multiple range test (DMRT) was used to separate the means.

(Table 1) shows the effect of Oryclotagus cuniculus blood and liver enzymes (ACP, ALT and AST) when exposed to 2,4-D (aminoforce) for 21 days. The concentration of AST in the liver were statistically different (P<0.05) across the various concentration of the toxicant. Values progress down the experimental group, albeit not in a dose dependent pattern, while blood AST values receded down the experimental group. The lowest blood AST value was recorded at 2.0 mgl^-1 (54.65±2.01 µ/l) compared to the control that had 96.59±1.10 µ/l. Liver ALT values showed a remarkable deviation from the control, which progressed in a dose dependent pattern down the experimental group. The highest value (697.99 µ/l) of liver ALT was recorded at the highest concentration (6.00 mgl^-1) (Table 1). Blood ALT values showed significant. However, multiple comparison showed no significant variation between 0.00 mg/l and 2.00 mg/l, and between 4.00 mg/l and 6.00 mg/l concentration of the toxicant. The blood ALT recorded were diminutive compared to the control, albeit values appreciated down the experimental group in a dose dependent pattern. Retrogressive values were recorded in liver ACP (albeit not dose dependent) quite unlike blood ACP that recorded elevated values at the last two concentrations (4.00 and 6.00 mgl^-1).

Many researchers have reported effects of xenobiotics on organism’s enzymes 23242526. According to Edsall 27, these biochemical changes or characteristics of blood or organs are among the most important indices used in assessing the status of the internal environment of a fish. Luskova et al. 28 added that changes in metabolites and biochemical processes of the organism resulting from the effect of various pollutants, makes it possible to study the mechanisms of the effect of these substances.

The elevated values of liver AST in this study have some trend with the reports of Inyang et al. 9, Aly and El-Gendy 15, Edori 29, Inyang and Williams 30. This elevation of enzymes in the experimental group compared to control is a clear indication of the effect of 2,4 –D on the Oryclotagus cuniculus. According to Ambali et al. 31, the disruption of transaminases from the normal values denotes biochemical impairment and lesions of tissues and cellular function because they are involved in the detoxification process, metabolism and biosynthesis of energetic macromolecules for different essential functions. Desmet and Blust 32 exposed Cryprinus carpio to cadmium for 21 days and observed elevation of the activities of AST and ALT in the liver, kidney and gill and opined that the elevation is a result of proteolysis which is intended to increase the role of proteins in the energy production during cadmium stress. Kaur and Dhauju 33 reported a significant increase in the activities of AST, ALT and ALP in the liver of albino rats exposed to monoclotophos, methyl parathion and dimethoate given orally for 90 days, and inferred that such increase is an indication of cellular toxicity of these organophosphate causing a release of the enzymes into the blood. Additionally, Kumar et al. 34 suggested that such increase might have resulted from stepped-up transamination, where amino acids are used to generate intermediate for tricarboxylic acid cycle in an attempt to cope with the energy crises during stress cause by the toxicant.

Liver ACP values decreased significantly down the experimental group compared to the control. Inyang 23, Inyang et al. 9 reported decreased concentration of ACP when diazinon and aluminum phosphide were exposed to Clarias gariepinus and Paraphiocephalus obscures respectively. The overt diminutive values recorded are caused by the toxicant and is significantly a clear sign of inhibition. Inhibition of the effect of the toxicant on the ACP infers a breakdown in the kreb’s cycle (TCA) intermediates, since transamination which provides the keto acid (α-ketogulatarate) is inactive and α-ketogulatarate is one of the intermediates in the TCA cycle 29. According to Jawale 26, Sherekar and Kulkarni 35, ACP is hydrolytic lysosome of foreign materials and the enzyme can hydrolyze the phosphorus ester in acidic medium. The authors added that increase or decrease in the lysosome enzyme activity depends upon the concentration of the pesticide the probe organisms are exposed to.

The values observed in this study indicates that some complications arising from the effect of the toxicant in the liver cells. Liver AST unveiled elevated values, while blood AST recorded decreased values denoting tissue damage and inhibition occurring concurrently in the tissues of the probe organism. Kalender et al. 36 reasoned that the susceptibility of liver tissues to the stress resulting from exposure to pesticides is a function of overall cellular balance between the degree of oxidative stress and the antioxidant capacity.

(Table 2) shows the effect of Oryclotagus cuniculus muscle electrolytes when exposed to 2,4-D (aminoforce) for 21 days. Sodium (Na⁺) values in the muscles were significant (p<0.05), and progressed down the experimental group compared with the control and it was dose dependent. Potassium (K⁺) values unlike sodium retrogresses down the experimental group in a dose dependent pattern. The least was recorded at the highest concentration (6.0 mgl^-1) i.e. 8.62 ± 0.06 mmol/l compared to the control that recorded 13.30±0.01 mmol/l. Chloride values were not significant (p>0.05) while Mg²⁺ showed a slight decrease down the experimental group (not in a dose dependent pattern).

Minerals are mainly responsible for the maintenance of osmotic pressure in the blood and proper functioning of all types of tissues 37. Christensen and Tucker 38 opined that the basic function of electrolytes in the body lies in controlling fluid distribution, inter and extra cellular acid-basic equilibrium, maintaining osmotic pressure of body fluids and normal neuromuscular irritability. The elevated value of Na⁺ in this study is caused by the toxicant. A slight shift in the values of electrolytes from normal may lead to serious physiological defect. Toxic metals and pesticides can alter the electrolytes in the blood, tissue and organs of organisms 39. Ca²⁺ K⁺ and Na⁺ ions functionally participate in maintaining normal irritability of the heart, muscles and nerves as well as selective permeability of the cell membrane 40. Therefore, significant elevation of Na⁺ can lead to hyperfunction of the muscle tissues (also known as hypernatraemia). This progressive value recorded for sodium in the muscle fibres may lead to loss of weight occasioned by excessive dehydration overtly attributed to increased muscular activity and stress.

A significant decline in the values of K⁺ unveiled 2,4-D effect on muscle tissue. Low levels of K⁺ could lead to a condition known as hypokalemia. According to Weiner et al. 41, hypokalemia can exacerbate hepatic encephalopathy by increasing renal ammoniagensis and hence increasing systemic ammonia level. Additionally, Na⁺ and K⁺ are constantly diffusing through the plasma membrane, moving from regions of high concentration to regions of lower concentration. However, the concentration of Na⁺ and K⁺ on the two sides of the membrane remains constant due to the action of the sodium-potassium pump which is provided by ATP 42. Retrogressive values as recorded in this study will surely affect membrane potential (electrolytes balance) and alter Na⁺ and K⁺ ion channel and also neuron communication in cells.

Stabilization of values were recorded in Cl^- while Mg²⁺ values showed a marked concentration (4.00 mgl^-1 and 6.00 mgl^-1). These results were also obtained by Inyang et al. 43 when the authors exposed Clarias gariepinus (juveniles) to dichlorvos. Luskova et al. 28 also recorded similar results when the author exposed common carp to diazinon. The toxicant used in this study may be less toxic to Cl^- and Mg²⁺ ions due to the concentration of the toxicant used. The stabilization of some electrolyte according to Ogamba et al. 44 could be a stress induced response occasioned by the effect of animals exposed to toxicant which could activate certain physiological and metabolic mechanisms that may lead to a rapid uptake of electrolytes from water and food material or a reduction of ion influx.