Effects of Glyphosate Herbicide on Physiological Parameters of Koi Carp, Cyprinus Carpio (Linnaeus, 1758) Fingerlings 

Herbicides used in agricultural pose a great threat to non-targeted organisms. The response of an aquatic organism to environmental pollutants which are resulting from industrial processes and agricultural runoff induces damage at cellular and biochemical levels, ultimately leading to changes in function of cells, tissues, physiology and behavior of the organism (Parvez and Raisuddin, 2005).
 
Most herbicides used in agriculture, except biocides such as disinfectant chlorine and the wood preservative, pentachlorophenol etc., are specific to a group of pests because of their specificity of mode of action. This is particularly the case for the herbicides that are designed to control plants. For example, photosynthetic inhibitors such as urea and triazine herbicides specifically target components of the photosynthetic apparatus (Devine et al., 1993), which are not found in animals. Therefore, animals are usually less sensitive to herbicides than plants. However, at significantly large concentrations, herbicides can be toxic to animals through narcosis (Solomon et al., 2013). Many herbicides are toxic to fish at very minimal concentration and several herbicides are recognized for causing renal and hepatic lesions in fish (Hogan and Draggan 2014).
 
Glyphosate, an isopropyl amine salt of N-(phosphonomethyl) glycine is one of the most important post-emergent herbicides used in agriculture and industry. Numerous commercial formulations containing glyphosate as the active ingredient have become popular around the world due to their effective action and low toxicity to mammals. It controls weeds by inhibiting the synthesis of aromatic amino acids necessary for protein formation in susceptible plants (Tu et al., 2001).
 
Asia-origin koi carp are currently listed among the most important ornamental species as it can be reared in all countries throughout the world ( Hekimoglu et al., 2014). It is a high value ornamental fish which fetch a good price on the market. The fish is also used as food fish when caught from the wild. The current toxicological study on the effects of glyphosate was conducted to evaluate the physiological parameter changes in Koi carp to ensure its sustainability, since, grow out is conducted in ponds and brood stock are captured from the wild.

Test animals
 
Koi carp (Cyprinus carpio) fingerlings with mean length 8.06 ± 0.99 cm were obtained from a freshwater fish seed hatchery of the university. The fishes were acclimatized for a period of two weeks prior to the start of the bioassay experiments. Water temperature and pH were recorded daily and ranged from 28 to 29°C and 6.5 to 7.0 respectively. Feeding was done twice a day during the acclimatization period using commercial pelleted feed.

Test solution 
 
Glyphosate 41% SL herbicide (Trade name: CEDAAR) was procured from a local agro-dealer in Ratnagiri market. Its chemical name is (N-(phosphonomethyl)-glycine). The test liquid was measured using a volumetric pipette for making stock solution. The test solution was prepared immediately prior to commencement of each experiment.
 
Physiological parameters
 
Oxygen consumption rate
 
Experiments on oxygen consumption rate by Koi carp exposed to glyphosate were conducted in glass aquarium tanks having 40 L capacity each. The test solution was exchanged after 24 hours and test fishes were fed during the period of the experiments. Respiratory apparatus were fabricated in the laboratory using two litre conical flask. The top was covered using thermocol to prevent gas exchange with the atmosphere. An aeration tube was inserted in the flask and the other end was closed.
 
After seven days of exposure, five fishes were transferred into the respiratory apparatus with the same concentration of glyphosate as those of the glass aquarium tanks (1/10^th and 1/5^th). The fishes were allowed to stabilize briefly and the experiment was run for three hours. Oxygen concentration before and after the three hours duration was determined as by Winkler’s method (Weish and Smith, 1960). The process was repeated on 14^th, 21^st and 28^th day of the experiment. Oxygen consumption rate by Koi carp exposed to glyphosate was determined as outlined by Zhen et al., (2010) and calculated as follows:  
Where,
DO₀  = O₂ of the water at the start of the experiment (mgL^-1).
DO_t   = O₂ of the water at the end of experiment.
W      = Weight of live fish (g).
t        = the experimental time (h).
V       = Volume of water.
 
Ammonia N excretion rate
 
Ammonia excretion rate calculation was based on the method described by Zhen et al., (2010) and expressed as NH4-N/g body weight of fish/h. Initial and final ammonia readings were recorded using a spectrophotometer with absorption set at 630 nm. Ammonia-N excretion rate (mg/L/g/h) was calculated as follows:
Where,
No = NH4-N of the water at the start of the experiment (mg/l).
Nt = NH4-N of the water at the end of experiment.
V    = Volume of water.
W   = Weight of live fish (g).
t     = the experimental time (h).
 
Oxygen nitrogen ratio         
       
The Oxygen : Nitrogen ratios was calculated from the rates of oxygen consumption and rates of ammonia excretion in Koi carp, Cyprinus carpio (Linnaeus, 1758). The Oxygen: Nitrogen ratio was calculated as the ratio of atoms of oxygen consumed to atoms of nitrogen excreted in a given time interval (Widdows, 1985).  
Where,
R = Respiratory rate (O₂ mg g^-1 h^-1).
E = Ammonia excretion rate (NH_4-N mg g^-1 h^-1).
 
Food consumption rate
 
Food consumption rate was estimated as the difference between given and recovered feed. The test organisms were fed once in 24 h using pelleted feed. The remaining feed was siphoned out and dried in an oven at 60°C overnight and measured to constant weight using desiccators. The experiment was repeated on the 7^th, 14^th, 21^st and 28^th day of the experiment using 2 sub-lethal concentrations of 1/5^th and 1/10 of the LC₅₀ value.

Oxygen consumption
 
Exposure of fishes to 1/10^th and 1/5^th of LC₅₀ concentration (3.3 and 6.6 mgL^-1 respectively) led to decrease in oxygen consumption rate (OCR). There was decrease from 0.1844±0.000 to 0.0737±0.003 and from 0.1793±0.005 to 0.0572±0.008 in 1/10^th and 1/5^th concentrations respectively. There was no significant change (P>0.05) in OCR in control on 7, 14. 21 and 28^th days of the exposure period. However, there were significant differences (P<0.05) in oxygen consumption rate in control, 1/10^th and 1/5^th concentrations of glyphosate on 7, 14. 21 and 28^th day of the exposure period (Table 1).
 

 
Oxygen consumption is widely accepted and commonly used as a good stress indication of an organism under pesticide poisoning (Vosloo et al., 2002). Results of the present study on oxygen consumption indicated significant decrease (P<0.05) during the 28 days of exposure period. Chinni et al., 2002; Wu and Chen 2004 and Barbieri 2007 also reported similar observations when fishes were exposed to different toxic substances. The significant decrease in the consumption of oxygen may be a result of alterations of energy metabolism (Olsen et al., 2006) caused by increased energy requirements to counteract stress. Studies by Misra et al., (1985); Huang and Wang (1995) and Barbieri et al., (2002) have also attributed the decrease in oxygen consumption to pathological effects caused by chronic exposure to chemical substances which leads to gradual destruction of gill filaments resulting in death of Koi carp due to asphyxia.
 
Ammonia-N excretion rate
 
Ammonia-N excretion rate increased significantly (P< 0.05) in fingerlings exposed to glyphosate herbicide. The excretion rate increased from 0.0033±0.0000 to 0.0035±0.0000, 0.0040±0.0002 to 0.0046±0.0000 and 0.0050±0.0003 to 0.0060±0.0000 in control, 1/10^th and 1/5^th concentrations of LC₅₀ of glyphosate respectively for a period of 28 days (Table 2). The results indicated more increase in ammonia-N excretion rate in 1/5^th concentration followed by 1/10^th concentration. There was gradual increase but no significant change (P>0.05) in ammonia-N excretion in control as the period of exposure increased.
 


During the present study, findings indicated significant increase (P<0.05) in ammonia-N excretion. Zhen et al., (2010) reported that ammonia excretion increased rapidly after 48 h exposure of Mytilus edulis to different concentrations of methamidophos and omethoate as compared to control. The study concluded that the increase in ammonia excretion rate could be attributed to a greater catabolism of amino acids or other nitrogenous compounds in comparison with the control group animals.
 
The increase in ammonium excretion may be due to an increase in catabolism of amino acids. This increase may be thought as a response of Koi carp fingerlings to get rid of glyphosate herbicide. It may also be explained as effects of toxicants on gill epithelium leading to favouring a shift in excretion of nitrogen as reported by Mayzaud and Conover, (1988). Similar results to the current study were also reported by Barbieri (2008) when Geophagus brasiliensis were exposed to 25 and 40 mgL^-1 of 2,4-D.
 
Oxygen : Nitrogen ratio
 
Oxygen: Nitrogen (O:N ratio) ratio for Koi carp fingerlings exposed to different concentrations of glyphosate herbicide is presented in Table 3.
 

 
Results indicated significant decrease (P<0.05) in O:N ratio in 1/10^th and 1/5^th concentrations. The ratio decreased from 17.46±0.000 to 6.11±0.000 and 13.72±0.0013 to 3.63±0.000 in fishes exposed to 1/10^th and 1/5^th of the LC₅₀ value. However, there was no significant change (P>0.05) in O: N ratio in control group.
 
Oxygen: Nitrogen ratio was always high in control throughout the study period. However, there was significant decrease (P<0.05) in O:N ratio in fingerlings exposed to glyphosate. This decrease may be attributed to change in substrate for catabolism. A high O:N ratio may suggest primarily lipid or carbohydrate metabolism, while a low O:N ratio indicated a protein metabolism. The use of protein as an energy source is generally indicative of stressful conditions (Zhen et al., 2010).
 
Food consumption rate
 
Food consumption rate (FCR) in fishes exposed to glyphosate decreased significantly and differed from the control group. However, there was no significant difference (P>0.05) in FCR in fishes exposed to 1/10 and 1/5^th concentrations of glyphosate. In 1/10^th and 1/5^th concentrations, food consumption decreased from 0.0125±0.0002 to 0.0082±0.0004 and 0.0124±0.0010 to 0.0071±0.0003 respectively. Contrary, food consumption increased significantly (P<0.05) in control from 0.0145±0.0032 to 0.0184±0.0003 (Table 4).
 

 
Food consumption is another widely used indicator of stress used in toxicity testing. In the present study, food consumption increased in control but decreased significantly (P<0.05) in 1/10^th and 1/5^th concentrations. The decrease in food consumption rate may be due to loss of appetite. Tissue damage may partially be a cause of reduced appetite in fish under stress conditions (Boeck and Blust, 1997). Similar results have also been reported by Giaquinto et al., (2017) in Pacu (Piaractus mesopotamicus) exposed to three glyphosate concentrations (0.2, 0.6 and 1.8 mgL^-1).

Glyphosate, a commonly used herbicide worldwide, has been found to cause negative impacts on Koi carp fish. Fingerlings exposed to glyphosate exhibited decrease in oxygen consumption rate, Oxygen : Nitrogen ratio and food consumption rate and increase in Ammonia-Nitrogen excretion rate. The results indicated that glyphosate is toxic to fish even at sub-lethal concentrations. Therefore, use of glyphosate for control of weeds in agricultural fields should be regulated to ensure appropriate use.