Effects of glyphosate-based herbicide on gametes fertilization and four developmental stages in Clarias gariepinus

Abstract

Comparative toxicology continues to provide information on how the age of every living organism affects the frequency, severity, and nature of the potentially toxic agent. We investigated the effect of glyphosate-based herbicide (GBH) exposure on gametes and four developmental stages of Clarius gariepinus (C. gariepinus) (African Catfish). Gametes from healthy gravid female and mature male C. gariepinus were exposed to GBH in sublethal concentrations of 0.0 (G1, control), 0.02 (G2), 0.05 (G3), 0.1 (G4), 0.5 (G5), and 1.0 (G6) mg/L for 24 h at the standard conditions of temperature and water quality parameters. The surviving embryos were examined microscopically for malformation rate and edema occurrence post-GBH exposure. In a separate experiment; postfryer, fingerling, posfingerling and juvenile C. gariepinus were exposed to G1, G2, G3, G4, G5 and G6 of GBH concentrations daily consecutively for 28 days. Fish growth performance, behavioural changes, haematology, oxidative stress, and histology were assessed. From our results, GBH showed altered morphology 24 h post-fertilization, decreased body weight, growth parameters, behavioural indices, and survival rate in the various developmental stages. Oxidative stress metabolite, malondialdehyde levels, increases in the postfryer > postfingerlin > fingerling > juvenile C. gariepinus following GBH exposure. Leukopenia and thrombocytosis were observed in the postfingerlings and juvenile fish and decrease in the levels of reduced glutathione and activity of superoxide dismutase compared with the control. Histology showed gross necrosis of the fish gills, liver, brain, and cardiac myocytes in the exposed fish. Hence, our findings provide an insight into C. gariepinus developmental toxicity due to GBH, although continuous measurement of glyphosate levels in the fish and fish environment is essential.

1. Introduction

Currently, farmers worldwide have increased their reliance on pesticides and chemical fertilizers to improve crop yields [1,2]. It is now being increasingly used in many parts of the world to replace tillage to improve environmental conditions [2]. But, their non-compliance application has created a widespread emergence of pesticides-resistant weeds. Also, reports have suggested the potential toxic doses of pesticides at concentrations relevant to human exposure [3]. Various farmers have different reasons for its application, for instance, fish farmers purposely employ pesticides to affect plants around the fish farms. However, the majority of it is taken by drainage into the pond which affects non-target organisms such as animals, including aquatic organisms [4,5]. Various studies have opined that pesticide use in most developed and developing countries in West Africa is a leading factor mitigating against high yield in fish farming. Herbicides such as glyphosate, atrazine, and paraquat are popular weed killers used around fish farms [[5], [6], [7]]. Herbicides are chemical compounds used mostly to control undesirable vegetation in agriculture and forestry [2,3]. Glyphosate commercial formulation, Glyphosate-based herbicide (GBH), is the most widely used herbicide worldwide, and its usage has increased geometrically [1,3,8]. Recent studies have implicated glyphosate and GBH as major risk factors for food poisoning, water pollution, metabolic and endocrine disruption among others [8,9]. Evidence to support glyphosate and its compound mixture toxicities were obtained from assays including cytotoxicity, genotoxicity, endocrine disruption, oxidative stress, induced pluripotent stem cells, and modification of adipogenesis [3,[10], [11], [12]]. Studies of increasing toxicological reports following occupational and non-occupational exposure made International Agency for Research on Cancer classify glyphosate and GBH as probably carcinogenic to humans, mainly for non-Hodgkin lymphoma [10]. When GBH is used to control weeds on the farm area may find its way into the water and affect the fish life cycle due to poor irrigation and monitoring. Now, different methods are being proposed using state-of-the-art facilities for the analysis and automated detection of pesticides in fruits and vegetables [13]. Accumulation of these risks involved constitutes a general concern on the growth and development of fish amongst others. Though animals were assumed to be safe from glyphosate toxicity, several recent studies have challenged this proposal [14,15]. Thus, studies on the toxicological effects of GBH have raised serious divergent findings in the literature [[15], [16], [17],[18], [19]]. Reports have shown that GBH once ingested can impact protective microbial communities; alter fish behavior and impair nutritional uptake and growth [[19], [20], [21]]. Mechanistically, glyphosate, the active ingredient in GBH, dislodges the shikimic acid of plant biochemical pathway which serves as machinery for the biosynthesis of amino acids [14,22]. Further, this can affect the offsprings pattern of aquatic life and impact subsequently transgenerational problems [6,23].

There is a paucity of data that informs on the risks associated with herbicide toxicity on the growth and developmental status of African catfish. The African catfish (Clarias gariepinus) is widely distributed throughout the African continent [[24], [25], [26]]. The high reliance on these opportunistic and omnivorous ocean foods demands maintaining the structure and stability of food webs in the ecosystem [[18], [27]]. It is used for spiritual purposes amongst Africans [26].

Catfish are locally produced and one of the most popular fishes in Western Africa, particularly in Nigeria [4,28]. The local fish farmers produce over 5 million catfish for sale daily in Africa [28]. Their demands and prices vary depending on its size; hence, the farmers show very concern about the growth performance. Fish diets, fish exposure, and management are influential and can greatly affect fish size, maturity, habitat responses, and yield [29]. Though glyphosate/GBH accumulation persists in the environment food web, issues related to few data available in the population has limited conclusion on tolerable levels of glyphosate/GBH for farm products, fishes, birds, and other mammals [[[18], [30]]]. Comparative toxicologists have not kept aloof from this problem; especially as the unexpected development of new diseases emerges. Moreover, lack of development of vital enzyme systems and or defensive mechanism in early life can make them more susceptible, whereas in the old, loss of contractility, degenerative processes, and diseases can precipitate unusual substance toxicological effects [1,12,31,32]. This is because catfish are sensitive to epigenetic, anthropogenic, and other biological factors that result in toxicity and animal health issues [29]. These data can be used to improve aquaculture practices and preserve environments at risk. Since a very large population in Africa rely on this fish for food, focusing on the general toxicological potentials of GBH and its congeners on the growth performance, biochemical, heamatological, behavioural, and organ-system functions matters [12,17]. One major reason for this study is a need to increase our understanding to predict the potential toxicity of GBH in fish and subsequently on the population. Also, to comment based on direct application as related to the fish farmers in the nearby local community. These will help the community with the possibility of adopting interventions that will encourage optimal fish production. Therefore, this study assessed the effects of Glyphosate-based herbicide on gametes fertilization and four developmental stages in Clarias gariepinus to ascertain their effects on fish biology as well as to adopt a One Health intervention approach.

2. Materials and methods

2.1. Drugs and chemicals

Ovulin® (Menotropin; Follicle Stimulating Hormone and Luteinizing Hormone in a ratio of 1: 1) was obtained from Ningbo Second Hormone Factory, 28 Renjian Road, Zonghan Street, Cixi City, Zhejiang Province, China. Glyphosate in the commercial product Forceup® was used (480 g/L Glyphosate-Isopropylamine salt) SL Sino Agrochemicals, China. All solvents and reagents used were of analytical grade.

2.2. Fish husbandry

All tanks were provided with adequate aeration according to standard protocols for fish management [33]. The experiment was conducted under the Guide for the Care and Use of Laboratory Animals approved by the College of Health Sciences, Olabisi Onabanjo University, Nigeria.

2.3. Experimental design and ethics

Healthy gravid female (n = 5) and mature (n = 3) male fish, as well as early stages of C. gariepinus were obtained from Wazhaq Agro Farms, Ikenne-Remo, Ogun State, Nigeria and acclimatized to laboratory conditions at the College of Health Science, Department of Pharmacology, Toxicology Unit, Olabisi Onabanjo University, Ogun State, Nigeria in aquarium tanks (1.4 × 0.7 × 0.7 m) for 7 days in aquaria (4 × 4 × 2 m) containing sufficient tap water (pH 7.6) at 26 °C. Breeding was carried out following the method of Sarkar, & Borah et al. [34]. Gravid females were injected an ovulin® (0.5 per kg fish body weight) intraperitoneally. About 10.0 ± 0.5 h after ovulin® injection, the ripe eggs were squeezed directly into Petri dishes (10 h post-injection). The testes was dissected and gently mixed with the eggs very quickly by hand swirling and kept for 5 min. The gamete mixture was washed in normal saline (0.6 NaCl) to remove the sperm suspension. For gametes, some amount (n = 60 mL eggs) was introduced into each pond containing tap water. GBH in different concentrations of 0.0 (G1, control), 0.02 (G2), 0.05 (G3), 0.1 (G4), 0.5 (G5), and 1.0 (G6) mg/L were administered into the ponds covered with kakaban for 24 h. The GBH and water in the control (G1) group were washed out after 24 h by gently draining each chamber and adding tap water slowly each day to avoid disturbing embryos, and the temperature was maintained at 26 ± 1 °C. The daily photoperiod consisted of 12 h of light and 12 h of darkness. Embryos were observed after 24 h for mortality, hatching rate, and occurrence of lethality. The embryonic acute toxicity of glyphosate was used to evaluate the cumulative mortality and number of the fertilized population due to GBH exposure (24 h post fertilization, hpf), in the control and test groups of catfish fry exposed to glyphosate. The surviving embryos were examined under the microscope for malformation rate and edema occurrence post-GBH exposure. The GBH was used directly with the concentrations deduced from the manufacturer instructions (480 g/L) for different ponds. The current maximum contaminant level (MCL) set by the U.S. Environmental Protection Agency (EPA) for glyphosate in drinking water in the United States is 0.7 mg/L Battaglin et al. [35]. The tolerable window for humans (0.7 mg/L) was considered to carefully select the doses of 0.02–1.0 mL (0.001 g–0.48 g of GBH). Each group was allowed to develop at 25 °C for measuring embryonic development. The fertilized embryo was viewed under a microscope (4X magnification). Microscopic observation of 24 hpf was performed using a Celestron digital microscope (4.3 LCD, 5 MP CMOS built-in digital camera). In a separate study, the GBH treatment was evaluated for a subchronic administration. The four different stages of catfish of different age groups of the same breed were observed in different pond water and GBH was introduced daily by gently draining each chamber, at the temperature of 26 ± 1 °C and maintained at 12 h light/12 h darkness. The postfry (n = 55 per aquarium), fingerlings (n = 48 per aquarium), post-fingerlings (n = 36 per aquarium), and juvenile groups (n = 34 per aquarium) were introduced into the tap water of control (G1) or in different doses of GBH, 0.02 (G2), 0.05 (G3), 0.1 (G4), 0.5 (G5), and 1.0 (G6) mg/L. The different groups were examined daily for 28 days for growth performance and behavioural responses. The fish were fed (Fish pellets from 24, Le Pontde Pierre, Skretting France) different diet sizes (0.3 mm, 0.5 mm, 1.5 mm, and 2 mm) two times daily (8:00–17:00 h) at the rate of 2 ± 0.5% of mean body wet weight of fish. The water quality parameters of the experimental water were determined according to APHA (2005) and mean ± SD values ranged from 26.10 ± 1.2 °C (temperature), pH (5.93 ± 0.42), conductivity (15.33 ± 5.03 μs/cm), dissolved oxygen (6.30 ± 0.26 mg/L), water hardness (9.67 ± 1.53 mg/L), salinity (0.004 ± 0.002) mg/L, and particulates (28.33 ± 15.5 mg/L). The dietary feeding rate of fish was adjusted weekly by the weight of the growing fish. Fish were collected randomly with a kitchen hand net 24 h after the last administration, and 10 healthy fish were selected randomly. The fish were sacrificed following the method by Blessing et al. [36]. Twenty-four hours after the last administration, whole blood was obtained into EDTA. Further, vital fish tissues (gill, brain, heart, and liver) were carefully excised, cleared of adhering tissues, and weighed. A small portion of the excised tissue was fixed in 10% formaldehyde and subsequently prepared for histology. The remaining portion of the excised gill was weighed and homogenized in four volumes of 100 mM of phosphate buffer (pH 7.4). The whole blood and gill homogenate obtained from each fish were then analyzed to assess haematological parameters and biochemical parameters respectively.

All the animal experiments and protocol were maintained according to the rules and guidelines of National Institute of Health (NIH, 2000) for laboratory animal care and use. All procedures followed the protocols of the Faculty of Basic Medical Sciences, College of Health Science, Olabisi Onabanjo University, Ogun State, Nigeria Institutional Animal Care and Use Committee. All methods reported were in accordance with ARRIVE guidelines for the reporting of animal experiments.

2.4. Fish exploratory behavior

The fish exploratory behaviour (FEB) of the control and GBH-treated was based on Altenhofen et al. [19]. This was performed in a temperature-controlled room (26 ± 1 °C) between 8 a.m. and 2 p.m. Animals were placed individually in experimental tanks (40 cm length × 20 cm height × 20 cm width), and after 60 s of habituation, their locomotor behavior and immobility were recorded for 10 min once in four weeks. The videos were analyzed using the Digital Camera (Kodac Pixpro 42× wide 24–1008 mm) software. The numbers of line crossed, and immobility time spent in water (anxiolytic-like behavior) were analyzed.

2.5. Morphometric analysis

On a weekly basis, counting fish per pond, randomized sampling length and general weight measurement (n = total weight of fish per pound) were assessed [25].

2.6. Growth performance

The direct weight (balance) and length (transparent meter rule) of the experimental fish were measured at the start (baseline), week 1, week 2, week 3, and week 4 respectively. The obtained weight and length were used to calculate growth index (GI), weight gain (WG), and specific growth rate (SGR), and growth performance was calculated using the following formulae [25]:

$$\mathrm{G}\mathrm{I}=(\mathrm{F}\mathrm{W}-\mathrm{I}\mathrm{W})/\mathrm{I}\mathrm{W}$$

WG = FW–IW; where FW is the final weight and IW is the initial weight

$$\mathrm{S}\mathrm{G}\mathrm{R}(\%\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{d}\mathrm{a}\mathrm{y})=100\mathrm{x}(\mathrm{F}\mathrm{W}-\mathrm{I}\mathrm{W})/\mathrm{T}$$

Relative growth rate (RGR, %) = (FW–IW)/(IW x 100)

Survival (%) = number of fish remaining/number of fish at initial stock

$$\mathrm{O}\mathrm{S}\mathrm{I}=(\mathrm{O}\mathrm{r}\mathrm{g}\mathrm{a}\mathrm{n}\mathrm{o}\mathrm{r}\mathrm{c}\mathrm{a}\mathrm{r}\mathrm{c}\mathrm{a}\mathrm{s}\mathrm{s}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}/\mathrm{w}\mathrm{h}\mathrm{o}\mathrm{l}\mathrm{e}\mathrm{b}\mathrm{o}\mathrm{d}\mathrm{y}\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t})\mathrm{x}100(\mathrm{L}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{t}\mathrm{h}\mathrm{g}\mathrm{a}\mathrm{i}\mathrm{n},\mathrm{LG})$$

$$=\mathrm{F}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{l}\mathrm{L}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{t}\mathrm{h}-\mathrm{I}\mathrm{n}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}\mathrm{L}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{t}\mathrm{h}$$

LG was used to calculate the percentage change in body length as follows:

% Survival = n/number initially in stock.

2.7. Biochemical analyses

2.7.1. Preparation of supernatant

At the end of the exposure, five pools of whole body fryers or gill were frozen until analysis. The sample was homogenized in a phosphate buffer (0.1 M; pH 7.5; 4 °C) using an UltraTurrax® tissue homogenizer fitted with a potter at 4,000 rpm for 10 min at room temperature. The supernatant fractions obtained were placed in different tubes for total protein and oxidative stress and antioxidant biomarkers assays.

2.7.2. The total protein

The total protein concentration was determined using the method of Lowry et al. [37] on fish aliquots fraction. Bovine Serum Albumin (BSA) was used as a standard. Measurement was performed using a spectrophotometer.

2.7.3. Oxidative stress and antioxidant status

Following the sacrifice, tissue samples (five samples from each experimental group) were obtained from the gills of all experimental groups, to determine oxidative stress markers and antioxidant status of the four different stages of C. gariepinus following the exposure to GBH herbicide. All collected tissues were homogenized in phosphate-buffered saline (pH 7.2) and centrifuged. The supernatant obtained was transferred into clean tubes and stored at −4 °C and used for these assays. Lipid peroxidation was also estimated spectrophotometrically in the gill tissue by the thiobarbituric acid-reactive substance (TBARS) using the method described by Varshney and Kale [38] and expressed in terms of malondialdehyde (MDA) formed per mg protein. Reduced glutathione (GSH) level in the same was estimated using the method described by Beutler et al. [39]. Assessment of gill superoxide dismutase (SOD) activities followed the methods of Misra and Fridovich [40]. An automated haematology analyzer (Pentra-XL 80, Horiba ABX, USA) was used to estimate whole blood parameters indices.

2.7.4. Haematology

An automated haematology analyzer (Pentra-XL 80, Horiba ABX, USA) was used to estimate whole blood parameters indices.

2.8. Statistical analysis

Statistical analysis was performed using SPSS and GraphPad Prism (version 6) software, respectively. Data was subjected to one-way analysis of variance (ANOVA), followed by T-test and Tukey's post hoc multiple comparison test. A p < 0.05 was considered significant.

3. Results

3.1. Acute toxicity of GBH to hatching of C. gariepinus embryos

Acute toxicity of GBH to hatching of C. gariepinus embryos exposed to GBH (Hours Post Fertilization, hpf). The percentage hatching rates (n = 92 ± 3.8 fish, n = 60 mL) following 24 h were 98.7, 95, 77, 61 and 43 in the 0.02, 0.05, 0.1, 0.5 and 1.0 of GBH administration when compared with control normal (100%) group. Significant differences (p < 0.05) were obtained in the 77%, 61% and 55% versus the control normal (100%) group yield.

3.2. Body weight gain

GBH decreased body weight gain (BWG) in the postfryers C. gariepinus administered G2 or G3 (52.5%), G5 (59.3%), and G6 (88.1%) when compared with the control untreated group (Fig. 2). Also, similar decreased were obtained in the Fingerlings C. gariepinus G4 (37.7%) and G6 (32%) respectively. The administration of GBH decreased BWG in the Postfingerlings that received G3 (42.9%), G4 (50.3%) and G6 (54.7%). Further, only the G3 (41.2%) shows decreased BWG in the juvenile C. gariepinus treatment groups.

Fig. 2.

Effects of sub-lethal concentration of glyphosate-based herbicide (GBH) on body weight gain in four stages developmental stages Clarias gariepinus (African Catfish).Results represented as Mean ± Standard Error of Mean (SEM). N = 15.). G1 (tap water of control), G2 (GBH, 0.02 mg/L), G3 (GBH, 0.05 mg/L), G4 (GBH, 0.1 mg/L), G5 (GBH, 0.5 mg/L), and G6 (GBH, 1.0 mg/L). *p < 0.01 or ^#p < 0.001 when compared with control normal (G1) group.

3.3. Growth index, relative growth rate and specific growth index

An administration of GBH to C. gariepinus decreases growth index (GI) in the Postfryer and Postfingerlings by 66.8% (G3), 69.2% (G4), 78.3% (G5), 93.3% (G6) and 48.2% (G2), 54.4% (G3), 58.8% (G4), 68.8% (G5), 68.6% (G6) when compared with the control untreated group (Fig. 3). Also, both C. gariepinus fingerlings G5 (40.5%), G6 (30.1%) and juvenile G3 (44.4%), G4 (33.3%), G6 (25%) showed decrease GI as well. At the Postfryer C. gariepinus stage, GBH reduces relative growth rate (RGR) by G5 (36.4%), G6 (82.2%) and in the fingerlings by G4 (39%), G5 (44%) and G6 (35.6%) when compared with the control untreated group (Fig. 4). Similarly, GBH administrations lowered RGR in the Postfingerlings and Juvenile C. gariepinus by G4 (53.3%), G5 (51.3%) and G4 (40.6%), G5 (33.4%), G6 (46.6%) in the treated groups. GBH administrations in the Postfryer C. gariepinus lowered specific growth index (SGI) in the G2 (52.6%), G3 (51.7%), G5 (59.8), G6 (88.5%) when compared with the control untreated group (Fig. 5). While fingerlings C. gariepinus did not show any significant change, the postfingerlings and juvenile had lowered SGI in the G4 (42.8%), G5 (54.8%), G6 (50.4%) and G3 (40.7%), G4 (33.3%) when compared with the control untreated group.

Fig. 3.

Effects of sub-lethal concentration of glyphosate-based herbicide (GBH) on growth index in four stages developmental stages of Juvenile Clarias gariepinus (African Catfish). Results represented as Mean ± Standard Error of Mean (SEM). N = 15. G1 (tap water of control), G2 (GBH, 0.02 mg/L), G3 (GBH, 0.05 mg/L), G4 (GBH, 0.1 mg/L), G5 (GBH, 0.5 mg/L), and G6 (GBH, 1.0 mg/L). *p < 0.01 or ^#p < 0.001 when compared with control normal (G1) group.

Fig. 4.

Effects of sub-lethal concentration of glyphosate-based herbicide (GBH) on relative growth rate in four stages developmental stages of Juvenile Clarias gariepinus (African Catfish). Results represented as Mean ± Standard Error of Mean (SEM). N = 15. G1 (tap water of control), G2 (GBH, 0.02 mg/L), G3 (GBH, 0.05 mg/L), G4 (GBH, 0.1 mg/L), G5 (GBH, 0.5 mg/L), and G6 (GBH, 1.0 mg/L). *p < 0.01 or ^#p < 0.001 when compared with control normal (G1) group.

Fig. 5.

Effects of sub-lethal concentration of glyphosate-based herbicide (GBH) on specific growth rate in four stages developmental stages of Juvenile Clarias gariepinus (African Catfish). Results represented as Mean ± Standard Error of Mean (SEM). N = 15. G1 (tap water of control), G2 (GBH, 0.02 mg/L), G3 (GBH, 0.05 mg/L), G4 (GBH, 0.1 mg/L), G5 (GBH, 0.5 mg/L), and G6 (GBH, 1.0 mg/L). *p < 0.01 when compared with control normal (G1) group.

3.4. Survival

The administrations of GBH alter cause significant loss in the Postfryer and Fingerlings population by 17% and 11% (Fig. 6). There was no change in the postfingerlings and juvenile population.

Fig. 6.

Effects of sub-lethal concentration of glyphosate-based herbicide (GBH) on survival rate in four stages developmental stages of Juvenile Clarias gariepinus (African Catfish). Results represented as Mean ± Standard Error of Mean (SEM). G1 (tap water of control), G2 (GBH, 0.02 mg/L), G3 (GBH, 0.05 mg/L), G4 (GBH, 0.1 mg/L), G5 (GBH, 0.5 mg/L), and G6 (GBH, 1.0 mg/L). *p < 0.01 when compared with control normal (G1) group.

3.5. Body length change

GBH administrations produce a significant decrease in the body length change (BLG) of the postfryer C. gariepinus that received G2 (39.4%) and G5 (32.7%) (Fig. 7). Also, there was a significantly reduced change in length in the fingerlings C. gariepinus by 40.3% (G3), 34.3% (G4), and 35.2% (G6) when compared with the control untreated group. However, in contrast, the postfingerlings and juvenile C. gariepinus had a length decrease in the grouped administered G3 (31.4%), G4 (37.3%), and G3 (194.2%), G4 (159.4%) when compared with control untreated group.

Fig. 7.

Effects of sub-lethal concentration of glyphosate-based herbicide (GBH) on body length in four stages developmental stages of Juvenile Clarias gariepinus (African Catfish). Results represented as Mean ± Standard Error of Mean (SEM). N = 10. G1 (tap water of control), G2 (GBH, 0.02 mg/L), G3 (GBH, 0.05 mg/L), G4 (GBH, 0.1 mg/L), G5 (GBH, 0.5 mg/L), and G6 (GBH, 1.0 mg/L). *p < 0.01 or ^#p < 0.001 when compared with control normal (G1) group.

3.6. Line crossing

The administrations of GBH significantly reduced locomotor and anxiety explorative line crossing test in the postfryer C. gariepinus that received the highest dose G6 by 38.5% (Fig. 8). Also, postfingerlings C. gariepinus had reduce numbers in the groups administered G3 (37.5%) and G4 (25.4%). The GBH administered to Juvenile C. gariepinus cause reduced length in the groups that received G5 (31.5%) and G6 (34%) when compared with the control untreated group.

Fig. 8.

Effects of sub-lethal concentration of glyphosate-based herbicide (GBH) on exploratory line crossing test in four stages developmental stages of Juvenile Clarias gariepinus (African Catfish). Results represented as Mean ± Standard Error of Mean (SEM). N = 10. G1 (tap water of control), G2 (GBH, 0.02 mg/L), G3 (GBH, 0.05 mg/L), G4 (GBH, 0.1 mg/L), G5 (GBH, 0.5 mg/L), and G6 (GBH, 1.0 mg/L). *p < 0.01 when compared with control normal (G1) group.

3.7. Swimming test

GBH administered postfryers C. gariepinus shows increased immobility time in the groups of G2 (34.1%), G4 (35%), G5 (42%), and G6 (37.3%) when compared with the control (Fig. 9). Also, the treated fingerlings C. gariepinus had increase immobility by G3 (42.6%), G4 (46.1%), G5 (37.3%) and G6 (61.9%) respectively. Additionally, both postfingerlings and juvenile C. gariepinus that received G4, G5, and G6 showed increased immobility time by 49.8%, 102.8%, 80% and 66.2%, 51.5%, 99.8% when compared with the control untreated group.

Fig. 9.

Effects of sub-lethal concentration of glyphosate-based herbicide (GBH) on Force Swim Test in four stages developmental stages of Juvenile Clarias gariepinus (African Catfish). Results represented as Mean ± Standard Error of Mean (SEM). N = 10. G1 (tap water of control), G2 (GBH, 0.02 mg/L), G3 (GBH, 0.05 mg/L), G4 (GBH, 0.1 mg/L), G5 (GBH, 0.5 mg/L), and G6 (GBH, 1.0 mg/L). *p < 0.01 or ^#p < 0.001 when compared with control normal (G1) group.

3.8. MDA gills assay

The lipid peroxidation measured as MDA level increased in the postfryer C. gariepinus groups that received GBH by 50% (G3), 78.6% (G4), 92.9% (G5), and 157.1% (G6) when compared with the control untreated group (Fig. 10). Also, similar increase where obtained for the fingerlings and postfingers in the G4 (62.5%), G5 (71.9%), G6 (125%) and G5 (96.7%), G6 (123.3%) respectively. GBH administrations cause MDA elevation in the juvenile C. gariepinus groups that received G3 (30.2%), G5 (37.2%) and G6 (58.1%).

Fig. 10.

Effects of sub-lethal concentration of glyphosate-based herbicide (GBH) on gill Malondialdehyde (lipid peroxidation) Levels in four stages developmental stages of Juvenile Clarias gariepinus (African Catfish). Results represented as Mean ± Standard Error of Mean (SEM). N = 5. G1 (tap water of control), G2 (GBH, 0.02 mg/L), G3 (GBH, 0.05 mg/L), G4 (GBH, 0.1 mg/L), G5 (GBH, 0.5 mg/L), and G6 (GBH, 1.0 mg/L). *p < 0.01 or ^#p < 0.001 when compared with control normal (G1) group.

3.9. GSH gills assay

GSH decreases significantly in the fingerlings, postfingerlings, and juvenile C. gariepinus that received G6 (31.5%) and G4 (31%), G5 (40.5%), G6 (42.9%) and G5 (35.5%), G6 (58.1%) when compared with the control untreated group (Fig. 11).

Fig. 11.

Effects of sub-lethal concentration of glyphosate-based herbicide (GBH) on gill Reduced Glutathione (GSH) in four stages developmental stages of Juvenile Clarias gariepinus (African Catfish). Results represented as Mean ± Standard Error of Mean (SEM). N = 5. G1 (tap water of control), G2 (GBH, 0.02 mg/L), G3 (GBH, 0.05 mg/L), G4 (GBH, 0.1 mg/L), G5 (GBH, 0.5 mg/L), and G6 (GBH, 1.0 mg/L). *p < 0.01 when compared with control normal (G1) group.

3.10. SOD gills assay

The Administration of GBH reduced the activity of SOD in the postfryer C. gariepinus by G5 (25%), G6 (50%) (Fig. 12). Also, SOD activity in the postfingerlings C. gariepinus was elevated in the groups that received G2 (122.2%) while it reduces in the G6 (38.9%) when compared with the control. However, the activity of SOD in the fingerlings and juvenile remained unaltered.

Fig. 12.

Effects of sub-lethal concentration of glyphosate-based herbicide (GBH) on gill Superoxide Dismutase (SOD) in four stages developmental stages of Juvenile Clarias gariepinus (African Catfish). Results represented as Mean ± Standard Error of Mean (SEM). N = 5. G1 (tap water of control), G2 (GBH, 0.02 mg/L), G3 (GBH, 0.05 mg/L), G4 (GBH, 0.1 mg/L), G5 (GBH, 0.5 mg/L), and G6 (GBH, 1.0 mg/L). *p < 0.01 when compared with control normal (G1) group.

3.11. Postfingerlings haematology

The WBC reduces in the GBH treated postfingerlings C. gariepinus groups that received G4 (38.5%), G5 (42%), G6 (35.6%) when compared with control (Table 1). Also, GBH lowered HGB levels significantly in this group that received the lowest dose by 20.1% (G2). The postfingerlings C. gariepinus had elevated platelet levels in the G2 (29.3%) while the same was lowered in the G5 (47.2%) and G6 (53.9%) when compared with the control untreated group.

Table 1.

Effects of sub-lethal concentration of glyphosate-based herbicide on some haematological parameters of Postfingerlings Clarias gariepinus (African Catfish).

PostFingerlings Haematology	G1	G2	G3	G4	G5	G6
WBC	13.35 ± 3.3	11.90 ± 3.70	10.85 ± 3.55	8.25 ± 0.55*	7.75 ± 0.45*	8.60 ± 1.90*
LYMPH	4.15 ± .05	4.05 ± 1.55	4.85 ± 0.75	3.85 ± 0.85	3.35 ± 0.55	4.35 ± 1.15
MID	0.65 ± .05	1.75 ± .750#	1.15 ± .050#	1.15 ± .350#	0.90 ± .30	1.20 ± .500*
GRAN	3.50 ± .50	7.55 ± .950*	9.85 ± 7.85*	6.90 ± 2.50*	3.50 ± .40	3.05 ± .25
LYMPH%	49.90 ± 3.10	29.05 ± 4.55*	47.55 ± 8.95	33.40 ± 3.20*	42.60 ± 4.60	50.30 ± 2.30
MID%	7.95 ± .05	12.70 ± 2.60	12.25 ± 4.25*	10.00 ± .20	11.25 ± 3.15*	13.45 ± 2.65*
GRAN%	45.15 ± .15	58.25 ± 7.15	40.10 ± 13.10	56.60 ± 3.40	46.15 ± 7.75	36.25 ± 4.95*
HGB	14.90 ± .30	11.90 ± 2.00*	14.45 ± .05	13.75 ± .15	13.65 ± .85	12.80 ± 3.30
RBC	7.93 ± .16	6.94 ± .90	8.44 ± .19	7.39 ± .02	6.09 ± .87	6.38 ± 2.13
HCT	45.10 ± 1.50	35.45 ± 5.95*	42.15 ± .05	40.10 ± .60	40.65 ± 2.25	38.65 ± 7.95
MCV	56.95 ± .75	50.95 ± 1.95	50.05 ± 1.05	54.35 ± .75	68.75 ± 13.45	63.55 ± 8.75
MCH	18.75 ± .05	17.00 ± .70	17.10 ± .30	18.55 ± .15	23.05 ± 4.65*	20.60 ± 1.70
MCHC	33.00 ± .40	33.50 ± .00	34.25 ± .05	34.25 ± .15	33.50 ± .20	32.70 ± 1.80
RDWCV	14.80 ± .50	16.00 ± .30	14.90 ± .90	15.20 ± .00	14.00 ± .50	15.45 ± 1.15
DWSD	28.80 ± .10	27.55 ± 1.95	25.60 ± 1.60	28.75 ± 0.75	34.05 ± 6.05*	35.20 ± 7.20
PLT	982.50 ± 218.50	1270.50 ± 101.50*	911.00 ± 121.00	835.00 ± 98.00	518.50 ± 312.50*	453.00 ± 78.00*
PCV	6.15 ± .15	6.75 ± .050	7.30 ± .100	6.55 ± .45	7.55 ± .75	8.85 ± 1.15
PDW	14.90 ± .30	15.50 ± .10	16.35 ± .05	15.25 ± .15	15.70 ± .20	15.90 ± .10
PCT	0.56 ± 0.06	0.52 ± 0.06	0.61 ± 0.08	0.45 ± 0.01	0.37 ± 0.20*	0.37 ± 0.19*

Results represented as Mean ± Standard Error of Mean (SEM). N = 10. G1 (tap water of control), G2 (GBH, 0.02 mg/L), G3 (GBH, 0.05 mg/L), G4 (GBH, 0.1 mg/L), G5 (GBH, 0.5 mg/L), and G6 (GBH, 1.0 mg/L). WBC × 103 (/mL) White Blood cell, LYMPH × 103 (/mL): Lymphocyte, GRAN (%): Granulocytes, HGB (g/dL): Hemoglobin, RBC × 106/mL: Red Blood Cell, HCT (%): Hematocrit, PLT (/L): Platelet, PCT (%): Plateletcrit. *p < 0.01 or ^#p < 0.001 when compared with control normal (G1) group.

3.12. Juvenile haematology

The WBC reduces in the GBH-treated Juvenile C. gariepinus groups that received G4 (41.5%), G5 (42.9%), G6 (49.1%) when compared with control (Table 2). Also, GBH increased HGB levels significantly in this group that received the G5 (56.8%) and G6 (37.9%). RBC was elevated (p < 0.05) in the G2 group by 40%. The Juvenile C. gariepinus had elevated PLT levels in the G2 (26.6%) while the same was lowered in the G5 (33.5%) and G6 (40.4%) when compared with the control untreated group.

Table 2.

Effects of sub-lethal concentration of glyphosate-based herbicide on some haematological parameters of Juvenile Clarias gariepinus (African Catfish).

Juvenile Haematology	G1	G2	G3	G4	G5	G6
WBC	11.20 ± .60	12.10 ± 1.30	9.20 ± 2.60	6.55 ± 3.05*	6.40 ± 3.00*	5.70 ± 2.70*
LYMPH	3.40 ± 1.200	6.00 ± .00*	2.50 ± 1.200	5.00 ± 1.00*	3.30 ± 1.40	2.65 ± 1.2
MID	0.80 ± 0.30	1.45 ± 0.45*	0.75 ± 0.45	1.05 ± 0.15*	0.55 ± 0.25	0.65 ± 0.35
GRAN	5.00 ± 1.10	4.65 ± .85	3.30 ± 1.40	5.15 ± 0.55	2.55 ± 1.35*	2.40 ± 1.20*
LYMPH%	35.95 ± 2.95	50.05 ± 5.65*	37.65 ± .65	44.55 ± 6.45	52.65 ± 2.75	47.20 ± 2.40
MID%	8.45 ± 0.65	11.40 ± 2.60	11.45 ± 1.55	9.40 ± 0.60	8.75 ± 0.25	11.00 ± 1.00
GRAN%	55.60 ± 3.6	38.40 ± 2.90*	50.85 ± 2.25	45.90 ± 7.20	38.60 ± 2.50*	41.25 ± 1.45
HGB	10.30 ± .400	13.70 ± 0.80	13.15 ± 2.35	12.40 ± 2.30	16.15 ± 0.05*	14.20 ± 0.30*
RBC	5.38 ± 0.93	7.53 ± .42*	6.63 ± 1.97	6.25 ± 1.60	7.38 ± 1.72*	6.19 ± 1.31
HCT	33.10 ± .30	41.25 ± 1.35	39.20 ± 6.40	37.20 ± 6.00	48.05 ± 1.75*	41.80 ± .90*
MCV	63.45 ± 10.35	54.95 ± 1.25	61.80 ± 8.70	61.15 ± 6.05	69.45 ± 18.55	71.15 ± 16.55*
MCH	19.55 ± 2.65	18.15 ± .05	20.55 ± 2.55	20.20 ± 1.50	23.10 ± 5.50	24.10 ± 5.60
MCHC	31.05 ± 0.95	33.15 ± 0.85	33.40 ± 0.50	33.15 ± 0.85	33.60 ± 1.10	33.90 ± 0.50
RDWCV	23.60 ± 3.40	16.75 ± 1.55	13.90 ± 0.90	15.50 ± 1.90	13.55 ± 0.55*	13.85 ± 0.45*
DWSD	49.75 ± 14.45	31.6 ± 2.80	31.20 ± 6.50	34.80 ± 7.60	34.45 ± 8.85	34.80 ± 6.80
PLT	747.50 ± 537.50	946.00 ± 56.0*	652.50 ± 405.50	648.50 ± 193.50	497.00 ± 314.00*	445.50 ± 322.50*
PCV	7.85 ± 0.85	6.55 ± 0.25	7.40 ± 1.10	7.35 ± 0.55	8.15 ± 1.15	8.00 ± 1.30
PDW	15.00 ± 0.30	15.40 ± 0.10	15.30 ± 0.10	15.35 ± 0.05	15.45 ± 0.15	15.75 ± 0.15
PCT	0.38 ± .01	0.62 ± .06*	0.44 ± 0.23	0.47 ± 0.11	0.37 ± 0.20	0.31 ± 0.20

Results represented as Mean ± Standard Error of Mean (SEM). N = 10. G1 (tap water of control), G2 (GBH, 0.02 mg/L), G3 (GBH, 0.05 mg/L), G4 (GBH, 0.1 mg/L), G5 (GBH, 0.5 mg/L), and G6 (GBH, 1.0 mg/L). WBC × 103 (/mL) White Blood cell, LYMPH × 103 (/mL): Lymphocyte, GRAN (%): Granulocytes, HGB (g/dL): Hemoglobin, RBC × 106/mL: Red Blood Cell, HCT (%): Hematocrit, PLT (/L): Platelet, PCT (%): Plateletcrit. *p < 0.01 or ^#p < 0.001 when compared with control normal (G1) group.

3.13. Histology

Histology of GBH-treated C. gariepinus following H & E stain showed grossly necrosis and destruction of both primary and secondary lamellae of the gills in all treated fish (Fig. 15, Fig. 17, Fig. 19, Fig. 23). Additionally, the highest dose causes extensive damage to brain tissue with characteristics of neuronal cell bodies disposed on a background of neuropil and interlacing fascicles of myocytes in the exposed fish plus extensive inflammation by aggregates of red blood cells (Fig. 14, Fig. 16, Fig. 18, Fig. 21). The liver was moderately altered in the treated fish (Fig. 22), although, GBH doses used were toxic to the heart (see Fig. 20).

Fig. 15.

Histologic sections of Glyphosate-based herbicide (GBH) exposured postfryer C. gariepinus (African Catfish) gill tissue shows G1 and G2 necrosis and destruction of both primary and secondary lamellae with mild lamellar necrosis; G3 and G4 necrosis and destruction of both primary and secondary lamellae with moderate lamellar necrosis; G5 necrosis and destruction of both primary and secondary lamellae with severe lamellar necrosis. G1 (tap water of control), G2 (GBH, 0.02 mg/L), G3 (GBH, 0.05 mg/L), G4 (GBH, 0.1 mg/L), G5 (GBH, 0.5 mg/L), and G6 (GBH, 1.0 mg/L) (H & E, X100).

Fig. 17.

Histologic sections of Glyphosate-based herbicide (GBH) exposured fingerling C. gariepinus (African Catfish) tissue shows immature gills with mild necrosis (G1) in the primary and secondary lamellae with mild lamellar necrosis; G2 and G3 and G4 and G5 tissue showed immature gills with mild necrosis in the primary and secondary lamellae with moderate lamellar necrosis; G6 tissue shows necrosis and destruction of both primary and secondary lamellae with severe lamellar necrosis. G1 (tap water of control), G2 (GBH, 0.02 mg/L), G3 (GBH, 0.05 mg/L), G4 (GBH, 0.1 mg/L), G5 (GBH, 0.5 mg/L), and G6 (GBH, 1.0 mg/L) (H & E X100).

Fig. 19.

Histologic sections of Glyphosate-based herbicide (GBH) exposured postfingerling C. gariepinus (African Catfish) tissue show in the G1 and G2 necrosis and destruction of both primary and secondary lamellae mild lamellar necrosis; G3 and G4 showed necrosis and destruction of both primary and secondary lamellae with moderate lamellar necrosis; G5 and G6 tissue showed necrosis and destruction of both primary and secondary lamellae with severe lamellar necrosis. G1 (tap water of control), G2 (GBH, 0.02 mg/L), G3 (GBH, 0.05 mg/L), G4 (GBH, 0.1 mg/L), G5 (GBH, 0.5 mg/L), and G6 (GBH, 1.0 mg/L) (H & E, X100).

Fig. 23.

Histologic sections of Glyphosate-based herbicide (GBH) exposured juvenile C. gariepinus (African Catfish) gills shows presence of primary and secondary lamellae. G1 shows no areas of necrosis or inflammation are seen (normal gills). G2 and G3 tissues showed necrosis and destruction of both primary and secondary lamellae (mild lamellar necrosis). G4 tissue shows necrosis and destruction of both primary and secondary lamellae (moderate lamellar necrosis). G5 and G6 tissues showed severe necrosis and destruction of both primary and secondary lamellae (severe lamellar necrosis). G1 (tap water of control), G2 (GBH, 0.02 mg/L), G3 (GBH, 0.05 mg/L), G4 (GBH, 0.1 mg/L), G5 (GBH, 0.5 mg/L), and G6 (GBH, 1.0 mg/L) (H & E Stain, X100).

Fig. 14.

Histologic section of postfryer C. gariepinus (African Catfish) brain tissue showed neuronal cells on a background of neuropil following Glyphosate-based herbicide (GBH) exposure. No abnormalities are seen in G1 - G6. G1 (tap water of control), G2 (GBH, 0.02 mg/L), G3 (GBH, 0.05 mg/L), G4 (GBH, 0.1 mg/L), G5 (GBH, 0.5 mg/L), and G6 (GBH, 1.0 mg/L) (H & E, X100).

Fig. 16.

Histologic section of Glyphosate-based herbicide (GBH) exposured fingerling C. gariepinus (African Catfish) brain tissue showed neuronal cells on a background of neuropil. G1 (tap water of control), G2 (GBH, 0.02 mg/L), G3 (GBH, 0.05 mg/L), G4 (GBH, 0.1 mg/L), G5 (GBH, 0.5 mg/L), and G6 (GBH, 1.0 mg/L). No abnormalities are seen (X100H & E stain).

Fig. 18.

Histologic section of Glyphosate-based herbicide (GBH) exposured postfingerling C. gariepinus (African Catfish) brain tissue showed neuronal cells on a background of neuropil. No abnormalities are seen. G1 (tap water of control), G2 (GBH, 0.02 mg/L), G3 (GBH, 0.05 mg/L), G4 (GBH, 0.1 mg/L), G5 (GBH, 0.5 mg/L), and G6 (GBH, 1.0 mg/L) (X100H & E stain).

Fig. 21.

Histologic sections of Glyphosate-based herbicide (GBH) exposured juvenile C. gariepinus (African Catfish) brain tissue shows neuronal cell bodies disposed on a background of neuropil. G1, G2, G3, G4 and G5 with no abnormality seen (normal brain). G6 showed areas of inflammation showing accumulated aggregates of red blood cells are also seen (encephalitis). G1 (tap water of control), G2 (GBH, 0.02 mg/L), G3 (GBH, 0.05 mg/L), G4 (GBH, 0.1 mg/L), G5 (GBH, 0.5 mg/L), and G6 (GBH, 1.0 mg/L) (H & E STAIN X100). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 22.

Histologic sections of Glyphosate-based herbicide (GBH) exposured juvenile C. gariepinus (African Catfish) liver tissue (G1 - G5) shows parallel radially arranged plates of hepatocytes with Central vein (CV), portal vein (PV) and the basophilic portion with nucleus and the acidophilic cytoplasm of the acinar cells (No abnormalities are seen). G6 showed inflammatory hepatocytes and distended PV. G1 (tap water of control), G2 (GBH, 0.02 mg/L), G3 (GBH, 0.05 mg/L), G4 (GBH, 0.1 mg/L), G5 (GBH, 0.5 mg/L), and G6 (GBH, 1.0 mg/L) (H & E STAIN X100).

Fig. 20.

Histologic sections of Glyphosate-based herbicide (GBH) exposured juvenile C. gariepinus (African Catfish) heart muscle shows interlacing fascicles of cardiac myocytes/myocardial cells with extensive inflammation seen by aggregates of red blood cells with myocardial inflammation. G1 (tap water of control), G2 (GBH, 0.02 mg/L), G3 (GBH, 0.05 mg/L), G4 (GBH, 0.1 mg/L), G5 (GBH, 0.5 mg/L), and G6 (GBH, 1.0 mg/L) (H & E STAIN X100). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

4. Discussion

The use of glyphosate-based herbicides (GBH) has increased geometrically in the last three decades [3,5]. Glyphosate is a synthetic and broad-spectrum herbicide commonly used around the world [12]. Thus, increasing levels of glyphosate, GBH, and their metabolites have constituted unwanted substances in soil, air, and water, as well as in disease [10,12]. Several efforts have been made to collate data on pesticides to examine relations among the many interfaces that pesticides transverses [41,42]. Early developments were targeted for pesticides to be persistent which has led to some of them being banned [43]. Studies on the effects of glyphosate and GBH on human health have been reported at the cellular level based on their toxicity to different organ systems [11,12]. This has informed efforts to revisit the previous postulation of the non-toxic action of glyphosate/GBH on rodents or humans. We now understand how glyphosate affects plant biochemical pathways, although its impacts on rodent and or aquatic communities are emerging [14,22]. Pesticides used in agricultural or urban settings have been detected as runoff into water bodies, and soil and volatilizing into the atmosphere [2]. The goal of one health intervention approach is to evaluate the popular aphorism that we are what we eat. Studies have been published on some aspects of the biology of GBH in catfish, but we find little information on the relationship between GBH exposure and the developmental stages of C. gariepinus [6,25,32]. This study, therefore, evaluated the toxicological potential of GBH exposure during gametes fertilization and four developmental stages of C. gariepinus (Fig. 1). Techniques for comparing actual measurements of pesticide residues in stream water with estimates of acute toxicity are a direct evaluation of contaminant toxicity levels [31]. Here, we recorded the morphological indices, developmental status, growth rate, biochemical, haematological, and histology which are predictors of biological changes. Although, several of the toxicological evaluation or measurements related to behavior in this present study are usually limited to the laboratory, however, such changes which arise from this exposure were confirmed through comparisons with controls or with responses observed given the exposure period. These laboratory events are supposed to correlate aquatic environments as the basis for direct field works to enable translation into what may be obtained in humans. From our results in this study, GBH decreased BWG, GI, RGR, and SGI in the four developmental stages when compared with the control untreated group. Although, growth performance showed no homogeneity in fish weight at the beginning of this experiment given the different levels of doses of GBH exposure (Fig. 2, Fig. 3, Fig. 4, Fig. 5). Also, RGR was less in the postfryer and fingerlings C. gariepinus stage than in the postfingerlings and juvenile C. gariepinus when compared with the control (Fig. 4). There were significant losses in the postfryers and fingerlings population due to GBH intoxication. Pesticides inhibition of fertilization of eggs fishes has been observed at different doses relevant to aquatic bodies [16,43]. Also, the decrease survival of developing eggs and percentage of hatchings of the eggs of catfish correlates with increasing concentrations of different GBH up to 1.0 part per million (Fig. 13). The present study would benefit from the assessment of glyphosate levels in the tissues of the different developmental stages in the future study. It has been confirmed that once these pesticides leave their point of application, they change from being plant or pest control chemicals to being environmental contaminants that are suspected sources of stress to aquatic plants and animals [5,43]. Our findings indicate that the very early developmental stages of C. gariepinus are very chemically sensitive, in particular to GBH toxicity, as we observed altered growth indices. Similarly, GBH administrations produce a significant decrease in BLC in the postfryer, fingerlings, then in the postfingerlings and juvenile C. gariepinus when compared with the control untreated group (Fig. 7). By implications, the postfryers and fngerlins are both formative stages that are rate limiting for the further development. This may be because all the body organs are fully developed in situ positioned. Nexus of behavioural models have been developed to understand the study of toxicants in aquatic toxicology [19,20]. Many of these endpoints have been applied to integrate how alterations in behavior may be related to ecologically-relevant issues such as predation avoidance, prey capture, growth, stress resistance, reproduction, and longevity [20,44]. They are valuable tools to discern and evaluate the effects of exposure to environmental stressors [44]. The administrations of GBH significantly reduced locomotor and anxiety explorative line crossing test in the postfryer, postfingerlings, juvenile when compared with the control untreated group (Fig. 8). All the treated C. gariepinus fish had increased immobility (Fig. 9) against the control untreated group. Reports have posited that alterations in fish behavior, particularly in non-migratory species, could be a fingerprint for an important signal for ecosystem assessment [20,45]. From our study, it appears both locomotor and fish immobility showed a discernible effect that was influenced by the introduction of GBH. Immobility is used to score neurotoxicity challenges frequently observed in changes in form, frequency, or posture of swimming movements before mortality [46]. Altered swimming behaviors have been associated with subchronic doses of pesticides and serve as models for additional stressors [47,48]. Studies in the future would be to direct efforts towards effective quantification of glyphosate levels prior to and post fertilization and during growth and development in the C. gariepinus fish onsite in their natural environment and then evaluate its potential toxicity levels on the behavioural indices. In addition, we measured the oxidative stress levels in the treated fish. Oxidative damage to the body is an exercise that is unavoidable in aerobic life [15]. But, reactive oxygen species (ROS) emanate from an imbalance between the pro-oxidative species and the antioxidant system that protect cells. Some generation of ROS arises by many mechanisms in an organism even under physiological conditions while others are induced [49]. In the organism, ROS plays a phagocytic role related to cell-to-cell activity by harnessing oxygen consumption whether in fishes or mammals [50]. Pesticides enhancing pro-oxidant production of ROS by several biochemical processes via the inactivation of antioxidant enzymes, depletion of free radical scavengers, membrane destabilization, and faulty electron transportation have been reported [33]. Wherefore, the actions of ROS result in oxidative processes that attenuate lipids, proteins, gene expression modification, and altered redox status have been documented [49,50]. In this study, the lipid peroxidation product measured as MDA level increased mostly in all fish that received GBH doses of G3, G4, G5 and G6 and this occurred at all the four developmental stages of C. gariepinus treated groups (Fig. 10). On the other hand, antioxidant GSH levels decreases significantly in the fingerlings, postfingerlings and juvenile C. gariepinus that received GBH intoxication (Fig. 11). Also, the GBH reduced the activity of SOD in the postfryer C. gariepinus. Contrastingly, SOD activity in the postfingerlings C. gariepinus was elevated while activities of SOD in the fingerlings and juvenile remained unaltered (Fig. 12). Consequently, our results support the hypothesis that an increasing age confers improving antioxidant status that may rescue the exposed fish from the environmental hazard [51]. Nevertheless, the most affected are the postfryer because they have low antioxidant power and or immune system. Further, we assessed haematological effects of GBH in the postfingerlings and juvenile C. gariepinus for any possible benign and malignant disorders due to GBH. Haematology has been performed in aquatic toxicology, but, few studies correlated the presence of pesticides on fish haematology and the natural immune system [50,52]. These are considered pathophysiological indices of the fish system and are important in evaluating the body status of fish exposed to toxicants [3,11,53]. Interestingly, haematological distortion characterizes the GBH-exposed fish as seen in this experiment. Following a subchronic exposure, reductions in some hematological parameters were observed in GBH-exposed fish. In the GBH-treated postfingerlings and juvenile C. gariepinus, the WBC reduces when compared with the control (Table 1). While the various GBH-treatments lowered HGB levels in the postfingerlings, it increases in the juvenile (Table 2). This observation may support the basis for studies of how GBH interacts with fish. The higher doses, G5 and G6, lowered platelets levels in both postfingerlings and juvenile treated C. gariepinus group. Whether our observed GBH-induced leukopenia and thrombocytosis at this development would persist in the adult fish and or would translate into One Health advert effects in the food chain is very hard to tell. Thus, this may serve as a basis for further assessment of GBH-exposed fish for a One-health epigenomic study. The GBH-induced growth retardation, behavioural changes, and biochemical toxicities observed in our results were accompanied by damage to the fish gills in all treated fish. Further, there was also inflammation of brain tissue associated with increasing doses of GBH (Fig. 14, Fig. 16, Fig. 18, Fig. 21). Histology shows intoxication as observed in the pathological alterations of inflammatory hepatocytes and portal vein congestion at the moderate to highest dose used in this study (Fig. 22) in the juvenile. Although our results provide valuable data for noticing the deleterious effects of GBH on non-target organisms, in particular C. gariepinus, we are limited by facilities to determine the exact concentration of glyphosate and its metabolite aminomethyl-phosphonic acid in the fish tissue and each pond. Studies to assess the molecular mechanism of glyphosate and its combination directly in the fish during growth and development are ongoing.

Fig. 1.

Glyphosate based herbicide (GBH) induction age of Clarias gariepinus gametes fertilization programing and transitions. dpf: per day post fertilization; hr: hour.

Fig. 13.

Microscopic morphological assessments of African Catfish C. gariepinus showing deformities observed following exposure to Glyphosate-based herbicide (GBH) (0, 0.02, 0.05, 0.5, 0.1 and 1.0 mg/L) at 24 hpf. Deformities included poor eye location (E), yolk sac edema (YE), pericardial edema (PE), spinal curvature (SC) and growth retardation (GR). G1 (tap water of control), G2 (GBH, 0.02 mg/L), G3 (GBH, 0.05 mg/L), G4 (GBH, 0.1 mg/L), G5 (GBH, 0.5 mg/L), and G6 (GBH, 1.0 mg/L). Two experiments were conducted at two different concentration ranges (Microscopic View of C. gariepinus embryo 24 hpf, scale: 1: 400 μm; Mag. x40).

5. Conclusion

Overall, evidenced by our findings are tendencies of GBH to cause toxicity in the various developmental stages of C. gariepinus. Also, there is a need to investigate the molecular mechanisms through GBH interaction with C. gariepinus. Additionally, the environmental protection agency needs to promote the campaign on the cautions regarding the potential of GBH to provide safe sea foods.

Ethical aspects

All experimental procedures were performed according to the ethical standards for animal experimentation, and meticulous efforts were made to ensure that the animals suffered as little as possible and reduce external sources of stress, pain, and discomfort. The current study has not exceeded the number of animals needed to produce reliable scientific data. This article does not refer to any study with human participants performed by any authors.

Author contribution section

Kale Ezekiel Kale,Temitope Funmi Kale, Ifabunmi Oduyemi Osonuga: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Adaeze Ngozi Adebesin: Performed the experiments; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Oluwatosin Omobola Soyinka: Conceived and designed the experiments; Performed the experiments; Wrote the paper.

Oladoja Farouk: Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Deborah Uwaezuoke, Oluwadunsin Olajide, Victor Akinloye, Olatoun Adedugbe, Faith Odibosa, Bolaji Oladele, Mariam Wahab, Chukwuemeka Cinderella Ebele: Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Funding statement

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. The authors solely funded this project.

Data availability statement

Data will be made available on request.

Declaration of interest’s statement

The authors declare no conflict of interest.