Assessment of multifaceted toxicity induced by heavy metal contamination in the Gelevera stream using the Allium test

Abstract

Determining the effects of pollution in a water source on life is critical for the balance of nature. The aim of this study was to determine heavy metal pollution in Gelevera Stream and its effect on Allium cepa, an agricultural organism. Germination percentage, root elongation and weight gain were used as physiological parameters, mitotic index (MI), micronucleus (MN), chromosomal abnormalities (CAs) were used as cytogenetic parameters, malondialdehyde (MDA), proline, superoxide dismutase (SOD), catalase (CAT) were used as biochemical parameters and meristematic cell damage was used as anatomical parameters. The amount of DNA damage was assessed using the comet test. Four stations, namely A1, A2, A3 and A4, were identified in Gelevera stream to determine heavy metal pollution in water. ICP-MS was used to measure the heavy metals in water samples that were taken from each station. In the water samples taken from each station, bulbs were germinated for 72 h and the collected root samples were used in the analysis. As a result, the highest heavy metal pollution was measured in water samples collected from stations A4 > A3 > A2 > A1. Concentrations of elements such as Al, Hg, Ba, Ti, Ti, RB Cd, Mn, Sr, U and Co measured in water samples collected from station A4 were found to be above the reference values set by the Turkish Standards Institution (TSI), European Union (EU) and World Health Organization (WHO). The germination parameters of bulbs exposed to Gelevera stream water decreased. In Group V germinated with river water sample, in which the most pollution was detected, germination decreased by 45%, root length decreased by about 3.0 times and weight decreased by about 4.8 times. In Group III, Group IV and Group V, which were germinated with water samples collected from stations A2, A3 and A4 where heavy metal pollution was high, statistically significant (p < 0.05) decreases in MI and DNA percentages were found. Compared to Group I (control), MI decreased by 0.54% in Group II, 1.25% in Group III and 1.77% in Group V. In addition, statistically significant changes (p < 0.05) were found in MN and CAs frequency, MDA levels, proline levels and SOD and CAT enzyme activities of these groups. Different kinds of CAs and anatomical damage in root meristem cells were encouraged by heavy metal ions in Gelevera stream water. As heavy metal pollution increased, comet test findings indicated a decrease in the amount of head DNA and an increase in the percentage of tail DNA. As a result, it was determined that there is heavy metal pollution in Gelevera stream originating from different sources and far above the reference values, which promotes multifaceted toxicity in A. cepa, a non-target eukaryotic organism.

Introduction

Water pollution is one of the most common types of environmental pollution and the most important causes of this pollution are the discharge of wastewater from various industrial facilities, agricultural activities, mining and residential areas into the natural water environment. Especially in water pollution caused by wastewater from industrial activities, the ecological balance can be irreversibly disrupted^1,2. Heavy metals, chemicals and toxic substances in these wastewaters can cause serious deterioration in the quality of water resources and directly affect aquatic life. One of the most important negative effects is that it reduces the quality of water used as drinking and irrigation water, making it harmful to the health of humans and other living things. This restricts the use of water in agricultural activities, drinking water supply, industry and energy production^3,4. Rivers, which are important water resources, are significantly polluted due to human activities such as urbanization, agricultural activities and production discharges⁵. Increasing population growth and intensive urbanization are taking water pollution to advanced levels⁶. Inadequate waste management in sewer systems and the application of pesticides and fertilisers resulting from agricultural activities contribute to river pollution. In particular, discharges from polluted areas, leaking and problematic sewage infrastructure and floods are constantly entering rivers with pollutants. Such pollution can threaten the aquatic ecosystem and the health of living beings that use these waters^7,8.

The levels of heavy metals in stream pollution are quite significant and of the approximately 60 known heavy metals, lead (Pb), arsenic (As), mercury (Hg) and cadmium (Cd) are listed as hazardous and toxic substances. Globally, the problem of heavy metal pollution in water is a major concern. Heavy metals can enter the water from pollutants from chemical plants and thermal power plants, urban and industrial wastes and pesticides used in agricultural activities. The aquatic environment contains ionic forms of heavy metals and due to their properties, they can precipitate and accumulate. This accumulation poses a threat to aquatic organisms and poses a health risk to humans and other organisms when used as drinking water or ingested through the food chain^9,10. Altunkaynak et al.¹¹ detected the presence of heavy metals, including Al, Ti, and Co, as well as radioactive heavy metals such as Rb, Sr, Sb, and Ba, in the Batlama Stream (Türkiye) flowing into the Black Sea, with concentrations that exceeded the acceptable parameters. Doğan et al.¹² reported that heavy metal pollution in Pazarsuyu Stream, another river flowing into the Black Sea, causes toxic effects on animals and contains heavy metals such as Ni, Fe, Ba, Mo, Be, Sr.

Gelevera stream is one of the most important rivers in Turkey. The stream takes its name from the Gelevera plateau. Its source is the Akılbaba mountain on the border of Gümüşhane. It empties into the Black Sea from the east of Espiye district of Giresun province of Turkey. Its length is approximately 80 km. It has an annual water supply of 668 cubic hectometers. Gelevera stream is formed by the merger of Boynuyogun, Karadogan and Karaovacik streams. These streams are the main sources of the water of the Gelevera stream. There is Gökçebel dam on the Gelevera stream. There is also Alaçamdere dam on Karaovacık stream, which is an important tributary of Gelevera stream. There are also hydroelectric power plants and regulators established on the stream for energy generation and agricultural irrigation activities. In recent years, agricultural and domestic pollution has been observed in Gelevera stream. The annual average nitrate values were measured between 6.5 and 6.75 mg/L¹³. According to a study by Yıldız⁸, it was reported that Gelevera stream was classified as polluted water in terms of phosphorus content.

Water resources are limited and valuable natural resources and the pollution in water resources has reached high levels as a result of the discharge of wastes from domestic, agricultural activities, industry and urbanization into rivers and streams. Considering that stream and river waters are consumed as drinking water and water for various purposes, it is very important to monitor the pollution level in these waters at regular intervals. Detection and especially prevention of pollution ensures the long-term usability of the resources. In this study, the extent of heavy metal pollution in Gelevera Stream and the toxicity caused by this pollution were investigated multidimensionally with the help of different parameters.It is as essential to determine the effects of pollution on living organisms as it is to determine the pollution in water resources. Determining the effects of pollution in the water source on vitality is critical for both the balance of nature and vitality. The aim of this study was to draw attention to both heavy metal pollution and the effects of this pollution on A.cepa, an agricultural organism, at different stations in Gelevera Stream.

Material and methods

Sampling stations and sampling process

Sampling was conducted from the Gelevera stream, which originates from the Balaban Mountains of Gümüşhane province and flows into the sea in the east of Espiye district of Giresun province. Water samples were taken on September 11, 2022 from four different stations (A1, A2, A3, A4) shown in Fig. 1. Table 1 provides comprehensive details about the sample stations. Samples of water were taken from various depths (bottom, middle and top), from regions with both water flow and stagnation with Swing Sampler (Field Tech Saolutions, 1.2 L). For heavy metal measurements, a total of six water samples were taken from each station in 250 mL sterile polyethylene bottles bottles. Water samples were collected and transported to laboratories in accordance with standard methods¹⁴. The sample bottles were labelled with the date and station number. The water samples were stored in an ice chest and transported to the laboratory via the cold chain in a dark environment, with analysis carried out on the same day.

Fig. 1.

Stations taking water from Gelevera stream. The map showing the sampled stations was created using Google.com/maps-2025. The coordinates of the sampled areas were entered into the Google map application and satellite images of the areas were taken¹⁵.

Table 1.

Locations and characteristics of water sampling stations.

Station	Sampling area	Latitude	Longitude
A1	500 m above Gelevera village (Gumushane), where the Gelevera stream originates	40°35′24″	38°51′42″
A2	Giresun province, Güce district, Tekkekoy village, Akılbaba construction site plateau location	40°39′17″	38°52′28″
A3	After HES, Guce district (Giresun) location	40°53′24″	38°47′08″
A4	500 m away from where the Gelevera stream flows into the Black Sea	40°56′23″	38°43′20″

Heavy metals measurements

ICP-MS (Inductively Coupled Plasma Mass Spectrometry) was utilised to assess the concentrations of heavy metals in the water samples at Sinop University Research Center (Agilent Technologies 7700X Systems). All measurements were conducted on three occasions, and the mean value was calculated. The water samples were subjected to filtration in the laboratory, employing a 0.45 µM diameter Whatman filter (Merck-Millipore). 10 mL of each water sample was collected and subsequently diluted by the addition of 0.1 mL of concentrated nitric acid. The quality assurance and control procedures employed in this study involved the use of triple measurement and certified reference materials (UME CRM 1201 spring water). The analytical precision was within ± 10% of the target value. Samples of the 1 ppm internal standard (Agilent 5188–6525) were analysed at regular intervals throughout the study.

Test material and groups

To assess toxicity, Allium cepa var. aggregatum L. bulbs (n = 16) were employed as the test organism. A. cepa, used in this study, is an important bioindicator plant favored for its sensitivity to chemical toxicity. Its exposure to river waters during agricultural irrigation makes it an important indicator of the impact of pollution on living organisms. It is particularly noteworthy that the organism has a limited number of large chromosomes (2n = 16), a feature that gives it a significant advantage in determining genotoxic effects. However, notable advantages of the assay include its high sensitivity, affordability and accessibility. Furthermore, the high correlation of A. cepa with other toxicity tests, especially those performed on mammals, further supports its use as an indicator in research and development¹⁶. 5 groups of bulbs were created for treatment and control. The identification of A.cepa was carried out at the Department of Botany, Giresun University by Prof. Zafer TURKMEN. One specimen with voucher number BIO-A.ce/2023 was deposited in the herbarium of the Department of Biology.

Group I: Control

Group II: Stream water from the first station (A1)

Group III: Stream water taken from the second station (A2)

Group IV: Stream water taken from the third station (A3)

Group V: Stream water taken from the fourth station (A4)

The bulbs from each group were transferred into sterilised glass beakers. Tap water was utilised for the germination of the bulbs in the control group, while stream water collected from stations A1, A2, A3, and A4 was employed for the germination of the bulbs in the treatment group. The germination process for all groups was conducted for 72 h at room temperature. At the conclusion of the designated period, the bulbs were cleaned using distilled water, meticulously prepared for analysis in accordance with standard methodologies¹⁶. The supply of plant material, the collection of water samples, and the experimental techniques performed on the samples were all carried out with consideration for institutional, national, and international rules and regulations.

Analysis of physiological parameters

The effect of heavy metal pollution in Gelevera stream water on A. cepa root growth was determined by measuring the length of the radicle, the structure responsible for root formation, with a millimeter ruler. By using a precision scale to weigh the bulb weights before and after the application, the impact on live weight was ascertained. Equation (1) was used to determine the influence on germination as a percentage¹⁷.

1

Analysis of genotoxicity parameters

CAs, MN frequency, MI and DNA damage were used as biomarkers of genotoxicity. In order to observe the processes of CAs and MN formation, root tips were cut into segments measuring 1 cm. Following a two-hour soak in Clarke’s solution, the samples were rinsed in 96% ethanol for a period of 15 min. Thereafter, they underwent a hydrolysis process in 1 N HCl for 17 min at a temperature of 60 °C. The samples were then preserved in 45% glacial acetic acid for duration of 30 min. The dyeing process involved the application of acetocarmine for a period of 24 h. Subsequent to the staining the root tips were meticulously placed on slides, crushed with 45% glacial acetic acid, observed under an Irmeco and IM-450 TI research microscope, and captured on camera at a magnification of × 50¹⁶.

MN detection was based on the criteria proposed by Fenech et al.¹⁸.

• One-third of the nucleus should be the MN diameter,

• MN need to be oval or spherical in shape,

• The boundary between the MN and the nucleus should be easily discernible if they come into touch.

Equation (2) was used to determine MI as a percentage.

2

Using the technique suggested by Sharma et al.¹⁹, DNA was extracted from A. cepa root cells for the Comet assay. The approach suggested by Dikilitaş and Koçyiğit²⁰ was applied to the Comet assay. Comet assays were performed using the “TriTek 2.0.0.0.38 Automatic Comet Assay” software program. The slides were soaked in ethanol overnight, dried in an oven and prepared. 100% µL agarose [1% melting point (NMP) dissolved in distilled water at 50 °C and 1% melting point] was poured onto the slides with a pipette, covered with coverslips and allowed to stratify. The slides were placed in the refrigerator for 5 min to allow the gel to set. A second layer was prepared from a mixture of 1% low melting point (LMP) agarose and freshly isolated cell suspension. LMP agarose and cell suspension in distilled water at 40 °C were mixed thoroughly on the slide at a ratio of 1/8. The mixture was spread on the first layer as 100 µL and covered with coverslip again. The preparations were cooled in the refrigerator for 5 min and the coverslips were removed. The preparations were placed in the buffer solution in the electrophoresis tank and incubated for 40 min to allow the DNA to dissolve and relax. The preparations were then subjected to electrophoresis. After electrophoresis, the slides were washed three times for 5 min each at room temperature with 0.4 M Tris–HCl solution to make the slides more visible under the microscope and to neutralize the alkaline environment. After neutralization, the slides were stained with 100 µL ethidium bromide, covered with coverslips, examined under a fluorescence microscope, counted and photographed. In each group, a total of 1.000 cells were examined. DNA amounts in DNA fragments determined as head and tail were analyzed as percentages. Tail DNA percentages were used to calculate the degree of DNA damage using the Pereira et al.²¹ scale.

Analysis of biochemical parameters

MDA measurement

0.5 g of root tips were homogenised in 1 mL of 5% trichloroacetic acid. The homogenate was subjected to a centrifugation process at 12.000 g for duration of 10 min. The mixture was then transferred to a new tube, with an equal volume of 0.5% thiobarbituric acid. The mixture was then subjected to an incubation process, involving the addition of 20% trichloroacetic acid solution at a temperature of 96 °C for duration of 30 min. Subsequent to the process of incubation, the tubes were subjected to cooling. Subsequently, the samples were subjected to a centrifugal process at a speed of 10.000 revolutions per minute for duration of 5 min. The absorbances of the extracts were measured at a wavelength of 532 nm, and the levels of malondialdehyde (MDA) were expressed as micromoles per gram of fresh weight (FW)²².

Proline measurement

A quantity of 0.5 g of root tips was homogenised in 10 mL of 3% aqueous sulfosalicylic acid. The homogenate obtained was filtered through filter paper. A volume of 2 mL of the filtrate was transferred into a test tube, together with 2 mL of acid ninhydrin and 2 mL of glacial acetic acid. The mixture was then subjected to heat at a temperature of 100 °C for duration of 1 h. The reaction was brought to a halt by immersing the test tube in an ice bath. The reaction mixture was then extracted with 4 mL of toluene, followed by vortex mixing for duration of 20 s. The toluene-containing chromophore was extracted from the aqueous phase, subsequently cooled to room temperature, and measured at 520 nm on a spectrophotometer. Toluene was used as a blind. Equation 3 was utilised to calculate the proline concentration from a standard curve²³.

3

Enzyme extraction and analysis

At + 4 °C, enzyme extraction was carried out. In short, a mortar and pestle were used to homogenize 0.5 g of root tips in 5 mL of cold sodium phosphate buffer. For 20 min, the homogenate was centrifuged at 10.500 g. Until it was examined, the supernatant was stored at + 4°C²⁴. A total of 3 mL of reaction solution was utilised, comprising 1.5 mL of 0.05 M sodium phosphate buffer (pH 7.8), 0.3 mL of 130 mM methionine, 0.3 mL of 750 μM nitroblue tetrazolium chloride, and 0.3 mL of 0.1 mM EDTA-Na2.The following solution was prepared for the measurement of SOD activity: 3 mL of 20 μM riboflavin, 0.01 mL of 4% (w/v) insoluble polyvinylpyrrolidone, 0.01 mL of enzyme extract and 0.28 mL of de-ionized water. The reaction was initiated by subjecting the tubes to a 15 W fluorescent lamp for a period of 10 min, followed by a 10-min period of darkness. Absorbance was measured at a wavelength of 560 nm. The activity of one unit of SOD was defined as the amount of SOD that was required to produce 50% inhibition of the reduction of nitroblue tetrazolium chloride under experimental conditions²⁵. The CAT activity was determined in a total volume of 2.8 mL of reaction mixture, which comprised 1.5 mL of 200 mM monosodium phosphate buffer (pH 7.8), 1.0 mL of distilled water, and 0.3 mL of 0.1 M H₂O₂. The UV–VIS spectrophotometer was used to determine the CAT activity. The commencement of the reaction process was initiated by the addition of 0.2 mL of enzyme extract. The level of CAT activity was measured by observing the decrease in light absorption at a wavelength of 240 nm resulting from H₂O₂ consumption. This decrease is expressed as the change in OD₂₄₀ nm per minute per gram of sample²⁶.

Detection of anatomical damage to the root

The tops of the roots were cut into 1 cm lengths and sandwiched between pieces of Styrofoam. A sharp razor blade was used to cut transverse pieces in a single stroke. A research microscope (Irmeco IM-450 TI) was used to analyze the sections after they were put on a slide, stained with 5% methylene blue, covered with a coverslip, and photographed at × 200 magnification²⁷.

Statistical analysis

The values were statistically analyzed using the SPSS Statistics 22 (IBM SPSS, Turkey) program, and the findings were shown as mean ± SD. The data’s statistical significance was evaluated using the “One-way Anova” and “Duncan” tests. Statistical significance was defined as a P value of less than 0.05.

Results and discussion

Heavy metal pollution

Table 2 displays the levels of heavy metals in water samples taken from the Gelevera stream. The highest heavy metal contamination was found at station 4 (A4) > station 3 (A3) > station 2 (A2) > station 1 (A1). At station 1, the least heavy metal pollution was detected because this station is very close to the area where the Gelevera stream originates, i.e. it is quite far from pollutant sources. Therefore, the concentrations of all heavy metals measured at this station are within the maximum parametric value ranges set by the Turkish Standards Institute, European Union and World Health Organization for drinking and potable water. The primary cause of the greatest levels of heavy metal pollution in the water of the 4. station is that this station is located near where the Gelevera stream flows into the sea. In other words, the water samples collected from this station also contain the water of the 2nd and 3rd stations, which are exposed to many pollutants. The heavy metal with the highest concentration in Gelevera stream water was determined as Al. The concentrations of heavy metals such as Al, Ti, Mn and Hg and radioactive elements such as Rb and Sr measured especially in the 3rd and 4th stations were found to be above the maximum parametric values determined by the above mentioned National and International organizations for drinking and utility water. For example, while the reference value determined for Al according to TSI, EU and WHO 200 µg/L is, this value was measured as 243 ± 0.04 µg/L at the 4th station where the highest pollution was detected. Similarly, while the reference values for Ti and Hg were 0 µg/L, the levels measured at the 4th station were 4.61 ± 0.71 and 0.05 ± 0.001 µg/L, respectively. While the reference value for Mn was 50 µg/L, the measurement value was 82.6 ± 0.02 µg/L. Despite this, the levels measured in metals such as Cr, Ni, and Cu were measured below the reference values. For example, Cr and Ni were determined as 0.65 ± 0.02 and 0.32 ± 0.07 µg/L at station 4, respectively, while the reference values are accepted as 50 and 20 µg/L. The situation where the metal levels are below or above the reference values can be associated with the type of pollutants on the stream.

Table 2.

Concentrations of heavy metals in stream water and the refecnce values of the TSI, EU and WHO.

Elements	Control (Tap water)	1. Station (A1) (µg/L)	2. Station (A1) (µg/L)	3. Station (A1) (µg/L)	4. Station (A1) (µg/L)	Maximum parametric values for drinking/potable water (µg/L)			Recovery (%)
						Turkish standards institution (TSI)	European union (EU)	World Health Organization (WHO)
Alüminyum (Al)	1.16 ± 0.03	3.14 ± 0.33	40.5 ± 0.04	173 ± 0.03	243 ± 0.04	200	200	200	97.81
Titanyum (Ti)	0.00 ± 0.00	0.01 ± 0.02	0.00 ± 0.00	2.60 ± 0.54	4.61 ± 0.71	0	0	0	–
Krom (Cr)	0.05 ± 0.02	0.43 ± 0.03	0.44 ± 0.02	0.52 ± 0.12	0.65 ± 0.02	50	50	50	97.05
Manganez (Mn)	0.76 ± 0.04	0.95 ± 0.07	13.0 ± 0.09	32.8 ± 0.04	82.6 ± 0.02	50	50	50	98.80
Kobalt (Co)	0.00 ± 0.00	0.03 ± 0.01	0.03 ± 0.02	0.10 ± 0.02	0.09 ± 0.01	0	0	0	99.80
Nikel (Ni)	0.06 ± 0.00	0.13 ± 0.02	0.25 ± 0.04	0.22 ± 0.07	0.32 ± 0.07	20	20	20	97.92
Bakır (Cu)	0.96 ± 0.03	1.50 ± 0.08	3.27 ± 0.12	6.60 ± 0.05	26.5 ± 0.27	2000	2000	2000	99.29
Arsenik (As)	0.03 ± 0.01	0.10 ± 0.06	0.17 ± 0.12	0.68 ± 0.03	0.65 ± 0.13	10	10	10	99.32
Kadmiyum (Cd)	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.20 ± 0.01	5	5	3	98.31
Cıva (Hg)	0.00 ± 0.00	0.11 ± 0.02	0.07 ± 0.01	0.06 ± 0.001	0.05 ± 0.001	0	0	0	–
Kurşun (Pb)	0.00 ± 0.00	0.00 ± 0.00	0.04 ± 0.02	0.10 ± 0.03	0.72 ± 0.05	10	10	10	98.87
Rubidyum (Rb)	0.00 ± 0.00	0.59 ± 0.03	0.73 ± 0.03	0.91 ± 0.02	0.95 ± 0.03	0	0	0	–
Stronsiyum (Sr)	0.00 ± 0.00	61.6 ± 0.01	67.2 ± 0.03	80.4 ± 0.01	85.7 ± 0.01	0	0	0	99.19
Antimon (Sb)	0.005 ± 0.002	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.01 ± 0.02	5	5	5	98.87
Baryum (Ba)	0.00 ± 0.00	29.3 ± 0.03	26.1 ± 0.02	24.4 ± 0.01	30.1 ± 0.02	0	0	0	98.46
Godolinyum (Gd)	0.00 ± 0.00	0.05 ± 0.01	0.08 ± 0.01	0.08 ± 0.01	0.19 ± 0.13	0	0	0	–
Uranyum (U)	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.05 ± 0.006	0	0	0	–

Dates are given as mean ± SD. The monomer residual concentration in water that results from the polymer coming into contact with water is known as the parametric value.

This heavy metal pollution observed in Gelevera stream is thought to be caused by domestic wastes due to the settlement established near the stream, pesticides from hazelnut agriculture, hydroelectric power plants established on the stream, industrial wastes released into the stream and mining activities. The literature contains a few study findings that bolster our arguments. Le and Nguyen²⁸ determined the presence of Fe, Zn, As, Cu, Pb, Cr and Cd pollution in surface water samples collected from Khanh Hoa province of Vietnam, which is a residential area, in their study covering the years 2016–2020. Mathys²⁹ revealed the presence of pesticide and nitrate pollution in public drinking water, groundwater and surface water samples collected from an intensively agricultural area between 1987 and 1992. Jourtani et al.³⁰ reported that petrochemical wastes in the Moussa estuary in the Northwest of the Persian Gulf caused As, Mo and V pollution in the water and the quality of seawater and sediment posed a potential threat to the environment and public health. According to Toczyk et al.³¹, sediment samples taken from Poland’s Ślęza River—where a hydroelectric power station was constructed—showed heavy metal contamination of Cu, Ni, Cr, Zn, and Pb.

Physiological parameters

Table 3 displays the physiological alterations brought on by heavy metal contamination in the Gelevera stream. The highest germination, root elongation and weight gain were measured in Group I (control) and Group II, which were treated with water samples taken from A1, the closest station to where Gelevera stream originates. Between these groups, there was no statistically significant (p > 0.05) variation in the physiological parameter values. Statistically significant (p < 0.05) decreases were detected in all physiological parameter values investigated in Groups III, IV and V treated with water samples collected from stations A2, A3 and A4, where heavy metal pollution was measured above the reference values, compared to the control group. These decreases were more pronounced in Group V treated with water samples collected from station A4. Compared to Group I (control), germination decreased by 45%, root length by about 3.0 times and weight by about 4.8 times in Group V exposed to stream water.

Table 3.

Physiological changes in A.cepa germinating in water samples taken from the stations of Gelevera stream.

Groups	Germination (%)	Root length (cm)	Initial weight (g)	Final weight (g)	Weight gain (g)
Group I	100	7.98 ± 0.47a	5.96 ± 0.59	11.2 ± 0.88	+ 5.24a
Group II	100	8.04 ± 0.75a	6.05 ± 0.60	11.1 ± 1.05	+ 5.05a
Group III	80	6.10 ± 0.82b	5.97 ± 0.58	9.88 ± 1.24	+ 3.91b
Group IV	69	4.25 ± 1.04c	5.96 ± 0.55	8.80 ± 1.08	+ 2.84c
Group V	55	2.65 ± 0.60d	6.00 ± 0.59	7.10 ± 0.74	+ 1.10d

Group I: Control, Group II: A1, Group III: A2, Group IV: A3, Group V: A4. Data are presented as mean ± SD. n = 10/50 (50 bulbs for germination and 10 bulbs for length/weight). In the same column, means denoted by different letters^(a-d) are statistically significant at p < 0.05.

Some research in the literature support our findings by demonstrating that various plant species experience physiological toxicity when exposed to water contaminated with heavy metal ions. Cavusoglu et al.³² reported that Kızılırmak (Kırıkkale-Turkey) river water contaminated with heavy metal-containing refinery effluents caused significant reductions in germination, root elongation and weight gain of Zea mays L. (maize) seeds. In a similar study, Cavusoglu et al.³³ found that heavy metal pollution (Pb, Ni, Al, Fe, Cr, Zn, Cu, Cd) caused by petroleum wastewater in the Melet river flowing into the Black Sea from Ordu province (Turkey) caused decreases in germination percentage, root length and weight gain of Vicia faba L. (broad bean) seeds. Dogan et al.¹⁷ observed that heavy metal ions such as Cr, Fe, V, Zn, Ni, Co, Pb, As, Cu in Civil (Ordu-Turkey) stream water caused significant decreases in germination percentage, root elongation and weight gain of A. cepa bulbs. According to Altunkaynak et al.¹¹, heavy metal contamination in the Batlama stream in Giresun, Turkey, significantly reduced the germination, weight increase, and root elongation of A. cepa bulbs.

The main reasons for these decreases observed in the physiology of A. cepa bulbs as a result of exposure to stream water were thought to be that heavy metal ions in the stream water restricted the uptake of water and inorganic substances, i.e. nutrients, decreased mitotic activity and caused damage to the roots. This notion is supported by some information found in the literature. For example, it was observed that Pb heavy metal ion inhibited seed germination, root/shoot length and plant growth by disrupting nutrient uptake through roots, altering water balance, damaging plasma membrane structure and permeability, and generating reactive oxygen species (ROS)³⁴. Root growth is directly related to cell division. In this context, exposure to heavy metals has been reported to cause a suppressed root growth by reducing mitotic activity in various plant species. Similarly, Cr(VI) has been found to cause a delay in the cell cycle, leading to inhibition of cell division and reduced root growth³⁵. The current study showed that A. cepa bulbs exposed to stream water tainted with heavy metals showed a marked reduction in root mitotic activity. On the other hand, heavy metal ions are known to promote structural damages such as epidermis cell damage, cortex cell damage and necrosis in A. cepa roots and cause a decrease in germination, root elongation and weight gain by restricting water and mineral uptake of roots^36–38. In this study, epidermis and cortex cell damages were observed in meristematic cells of A. cepa roots exposed to heavy metal contaminated stream water.

Genotoxicity parameters

Tables 4 and 5 as well as Fig. 2 demonstrate the genotoxicity that heavy metal ions in Gelevera stream water produce in A. cepa root meristem cells. The results revealed that heavy metal pollution in the stream water caused serious damage to cell division and genetic structure. The highest MI value and DNA amount and the lowest numbers of MN and CAs were observed in Group I (control) and Group II treated with water samples taken from station A1, which was established near the area where Gelevera stream originates. These genotoxicity data found in Group I and Group II did not differ statistically significantly (p > 0.05). A statistically significant (p < 0.05) decrease in MI values and DNA amounts was detected in Group III, Group IV and Group V treated with water samples collected from stations A2, A3 and A4. Compared to Group I (control), MI decreased by 0.54% in Group II, 1.25% in Group III and 1.77% in Group V. Heavy metal-induced DNA damage was assessed using the very sensitive and reliable Comet assay. Comet test results showed a decrease in the amount of head DNA and an increase in the amount of tail DNA with the increase in heavy metal concentration. In Group V, where the most intense heavy metal contamination was measured, the amount of head DNA decreased by 43.6% compared to Group I (control). Exposure to heavy metal ions in stream water collected from stations A2, A3 and A4 caused statistically significant (p < 0.05) increases in the number of MN and CAs in Group III, Group IV and Group V. Heavy metal ions in stream water promoted CAs in A. cepa root meristem cells such as sticky chromosome, vagrant chromosome, unequal distribution of chromatin, bridge, fragment, nucleus with vacuole and irregular mitosis. The number of these damages was found to increase in direct proportion to the increase in heavy metal concentration. In this context, the highest number of MN and CAs were observed in Group V > Group IV > Group III, respectively. The biggest effect of heavy metal ions in the stream water on chromosomes is the formation of sticky chromosomes.

Table 4.

Genotoxic effects of Gelevera stream water samples on A.cepa.

Abnormalities	Group I	Group II	Group III	Group IV	Group V
MI %	833 ± 12.1a (8.33)	835 ± 11.8a (8.35)	779 ± 14.0b (7.79)	708 ± 17.1c (7.08)	656 ± 12.1d (6.56)
MN	0.20 ± 0.42d	0.10 ± 0.32d	24.2 ± 4.92c	50.9 ± 8.96b	96.5 ± 8.20a
Sticky chromosome	0.30 ± 0.48d	0.20 ± 0.42d	19.7 ± 2.95c	45.0 ± 8.65b	73.7 ± 7.27a
Vagrant chromosome	0.20 ± 0.42d	0.30 ± 0.48d	16.0 ± 4.08c	36.1 ± 3.96b	61.3 ± 5.62a
Unequal distribution of chromatin	0.00 ± 0.00d	0.00 ± 0.00d	14.7 ± 2.83c	35.2 ± 4.85b	51.9 ± 6.15a
Bridge	0.00 ± 0.00d	0.00 ± 0.00d	12.4 ± 2.99c	26.4 ± 3.60b	40.4 ± 4.09a
Fragment	0.00 ± 0.00d	0.00 ± 0.00d	9.70 ± 2.16c	18.8 ± 3.58b	34.0 ± 4.35a
Vocuolated nucleus	0.00 ± 0.00d	0.00 ± 0.00d	6.70 ± 1.77c	12.5 ± 2.59b	26.6 ± 3.89a
Irregular mitosis	0.00 ± 0.00d	0.00 ± 0.00d	3.60 ± 1.84c	9.40 ± 2.22b	16.9 ± 2.02a

Group I: Control, Group II: A1, Group III: A2, Group IV: A3, Group V: A4. Each group’s MN and CAs were determined by studying 1.000 cells, while each group’s MI was determined by analyzing 10.000 cells. In the same line, means denoted by different letters^(a-d) are statistically significant at p < 0.05.

Table 5.

DNA fragmentation induced by radioactive elements and heavy metals in the Gelevera stream.

Parameters	Group I	Group II	Group III	Group IV	Group V
Head diameter (px)	24.000	24.000	22.000	20.000	16.000
Head density	126.866	114.831	113.730	119.326	124.556
Head DNA (%)	95,9 ± 1.79a	95.3 ± 1.60a	77.3 ± 1.57b	67.0 ± 1.63c	54.1 ± 1.52d
Tail length (px)	3.000	2.000	16.000	26.000	44.000
Tail density	5.487	5.690	33.459	58.749	105.603
Tail DNA (%)	4.10 ± 1.79d	4.70 ± 1.60d	22.7 ± 1.57c	33.0 ± 1.63b	45.9 ± 1.52a
Tail moment	0.124372	0.094423	3.637	8.577	20.188

Group I: Control, Group II: A1, Group III: A2, Group IV: A3, Group V: A4. Data are presented as mean ± SD (n = 10). Each group’s 1.000 cells were examined for DNA damage. In the same line, means denoted by different letters^(a-d) are statistically significant at p < 0.05. Based on the percentage of tail DNA, a comet scale illustrating DNA damage was produced. Damage classified as minimal or nonexistent (≤ 5%), low (5–20%), moderate (20–40%), high (40–75%), or severe (≥ 75%).

Fig. 2.

CAs caused by radioactive elements and heavy metals in the Gelevera stream. Bar = 10 µm.

Our findings are consistent with those of other researches examining the genotoxic consequences of water pollution from heavy metals. Cavusoglu et al.³⁹ reported that Pb, Zn, Fe, Cu, Ni, Cd, Cd and Hg pollution caused by petroleum wastewater in Kızılırmak river (Turkey) increased MN formation in Vicia faba L. root tip cells. Kanev et al.⁴⁰ found that heavy metal pollution in the Ergene river (Turkey) caused by increasing industrial activities in the Thrace region led to decreased mitotic activity and CAs such as chromosome asynchrony and mispolarization in A. cepa root tip cells. Siddiqui et al.⁴¹ observed that heavy metal pollution from Cd, Zn, Ni, Cu and Fe in the Yamuna river flowing through Delhi, India caused a significant decrease in the MI value of A. cepa root tip cells and a significant increase in the number of CAs and cellular death (apoptosis) rate. Altunkaynak et al.¹¹ showed that water contaminated with heavy metal ions such as Al, Ti and Co in Batlama stream in Giresun province (Turkey) caused a decrease in the MI value of A. cepa root tip meristem cells and an increase in the number of MN and CAs. The researchers also determined that heavy metal contamination in water caused a decrease in the amount of head DNA and an increase in the amount of tail DNA, based on Comet assay results indicating DNA damage. In the Citarum River, the biggest river in West Java, Annisa et al.⁴² found that heavy metal pollution from Cr, Cd, Pb, and Hg led to a decrease in MI and an increase in CAs (such as stickiness, chromosome loss, bridging, fracture, multipolarity, binuclear cells and c-mitosis) in A. cepa.

The decrease in the MI value, i.e. mitotic activity, observed as a result of exposure to heavy metal ions in Gelevera stream water can be explained by damage to the polymerization of tubulin proteins that form the structure of microtubules responsible for the formation of spindle threads during cell division. In other words, it may be due to the inhibition of the organization of the microtubule skeleton. Some data in the literature support this idea. For example, Zhao et al.⁴³ reported that As ions cause inhibition of microtubule polymerization by reducing tubulin binding. Liliom et al.⁴⁴ observed that Cr ions inhibit tubulin polymerization, reduce microtubule formation and promote the formation of abnormal oligomers. Frantzios et al.⁴⁵ discovered that Al disrupted the processes governing tubulin polymerization and the architecture of the microtubule cytoskeleton, resulting in aberrant mitotic cells. There may be two main reasons for the decrease in the amount of DNA, i.e. DNA damage, and the increase in the number of MN and CAs as a result of exposure to stream water. The first one is that heavy metal ions bind directly to DNA and promote these damages. The second is that heavy metal ions indirectly promote these damages by increasing free radical production. Some information in the literature supports this idea. For example, Kocadal et al.⁴⁶ reported that heavy metals can directly cause single- or double-stranded DNA breaks in DNA. Briffa et al.⁴⁷ reported that heavy metals interact with nuclear proteins as well as DNA, causing direct site-specific damage. Dourado et al.⁴⁸ studied the genotoxicity of the water of the Água Boa stream (Brazil) with comet assay and reported that the highest DNA damage in fish occurred at stations with high metal (Cu, Pb, Cd, Ni) presence. Valko et al.⁴⁹ reported that heavy metal-mediated free radical formation causes various changes in DNA bases and increased lipid peroxidation. Arya and Mukherjee⁵⁰ showed that metabolic products of Cd, namely ROS, play a major role in the formation of DNA damage. Matos et al.⁵¹ reported that heavy metals are potential inducers of oxidative stress and cause DNA damage and cell death by promoting ROS production.

Biochemical parameters

Table 6 displays the impact of heavy metals in Gelevera stream water on the biochemical characteristics of A. cepa root meristem cells. The lowest MDA and proline levels and SOD and CAT enzyme activities were measured in Group I (control) and Group II treated with water samples taken from station A1, which was established near the area where Gelevera stream originates. These biochemical parameters assessed in Group I and Group II did not differ statistically significantly (p > 0.05). Statistically significant (p < 0.05) increases in MDA and proline levels and SOD and CAT enzyme activities were determined in Group III, Group IV and Group V treated with water samples collected from stations A2, A3 and A4 where heavy metal pollution was high. These increases were more remarkable in Group V, where the highest heavy metal contamination was measured. Compared to Group I (control), MDA level increased approximately 1.75 times, proline level increased approximately 2.84 times, SOD activity increased approximately 1.46 times and CAT activity increased 1.92 times in Group V. In conclusion, heavy metal pollution in water caused significant changes in the biochemical processes of A. cepa roots.

Table 6.

Biochemical toxicity caused by radioactive elements and heavy metals in the Gelevera stream.

Groups	MDA (µM/g FW)	Proline (µmol/g FW)	SOD (U/mg FW)	CAT (OD240 nm min./g FW)
Group I	21.2 ± 1.28d	10.5 ± 0.92d	138 ± 30.8c	1.55 ± 0.11d
Group II	21.1 ± 1.27d	10.4 ± 0.92d	139 ± 30.4c	1.53 ± 0.11d
Group III	25.6 ± 0.81c	15.8 ± 0.66c	156 ± 5.38bc	1.85 ± 0.08c
Group IV	30.3 ± 1.08b	21.6 ± 0.81b	173 ± 6.57b	2.32 ± 0.14b
Group V	37.2 ± 0.87a	29.8 ± 1.01a	202 ± 7.37a	2.97 ± 0.11a

Group I: Control, Group II: A1, Group III: A2, Group IV: A3, Group V: A4. Data are presented as mean ± SD (n = 10). In the same column, means denoted by different letters(a-d) are statistically significant at p < 0.05.

Our findings are consistent with research examining biochemical alterations in plants exposed to heavy metal-ion-contaminated water. Altunkaynak et al.¹¹ reported that heavy metal pollution in Batlama stream, which flows into the Black Sea from Giresun (Turkiye) caused a decrease in chlorophyll content of root and leaf tissues of A. cepa and an increase in MDA and proline levels, SOD and CAT enzyme activities. In the Pazarsuyu stream that flows into the Black Sea from Giresun province (Turkey), Doğan et al.¹² found that heavy metal pollution (Ni, Fe, Ba, Mo, Be, Sr) increased MDA levels and the activity of the SOD and CAT enzymes in A. cepa root cells. Aljahdali and Alhassan⁵² found significant increases in SOD, CAT, Glutathione S-transferase (GST) and Acetylcholinesterase (AChE) enzyme activities of seagrass (Cymodocea serrulata (R.Brown) Ascherson & Magnus) grown in seawater polluted with heavy metals such as Fe, Mn, Cu, Zn, Cd, Cr, Pb and Ni. MDA levels of A. cepa roots significantly increased as a result of heavy metal contamination from oil wastewater discharge in the Melet River, which empties into the Black Sea from Ordu province (Turkey), according to Turkmen et al.⁵³.

The increases observed in the MDA levels of A. cepa root cells as a result of exposure to Gelevera stream water may have been caused by the heavy metal ions in the stream water damaging the structure of the stem cell membranes, causing lipid destruction, i.e. lipid peroxidation, and as a result, increasing MDA production as an intermediate product. Because lipid peroxidation is a process that disrupts the structural integrity of the cell membrane and negatively affects cell functions. This notion is supported by some information found in the literature. Kaya et al.²⁷ reported that heavy metals cause MDA production by increasing free radical production in the cell, damaging stem cell membranes and increasing lipid destruction. Similarly, Rizvi et al.⁵⁴ showed that heavy metals cause an increase in lipid peroxidation in the cell, promote the conversion of unsaturated fatty acids into small hydrocarbon fragments such as MDA, and can also lead to cell death by changing the basic properties of the cell membrane such as fluidity and permeability. Therefore, the increase in MDA level is considered as a critical parameter to assess the degree of oxidative damage to the cell membrane⁵⁵.

The increase in proline levels of A. cepa roots exposed to Gelevera stream water is thought to be a defense mechanism developed by the plant to cope with heavy metal stress. According to reports in the literature, proline, one of the amino acids abundant in higher structured plants, accumulates excessively under stress conditions such as heavy metal exposure, salinity and drought. It has also been reported that the increase in proline level plays a role in protecting plants from the harmful effects of oxidative and osmotic stress, alleviating oxidative stress by scavenging free radicals and maintaining the stability of protein and membrane structures in the cell^56,57.

The increases observed in SOD and CAT enzyme activities in A. cepa roots exposed to Gelevera stream water suggest that the heavy metals in the water may be due to the fact that the heavy metals in the water promote free radical formation in the cell and the root cells increase the synthesis of SOD and CAT enzymes to eliminate the effect of these free radicals and to initiate the detoxification process. This notion is supported by some information found in the literature. Aydın et al.⁵⁸ reported that heavy metals cause the formation of different forms of ROS in the cell and as a result, cells increase SOD and CAT enzyme synthesis in order to reduce the effect of these ROS. Similarly, Kamiński et al.⁵⁹ reported that heavy metals can cause an increase in the production of ROS, which can damage lipids, proteins and DNA, leading to lesions and various dysfunctions in the cell, and that cells increase SOD and CAT enzyme activity to protect against these effects of ROS. Sedeño-Díaz et al.⁶⁰ evaluated the oxidative stress in the freshwater mollusc Physella acuta induced by potential toxicity in water from two rivers in the Mexican Atlantic Slope (Tecolutla and Tuxpan rivers), reporting the induction of CAT and GPx activities.

Anatomical observations

Figure 3 and Table 7 depict the anatomical alterations in A. cepa root tip cells brought on by heavy metal contamination in the Gelevera stream. No anatomical damage was observed in the root tip meristem cells of the bulbs in the control group (Group I) and Group II, which were treated with water samples taken from station A1, which was established near the area where the Gelevera stream originates. Anatomical damages such as epidermis and cortex cell damage, flattened cell nucleus and conduction tissue thickening were detected in the root tip meristematic cells of bulbs in Group III, Group IV and Group V treated with water samples collected from stations A2, A3 and A4 where high levels of heavy metal pollution were measured. The severity of these damages was determined as Group V > Group IV > III.

Fig. 3.

Anatomical damages caused by radioactive elements and heavy metals contained in the Gelevera stream.

Table 7.

Severity of anatomical damage induced by heavy metal pollution.

Groups	Epidermis cell damage	Cortex cell damage	Flattened cell nucleus	CTT
Group I	-	-	-	-
Group II	-	-	-	-
Group III	●	●	●	●
Group IV	●●	●●	●●	●●
Group V	●●●	●●●	●●●	●●

Group I: Control, Group II: A1, Group III: A2, Group IV: A3, Group V: A4, FCN:, CTT: conduction tissue thickening. No damage (-), minor damage (●), moderate damage (●●), and severe damage (●●●).

Our results are in line with other studies in the literature which revealed that heavy metal pollution in water causes different types of anatomical damages in A. cepa root tip meristem cells. Altunkaynak et al.¹¹ reported that heavy metal pollution in Batlama stream (Giresun-Turkey) caused anatomical changes such as epidermis and cortex cell damage, thickening of the cortex cell wall and necrosis in A. cepa root tip meristem cells. Doğan et al.¹² observed epidermis and cortex cell damage, flattened cell nucleus and thickening of the conduction tissue in onion root tips germinated with water samples collected from Pazarsuyu stream (Giresun-Turkey) polluted with heavy metal ions. In another study conducted by Doğan et al.¹⁷, heavy metal pollution in Civil stream (Ordu-Turkey) promoted anatomical damages such as cell deformation, thickening of cortex cell wall, necrosis, thickening of conduction tissue, flattened cell nucleus and necrosis in A. cepa root tip meristem cells.

It is believed that the defense mechanisms established by the roots to keep heavy metals out of the cell are the cause of the anatomical damage seen in A. cepa root tip meristem cells after exposure to Gelevera stream water. Indeed, microscopic examinations showed that roots treated with Gelevera stream water had a markedly higher number of cortex and epidermis cells. While these increases act as a barrier that restricts the entry of heavy metals into the cell, it also increases the contact of the cells with each other and creates a mechanical pressure. Their nucleus with the epidermis and cortical cells may distort as a result of this pressure. Another cause of cell deformation detected in root cells may be the disruption of the structural stability of the cell membrane. Because root tissue is the first to be affected in plants exposed to heavy metals. Therefore, heavy metals may also cause lipid peroxidation in root cell membranes, leading to loss of membrane integrity and consequently cell deformations. In addition, another reason for the flattening observed in the cell nucleus may be the change in intracellular pressure after the entry of heavy metals into the root cells. Because the pressure change may affect DNA density and nuclear protein concentrations and cause deformations in the cell nucleus. An additional defense mechanism created by plant roots to stop heavy metals from entering and moving through cells is the thickening of the conduction tissue. Similar possible mechanisms to the above-mentioned anatomical damages were also reported by Yılmaz et al.³⁸, Üstündağ et al.⁶¹, Sipahi Kuloğlu et al.⁶² and Özkan et al.⁶³, who studied the anatomical changes caused by heavy metals in A. cepa roots.

Conclusion

It has been determined that the Gelevera stream is exposed to heavy metal pollution caused by agricultural, industrial and domestic wastes on the routes it passes from the region where it is born until it flows into the Black Sea. It has been determined that this heavy metal pollution increases continuously as it approaches the Black Sea. In addition, some metals were found to be much higher than the reference values permitted by national and international organizations. This heavy metal contamination, measured above reference values in Gelevera stream, caused multifaceted toxicity in A. cepa, a non-target eukaryotic organism. In particular, it was determined that it retarded physiological development and caused cytogenetic abnormalities. In addition, it caused abnormalities in biochemical parameters and changes in anatomical structure by inducing oxidative stress in the organism. Therefore, water resources used especially for agricultural irrigation should be regularly controlled and their pollution should be prevented. In order to effectively combat water pollution, industrial facilities, urbanization and agricultural pesticides should not be allowed near water resources used for agricultural irrigation and drinking water. In addition, wastes should be prevented from reaching and discharging into water, strict controls should be carried out in wastewater discharges of industrial enterprises, and heavy metal and pesticide measurements should be carried out at certain intervals. In this context, representatives and employees of industrial organizations, farmers and households should be made aware of water pollution and its possible effects, and the value of water resources should be explained. The number of studies on Gelevera Stream in the literature is quite limited. These studies are mostly related to the microorganism load and heavy metal pollution of the stream. In this study, not only heavy metal pollution but also the possible toxic effects of this pollution on A. cepa, a non-target organism, were addressed with the help of many different parameters. We believe that our study will make a great contribution to the literature in this respect.

Author contributions

F. A: investigation; methodology; visualization; writing-review and editing. E.Y: conceptualization; methodology; data curation; software; visualization; writing-review and editing. K.Ç: conceptualization; data curation; investigation; methodology; visualization; writing-review.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.