Growth performance of major carps during exposure of zinc and bioaccumulation in fish body organs

Growth is an index of metals influence on a fish. This investigation aimed to determine growth responses of major carps viz. Catla catla , Labeo rohita and Cirrhina mrigala during exposure to sublethal concentration to zinc. The variations among fish age, species, treatment (zinc) of growth parameters viz. weight increment, fork lengths, and condition factor were statistically significant at P<0.01. The control fish age group 90,120,150 and 180 day showed better feed conversion ratio 0.234, 0.168, 0.132 and 0.29 as compared to that of zinc exposed fish which showed range 0.01 to 0.23. Three fish species showed significantly higher amount of zinc in liver than other organs. Labeo rohita showed significantly higher trend for zinc accumulation in body organs than as compared to Cirrhina mrigala and Catla catla . Among body organs; skin, muscle, scales displayed significantly lesser trend for zinc accumulation. It is concluded that metal intoxication significantly reduced the growth of all three fish species and may also show good indicator of metals present in surrounding environment.


Introduction
Recent era is well aware about pollution and its effect especially the river pollution with heavy metal is affecting aquatic life badly.Many awareness programs have been started to observe and abatement of river pollution with heavy metal.Discharging of all sorts of highly obnoxious matter into freshwater is an affront to civilized values and damaging sustainability to the environment.In aquatic ecosystem, Fish can be used as an indicator of freshwater pollution through heavy metals as they inhabit diverse tropic levels.Fish absorbs dissolved heavy metals from the ambient water that can accumulate in several tissues and organs and even be bio-magnified in the food web.Environmental and health issues resulting from metals present in aquatic ecosystem are very well known (Shah et al., 2020).Decrease in fish species can be linked with industrial and domestic pollutants, ecological degradation and change in habitat in rivers (Atique and An, 2019).In the absorption procedure, there are four likely ways for the substance (such as metals) to come in a fish; as food ingested, simple circulation of the metals in ionic form over gill pores, drinking water and through skin absorption (Frank et al., 1998;Khan et al., 2018).Most abundant vertebrate are fish which affect human through food intake (Lee et al., 2019).The accumulation of heavy metals is dose and time dependent (Annabi et al., 2013).The trace metals are vital for common physiological procedures; unusually high concentration can be lethal to aquatic organisms (Javed, 2003).The most common pollutants include manganese, Iron, nickel and zinc which are found in Punjab Province Rivers (Javed and Mahmood, 2001).Feed intake and fish productivity decreases in the presence of heavy metals (Sfakianakis et al., 2015).Alvarado et al. (2021) determined the heavy metal concentrations in the organs/tissues of carp and Catfish.Results showed that metal concentrations (Zn, Cu, Cd and Pb) were highest in liver whereas metal concentrations (Hg and As) were highest both in liver and muscle tissue.Lorenten and Maage (1999) studied the bioaccumulation of trace elements zinc, manganese, iron and copper from first feeding of fingerling of Atlantic salmon (Salmo salar).Selenium become highly toxic when present in high amount in aquatic environment (Li et al., 2020).Zinc, Molybdenum and copper was proved to be the most toxic heavy metals causing increased hazard rate to mortality up to 96 hr and shorter life expectancy (Gouva et al., 2020).Zinc and iron were deposited to body with growth at all stages.High influx of industrial and urban effluents into the Riverine system of Pakistan necessitates the importance to investigate the growth responses of major carps, which are adversely affected due to heavy loads of metals and bioaccumulation of zinc during chronic exposures to suggest measures for the rehabilitation of the aquatic habitats.

Material and Methods
The research was conducted at Fisheries Research Farms, University of Agriculture, Faisalabad.The experimental organisms were fingerlings major carps viz.Catla catla, Labeo rohita and Cirrhina mrigala starting from the age of 90, 120, 150 and 180 days.Four age groups of three fish species were reared separately for 30-day interval under sub-lethal concentration of zinc with three replications.The fish were kept under laboratory conditions in 500 liter cemented tank for two weeks prior to the experiment.After this acclimatization period, the fish were transferred in 50 liter glass aquaria for growth trials.Five fish of each species were kept in each aquarium containing sublethal concentration of zinc.Chemically pure chloride of zinc (ZnCl 2 ) was used to prepare stock solutions for desired dilutions.In control aquarium no metal was added.The condition factors (weight-length relationship) of the fish were examined before starting the experiment.The fish in good state of health, after acclimatization period, were exposed to the following sub-lethal levels of zinc as mentioned (Table 1) for a 30-day growth trial.(Javed and Abdullah, 2003) Fish growth studies Prior to the experiment, initially the growth parameters viz.wet body weight, fork and total length of fingerlings were measured and recorded.
After sublethal metal (zinc) exposures of 30-days for each age group, the feed intake, increase or decrease in average weight, length and feed conversion ratios were measured.During each trial, fish was feed to satiation daily (6 days a week) at 9:00 and 17:00 hours with the feed of digestible energy 2.90 Kcal g-1 ( 35 percent digestible protein).

Determination of Zinc in fish
A pre-weighed fish body organ samples were transferred to a beaker of 100ml and concentrated HNO 3 of 20ml was added to it.The beaker was enclosed with a watch glass and low heat was applied on a hot plate until the solid material dissolved.Perchloric acid (72%) was added @ 10 ml per sample after cooling and heated gently again initially and then vigorously until a clear, colourless solution resulted.When the volume was reduced to about 3ml, cooled and transferred through filter paper to a 50ml volumetric flask and made up the volume.The filtrate was stored in Teflon bottles for the determination of zinc concentration through Atomic Absorption Spectrophotometer (Perkin Elmer-5100) by following the method as defined in SMEWW (1989).

Statistical analysis
The data obtained from this experiment, were analyzed statistically using MSTAT software (Nissen, 1989).Analysis of variance was accomplished to find out statistical differences between variables of study.

Growth performance of three fish species in chronic exposure of the Zinc
The experiment was conducted to observe the growth performance of three fish species includes Catla catla, Labeo rohita and Cirrhina mrigala reared in chronic exposure of zinc at sub-lethal concentrations for four age groups.

i. 90-day fish
Table 2 represents growth performance of 90-day fish after treatment in comparison of control group.During this trial, Catla catla, Labeo rohita and Cirrhina mrigala gained the weights of 0.59, 1.03 and 1.05 g respectively.The average total feed intake by Catla catla was maximum as this fish consumed 66.95 g feed.Regarding the fork length, the positive increment was found in all three fish species.There was increase in total length in all three fish species with the highest average increment of 0.80 mm in both Catla catla and Cirrhina mrigala.ii.120-day fish During this trial both Labeo rohita and Cirrhina mrigala gained the average weight of 0.68 g.The total feed intake by Catla catla was maximum by consuming 47.99 g feed.The fork length increments were similar in both Catla catla and Cirrhina mrigala (0.60 mm) while in Labeo rohita it was 0.40 mm.However, total length increment was positive in all three fish species with highest increase in total length of 1.80 mm for Catla catla (Table 3).iii.150-day fish A negative growth performance was observed for the three fish species and maximum weight loss was observed in Catla catla i.e. -2.72 g.The total feed intake was maximum for Labeo rohita by consuming 59.84g.Fork length of Catla catla and Cirrhina mrigala did not change while the same for Labeo rohita decrease as -0.20 mm.regarding the total length, there were observed no change in all the three fish species (Table 4).

iv.
180-day fish During this growth trial a decline in weight were observed for all the three fish species.The maximum decrease was observed by Catla catla i.e. -1.94 g.Maximum feed consumption was observed by Cirrhina mrigala consuming 98.72 g.Fork length of Cirrhina mrigala did not change while the same for Catla catla and Labeo rohita were -0.60 and -0.20 mm respectively.Labeo rohita and Cirrhina mrigala did not show change in term of total length gain (Table 5).Analysis of variance was carried out the difference for growth parameters include weight, fork length, and total length of fish species under chronic exposure to zinc.The results of ANOVA (Table 6) on weight(g) and fork length(mm) showed significant response towards fish species, age and treatment at p<0.01.However for total length (mm) responses were significant towards fish species and age at p<0.01, whereas non-significant for treatment.Most important is that the interaction (species*treatment*age) is strongly significant at the level of 0.01, which shows that with change in any parameter there were significant differences in growth parameters.**Significant at the level of 0.01, *Significant at the level 0.05 Condition factor for three fish species during these trials varied significantly at the level of 0.01.The interaction (species ҳ age) was significant at the level of 0.01 also.Condition factor for 180-day zinc exposed fish was 2.326 while the same lowest was 2.009 for 90-day fish (Table 7).

Accumulation of metal in body organs of fish
Table 8 displays zinc accumulation patterns in body organs of three fish species during growth trials for four age groups.

i. 90-day fish
During 30-day trial liver was the organ which showed significantly highest accumulation of zinc as 835.00, 1100.00,756.25 ug g -1 in Catla catla Labeo rohita and Cirrhina mrigala correspondingly.As far as the mean accumulation of zinc in all three fish species was concerned Labeo rohita displayed the maximum average accumulation (477.87±384.12 ug g -1 ).The differences among all the fish species were statistically significant (Table 8).ii.120-day fish Both liver and kidney appeared as organs for maximum zinc accumulation while the same were minimum for muscle in three fish species.Cirrhina mrigala of this age group showed maximum tendency for zinc accumulation (Table 8).
iii. 150-day fish Liver of 150 day Labeo rohita showed the highest accumulation of zinc (1834.53ug g -1 ).The muscle of all three fish species shows the lowest levels of zinc accumulation.However, the mean accumulation patterns in three fish species varied significantly with the highest concentration (594.01±722.32 ug g -1 ) of Labeo rohita (Table 9).

iv. 180-day fish
Catla catla and Cirrhina mrigala were the species that showed maximum accumulation of zinc in liver while kidney of Labeo rohita appeared as organ that concentrate maximum zinc as 793.62 ug g -1 .However, fish muscle showed minimum accumulation of zinc as 130.50, 96.19, 46.08 ug g -1 in Cirrhina mrigala, Labeo rohita and Catla catla correspondingly (Table 9).

Discussion
During the course of the experiments three fish species in the treated group showed a reduction in their growth at all age groups in comparison to the control.Fish growth was found reduced significantly in Nile tilapia when exposed to sublethal concentration of Zinc (Abdel-Tawwab et al., 2016).Heavy metal intoxication was found to results in toxic impacts and ecological damages (Moon et al., 2019).Feed intake and fish productivity decreases in the presence of heavy metals (Sfakianakis et al., 2015).The adrenergic responses consequences in the raise of circulating adrenaline which organizes the body for exercise by increasing ventilation rate, cardiac output and blood flow to muscle, and mobilizing substrates aimed at aerobic metabolism, and second common reaction to stress is the reduced growth.Perhaps this is not surprising that metals can cause reduction in growth (Elling et al., 1999).
Cirrhina mrigala and Labeo rohita showed better growth performance when compared with fish of all four age groups i.e.Catla catla showed higher sensitivity towards zinc contamination as far as growth performance is concerned.khanom et al.
(2020) studied muscular tissue bioaccumulation of heavy metals including Pb, Cd, Cr, Zn, Cu and Mn in two fish species (Gudusia chapra and Eutropiichthys vacha) and found that Zn was at a higher quantity, while Cd at a lower amount in both species in Padma River.
During chronic exposure to zinc for all trials, bioaccumulation of zinc occurred maximum in liver for all fish age group except 180-day Labeo rohita and lowest concentration of zinc observed in muscle and as described by Zhao et al that higher accumulation of metals in liver related to its function of metabolism that required to maintain life (Zhao et al., 2012), which is in confirmatory to following results.Abdel-Tawwab et al. (2016) also found that Zinc was found maximum in liver and kidney which was lowest in muscle.Alvarado et al. (2021) determined the heavy metal concentrations in the organs/tissues of Carp and Catfish cultured in Lake Chapala and found that liver had a higher concentrating capacity for Zn, Cu, Cd and Pb were highest in liver and muscle tissue.According to Bervoets et al. (2001) concentrations of zinc, lead, copper, and cadmium was measured in food, water, sediment, and Gasterosteus aculeatus tissues.Metal accumulation level in tissues was associated to the levels in water, sediment and food whereas gill/liver showed highest metal accumulation as compared to muscle.

Conclusion
Regarding metal toxicity, Zinc affect growth parameters (weight increment, fork lengths and condition factors) significantly (P<0.01).The control fish showed better feed conversion ratio as compared to that of zinc exposed fish.Liver showed significantly higher tendency to Zinc accumulation and significantly least trend in muscle than other organs of three fish species.Labeo rohita showed significantly higher trend to accumulation of zinc in body organs followed Cirrhina mrigala and Catla catla.It is concluded that metal intoxication significantly reduced the growth of all three fish species and may also show good indicator of metals present in surrounding environment.

Table - 7: Analysis of variance on condition factors (K) of three fish species under chronic exposure of zinc.
Imran Riaz Malik et al 5/7 Asian J Agric & Biol.2022(2).