By Yerima, R; Jacob, LT; Nazeef, S (2023). Greener Journal of Biological Sciences, 13(1): 16-23.

 

Greener Journal of Biological Sciences Vol. 13(1), pp. 16-23, 2023 ISSN: 2276-7762 Copyright ©2023, the copyright of this article is retained by the author(s) DOI: https://doi.org/10.5281/zenodo.8141211 https://gjournals.org/GJBS

Acute Toxicity of Glyphosate and Propanil on Clarias gariepinus Juveniles

Yerima R. ^1*, Jacob, LT. ², and Nazeef S.³

^{1 & 3}Department of Biological Science, Gombe State University, Gombe, Nigeria

²Department of Biology, Ahmadu Bello University, Zaria, Kaduna State, Nigeria.

ARTICLE INFO	ABSTRACT
Article No.: 070723060 Type: Research Full Text: PDF, HTML, PHP, EPUB, MP3 DOI: 10.5281/zenodo.8141211	The impact of short term exposure to the commonly used herbicides, glyphosate and propanil on Clarias gariepinus juveniles was evaluated using standard methods that assessed fish behavior and mortality. Fish exhibited some behavioral abnormalities such as hyperactivity, hitting of tails against the walls of the holding medium and abnormal swimming. The herbicides caused mortality of fish in a concentration dependent manner for the duration of exposure to acute concentrations of both herbicides for 96 hrs. However, the LC50 value for glyphosate by probit method/analyses was 2.09 mg/L while that of propanil was 4.57 mg/L, hence glyphosate was more toxic than propanil.
Accepted: 07/07/2023 Published: 13/07/2023
*Corresponding Author Nazeef S E-mail: ymsultan6@ gmail.com
Keywords: herbicides, Acute toxicity, Clarias gariepinus, mortality, Juveniles

INTRODUCTION

A great number of the Nigerian populace are into agriculture, the vocation accounting for a portion of the nation’s gross domestic product (GDP) (Ayanda et al., 2021). Some groups of pesticides are toxic to non-targeted organisms. Herbicides are one of the main methods for controlling noxious weeds in agricultural and non-agricultural lands all over the world and it presented more than 60% of total pesticides used in agricultural sectors (Elalfy et al., 2017). Herbicide is an agent that has the ability to cause death to plants. They are chemicals that are used for agricultural and industrial purposes, and are effective against humans, plants and aquatic organisms such as fish because of their toxicity (Adedeji and Okocha, 2012). Herbicides can be found in large amount in the environment including soil, aquatic and biotic environments. Many herbicides are toxic to fish at very minimal concentration. Several herbicides are recognized for causing renal and hepatic lesions in fish (Hogan, 2014). Therefore, the mass killing of fishes can be attributed to pesticides and herbicides (Adedeji and Okocha, 2012). Similarly, the effects of herbicides are based on solubility of herbicides, magnification of concentration upon entry into the food chain and persistence of herbicides and transformation to other harmful metabolites upon entry into soil, water and biota (Hogan, 2014). Hence, the application of herbicides is reported to have a negative effect on aquatic organisms such as fish. Previous studies describe the wide application of glyphosate as a threat to aquatic environment due to negligence, non-adherence to instructions, lack of knowledge about the negative implication of herbicides and laws that govern the use of herbicides. Clarias gariepinus is an important fish in Nigeria (Ayanda et al., 2021). It is highly prized by both farmers and consumers and distributed throughout all freshwater ecosystems. It is also being widely used as a sentinel organism in toxicity studies. Hence, this study was conceived to examine the toxicity of acute concentrations of two commonly used herbicides Glyphosate and Propanil in the African catfish, C. gariepinus.

 

MATERIALS AND METHODS

Experimental Fish

Juveniles of Clarias gariepinus of mixed sexes and fairly uniform sizes were obtained from a fish farm in Zaria, Kaduna State, Nigeria. The Clarias species averaging 11.99 ± 1.56cm standard length and body weight of 4.25 ± 1.17g were used for the study. The fish were then transported in oxygenated polythene bags to the Fisheries Laboratory, Department of Biology, Ahmadu Bello University, Zaria, Kaduna State, Nigeria. They were fed on a commercial pellet diet (3 % of body weight per day) and acclimatized for three weeks in 800 L rectangular tanks containing dechlorinated tap water (conductivity 2000 µs/cm; pH ≈ 7.5; Oxygen 90-95 % saturation; temperature 25 ºC; photoperiod 12:12 Light: Dark). The feeding stopped 24 hours prior to the commencement of the experiment.

Preparation of herbicides Test Solution

Two herbicides were used in the investigation. Both herbicides were purchased from a commercial outlet in Kaduna. Out of 360g/L glyphosate, a stock solution (5mg/L) of the toxicant was prepared by adding 1mL of the toxicant to 999mls of water (Reish and Oshida, 1987). Similarly, out of 276g/L propanil, stock solution of 1mg/L was prepared in the same way as glyphosate. The stock solutions were used for preparing different concentrations of the test solutions by diluting measured volumes (i.e. 0.36mL in 999.64mL of dechlorinated tap water for 0.36mg/L concentration). The dechlorinated tap water used had the same physical and chemical properties with the one used in acclimatizing the fish. The control solutions were made up of only dechlorinated tap water.

Experimental Design

Static bioassay was conducted in the Laboratory between July and November, 2020 following OECD (1992) guidelines to determine the toxicity of glyphosate and propanil to C. gariepinus. From freshly prepared stock solutions, five concentrations of 0.90, 1.80, 2.70, 3.50, 4.50 mg/L for glyphosate and 3.96, 4.32, 4.68, 5.04, 5.40 mg/L for propanil were dispensed with a 100mL measuring cylinder and 5mL syringe respectively for each concentration containing dechlorinated water of 20L to 25L tanks and control. Ten fishes were randomly distributed into each test tank in 3 replicates. The physicochemical parameters of the diluting water (temperature, pH, dissolved oxygen, total hardness, total alkalinity and conductivity) during the acute test were measured. The opercular ventilation count (OVC) and tail fin movement rates (TMR) were counted manually per minute, for three minutes and the mean recorded using a stop watch. The OVC and TMR were observed and recorded at 12, 24, 48, 72 and 96hr. Survival and mortality were noted during this period. Fishes were considered dead when the opercular movement ceased and there was no response to gentle probing. This was used as a measure of mortality. The LC₅₀ was determined by plotting a graph of the different concentrations of the toxicants against the number of dead fishes using the probit method. One fish species (C. gariepinus) was used for the study. Each of the herbicides was administered at five levels of concentration and a control. That is, 6 treatments, three replicates 18 experimental set ups or tanks for each herbicide.

Data Analysis

Probit analysis was used to determine the LC₅₀ value for the herbicides using minitab 15 statistical packages.

 

RESULTS

Diagnostic Behavioural Responses of C. gariepinus Juveniles Exposed to Acute Concentrations of Glyphosate

Opercular Ventilation Counts

Results of the opercular ventilation counts of C. gariepinus juveniles exposed to acute concentrations of glyphosate is presented in figure 1. At 12 hours, opercular ventilation counts of the exposed group of fish were higher than that of the control. The increase in opercular ventilation counts was dose-dependent. On the other hand, at 24, 48, 72 and 96^th hours the OVC of the control group was higher than that of the exposed group. Decrease in OVC with increased in concentration of toxicant was observed from 24 to 96 hours (Figure 1).

Tail Fin Movement Rate

Results of the tail fin movement rate of C. gariepinus juveniles exposed to acute concentrations of glyphosate is presented in figure 2. At 12th and 24th hours, tail fin movements of the control were lower than the exposed group. This increased and decreased of tail fin movement rate was time and dose-dependent (Figure 2).

Mortality/96-hr LC₅₀ Values

The mortality and probit values for fishes exposed to acute concentrations of glyphosate are shown in Table 1. Mortality was observed to increase with increasing concentrations of both herbicides, glyphosate, and the highest mortality was recorded against the highest concentration 4.50 mg/L. The 96hr LC₅₀ value for the herbicide (glyphosate) was calculated based on these values, and the mean value from the probit and log of concentrations was found to be (0.320197) (Figure 3). Taken the antilog of the mean, gives an LC₅₀ value of 2.09 mg/L (Table 1).

 

Figure 1: Time Course Interaction Effect of Acute Doses of Glyphosate with Time on C. gariepinus Juveniles Opercular Ventilation Count

Figure 2: Time Course Interaction Effect of Acute Dose of Glyphosate with Time on C.gariepinus Juveniles Tail fin movement Rate

 

Table 1: Mortality and Probit Values of Clarias gariepinus Juveniles Exposed to Acute Doses of Glyphosate After 96 hrs

Conc. (mg/L)	Log-conc.	NO. of fish exposed	Mortality	%Mortality	Probit Value	Mean	LC50 mg/L (Antilog of mean)
0.00 (Control)	0.000	30	0	0.00	0.00	0.320197	2.09
0.90	-0.046	30	8	26.7	4.30
1.80	0.255	30	12	40.0	4.76
2.70	0.431	30	15	50.0	5.00
3.60	0.556	30	22	73.3	5.61
4.50	0.653	30	24	80.0	5.84

Figure 3: 96-H LC₅₀ of C. gariepinus juveniles exposed to acute doses of glyphosate

 

Diagnostic Behavioural Responses of C. gariepinus Juveniles Exposed to Acute Concentrations of Propanil.

Opercular Ventilation Counts

Results of the opercular ventilation count of C. gariepinus juveniles exposed to acute concentrations of propanil is presented in figure 4. At 12th and 24th hours, opercular ventilation counts of the control were lower than the exposed group. However, at 48, 72 and 96 hours, the opercular ventilation count of the control group were higher than that exposed group to 4.32 mg/L to 5.40 mg/L respectively. This increased and decreased opercular was time and dose-dependent (Figure 4).

Tail Fin Movement Rate

Results of the tail fin movement rate of C. gariepinus juveniles exposed to acute concentrations of propanil is presented in figure 4.7. At 12 and 24 hours tail fin movement rate of the exposed group of fish were higher than that of the control. The increase in tail fin movement rate was dose-dependent. On the other hand, at 24, 48, 72 and 96^th hours the tail fin movement rate of the control group was lower than that of the exposed group. Decrease in Tail fin movement rate with increase in concentration of toxicant was observed from 24 to 96 hours (Figure 5)

Mortality/96-hr LC₅₀ Values

The results of propanil acute toxicity bioassay showing mean mortality of C. gariepinus juveniles are presented in Table 2. Mortality of fish was observed in all the treatment groups except the control. Mortality was first observed in concentrtions 3.96, 4.32, 4.68, 5.04 and 5.40mg/L concentrations respectively. The highest mortality of 25 was recorded in the highest concentration 5.40 mg/L of propanil while the least mortality of 8 was observed in the 3.96 mg/L treatment. Similarly, the total mortality, percentage mortalities, probit kill values and the LC₅₀ in mg/L of C. gariepinus exposed to acute nominal concentrations of propanil are presented in Table 2. Mean mortality was observed to be concentration-dependent. The 96-hour median lethal concentration (LC₅₀) of propernil for C. gariepinus juveniles using the minitab was found to be 4.57 mg/L from the antilog of the mean (0.659770) from Table 2 and Fig. 6 respectively.

 

Figure 4: Time Course Interaction Effect of Acute Doses of Propanil with Time on C. gariepinus Juveniles Opercular Ventilation Count

 

Figure 5: Time Course Interaction Effect of Acute Doses of Propanil with Time on C. gariepinus Juveniles Tail Fin Movement Rate

Table 2: Mortality and Probit Values of C. gariepinus Juveniles Exposed to Acute Dose of Propanil After 96 hrs

Conc. (mg/L)	Log-conc.	NO. of fish exposed	Mortality	%Mortality	Probit Value	Mean	LC50 mg/L (Antilog of mean)
0.00 (Control)	0.000	30	0	0.00	0.00	0.659770	4.57
3.96	0.598	30	8	26.7	4.30
4.32	0.635	30	10	33.3	4.50
4.68	0.670	30	15	50.0	5.00
5.04	0.702	30	22	73.3	5.61
5.40	0.732	30	25	83.3	5.95

Figure 6: 96-H LC₅₀ of C. gariepinus juveniles exposed to acute doses of propanil

 

DISCUSSION

Acute and sublethal toxicity tests are commonly used to assess the toxicity of chemicals on non-target animals (Santos et al., 2013). The 96 h LC₅₀ is one of the most important factors for evaluating the toxic effects of contaminants. The 96h LC₅₀ value of Glyphosate and Propanil in this study was found to be 2.09 mg/L and 4.57 mg/L respectively. This is proportional to increase in glyphosate and propanil herbicides concentration and duration of exposure. Hence, mortality is dose dependent, which suggest that the herbicides are toxic to fish. Varying LC₅₀ values have been obtained from different investigation on exposure of the same organisms to different herbicides. The differences in the values may be as a result of variation in the concentration of herbicide, age of organism and environmental condition Olorunfemi et al. (2014). A steady trend was generally observed in the percentage mortality rate of C. gariepinus which increases with an increase in concentrations of glyphosate and propanil which is consistent with observation from (Sani and Idris, 2016). Similarly, less mortality was found at 0.90mg/L concentration and higher mortality was found at 4.50 mg/L concentration of glyphosate, while for the fish exposed to propanil concentration, lower mortality was recorded at 3.96 mg/L while higher mortality was recorded at 5.40 mg/L. However, with highest concentration of glyphosate and propanil, various behavioural changes occur such as erratic swimming, gulping of air, loss of equilibrium and resting motionless at the bottom of the aquaria were observed, which was similar to observations made by Yaji et al. (2018). The erratic swimming, restlessness, gulping of air and resting motionless at the bottom of aquaria observed in the investigation are not only as a result of impaired metabolism but could be due to nervous disorder. This was because the behavioural changes were unnoticed before the application of glyphosate and propanil. This was also in line with previous studies of Ayanda et al. (2021). Low concentration of glyphosate and propanil herbicides (0.90mg/L and 3.96mg/L respectively) did not produce any serious change in the fish behaviour within 24 h. Meanwhile, higher concentrations of glyphosate (4.50 mg/L) and propanil (5.40 mg/L) at 96 h showed loss of response to stimulus and death. At the early stage of the toxicant introduction, all the fish survived the initial attack. This may be owing to their defensive adaptations as the respiratory mucosa on the inner walls of the air-sacs is thrown into folds and ridges for increasing the surface area for gas exchange. In contact with low oxygen level, C. gariepinus breathe through their skin and even use their air bladder as an emergency lung by gulping surface air. During the 48, 72, and 96 h of exposure, the fish displayed physiological malfunctions such as hyperventilation, motionless State, increase opercular ventilation, general body weakness, skin discoloration, loss of reflex, erratic swimming which were noticeable particularly among some fish in the highest concentrations of Glyphosate (4.50 mg/L) and Propanil (5.40 mg/L) in which 80% and 90% mortality was recorded. The physiological malfunctions are believed to weaken the organism’s resistance to toxins and consequently resulting in the significant death of almost 50% at the highest concentration. With progressive exposure, deaths become inevitable even at a lower concentration. This could be owing to stress and the cumulative impact of Glyphosate and Propanil toxicity. The mortality pattern recorded in this study agrees with that observed by Rand and Pectrocelli (1985) which stated that there should be less than 35% mortality in one of the concentrations and at least more than 65% mortality in the highest concentration. The mortality observed in the study was considered a result of stress-induced on the immune system of fish. Thus, slow toxic progress and long continuance can result in a chronic toxic response. The haematological parameters have been used as a sensitive indicator of stress in fish exposed to different aquatic contaminants and toxins of various types. Sub lethal concentrations of toxicants in the aquatic ecosystem will not certainly result in the outright death of aquatic organisms. However, the bioaccumulation of these contaminants over an era of time may create potential health threats not only to the aquatic animals like fish but also on higher trophic level particularly man. In the present study, the behaviour of C. gariepinus juveniles exposed to both herbicides Glyphosate and Propanil were observed to be similar. This could be as a result of the fact that both chemicals have the same mode of action since they belong to the same group of pesticides called herbicides. However, in the present study, Glyphosate was found to be twice more toxic than propanil, sincer LC₅₀ of glyphosate was found to be 2.09 mg/L and that of propanil was found to be 4.57 mg/L respectively.

 

CONCLUSION

The present study shows that mortality of C. gariepinus juveniles increases with increase in concentrations of Glyphosate and Propanil herbicides which has manifested in behavioral changes. Therefore, the higher the concentrations, the higher the mortality rate and change in behaviour. More so, the lower the concentrations, the lower the mortality rate and change in behaviour. Hence, Glyphosate and Propanil herbicides can induce mortality in Clarias gariepinus juveniles.

 

REFERENCES

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Olorunfemi, D. I., Olomukoro, J. O. and Anani, O. A. (2014). Acute toxicity of produced water on Clarias gariepinus juveniles. Studia Universitatis” Vasile Goldis” Arad. Seria Stiintele Vietii (Life Sciences Series), 24(3): 299.

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Yaji, A. J., Iheanacho, S. C. and Ogueji, E. O. (2018). Haematology and biochemical responses in Oreochromis niloticus exposed to subacute doses of Aronil in a flow through bioassay. Egyptian Journal of Aquatic Biology and Fisheries, 22(3): 89-98.

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Cite this Article: Yerima, R; Jacob, LT; Nazeef, S (2023). Acute Toxicity of Glyphosate and Propanil on Clarias gariepinus Juveniles. Greener Journal of Biological Sciences, 13(1): 16-23, https://doi.org/10.5281/zenodo.8141211.

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