ACUTE AND SOME CHRONIC EFFECTS OF NICKEL IN GAMBUSIA HOLBROOKI

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ACUTE AND SOME CHRONIC EFFECTS OF NICKEL IN

GAMBUSIA HOLBROOKI

Ahmet Burak Dumlu, Utku Guner^*

Trakya University Science Faculty Department of Biology, Balkan Campus, 22030 Edirne, Turkey

ABSTRACT

Nickel, which has a wide usage area throughout history, spreads throughout the ecosystem. Although Ni is low in the ecosystem, its usage continues to increase due to human use. Nickel is used in different industrial areas and released into nature from the atmosphere, from underground and above groundwater resources to aquatic ecosystems through erosion.

Acute and some chronic toxic effects of Ni on Gambusia holbrooki, mosquito biological control fish, were investigated. Acute toxicity of Ni was determined by the probit analysis method and 96- hour lethal dose test result was 6.811 mg/l for G.

holbrooki .During acute and chronic toxicity experiments, some behavioral changes in concentration, water column distribution, clustering and mobility and some physical changes such as blood supply to the gills and blackening of the outer epithelial tissue were observed in test animals.

KEYWORDS:

Nickel, mosquitofish, Gambusia holbrooki, acute toxicity, LC50, lethal dose, lethal concentration

INTRODUCTION

The current number of human-made chemicals added to the aquatic ecosystems in the world is esti- mated to be about nine millions. Toxic effects of heavy metals such as copper (Cu), cadmium (Cd), silver (Ag) and zinc (Zn) on fish have received con- siderable research attention [1, 2]. However, nickel (Ni) has received much less attention although it has long been known as an important chronic and acute toxicity agent. Nickel enters the environment from natural and anthropogenic sources and is distributed throughout all compartments using chemical and physical processes and biological transport by aquatic animals [3]. The continuous increase in the use and quantity of heavy metals is one of the biggest environmental problems. Different definitions have been proposed for heavy metals based on density, atomic number or atomic weight, and chemical properties or toxicity [4, 5]. Heavy metals can enter

aquatic systems from different natural and anthropogenic sources, including industrial, agricultural or/and domestic wastewater, stormwater flow, land- fill leaks, and transport [6, 7]. Heavy metals in such environments have been a threat to all aquatic organisms even at low levels [8]. The most important property of heavy metals is that they can accumulate in the exposed organism and the accumulation in aquatic organisms is 1.000-10.000 times more than water concentrations.

Heavy metals not only have acute but also chronic effects on the entire life cycle of aquatic organisms, especially as loss of growth and damage to reproductive functions. The type, age and sex of aquatic organisms affect aquatic toxicity [9, 10].

In aquatic ecosystems, fish are usually located at the top of the food chain and transfer some heavy metals from water to other food networks. Therefore, fish are the most decisive factor for estimating the risk potential of heavy metal pollution and human consumption in freshwater systems [11]. Some metals such as Cu and Zn which are important for normal metabolism of fish should be obtained from food or sediments. Non-essential metals are also known to be taken up by fish where they accumulate in tissues [12, 13].

Combating pests with biological control meth- ods is also a problem. Gambusia holbrooki (Girard, 1859) is one of the most widely used organism used in biological control of mosquitoes. At this point, it is very important to know how living organisms used in biological control are affected by pollution. Gam- busia holbrooki is commonly used in Thrace region in Turkey and is exposed to heavy metal pollution of agricultural and industrial origin. In this study, we determined the 96-hour acute toxicity and show some chronic toxicity of Ni on G. holbrooki.

MATERIALS AND METHODS

Gambusia holbrooki specimens were collected from Güllapoğlu Pond located in Balkan Campus of Trakya University (Edirne, Turkey) and brought to the laboratory. The collection was carried out fol- lowing the research permission obtained from the General Directorate of Nature Conservation and Na- tional Parks of the Ministry of Agriculture and For-

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estry. In order to adapt the fish to the laboratory environment and the physicochemical properties of the test water, they were placed in kept in stock aquariums for 14 days in a 12:12 day-night period. At the beginning of the acclimatization, fish were placed in aquariums with water taken from their natural environment. Then, the water of the aquariums partially changed with test water at a rate of 20%, 50%, 75%

at certain day intervals until the stock aquarium hav- ing the test water completely Physicochemical properties of the water taken from the natural habitats of the fish and the test water were given in Tables 1 and 2, respectively. All aquariums (stock and quarantine) were aired (3.5 l/hours) with an air motor (RS 628A®) during all experiments and their water was continuously cleaned with an internal filter (Sebo WP-850F®). Water temperatures of the aquariums were maintained at 22 ± 3 °C with the help of ther- mostat heaters. The physicochemical parameters of the test aquarium water were measured every day.

Fish were fed with flakes (TetraMin Flakes®) every 12 hours except the last 24 hours before the start of the experiment.

Physicochemical properties of test and water used in the experiment were shown in Table 1 and 2.

All experiments were conducted with the knowledge and approval of Trakya University Ani- mal Experiments Local Ethics Committee. Acute toxicity experiments were performed to determine

the LC50 value of Ni on G. holbrooki. Preliminary tests were performed to determine the selection of appropriate test doses before the actual tests to determine the LC50 value. The actual experiments with the appropriate doses determined were carried out in three replicates.

TEKKIM® brand (NiSO4.6H2O Extra Pure 99%) (Cat. No: TK.200221, CAS: 10101-97-0, UN 3077, EC 232-104-9) was used as Ni source. The stock Ni solution used in the LC50 experiments was prepared by dissolving 4.48 g NiSO4.6H2O in 1000 ml distilled water to obtain 1000 ppm Ni (1000 mg Ni/l).

In each experiment, 1 control and 5 dose groups were studied and in each experimental group aquariums containing 10 liters of test water were used. In order to provide continuous aeration of the experimental groups, a comparable amount of airflow was provided to all groups with the help of two outlet air pumps, 6 distributors and 6 air valves.

In the distribution of air, ceramic air stones and silicone aquarium air outletlocated at the bases of the aquariums were used. All experiments were carried out under the same conditions. Ten experimental animals were tested in each group, including the control. Gambusia holbrooki used in the experiments were 30 ± 10 mm in length and 0.2-0.6 g in weight.

TABLE 1

Physicochemical parameters of pond water at the time the fish were collected and the stable state (average during the experiment) of stock and quarantine aquariums.

TABLE 2

Physicochemical properties of water used in the experiment.

Parameter Value

Aluminum (Al) 0.0 µg/l

Ammonium (NH4) 0.0 mg/l

Chloride (Cl) 0.0 mg/l

Iron (Fe) 0.0 µg/l

Manganese (Mn) 0.0 µg/l

Sulfate (SO4) 0.0 mg/l

Sodium (Na) 0.9 mg/l

Total Hardness 2.5 mg/l CaCO3

pH 7.55 ± 0.25

Conductivity (EC) 15.64 µS/cm Oxidability (O2) 0.5 mg/l

Parameter Natural Water Values Aquarium Water Values

pH 6.08 7.12 ± 0.6

ORP 1.1 mV -15.7 ± 10 mV

Solve. Oxygen (LDO) 7.21 mg/l 7.39 ± 0.3 mg/l

Solve. Oxygen % (LDO%) %89 %93.8 ± 4.3

Temperature 24.6 ^oC 23.1 ± 2.7 ⁰C

Conductivity (EC) 349 µS/cm 97.8 ± 36.6 µS/cm

Salinity (TDS) 223 ppm 62.5 ± 37.5 ppm

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The equality of females or males was not considered in the selection of the fish and randomly selected specimens of both sexes were randomly assigned to the control and dose groups.

During the experiments, the physicochemical parameter measurements of the control and dose groups were performed with multi-device (Hach Lange Hq40d®) every 24 hours. The calibrations of the device were checked regularly with pH 4, 7 and 10 calibration solutions of the same company and conductivity calibrations with 1000 µS/cm NaCl liquid regularly. LDO (Luminescent Dissolved Oxygen) probe of the same brand was used to determine the amount of dissolved oxygen.

The average lethal concentration value (LC50) was calculated as a result of acute toxicity experiments. The data were statistically analyzed on a computer and LC50 calculations were made according to Probit Regression analysis in IBM®

SPSS Statistics 22 program. At the end of the experiments, the 24, 48, 72 and 96-hour LC50 values were calculated.

Behavioral and physical changes in the experimental groups were carefully examined and evaluated. The evaluation was performed

qualitatively on 10 experimental animals subjected to acute toxicity tests and the frequency of effects in individuals in the dose groups was shown as “+”

scoring. The effects were represented as “+” if the number of individuals in the experimental dose groups was less than half,“ ++” if close to half, and

“+++” if more than half.

RESULTS

The mortality rates of the nickel concentration studied in the main experiment determined after the preliminary test were shown in Table 3.

The three-replicated acute toxicity experiments and the mean time-dependent percentage mortality charts for 48, 72 and 96-hours were shown in Figure 1. No mortality was observed during the first 24 hours of acute toxicity experiments. In all experiments, the fish in the control group showed normal behavior and no death was observed in this group.

TABLE 3

96-hour mortality and percentages of G. holbrooki at different Ni concentrations.

Ni concentration (mg / l,

ppm) Number of

fish (n) Number of dead

fish Ratio%

0 (control) 30 0 0

5 30 0 0

10 30 7 23.33

20 30 15 50

30 30 27 90

50 30 30 100

FIGURE 1

Time-percentage mortality graphs in 48-72 and 96-hour concentration in acute toxicity experiments.

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In acute toxicity experiments, the highest mortality rate was in the 50 ppm Ni dose group after 48- 72 and 96-hours. At the end of the tests periods, Pearson correlation significance controls were performed to determine the relationship between dose and mortality and p values were 0.006; 0.002 and 0.001. However, at the end of the experiments, lethal concentration values and confidence intervals of 48, 72 and 96-hours for Nickel Gambusia holbrooki and confidence intervals were calculated by probit analysis (24 hours LC50 value could not be calculated due to lack of mortality) and nickel LC50 value for Gam- busia holbrooki 16.811 mg / It was determined as (Table 4). Significance values for probit analysis were p= 0.003 for 48 hours, p=0,000 for 72 hours and p=0,000 for 96-hours showing that the model and the obtained data had high levels of significance.

Gambusia holbrooki’s LC50 value for soft wa- ter according to WHO [14] is 4-14 mg/l which is close to the value of Poecilia reticulata in the same family (Poeciliidae) (P. reticulata Ni LC50: 15.62 mg/l [15]).

Acute toxicity studies with heavy metals for G.

holbrooki and the results of this study conducted with nickel LC50 values calculated for cadmium (Cd) 0.037 mg/l (soft water); 0.017 mg/l (soft water) for copper (Cu); 0.35 and 0.46 mg/l (soft water) for zinc (Zn); 0,36 mg/l (very hard water) for mercury (Hg) (Hardness indicator was accepted as 75 mg/l CaCO3), [16-19]. According to this, nickel toxicity was lower than Cd, Hg, Zn and Cu.

The physical and behavioral changes observed in the acute toxicity experiments were recorded and checked daily at 2-3 hour intervals. The evaluation was performed qualitatively on 10 experimental animals subjected to acute toxicity tests and the frequency of effects in individuals in the dose groups was shown Table 5.

In the observations made during the acute toxicity experiments, differences were observed in the distribution of fishes in water and swimming behav- iors among to dose groups. In the experiments, ho- mogenous distribution, surface swimming and mobility parameters of the fish tended to decrease with increasing doses of Ni while clustering, swimming at the bottom, standing close to the air source, color change in the skin and bleeding in the gills tended to increase. Although homogeneous distribution and calm swimming behavior were observed in the control and low dose (5 ppm Ni), individuals in the does groups with and higher than 10 ppm Ni exhibited a stressful attitude to each other shortly after the first introduction of Ni into the environment, and after a while individuals decreased their mobility’s and showed tendency to cluster with increasing dose amounts. At the same time, the placement of fish in the water column in the control and 5 ppm Ni dose groups showed a proportional spread, whereas individuals in the 10 ppm Ni group and other doses with

higher Ni concentrations preferred to be present at the bottom of the water.

There are several studies on physiological changes caused by Ni exposure. Nielsen [20] reported that the toxic effect of Ni may be due to the penetration into the epidermis after contact with the body surface and subsequent association with the body protein. Nickel also induced a series of histopathological changes including the fusion of gill lamellae, tissue hyperplasia and hypertrophy [21, 22].

Nath and Kumar [23] exposed Colisa fasciatus (trop- ical freshwater perch) in hard water (172 mg/l CaCO3) to a concentration of approximately 14 mg/l nickel and reported great damage to the gill structure. The gill cavities of the fish exposed to Ni compounds were filled with mucus and the lamellae were dark red. It was stated that the destruction of gill lamellae with ionic Ni may result in decreased venti- lation rate, and blood hypoxia which may cause death [24, 25]. In a study examining the effects of Cd, Cr and Ni on the diffusion capacity of the gills, only Ni was observed to harmful effect depending on the concentration [21].

CONCLUSION

Nickel can be found in air, water, soil, nutrients and household items making all organisms absorb Ni either by inhalation, ingestion, contact or other forms. Nickel can be found in low concentrations in many organisms but exposure to high concentrations is toxic. Several national and international organiza- tions have set limit values for Ni. For instance, the limit values are 1 mg/m³ for OSHA, 0.015 mg/m³ for CDC NIOSH and 0.2 mg/kg/day for EPA [28].

The average and maximum allowable levels for Ni in Turkey are determined by regulations. Accord- ing to the Regulation on Surface Water Quality, the annual average value of Ni and its compounds is 4 µg/l (0.004 ppm) in rivers and lakes, and 8.6 µg/l (0.0086 ppm) in coastal and transition areas, while the maximum permissible environmental quality standard values for both habitats is 34 µg/l (0.034 ppm) [29]. According to the provisions of the Regu- lation on Water for Human Consumption, the permissible parametric value of Ni in drinking-using water was determined as 20 µg/l (0.02 ppm) [30]. It was found that although the swimming mobility decreased in the 24-96-hours interval, operculum and mouth mobility increased in subjects subjected to the experiment at doses of 10 ppm Ni and above. Simi- larly, Khangarot and Ray's [26] study of Ni toxicity in P. reticulata suggests that the gill and mouth mo- tility of fish may be nickel-derived, but Athikevasan et al. [21] showed with Hypophthalmichthys molitrix (Silver carp) that the exposure to fish as a result of nickel agonistic behavior supports the observed effects. Also, various factors have been attributed to behavioral changes / abnormalities in fish exposed to

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heavy metals. Nerve disorders due to obstruction of nerve effector regions, respiratory disorders caused by enzyme dysfunction and low energy due to the effect of the energy formation process were thought to be the factors that cause behavioral effects [27].

In the present study, the average lethal concentration of Ni (96-hours LC50) for Gambusia holbrooki was determined as 16.811 mg/l. Gambusia species can rapidly adapt extreme environmental conditions and are highly tolerant of contaminants, which can be very difficult to eliminate by aggres- sive behavior in their habitat. Therefore, the uncon- trolled release of Ni and its compounds in LC50 con- centrations determined for G. holbrooki will have a fatal effect on many species living in both aquatic and terrestrial ecosystems which are more suscepti- ble to toxic substances than G. holbrooki.

Some behavioral changes in the case of feed- ing, reproduction and respiration etc. as a result of heavy metal exposure have been observed in fish [31]. During long-term chronic experiments, differences were observed between dose and control groups in various behavioral patterns including homogeneous distribution, clustering, surface swimming, bottom swimming, mobility and proximity to an air source and also in skin color change) (see Ta- ble 5).

As a result, research on negative effects of Ni and its compounds which have been increasingly used in various industries, should be increased and the factories, mines etc. using Ni and its compounds as a source should be decreased. The control of Ni emissions of companies should be ensured continuously. Increasing the presence of Ni in aquatic ecosystems and ignoring the dangers that may arise will cause the problems related with the food chain to increase day by day. The 96-hour LC50 value of Ni for G. holbrooki can be used as a reference for the crea- tion of new regulations and control and monitoring protocols for the release of wastewater into the environment. Mosquito fish is a biological control tool which can effectively be used in mosquito control.

Thrace region is a region with dense industrial areas acting as point pollution sources and intensive spray- ing and fertilization activities acting as non-point pollution sources. There is an increasing risk of industrial and agricultural heavy metal pollution in the region and it is important to know acute and chronic effects of Ni pollution on a fish species used in mos- quito control. In conclusion, G. holbrooki with a high ecological tolerance will be less affected by possible Ni pollution than native species found in the natural environment and the potential of spreading was high.

TABLE 4

95% confidence interval for 24, 48, 72 and 96-hour lethal dose values (mg / l, ppm).

Time LC point Value Low limit High limit

24 hour LC 50 Could not be calculated

48 hour LC ⁵⁰ 85.314 56.028 501.946

72 hour LC ⁵⁰ 40.378 33.511 53.927

96-hour

LC 10 8.695 6.326 10.649

LC 15 9.863 7.463 11.850

LC 20 10.903 8.495 12.924

LC 30 12.836 10.440 14.954

LC 40 14.756 12.367 17.057

LC ⁵⁰ 16.811 14.373 19.443

LC 80 25.920 22.207 32.032

LC 90 32.503 27.113 42.756

LC 95 39.183 31.759 54.633

LC 99.99 55.637 42.381 87.241

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TABLE 5

Evaluation of some physical and behavioral changes in acute toxicity experiments.

96-hour acute toxicity test (average of data obtained from all experiments)

Effect / Dose 0 ppm 5 ppm 10 ppm 20 ppm 30 ppm 50 ppm

Homogeneous +++ +++ ++ + + +

Distribution

Clustering + + ++ ++ +++ +++

Surface Swim-

ming +++ ++ ++ + + +

Bottom Swim-

ming + + ++ +++ +++ +++

Mobility +++ +++ ++ ++ + +

Proximity to Air

Source + + + ++ +++ +++

Bleeding in the

gills - - - +

Skin Color

Change - + + ++ ++ +++

“-“ : Not available.

“+” : Less than half of the total number of individuals.

“++” : The number of individuals close to half of the total number of individuals.

“+++”: more than half of the total number of individuals.

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Received: 26.08.2019 Accepted: 10.06.2020

CORRESPONDING AUTHOR Utku Guner

Trakya University Science Faculty Department of Biology,

Balkan Campus, 22030 Edirne – Turkey e-mail: uguner@trakya.edu.tr