Abstract: The lethal effects of Spent Lubricant Ooctober 16, 2009il (SLO) and dispersant acting singly and jointly as binary mixtures in predetermined (9:1) and equitoxic (3:1) ratios on Macrobrachium vollenhoevenii were evaluated and haematological parameters were used to assess effects of sub lethal exposure. The concentration-addition model (relative toxic units RTU) and Synergistic model (SR) were used to assess their interaction of the tested mixtures meant to simulate environmental control settings in aquatic ecosystems. On the basis of derived toxicity indices, the dispersant (96 h LC50 = 0.06 ml L-1) was found to be about 1.33 times more toxic than the SLO (96 h LC50 = 0.08 ml L-1) when acting singly against M. vollenhoevenii while the joint action response indicated that the equitoxic ratio (96 h LC50 = 0.56 ml L-1) was relatively more toxic. The evaluation of the binary mixtures based on RTU (with reference to SLO) revealed conformity to model of synergism (RTU = 5.66; 1.33) for predetermined and equitoxic ratios while the synergistic evaluation indicated antagonism (SR = 0.44) and synergism (SR = 1.33) for predetermined and equitoxic mixtures, respectively. The haematological parameters of M. vollenhoevenii exposed to sub lethal concentrations (1/10th, 1/100th and 1/1000th of 96 h LC50 values) of the tested mixtures indicated that the total leucocyte count and differential polymorpho-neutrophils count increased significantly (p>0.05) with exposure period and concentrations while the differential lymphocyte count had an inverse relationship. The differential eosinophils count appeared to have no significant changes (p>0.05). The environmental significance of the results in establishing safe limits for dispersant use in aquatic ecosystems are discussed.
INTRODUCTION
The possible toxic effects of dispersed oil in the water column are a primary consideration when assessing the use of dispersants to deal with an oil spill. The hydrocarbon types of dispersants used in the past have had adverse effects on the environment due to their toxicity. Modern dispersants used today are significantly less toxic than the oils they disperse. These dispersants are used when the impact of the floating oil has been determined to be greater than the impact of mixing of oil into the water column.
Although, dispersants have been found to be very useful in some oil spill clean-up operations, the applications of some dispersants to spilled oil have also been found to cause more damage to the living organisms inhabiting such ecosystems than the spilled oil acting singly (Otitoloju, 2003).
The potential impact of dispersed oil on aquatic ecosystem should be thoroughly considered prior to use. Earlier studies have indicated that acute tests gave little or no relevant information on sub-lethal effects of dispersants or oil/dispersant mixtures (Baklien et al., 1986; Oyewo, 1986). Dispersant toxicity can be affected by physiological and biochemical processes not normally tested for in acute tests on single life-stage (Singer et al., 1990; Volkman et al., 1994; Odiete, 2003). It is generally considered that the toxicity of mixtures of oil and dispersant is of greater concern than the dispersant alone (Samuel et al., 2008).
Many researchers have shown the surface molecules like dispersant in spite of being differentially toxic at times greatly synergise or enhance the toxicity of spilled oil on the aquatic organism (Adams et al., 1999; Chukwu, 2006). It is postulated that dispersants are known to synergize the toxic effects of oil during clean up by solubilising delicate membrane like those of the gills leading to enhanced entry or penetration of toxicants.
Furthermore, the effects of pollutants on physiological processes are relatively well-known and include respiratory, cardiovascular, osmoregulatory, neurological and endocrine disturbance (Wendelaar-Bonga and Lock, 1992; Randall et al., 1996; Bamber and Depledge, 1997; Chukwu, 2006; Velisek et al, 2006; Abdel-Moneim et al., 2008). Physiological assays attempts to measure responses within the normal physiological scope of the organism and correlate these with pollutant exposure or effect. A variety of haematology, respiratory, cardiovascular, osmoregulatory or neurological assays have been developed (Larsson et al., 1985; Baatrup et al., 1990; Bradbury et al., 1991; Depledge and Andersen, 1990; Aagaard et al., 1991). Physiological assays are especially useful for monitoring fluctuating exposures, acting as early warning systems for acute events, because the toxic response is usually instantaneous and or sensitive to low exposure concentrations.
Blood is a pathophysiological reflector of the body because it is highly susceptible to internal and external environmental fluctuations (Seth and Saxena, 2003). Physiomorphological changes in blood indicate the changes in the quality of the environment and therefore blood parameters are important in diagnosing the functional status of the animal exposed to toxicants. Haematological parameters act as a physiological indicator to changing external environment (Abdel-Moneim et al., 2008) as a result of their relationship with energetic (metabolic levels), respiration (haemoglobin levels) and defense mechanisms (leucocytes levels). There is a growing body of literature on the effects of pollutants on blood and other extracellular fluids; and this has largely been obtained during experimental exposures to identified toxicants (Velisek et al., 2006).
Macrobrachium sp. being an arthropod has an open circulatory system in which the blood comes in contact with the tissue fluid (lymph) to give the haemolymph. Crustacean haemocytes play important roles in a host’s immune response; however, there is no uniform classification scheme for crustacean haemocytes. The circulating haemocyte number is a stress indicator and haemocyte counts may be a valuable tool in monitoring the health status of crustacean species. Studies on the haemocytes of crustaceans contribute to the accumulation of the basic knowledge on haemocytes, especially with regard to the physiological condition of the animal.
Therefore, an evaluation of the toxicological and physiological effects of spent lubricant oil and dispersant in various mixing ratios before setting environmentally safe optimal dispersal limits for managing spills in fragile aquatic ecosystems will be very useful. The aim of the present study was to investigate the lethal effects of Spent Lubricant Oil (SLO) and dispersant (OSD 94-60) acting singly and jointly as binary mixtures in predetermined (9:1) and equitoxic (3:1) ratios on M. vollenhoevenii and use haematological parameters to assess effects of sub lethal exposure.
MATERIALS AND METHODS
Test Animals
The test organism, M. vollenhoeveni (Arthropoda, Decapoda, Crustacea)
was selected for this study because it is one of the organisms approved for
toxicity testing involving dispersants in Nigeria by the Department of Petroleum
Resources (DPR). They were obtained from Lagos lagoon with a scoop net and conveyed
in plastic bucket holding water from that habitat. The collection was made between
6-7 am in the morning to reduce stress on the organisms. The test animals collected
were of similar sizes ranging from 4.0 to 6.0 cm weight ranging from 225.0-243.0
mg. Efforts were made to obtain the animal from the same location over the experimental
period (January-June, 2007) to reduce variability in biotypes.
Acclimatization of Animals
The prawns, M. vollenhoevenii were kept in plastic holding tanks
(75x30x32 cm) which were three-quarter filled with water from lagoon and left
for a minimum of 7 days to allow them to adapt to experimental conditions (temperature
29.0±2°C, Relative Humidity, 78.9±3.0, Salinity, 16%). During
this period of acclimatization, the test animals were fed with dried and grounded
crayfish once daily. The lagoon water in the plastic holding tanks were aerated
with a 220V air pump and was changed once every 3 days to prevent accumulation
of toxic waste metabolite and decaying of food materials. Dead organisms were
removed with a hand net. Organisms were monitored until the population stabilized
before being used for experiment.
Test Chemicals
The test chemicals used were the spent lubricant oil and dispersant (OSD
94-60).
Spent Lubricant Oil (SLO)
The spent lubricant oil was collected from different mechanic workshops
around University of Lagos, Akoka Yaba, Lagos, Nigeria and stored in a 25 L
keg. The collection of the SLO in the different places was to simulate a natural
situation, since the spent lubricant oil that impact aquatic organisms come
from different sources.
Dispersant
The dispersant, OSD 94-60 is water based biodegradable hydrocarbon approved
for use in oil spill control by the Nigerian Regulatory Agency, Department of
Petroleum Resources (DPR).
Preparation of Test Media and Application of Toxicants
Lagoon water was used for the preparation of the test media. The bioassay
containers used were glass tanks (22x15x18 cm) which are observed to be advantageous
when compared to plastic containers as they minimize absorption of toxicants
and prevent risk of corrosion and chemical reactions. Some plastics are known
to react with some crude oil components.
A given volume of Lagos lagoon water was measured using a measuring cylinder into clean dry bioassay containers and a predetermined volume of Spent lubricant oil or dispersant or the mixture (9:1 and 3:1; SLO: dispersant) was added into the Lagos water to make it up to 1000 mL (total volume of test media) to achieve the desired test concentrations. The measurements of the SLO and dispersants were done using syringes of different volumes. The joint action tests were carried out based on the ratio 9:1 which represents the prescribed ratio for oil spill control in aquatic systems by the regulatory body in Nigeria while the ratio 3:1 serves as the equitoxic ratio of SLO and dispersant.
Bioassays
Single Action Toxicity of Spent Lubricant Oil against M. vollenhoevenii
Ten M. vollenhoeveni specimens of similar sizes were introduced
randomly into each of the 6 bioassays containers in three replicates per treatment
giving a total of 30 prawns including the untreated control. The prawns in the
bioassay container were exposed to varying concentration of SLO as follows:
1.0, 2.0, 3.0, 4.0 and 5.0 mL and untreated control. Mortality assessment was
carried out every 24 h for 4 days.
Single Action Toxicity of Dispersant against M. vollenhoevenii
Ten M. vollenhoeveni specimens of similar sizes were introduced
randomly into each of the 6 bioassays container. This was done in three replicates
per treatment giving a total of 30 prawns including the untreated control. The
prawns in the bioassay containers were exposed to varying concentrations of
dispersant as follows: 0.2, 0.4, 0.6, 0.8 and 1.0 mL and untreated control.
Mortality assessment was carried out every 24 h for 4 days.
Joint Action Toxicity of Mixtures of SLO and Dispersant against M. vollenhoevenii
Three replicates of 10 M. vollenhoevenii were introduced per
tank (6 tanks in all) including control tanks. For the predetermined test the
ratio of SLO and dispersant was in the ratio 9:1 and the prawns were then exposed
to varying concentration of the mixture as follows: 1.0, 2.0, 3.0, 5.0 and 6.0
mL and untreated control. For the equitoxic ratio 3:1, the prawns were exposed
to varying concentrations of the mixture as follows: 0.8, 1.0, 1.2, 1.4 and
1.6 mL and untreated control. Mortality assessment was carried out once every
24 h in 4 days.
Assessment of Quantal Response (Mortality)
The test animal, M. vollenhoevenii was considered dead if there was
no movement or response to stimulus even when prodded with a blunt glass rod.
Sublethal Studies
Acclimatization of the prawns for 7 days was also necessary before the start
of the sub-lethal studies. After the acclimatization period, twenty prawns were
exposed per tank to 1/10th 1/100th and 1/1000th of 96 h LC50 values
for the predetermined ratio, 9:1 and the equitoxic ratio, 3:1. For both the
predetermined and equitoxic experiments, there was a control. The exposure of
the prawns was done for 2 weeks (14 days) with the toxicants (SLO and dispersant)
replaced every 48 h before which the prawns were fed at least 2 h prior to toxicant
renewal.
Extraction of Haemolymph
Haemolymph was collected from three prawns using 1 mL syringe which was
injected at the base of the carapace and at the tail (telson). The haemolymph
was immediately transferred into an EDTA bottle (treated bottle to prevent the
coagulation of the haemolymph). Haemolymph extraction was done every 7 days
and the haemolymph samples were taken to the Haematological unit of Lagos University
Teaching Hospital (LUTH) at Idi-Araba, Lagos, Nigeria where they were analysed.
The tests carried out were Total leucocyte count, Differential polymorpho-neutrophils
count, Differential lymphocyte count and Differential eosinophil count.
Laboratory Analysis of Haemolymph
The materials that were used in the sub-lethal test were the commercially
prepared Leishman stain and distilled water. A drop of the blood sample was
placed on a clean glass slide and a cover spreader was used to make a thin film
of the blood. It was allowed to dry on a staining rack. The slide was flooded
with Leishman stain for 2 min. Staining was differentiated with 3-4 drops of
distilled water and left for 9 min. The slide was the rinsed with running water
and left to dry before examining under a microscope using immersion oil.
Statistical Analysis
Concentration Response Data Analysis
Toxicological concentration data involving quantal response (mortality) for both single and joint action studies were analysed by Probit analysis (Finney, 1971). The indices of toxicity measurement derived from these analyses was LC50 (median lethal concentration that causes 50% mortality of exposed organisms) and Toxicity factor (T.F) for relative potency measurements.
Analysis of Data and Measurement of Joint Action Toxicity of Binary and
Multiple Mixture of Test Compounds
The models used for the analysis of joint action toxicity data are the concentration-addition
model (Anderson and Weber, 1975) and the synergistic
ratios model. The concentration-addition model assumes that when similarly acting
toxicants are mixed in any proportion, they will add together to give the observed
response. In evaluating the joint-action, a predicted response value(s) e.g.,
LC50, is derived by summing up the LC50 values of separate
toxicants according to the proportion of their contribution in the mixture.
The predicted LC50 value(s) is then compared to the observed LC50
value of the mixture to classify the type of interaction among the components
of the mixture as follows:
| • | Additive if the observed LC50 value of the mixture is equal to the predicted LC50 value |
| • | Synergistic if the observed LC50 value of the mixture is less than the predicted LC50 value |
| • | Antagonistic if the observed LC50 value of the mixture is greater than the predicted LC50 value |
The relationship of derived LC50 values to predicted LC50 (RTU) is estimated as:
where, RTU = 1 describes additive action; RTU <1 describes antagonism; and RTU >1 describes synergism.
Measurement of joint-action toxicity by synergistic ratios was defined after Hewlett and Plackett (1959) as:
where, SR = 1 describes additive action; SR <1 describes antagonism; and SR >1 describes synergism.
Analysis of Variance (ANOVA), Student Newman Keuls (SNK) Tests and Duncan
Multiple Range Tests (DMRT)
One way Analysis of Variance (ANOVA) and comparison of means by Student
Neuman Keuls (SNK) (Zar, 1996) test were used to test
for statistical differences of the median lethal toxicity tests. One way analysis
of variance (ANOVA) and comparison of means by Duncan Multiple Range Test (DMRT)
(Zar, 1996) were used to test for statistical differences
between extrapolated fractions (1/10th, 1/100th and 1/1000th 96 h LC50)
and untreated control in the results (computed means) of the sub lethal tests
at 7 day and 14 day exposure periods.
RESULTS
Physico-Chemical Parameters of the Test Media
The mean values obtained for the physico-chemical parameters of the test
media (Lagoon water) throughout the period of the experiment were dissolved
oxygen (8.40 mg L-1), pH (6.84), Total dissolved solids (3.48 mg
L-1), Salinity (16‰) and temperature (29.0°C).
Single Action Toxicity of Spent Lubricant Oil and Dispersant against M.
vollenhoevenii
The result of dose mortality analysis of spent lubricant oil and dispersant
against M. vollenhoevenii reveals that the derived toxicity indices (LC50)
for spent lubricant oil ranged from 0.19 ml L-1 (24 h LC50)
to 0.08 ml L-1 (96 h LC50), while for dispersant the derived
toxicity indices (LC50) ranged from 0.12 mL (24 h LC50)/L
to 0.06 ml L-1 (96 h LC50) (Table 1).
| Table 1: | Relative toxicity of spent lubricant oil and dispersant against M. vollenhoevenii |
| CL: Confidence limit, LC: Lethal concentration, TF: Toxicity factor, TF = (LC50 of test compound at 48 h/LC50 of test compound at other h (72 and 96 h)), TF2: Toxicity factor, TF2 = (96 h LC50 of least toxic compound/96 h LC50 of test compounds) | |
On the basis of the computed toxicity factor (96-h LC50) ratios, the spent lubricant oil was found to be about 1.33 times more toxic against M. vollenhovenii than the dispersant. The randomized analysis of variance (ANOVA) showed that there was significant difference (p<0.05) in response to different treatments (concentrations) at 24, 48, 72 and 96 h of exposure.
Joint Action Toxicity of Binary Mixtures of Spent Lubricant Oil and Dispersant
(9:1) and (3:1) against M. vollenhoevenii
The analysis of concentration response data for the joint action toxicity
of SLO and dispersant in M. vollenhoevenii revealed from Table
2 that the median lethal concentrations of the binary mixture (9:1) and
(3:1) of SLO and dispersant were 0.18 and 0.56 ml L-1, respectively
indicating that the predetermined ratio was about 3 times more toxic than the
equitoxic ratio. In each case, it was observed that toxicity increases with
increase in exposure time.
Further analysis of the joint action toxicity data based on the concentration-addition model gave Relative Toxicity Unit (RTU) values of 5.66 and 1.33 for predetermined (9:1) and equitoxic (3:1) ratios, respectively indicating conformity with the model of synergism (Table 3). Furthermore, analysis of the joint action results based on the synergistic ratios model with reference to SLO shows that mixtures prepared based on ratio 9:1 was found to be less toxic (Sra = 0.44) (antagonism) than the spent lubricant oil when acting singly while the mixture prepared based on ratio 3:1 was found to conform with the model of synergis (Sra = 1.33) (Table 3). The interaction in SLO-dispersant (9:1 and 3:1) mixtures with reference to dispersant conformed to the model of antagonism (Srb = 0.33 and Srb = 0.11) (Table 3).
| Table 2: | Relative toxicity of the binary mixtures of SLO and dispersant against M. vollenhoevenii in predetermined ratio, 9:1 and equitoxic ratio:3:1 |
| CL: Confidence limit, LC: Lethal concentration, TF: Toxicity factor, TF = (LC50 of test compound at 48 h/LC50 of test compound at other h (72 and 96 h)), TF2: Toxicity factor, TF2 = (96 h LC50 of least toxic compound/96 h LC50 of test compounds) | |
| Table 3: | Analysis (Based on concentration-addition and synergistic ratio models) of the 96 h LC50 values of spent lubricant oil and dispersant when acting singly and jointly against M. vollenhoevenii |
| RTU: Relative toxic unit, RTU = Predicted LC50 value binary mixture/Observed LC50 value binary mixture, SR: Synergistic ratio, SR = LC50 of spent engine oil or dispersant/LC50 (spent engine oil+dispersant), TF: Toxicity factor, T.F = 96 h LC50 of least toxic compound/96 h LC50 of test compounds, SRa: Synergistic ratio (SLO), SRb: Synergistic ratio (dispersant) | |
| Table 4: | Mean haematological parameters of M. vollenhoevenii exposed to sublethal concentrations of binary mixtures of SLO and dispersant in pre-determined ratio (9:1) for 14 days |
| Means with the superscript letter in a column are not significantly different in the DMRT (p = 0.05) | |
| Table 5: | Mean haematological parameters of M. vollenhoevenii exposed to sublethal concentrations of binary mixtures of SLO and dispersant in equitoxic ratio (3:1) for 14 days |
| Means with the superscript letter in a column are not significantly different in the DMRT (p = 0.05) | |
Sub Lethal Effects of Binary Mixtures of SLO and Dispersant on Haematological
Parameters of M. vollenhoevenii
The haematological observations on the Total leucocyte count, Differential
polymorpho-neutrophils count, Differential lymphocyte count and the Differential
eosinophil count on M. vollenheovenii under sub lethal exposure to binary
mixtures of SLO-dispersant (9:1 and 3:1 ratios) are shown in Table
4 and 5. The results indicate a significant increase (p>0.05)
in the total leucocyte-count and differential polymorpho-neutrophils count of
M. vollenheovenii exposed for 14 days in the different treatments compared
to control. It was evident that the differential lymphocyte count of the M.
vollenheovenii exposed for 14 days resulted in a significant decrease (p>0.05)
in mean values in the different treatments. The randomized analysis of variance
(ANOVA) and Duncan test showed that there were significance differences (p>0.05)
in haematological parameters exposed to different sub lethal concentration at
7 and 14 days (Table 4, 5).
DISCUSSION
In this study, on the basis of derived toxicity indices, the dispersant (96 h LC50= 0.06 ml L-1) was found to be about 1.33 times more toxic than the SLO (96 h LC50= 0.08 ml L-1) when acting singly against M. vollenhoevenii while the joint action response indicated that the equitoxic ratio (96 h LC50 = 0.56 ml L-1) was relatively more toxic than the pre-determined ratio. This findings are in agreement with those of Otitoloju (2005) and Chukwu, 2006, who evaluated the joint action response of prawns against other dispersants approved for use by the Nigerian regulatory agency. However, the dispersant OSD 94-60 used in this study is more toxic than the biosolve dispersant used by Otitoloju (2005), where he gave the 96 h LC50 of biosolve against M. vollenhoevenii as 1.90 ml L-1.
From this study, the dispersant OSD 94-60 was found to be 3.2 times more toxic than the SLO against M. vollenhoevenii when acting singly. This observation is in agreement with the findings of Oyewo (1986) and Chukwu, 2006, who reported that some dispersants used in Nigeria were relatively highly toxic to some brackish water species. The different mixtures of SLO and dispersant in the joint-action toxicity in the ratios 9:1 and 3:1 tested against M. vollenhoevenii, showed that there were variations depending on the proportion of addition of the mixture components. The concentration addition model shows that the joint action of SLO and dispersant in the ratios 9:1 and 3:1 were both synergistic with the RTU values of 5.66 and 1.33, respectively. This means that at ratios 9:1 and 3:1 of the SLO and dispersant mixtures, the damages caused on organisms would be synergised when such ratios are applied during spent lubricant oil clean-up exercise.
When comparing the mixtures of SLO and dispersants at the different ratios ( 9:1 and 3:1) with SLO acting singly using the synergistic ratio model, it was found that the mixture of SLO and dispersant in the ratios of 9:1 and 3:1 was less toxic than when SLO was acting alone on M. vollenhoevenii. The findings also indicate that based on the synergistic ratios model with reference to SLO shows that mixtures prepared based on ratio 9:1 was found to be less toxic (Sra = 0.44) (antagonism) than the spent lubricant oil when acting singly while the mixture prepared based on ratio 3:1 was found to conform with the model of synergism (Sra = 1.33). Therefore, the use of the dispersant with the SLO in the ratio 9:1 would reduce toxic effect on M. vollenhoevenii when used in clean-up exercise of SLO spillage in the aquatic environment as the SLO acting singly has shown to be more toxic. However, comparing the mixtures of the SLO and dispersant in the ratio 9:1 and 3:1 to the dispersants acting alone shows that the mixtures in both ratio were less toxic. It is important to note that the SNK test results which show the statistical differences between all pairing concentrations indicated that there were significant differences in the mortality responses in the different concentrations of toxicants both in the single action and joint action toxicity.
Many studies have been conducted using isolated haematological parameters on fish especially (Blaxhall, 1972; Mawdesley-Thomas, 1971; Singer et al., 1990) but there is paucity of published data on prawn haematological parameters. It therefore becomes imperative to encourage haematological investigations of tropical prawns, especially the establishment of normal values together with the identifications of those environmental and disease conditions that may affect these values. If these are done, a bank of useful information would be available from which valuable and informative conclusions can be drawn. From results of haematological tests various derived values can be calculated such as the cellular stability, leucocyte count, haemocyte count, neutrophils count among many others.
In this study healthy prawns, M. vollenhoevenii were used to ascertain the sub lethal effects on the haemolymph exposed to binary mixtures of SLO and dispersant at various fractions of the 96 h LC50 of ratios 9:1 and 3:1. The haematological parameters of interest were the total leucocyte count, differential polymorpho-neutrophils count, differential lymphocyte counts and the differential eosinophil counts. Exposure of M. vollenhoevenii to sublethal concentrations of the binary mixture of SLO and dispersant in ratios 9:1 and 3:1 affected the total leucocyte count in that there was significant increase in values as the exposure time increased from day 0 to day 14. Higher total leucocytes values obtained with increase in sublethal concentrations and increase in number of days may be as a result of combating and coping with stress induced toxicants. These haematological responses of the prawn in this study are in agreement with the findings of Donsikova et al. (2006) reported that the haematological profile of carp, Cyprinus carpio showed significant (p<0.01) increase in the number of erythrocytes, segmented neutrophile granulocytes, developmental forms of myeloid sequence and eosinophils on exposure to sub lethal doses of pryrethoid, Cypermethrin. However, Svobodova et al. (2003) reported significantly lower values (p<0.01) of erythrocytes, haemoglobin and Packed Cell Volume (PVC) as a result of possible disruption of haemotopoiesis, but found no changes in white blood picture in carp after acute exposure to deltamethrin.
According to Johansson et al. (1992), the various haematogical parameters are closely attributed to the response of the organisms to its environment (test media in the experimental chambers). The leucocyte indicates any possibility of disease due to the pollutions or environmental conditions. Joshi et al. (2002) reported that the increase in white blood cell count can be correlated with an increase in antibody production which helps in survival and recovery of the fish exposed to sub lethal concentrations of pesticides.
Yamuna et al. (2002) stated that the immune defense of invertebrates is extremely efficient and like vertebrates, is mediated by complex cellular and hormonal interactions. They also reported that activation of immune functions worked well in test prawns, Macrobrachium malcolmsonii exposed to lowest sublethal concentrations of mercury and copper, hence total hemocytes number, percentile phagocytosis and superoxide anion production were found to be enhanced while higher sublethal concentrations of these metals have inhibitory effects on the hemocytes-mediated immune functions, therefore total hemocytes number, percentile phagocytosis and superoxide anion production were found to be declined.
There was an increase in the differential polymorpho-neutrophils count with increase exposure period from 0 to 14 days. Though there was no regular pattern between sublethal concentrations with differential polymorpho-neutrophils count, the longer the exposure period of the test organism, M. vollenhoevenii the higher the level of the differential polymorpho-neutrophils count. The differential polymorpho-neutrophils indicate level of stress on organisms. The differential lymphocyte count was found to decrease with increase in the number of days from day 0 to day 14, though there was no regular pattern between sublethal concentration and the differential lymphocyte count. The ANOVA and Duncan test revealed significant differences (p>0.05) in each of the haematological parameters in the different sublethal concentrations. The differential eosinophil count seems to be of no importance as no values were recorded in the test organism. Other authors who have identified blood parameters as potential tools for identification of stress in the environment include, Jusila et al. (2001) and Masson et al. (2002). According to these researchers, blood parameters such as levels of cortisol, glycemia, haemoglobin, hematocrit, Cl¯ content, haemolymph clotting time and total haemocyte counts are very useful indicators of chemical/physical stress in the environment.
In conclusion, the result of this present study suggests the use of dispersants for control of spent lubricant oil spillage may actually reduce biological damage if the varying mixtures of SLO and dispersants at certain ratios are taken into considerations. If this is done the immune functions of the test prawns, M. vollenhoevenii would be less susceptible to any disease and stress induced toxicant as indicated by the results obtained in varying sublethal concentrations on the tests prawns. Hence, pollution of natural aquatic environment by spent lubricant oil spillage should be strictly monitored.