Morphometric indices and Oxidative Stress Biomarkers of African Catfish Clarias gariepinus juveniles Exposed to Agricultural Fungicide Mancozeb

Introduction

The repeated and indiscriminate use of pesticides due to anthro- pogenic activities have resulted in a large build-up and subsequent discharge of surface water run-off, with damaging consequences to aquatic ecosystem [1]. Schulz reported that, 10% of pesticides re- leased to the soil reach non-target areas more especially in the farms and fields [2]. Sources of agricultural pollutants worldwide, 40% of pesticide is confined to herbicides, 10% to fungicides and the rest of pesticides fall into other classes of pesticides which are accumulated by fish from water, food sediments, and residues [3, 4].

Mancozeb is a synthetic ethylene bisdithiocarbamates belonging to a subclass dithiocarbamates of carbamates pesticides containing manganese Mn2+and Zinc2+ atoms [5]. Mancozeb is classified as a contact or broad spectrum fungicide having a preventive activity characteristic and one of the most used agrochemical on a global scale [6, 7]. Mancozeb has low acute toxicity to aquatic organisms and there are several concerns with degradation by products [8]. Morphometric indices such as condition factor and hepatosomatic indices can be used in environmental toxicological investigations to find out fish health status following exposure to toxicants [9, 10].

Oxidative stress has been identified as a major mechanism by which mancozebcause harmful effects to fish [11]. In fish species oxidative stress is mainly studied in environmental contamination by pollutions [12]. Specifically, oxidative stress induced bymanco- zeb in fish has been linked with manganese- derived reactive oxy- gen species (ROS) formation mechanism [13, 14]. Considering the effects of exposure of fish to different dosages of mancozeb (0.9, 1.5 and 3mg). Atamaniuk et al. evaluated the levels of oxidative stress markers and in the system of antioxidants of kidney, liver and brain of Goldfish Carasiusauratus [15]. The result showed collectively that exposure of this species to fungicides led to the development of mild oxidative stress and activation of antioxidant defense system in the tissues [15]. The present study investigat- ed the morphometric indices and oxidative stress biomarkers of African Catfish Clariasgariepinusjuveniles exposed to agricultural fungicide mancozeb

Method

Procurement of experimental fish

A total of 150 Clariasgariepinusjuveniles with standard length and weight that ranged from 9.8 cm to 17.5 cm and 11 g to 55 g were procured from Freedom Fisheries Limited, University market Road, Nsukka, Enugu, Nigeria and was transported to Fisheries Wet Laboratory, Department of Zoology and Environmental Biol- ogy, University of Nigeria, Nsukka. The Fishes were disinfected with 0.05% potassium permanganate (KMnO4) for 2minutes to avoid any dermal infections, were allowed to acclimatize in the laboratory conditions for 2weeks in plastic tanks of 300Litre (L) Capacity. Fishes were fed daily with food (Aqua-feed commercial feed size 3mm) contaminating 40% crude protein twice daily at 2-3% body weight. Food, faecal matter and other wastes will be siphoned off and water were changed weekly to reduce ammonia content in water. Dead fishes were removed with forceps to avoid possible deterioration of the water quality. During acclimatiza- tion, the water was changed weekly with well aerated tap water. The commercial formulation of Mancozeb 80% WP(Z-FORCE®) weighing 50g with batch number 01062018 marked by Jubaili Agrotec Limited Abuja, Nigeria were procured from Ogige Local Market Nsukka, Enugu State, Nigeria and stored at room tempera- ture.

Experimental design for sublethal exposure

The experimental design for sub-lethal exposure consist of 150 fish for four groups of 0.0, 20.55mg/L, 41.09mg/L and 82.18mg/L (A- D), each with three replicates. Each tank contained 10 litre dechlo- rinated tape water served as the control while the three other treat- ments were exposed to water containing 20.55mg/L, 41.09mg/L and 82.18mg/L of Mancozeb corresponding to1/20, 1/10 and 1/5 of the 96hrs LC50 value that were derived after acute toxicity ex- periment. The experimental duration lasted 28days during which the fish was fed with small quantity of feed approximately 1% of their total body weight about an hour before the test solution were renewed daily. The feeding was to avoid mortality and cannibal- ism.

On each sampling day (1, 7, 14, 21 and 28) three to five fishes from each of the treatment groups including the control were sacri- ficed after anesthesizing with Tricainemethanesulfonate (MS222) to minimize stress. The sacrificed fishes were weighed and blood was collected through caudal puncture while the organs such as Kidney, Liver, gill and Spleen were quickly rinsed in cold 0.9% Sodium chloride solution. Tissues of the Clariasgariepinusfrom each triplicate were homogenized immediately in pre-chilled Po- tassium phosphate buffer (1:10 w/v, 0.1 M, pH 7.0). One part of the homogenate was used for the estimation of lipid peroxidation while the other part was centrifuged for 20 minutes at 10,500 rpm under 4°C to obtain the supernatant which were stored at 4°C for further enzyme assay. After, the end of the sub-lethal exposure, the remaining fish in each of the concentrations were withdrawn from the exposure of chemical and were placed in chemical free water in which further observation were made after 7days of the withdrawal.

Determination of morphometric Indices

The body weight of each fish from the control and each treatment (Triplicate) group were sampled on day 1, 7, 14, 21 and 28days (7days recovery) of the experiment and recorded based on hep- atosomatic index and condition factor. Dissecting kits were used to dissect the liver of the fish. The liver of fish was weighed. The hepatosomatic index and condition factor were calculated based on White and Fletcher method as follows [16]: HSI= liver weight (g)/ body weight (g) x 100/1 CF = Body weight (g)/ standard length (cm)3 x 100/1

Assay on oxidative stress and antioxidant parameters

Tissue lipid peroxidation was measured by estimating the quantity of TBARS according to the method of Sharma and Krishna-Murti and was expressed as nanomoles of TBARS formed/mg protein [17]. Briefly, 1.0 mL of homogenate prepared in KCl solution was incubated at 37°C for 30 min. Proteins were precipitated by adding 1 mL of 10% trichloroacetic acid and then centrifuged at 2,000 × g for 15 min. One mL of supernatant was taken as an aliquot in a separate tube to which 1 mL of thiobarbituric acid reacting sub- stances solution was added. The tubes were kept in a boiling-water bath for 10 min. After the tubes were cooled, the optical density was read at 535 nm.

Tissue catalase was spectrophotometrically determined by mea- suring the rate of H2O2 breakdown based on the decrease in ab- sorbance at 240 nm; the activity was expressed as U/mg protein [18]. Superoxide dismutase (SOD) activity was assayed by the method of Misra and Fridovich (1972), the specific activity being expressed as U/mg protein in the liver and gill tissues [19]. The ac- tivity of glutathione peroxidase (GPx) was measured by the meth- od of Lawrence and Buck (1976), with the specific activity being determined using the extinction coefficient of 6.22 mM/cm [20]. The activity of glutathione reductase (GR) was assayed by mea- suring NADPH oxidation at 340 nM, the activity being expressed as U/mg protein [21]. The total protein contents were determined by the method of Lowry et al.

Statistical Analysis

Data was analysed with Statistical Packages for Social Scienc- es (SPSS) version 20.0 (IBM Corp, Armork, USA) and Statplus v5.9.8 (Analyst soft Inc., Walnut, Canada), probit regression anal- ysis using Finney method (lognormal distribution) for (LC) was recorded. Two-way analysis of variance (ANOVA) was used to compare concentration of Mancozeb and duration of exposure de- pendent effects. The mean was partitioned using DMRT (Duncan Multiple Range Test). Level of significance was set at p<0.05 re- spectively

Results

Physico- Chemical Parameters of the Testwater

The physico-chemical parameters of the test water used for sub-le- thal concentration for 5weeks were shown in table 1. The tempera- ture ranged from 29.4to29.8°C,pH ranged from 9.23 to9.43, Alka- linity ranged from 1.95 to 2.43, CO2ranged from 6.5 to 8.88mg/L, DO2 ranged from 7.0 to 7.15mg/L.

Table 1: Physico-chemical parameters of the water used for the experimentonClariasgariepinus lethal concentration

Behavioral changes and physiological abnormalities of Clarias gariepinus exposed to Mancozeb at different concentration lev- els for both acute and sub-lethal toxicity (Table 2). In the control duration of exposure, normal behavioral responses and no mor- tality were observed. Treatment groups with Mancozeb exhibited physiological and behavioral abnormalities based on increase in duration and concentration. The tanks with higher concentration of the test chemical fish displayed faster opercular movement, jerky movement, erratic swimming, skin coloration, convulsion, hyper- activity, gulping of air, hemorrhage and loss of equilibrium status were also observed. Clarias gariepinus lost equilibrium balance, became exhausted owing to respiratory complications and finally settled down at the bottom and mortality occurred.

                                                                                    Notes: None=-, Mild=+, Moderate=++, Strong=+++.

     Table 2: Effect of Mancozeb on behavioural characteristics of Clariasgariepinus at different concentration levels

                                            Table 3: Results of various toxicity investigations of mancozeb on some Fish species

Percentage mortality of Clarias gariepinus Juveniles exposed to graded concentration of Mancozeb at 96h increase in toxicant concentration (Table 4). Fishes exposed to 150mg/L, 300mg/L, 450mg/L, 600mg/L and 700mg/L had 5%, 20%, 55%, 70% and 85% which recorded the highest mortality compared to other con- centrations. While no mortality was recorded at the control group. Throughout the exposure duration no absolute (100%) mortality occurred (Table 4)

                                         Table 4: Mortality of Clarias gariepinus exposed to different concentration of Mancozeb

In sub- lethal concentration no mortality was observed and fish displayed abnormal behavioral changes throughout the experi- mental period. Result findings on different investigations of Man- cozeb on some fish species showed variations in LC50based on the pesticide type, duration of exposure and stage of maturity (Table 5). The toxicant concentration in all the groups exposed to Manco- zeb decreased as time progressed. LC50values of Mancozeb with 95% confidence limit of various concentration in Clarias gariepi- nus for the duration of 24, 48, 72 and 96h were 1740.58mg/L(95% cl, 14148.68 – 21088.18), 23044.02mg/L (95% cl,1833.49 – 2842.20), 6211.00mg/L (95% cl, 5129.85 – 7419.65) in Table 5.

                                                                                                   CI= confidence interval

                                                       Table 5: Lethal concentration of Mancozeb on Clarias gariepinus

The estimated safe level of Mancozeb values in Clarias gariepinus varied from 41.09 to 4.109 x 10^-3mg/L respectively (Table 6).

                                 * C= 48h LC₅₀ × 0.03S² Where C is the presumed harmless concentration and S=24h LC₅₀

                                    Table 6: Estimation of safe level for Clarias gariepinus after 96h Exposure

Morphometric indices (Hepatosomatic indices and Condi- tion factor)

In this work the result on condition factor and hepatosomatic in- dices revealed that no significant effect was observed between the control groups and fish treated with Mancozeb which also indi- cated that Mancozeb does not have any serious effect on the liver weight in comparism to the fish body weight. In our study, there was no significant effect of Mancozeb on hepatosomatic indices in Clariasgariepinus (Table 1).

Condition factor

The result on condition factor in this work revealed that no signifi- cant change was observed for the condition factor between control group and fish treated with Mancozeb. This indicated that condi- tion factor of the fish was not altered throughout the duration of the exposure (Table 7).

Table 7: Effect of Exposure to various Sub-Lethal levels of Mancozeb on Hepatosomatic Indices and Condition factor of Clar- iasgariepinus Hepatosomatic indices

Data were presented with mean ± standard deviation. Means with different alphabet superscripts along each column represents sig- nificant differences for the concentrations while Means with dif- ferent number superscripts along each row represent significant differences for the exposure duration.

Effects of different concentration of mancozeb on oxidative stress biomarkers

Oxidative stress biomarkers parameters such as LPO, SOD, GPx, GR and MDA had significant difference in different tissues (Kid- ney, liver and Gill) except CAT across treatment groups and ex- posure durations compared to their controls for 28days (Table 8). Our results showed duration and concentration dependant increase in LPO reflecting increased oxidative stress in Clariasgariepinus exposed to Mancozeb on day 21 was higher in the gills group C than Liver compared to day 28 (7days recovery) which had low LPO level in the kidney Control. CAT indicated that there was no significant difference on the Catfish tissue (Kidney, liver and gill) exposed to Mancozeb across different treatment groups and peri- ods for 28days. The pesticide had higher effect on day 7 Kidney control compared to day 1 for kidney group C. Our result on SOD showed significant increase and fluctuating trends on the catfish tissues (Kidney, liver and gills) exposed to Mancozeb across dif- ferent treatment groups and periods for 28days. The pesticide had higher effect on day 7 gills group C compared to day 21 for liver group A. SOD significantly decreased on 7, 14, and 21 compared to controls, but was as higher in tissues controls.

Our result on GPx showed significant reduction and fluctuation trends on the catfish tissues (Kidney, liver and gill) exposed to Mancozeb across different treatment groups and period for 28days compared to control. The pesticide had higher effect on day 1 gill group A compared to day 21day liver group C to control.

Our result with GR showed significant increase and fluctuation trend on the Catfish tissues (kidney, liver and gill) exposed to Man- cozeb across different treatment and periods for 28 days. The pes- ticide had higher effect on day 21 gills group C compared to day 1 kidney control. Our result on MDA showed significant increase and fluctuation trend on the Catfish tissues (Kidney, liver and gill) exposed to Mancozeb across different treatment groups and peri- ods for 28 days. The pesticide had higher effect on day 1 gill group C compared to day 28 kidney control.