Evaluation of Toxicity of Lead Salts (PbCl 2 and PbNO 3 ) Exposed Common Carp ( Cyprinus carpio ) in Two Seasons (Summer and Winter) of Different Temperature Conditions

The main objective of the intended research was to ascertain the toxic effects of Lead salts (PbCl 2 and PbNO 3 ) in two seasons of varying temperatures in the Indian subtropical region of Bhopal. The toxicity test was carried out at concentrations of 250, 300, 350, 400,450, and 500 mg/L for the determination of LC 50 of PbCl 2 and PbNO 4 in summer at high temperature (HT) and in winter with low temperature (LT) conditions in common carp ( Cyprinus carpio). The LC 50 values for PbCl 2 for 24 hours in winter (LT) were 487.98 ± 1.17 mg/L and decreased to 466.66 ± 1.31 mg/L, 436.11 ± 1.68 mg/L, 416.49 ± 1.61 mg/L, for 48, 72, and 96 hours respectively whereas in summer (HT) the LC 50 values were calculated as 396.83 ± 1.38 mg/L, 359.42 ± 1.71 mg/L, 345.95 ± 1.80 mg/L and 321.67 ± 1.24 mg/L for 24, 48, 72 and 96 hours respectively. The LC 50 values for PbNO 3 in winter (LT) were calculated as 427.76 ± 2.02 mg/L, 400.13 ± 1.01 mg/L, 373.42 ± 1.76 mg/L, and 359.92 ± 1.36 mg/L for 24, 48, 72, and 96 hours and in summer (HT) LC 50 values calculated as 357.93 ± 1.69 mg/L , 329.76 ± 0.83 mg/L , 300.47 ± 1.41 mg/L and 274.29 ± 2.06 mg/L for 24, 48, 72 and 96 hours respectively. This study discovered that the counterions play an important role in toxicity due to which PbNO 3 toxicity was high as compared to PbCl 2 in both seasons as well effect of temperature in enhancing the toxicity of both Lead salts (PbCl 2 and PbNO 3 ) in summer was studied.


Introduction
Toxicology is the science that recognizes the repercussions of toxic chemicals on humans, animals, and the environment as well as understands how a toxic substance will affect or doesn't affect an individual, the amount and time of exposure, sensitivity, as well age-dependent effect of a particular chemical substance (Bruckner,  To figure out the mechanisms by which substances exert toxic effects as well as to ascertain the presence of toxic substances in various samples, aquatic toxicology is a multidisciplinary science that syndicates the toxicology, the chemistry of water, and the ecology of water. Standardized tests estimate the acute to chronic toxicity with different exposure systems like static, recirculatory, renewal, and flow through as well the pathway whether water-borne toxicity or administered through feed provide qualitative and quantitative figures on deleterious effects of chemical substances on aquatic organisms with several standardized methods widely accepted by international agencies like American Public Health Association (APHA), U.S Environmental Protection Agency (EPA). The science of aquatic toxicology understands the detrimental impacts of toxic chemicals on aquatic organisms including fish, and shellfish. It is also well understood that the toxicity of a particular chemical or a substance differs among fish species, and their different developmental stages, and there can be a difference in toxicity level with temperature differences, and ionic composition of water ( . Dose significantly plays an important role in toxicology as stated by 'Paracelsus', the father of modern toxicology "Only the dose is the poison" meaning dose is the prime factor to assort the toxicity of various chemicals (Hunter, 2008). The terms LD 50 and LC 50 are significantly used by toxicologists to determine the amount of a substance that causes the death of 50% of the population of the tested group in a particular duration of time. J.W Trevan in 1927 first created a test to determine the median lethal dose of LD 50 which is usually performed on mice and expressed as a mass of chemical substance administered in milligrams per kg body weight or Nanograms for bacteria. LD 50 is an ethical procedure for evaluating acute toxicities, whereas LC 50 means the lethal concentration that determines 50 % of mortalities in water-related toxicities when exposed to chemical substances usually expressed as mg per cubic meter or ppm (parts per million). Lead (Pb) toxicity gets induced at high 14 temperatures leading to oxidative and metabolic stress in fish (Kumar et al., 2018). The objective of the study was to evaluate Lead (Pb) toxicity in different temperatures (seasonal) conditions as well the toxicity of Lead (Pb) and impact of counterions on toxicity at different seasonal temperatures is less studied.

Experimental fish
Common carp (Cyprinus carpio) was selected as an experimental animal. Common carp (Cyprinus carpio) is a freshwater fish belonging to the family Cyprinidae is a very resistible fish, can tolerate a wide range of temperatures as well as able to survive in low oxygen concentrations, can spawn throughout the year, and is one of the most cultivable species throughout the world (Froese and Pauly, 2002), making it a suitable candidate species for toxicological studies.

Acclimatization
Based on prior studies and laboratory prerequisites, a total of 2000 fish were brought from local aqua farms of Bhopal and were maintained under laboratory conditions for acclimatization for 20 days in de-chlorinated water by aging water for 6 days in circular tanks of capacity 500 liters. Fish were fed thrice 3 % of body weight with Growel GROWIN feed a day during the acclimatization period of 20 days with continuous aeration as each tank was fitted with air compressors. For this 1200 fish were used in two static experiments at different temperature conditions (summers and winters). Six replicates with 20 fish per replica with one control were maintained in 100 liters aquariums of dimensions 1m × 1m × 0.5m.

Chemicals
For toxicity analysis, PbCl2 was purchased from Fizmerk, India. A stock solution of PbCl2 was prepared in double distilled water. Other chemicals and reagents used were from Himedia, Pvt Ltd, Mumbai, Maharashtra, India.

Evaluation of LC 50 in winters (LT)
Evaluation of LC 50 at mean room temperature 23.33 ± 1.24 °C during the day and 9.33 ± 1.24°C at night, water temperature of 7.2 ± 0.75 °C fish of size 10.15 ± 0.54 cm and weight 12.63 ± 0.29 g were selected for static test in which no food was given to the fish. LC 50 at different concentrations of 250, 300, 350, 400, 450, and 500 mg/L of toxicant PbCl2 and PbNO3 were set for the evaluation of LC 50 values for 24, 48, 72, and 96 hours respectively using Probit analysis by Finney, (1952) and regression analysis in excel.

Evaluation of LC 50 in summers (HT)
Fish of size 10.2 ± 0.58cm and 12.46 ± 0.44g were selected. LC 50 values at a mean room temperature of 39 ± 0.81 °C during the day and 33.66 ± 0.47°C at night with a mean water temperature of 27.66 ± 0.47°C at the same concentrations of 250, 300, 350, 400, 450, and 500 mg/L of PbCl2 were evaluated.

Temperature, Ammonia, and Dissolved oxygen (DO) assessment
Ammonia levels and dissolved oxygen content (Winker's method) according to APHA, (1985). Water temperature was accessed using a mercury thermometer from LAB WORLD and the room temperature was measured using a LABPRO wall thermometer.

Analysis of data
Statistical analyses were performed using one-way ANOVA and Tukey post hoc pairwise comparison at P<0.05 significance level. Data are presented as mean with ± Standard deviation (S.D).

Morphological and behavioral changes
The toxicity of both salt of Lead induced behavioral changes like change in swimming movement with irregular moves, hyperactivity and loss of balance. The color of the fish faded with the increase in toxicity period, lots of mucous secretion, loosening of skin whereas in control no behavioral or morphological changes were observed.

Estimation of ammonia at different concentrations of PbCl2 and PbNO3 in summer
The ammonia levels in summers with increase in temperature were elevated with water temperature of 29.66 ± 0.47°C. Ammonia concentration in water was observed as 0.33, 0.42, 0.83, 0.83 and 1.33 ppm at the concentration of 300, 350, 400, 450, 500 mg/L PbCl2 respectively. As well ammonia excretion increased with PbNO3 salt as compared to PbCl2 at high temperature as 0.42, 0.83, 1.17, 1.33, 2.67 and 3.33 ppm at the concentration of 250, 300, 350,400, 450 and 500 mg/L respectively as depicted in Figure 2.

Evaluation of dissolved oxygen content at different concentrations of PbCl2 and PbNO3 in winter (LT)
The dissolved oxygen content at the average water temperature of 7.2 ± 0.75°C was estimated as 7.50 ± 0.10mg/L, 7.23 ± 0.06 mg/L, 6.80 ± 0.10mg/L, 6.50 ± 0.10 mg/L and 5.90 ± 0.10mg/L and 5.40 ± 0.10 mg/L at different concentrations of PbCl2 whereas the dissolved oxygen content with PbNO3 was estimated as 7.40 ± 0.10 mg/L, 7.20 ± 0.17 mg/L, 6.80 ± 0.10 mg/L, 6.40 ± 0.10 mg/L and 5.30 ± 0.44 mg/L and 5.23±0.25 mg/L at different concentrations with no significant difference with the dissolved oxygen content of both salts of Lead toxins in winter as depicted in Figure 3.

Discussion
Aquatic environment is polluted by toxic chemicals due to anthropogenic or natural activities because harmful chemicals especially heavy metals which tend to bio accumulate in fish tissues make the fish consumption risky (Javed and Usmani, 2019). Heavy metals affects the swimming performance as well causes alterations in the sensory perception of fish (Prashanth et al., 2011). Lead is toxic to both fish and humans as it affects the hematology, bio accumulates in muscles, bones as well creates disorders in children with low I.Q, stillbirth and birth defects in children (Lee et al., 2019; Krshisagar et al., 2015;  Shah et al., 2020; Wani et al., 2015). In this study, the toxicity of two salts of Lead was evaluated. The toxins PbCl2 and PbNO3 were accessed for the toxicity test in Cyprinus carpio in two different seasons of varied temperature conditions. The toxicity was evaluated in winter and summer. In this study at high temperature in summer, increased in the toxicity of both salts of Lead with behavioral and morphological changes in fish. In another study at high temperature and prolonged exposure of heavy metals to fish, mortalities were observed (Khanna et al., 2007). Also with the increase in the temperature Lead accumulation rate was high in estuarine teleost fish Gillichthys mirabilis (Somero et al., 1977). Relatively high temperatures can accelerate the toxicity of heavy metals leading to greater mortality of fish. The LC 50 of PbNO3 in the Pangasius hypothalamus was found to be 83.10mg/L, comparatively high at high temperatures than the normal temperature which was 84.93mg/L (Kumar et al., 2018). Also, thermal stress along with Lead (Pb) toxicity decreased survivability, affected antioxidant activity as well as body malformations in Zebrafish (Danio rerio) embryos (Park et al.,  2020). At a high temperature of 32°C, pesticide toxicity was increased in goldfish (Carassius auratus) with greater cellular damage to the liver and gills (Jacquin et al., 2019). In another finding, it was also observed that the toxicity of pesticides endosulfan, chlorpyrifos, endosulfan, and phenol was increased with the increase in temperature in both cold water fish Rainbow trout and warm water fish silver perch with rainbow trout evincing greater toxicity at 30 °C whereas silver perch evincing greater toxicity between 30 to 35 °C for the short duration of 24 and 96 hours (Patra et al., 2015). In this study PbNO3 was found to be more toxic than PbCl2. The role of counter ions in the toxicity of Lead was also studied in the spring tail Folsomia candida in soil in which PbNO3 was found to be more toxic than PbCl2, which affected the survivability of Folsomia candida in soil (Bongers et al., 2004). As well Lead with nitrate was more toxic than chloride in Lumbricid earthworm Eisenia fetida (Peredney and Williams, 2000). In our study at high temperature the toxicity of PbCl2 and PbNO3 increased with reduction in dissolved oxygen content and increase in ammonia levels with increase in the concentrations of toxicant. The dissolved oxygen content was recorded as 6.07 ± 0.10 mg/L, 6 ± 0.10 mg/L, 5.43 ± 0.03mg/L, 5.10 ± 0.10mg/L, 4.80 ± 0.10 mg/L and 4.50 ± 0.10 mg/L at different concentrations of PbCl2 .There was considerable reduction in the dissolved oxygen content with the toxicant PbNO3 as 6.07 ± 0.12 mg/L, 5.79 ± 0.44 mg/L, 5.41 ± 0.08mg/L, 4.87 ± 0.15 mg/L, 4.70 ± 0.10 mg/L and 4.60 ± 0.06 mg/L at different concentrations of PbNO3 in summer. Also in another study Increase in temperature caused stress in fish that resulted in the mortalities (Hobbs and Mcdonald, 2010). In rainbow trout (Salmo gairdnerii) the toxicity of Zinc sulphate caused reduction in dissolved oxygen content with increase in toxicity of Zinc sulphate (Lloyd, 1960). Increase in temperature caused increase in the oxygen consumption and increase in ammonia excretion in Artic charr (Lyytikainen and Jobling, 2005) also in Alepes djidaba increase in oxygen consumption and ammonia excretion was observed (Krishnamoorthy et al., 2008).  , 2015). In our study ammonia concentration in water during summer was observed as 0.33, 0.42, 0.83, 0.83 and 1.33 ppm at the concentration of 300, 350, 400, 450, 500 mg/L PbCl2 respectively as well ammonia excretion increased with PbNO3 salt as compared to PbCl2 at high temperature as 0.42, 0.83, 1.17, 1.33, 2.67 and 3.33 ppm. The toxicity of PbCl2 and PbNO3 was high in summers at high temperatures which might be the result of combined stress due to the toxicity of PbCl2 and PbNO3 as well increase in oxygen demand and decrease in oxygen availability to respiratory tissues of fish and excess ammonia excretion enhancing toxicity of PbCl2 and PbNO3 resulting in lower LC 50 values in summers at high-temperature conditions. One of the important and less addressed roles of counter ions in any type of metal toxicity in fish toxicology was studied and for these two different salts of Lead was used in our experiment and it was observed that NO3counter ion was more toxic at both temperatures than Cl-ions.

Conclusion
The LC50 of PbCl2 and PbNO3 differ in common carp with PbNO3 more toxic than PbCl2; this might be due to the effect of counter ion NO3 -which was found to be more toxic than Clions. The LC 50 of PbCl2 and PbNO3 in common carp varied seasonally in this experiment with greater toxicity in summers at high temperatures than in winters with low-temperature conditions as well enhanced toxicity in Cyprinus carpio might be due to combined metabolic stress created by high temperature conditions in summer which resulted in elevated toxicities of both Lead salts.