Analysis of pesticide and heavy metal bioaccumulation in earthworms from Central plain zone of Punjab

doi:10.21203/rs.3.rs-4719879/v1

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Analysis of pesticide and heavy metal bioaccumulation in earthworms from Central plain zone of Punjab

https://doi.org/10.21203/rs.3.rs-4719879/v1

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The present study was undertaken to estimate the pesticide residue and heavy metal contents of agricultural soil samples to analyse their bioaccumulation in earthworm tissues and genotoxic effects from different locations of central plain zone of Punjab, India. Monitoring of four locations of Punjab showed the concentrations of heavy metals in the soil and earthworm tissue followed similar trend, and were in the order of Mn > Zn > Cr > Cu > Pb > Co > As > Cd. Soil samples of Bhogiwal and Jassian village were detected with chlorpyrifos and Tebuconazole; and chlorpyrifos respectively. Bioaccumulation factor for heavy metals was found to be highest for Cadmium in earthworms of Bhogiwal village of Malerkotla (9.19 ± 0.55), followed by Zinc (1.43 ± 0.01 to 2.07 ± 0.12), and lowest for Chromium and Cobalt in village Bhogiwal of Malerkotla and in village Balrampur of Ropar respectively. Damaged DNA was observed in earthworm samples of Jassian and Bhogiwal villages, providing insights to the genotoxic effects of pesticide and heavy metal contamination.

Earthworms

heavy metal

pesticide

bioaccumulation

comet

soil

genotoxicity

Environmental pollution is concerned problem all around world due to rapid increase of industrialization and urbanization. Heavy metal pollution of terrestrial ecosystems has long been recognized as a serious environmental concern. Heavy metals are ubiquitous in the environment, as a result of both natural and anthropogenic activities, and humans are exposed to them through various pathways (Wilson and Pyatt, 2007). Earthworms are important organisms in terms of soil functionality and consequently play a key role in terrestrial eco- toxicological risk assessment (Weeks et al., 2004). One of the most peculiar and special behaviour of earthworms is to accumulate heavy metals in their tissues and are enough sensitive to contaminants, which make them good bioindicators (Nahmani and Lavelle, 2002). Heavy metals are considered serious pollutants because they are toxic and non-degradable, thus accumulation of heavy metals in soil poses many risks to humans and the ecosystem (Odoh et al., 2011). The main reason in most of the developing countries is discharge of untreated sewage and industrial waste on land or into the running water streams which is used for irrigating crops. In USA and other developed countries many metals in sewage wastes are removed by treatment before its application to land to prevent their entry in the sewage system (Liphadzi et al. 2003).

In order to protect their crops from losses due to pests and diseases, the farmers in Punjab use huge quantities of pesticides. Among various crops, the pesticide usage is highest in the cotton fields to protect the crop from attacks of the Helicoverpa (Mehrotra and Phokela 1992). As per the report of Thakur et al. (2008), 70% of Punjab’s population is directly or indirectly associated with agriculture and nearly the same trend is followed in the Malwa region. Cotton is a major crop in the Malwa region, which requires more use of pesticides to control the infestation of pests, as the pests have developed resistance to the pesticides (Shetty 2004). Also, it was reported that the farmers were using 30–35 pesticides spray for a single cotton crop as against 8–10 recommended by agriculture experts in some parts of the Malwa region (Thakur et al. 2008). Once a pesticide is applied to soil, it follows one of three pathways: 1) adhering to soil particles (mainly organic matter and clays); 2) degrading by organisms and/or free enzymes; and 3) moving through the soil with water. As according to Francia et al, 2006, pesticides have played a key role against food shortages and vector-borne diseases, and humankind would be vastly different without their use. However, non-target impacts from pesticide associated agricultural runoff persist. Punjab is the most productive agricultural state of the country where untreated sewage water is being used as a source of irrigation. Therefore, in view of the limited information available, accumulation of heavy metal and pesticides in soils in agricultural fields, and their bioaccumulation in earthworms and genotoxic effects were monitored.

Site selection and sample collection: The research was conducted in agricultural fields of two districts (Ludhiana and Malerkotla) of Central Plain Zone of Punjab. Soil and earthworms sampling was done from agricultural fields of four villages namely, Jassian and Manakwal from Ludhiana, Kupp and Bhogiwal from Malerkotla. For sampling of soil, digging was done to a depth of 30 cm to obtain soil samples from the aforementioned areas. These were run through a 2mm sieve after being packed in plastic bags for further analysis. For earthworm sampling, the soil was dug upto a depth of 30cm with a shovel and earthworms were collected manually with the appropriate amount of soil from the aforementioned areas from each district.

Estimation of heavy metals: To determine the metal content of soil, 2 grams of air-dried soil was mixed with 20 ml of aqua regia and kept the mixture overnight. After that, distilled water was used to dilute the digested matter to 50ml and Atomic Absorption Spectrophotometer (AAS) was put-to-use to analyse the solution. For heavy metal estimation in earthworm tissue, earthworms were dried and crushed into small pieces and were digested using 10ml of hydrochloric acid and 20ml of nitric acid. The digests were heated on a hot plate at 90-95^oC for four hours and the material was poured through Whatmann filter paper into 150ml flasks. The solutions were made to a 100 ml volume using distilled water and the heavy metal concentration of the filtrate was evaluated using an Atomic Absorption Spectrophotometer (AAS). Concentration of heavy metals in these samples was calculated by the following formula. Concentration of heavy metals (mg/kg) = Reading from ICAP-AES × Volume of digest /Weight of sample

Bioaccumulation factor (BAF): The indices of metal accumulation by organisms in their body tissue is known as BAF. It is the ratio of the level of metals in organisms to the soil substrate. BAF for earthworms were estimated for the metals in earthworm tissues and substrate materials using the method described by Mountouris et al (2002). The BAF was calculated using the formula:

BAF = Ce/Cs, where Ce and Cs were the total concentrations of heavy metals in earthworms and soil substrate respectively.

Estimation of pesticide residues in soil and earthworm samples: Pesticide estimation was done by preparing the working solutions (100, 10 and 1 g ml^− 1) and stock solutions (400 g ml^− 1) at the Pesticide Residue Analysis Lab which already has the technical grade certified reference standards of the pesticides utilized in this experiment. The stock solutions of pesticides were maintained at -20°C. In order to create working solutions for sample fortification, pesticides were diluted with hexane, and standards were stored at -4°C until analysis while estimation was done using HPLC.

Comet assay: Comet assay was performed using the methodology from Singh et al 1988 for DNA damage detection by extracting coelomocytes from earthworms. A programme CometScore2_0 was used to gather the data for the analysis of the comet assay test. The average tail length and tail DNA percentage of the comets derived from the study of the earthworm coelomocytes were measured.

Statistical analysis: The concentration of heavy metals in contaminated soil samples and in tissues of different earthworm species collected from respective sites were compared by using one-way Anova. All data was calculated by using SPSS version 20.0 program for windows.

Values recorded for eight heavy metals including Arsenic (As), Cadmium (Cd), Copper (Cu), Chromium (Cr), Lead (Pb), Manganese (Mn), and Zinc (Zn) in the soil and earthworm samples, from all the locations, were significantly different (p < 0.05). Mean values for heavy metal contents in soil from four villages of Central plain zone i.e. Ludhiana (Jassian, Manakwal) and Malerkotla (Kupp, Bhogiwal) are given in Table 1. Average concentration of heavy metals in soil from all the villages followed the trend: Mn > Zn > Cr > Cu > Pb > Co > As > Cd. Sutowska et al 2020 conducted a thorough investigation to determine the concentration of heavy metals and the distribution of their sources in the industrial region of Upper Silesia in Southern Poland. To ascertain the concentration of heavy metals in distinct soil strata, they employed diverse contamination indicators that were produced. According to their findings, the concentration of heavy metals in the shallow strata was Pb > Cd > Zn, in that order of highest to lowest concentration. While Zn > Cd > Pb was found in the deep strata. The significant toxicity of the examined area was shown by the concentration of all metals surpassing the geochemical background limits.

Table 1

Estimation of heavy metal contents (ppm) in soil from Central Plain Zone of Punjab
Metals	Ludhiana		Malerkotla		Permissible limit (Agarwal, 2009)/EC*
	Jassian	Manakwal	Kupp	Bhogiwal
Arsenic	5.36 ± 0.08	5.33 ± 0.06	4.78 ± 0.04	4.83 ± 0.03	20*
Cadmium	0.72 ± 0.01	0.73 ± 0.05	0.31 ± 0.01	0.31 ± 0.02	0.06
Chromium	35.07 ± 0.95	35.67 ± 1.21	25.53 ± 0.14	25.08 ± 0.12	100
Cobalt	13.15 ± 0.01	7.25 ± 0.08	11.42 ± 0.09	7.52 ± 0.56	8
Copper	25.62 ± 0.06	21.97 ± 0.14	19.63 ± 0.13	19.79 ± 0.15	20
Manganese	249.35 ± 3.65	248.15 ± 1.56	243.69 ± 1.66	268.47 ± 8.59	850
Lead	16.24 ± 0.17	16.13 ± 0.07	14.75 ± 0.01	14.95 ± 0.10	10
Zinc	64.83 ± 1.14	60.40 ± 2.27	58.77 ± 0.22	49.20 ± 0.12	50
Values are mean ± S.E of three replicates
EC*: European Community

Mean values for heavy metal contents in earthworms from four villages of Central plain zone i.e. Ludhiana (Jassian, Manakwal) and Malerkotla (Kupp, Bhogiwal) are given in Table 2. Average concentration of heavy metals in soil from all the villages followed the trend: Mn > Zn > Cr > Cu > Pb > Co > As > Cd. The concentrations of heavy metals in the tissues of all the earthworm species obtained from the four locations, was significantly lower (p < 0.05) than that in the soil samples of respective locations.

Table 2

Estimation of heavy metal contents (ppm) in earthworms from Central Plain Zone of Punjab
Metals	Ludhiana		Malerkotla
	Jassian	Manakwal	Kupp	Bhogiwal
Arsenic	1.89 ± 0.02	2.05 ± 0.22	3.76 ± 0.02	3.39 ± 0.22
Cadmium	2.48 ± 0.01	2.89 ± 0.13	2.63 ± 0.02	2.85 ± 0.15
Chromium	16.60 ± 0.23	18.01 ± 0.89	14.13 ± 0.08	14.65 ± 0.50
Cobalt	6.40 ± 0.05	5.27 ± 0.26	6.12 ± 0.08	3.24 ± 0.34
Copper	33.25 ± 0.25	25.97 ± 1.24	32.37 ± 0.23	26.33 ± 0.25
Manganese	86.76 ± 0.97	95.43 ± 4.28	133.03 ± 0.58	97.43 ± 3.08
Lead	12.14 ± 0.31	7.02 ± 0.62	11.59 ± 0.02	8.16 ± 0.52
Zinc	94.38 ± 0.22	91.77 ± 4.74	105.48 ± 0.49	85.05 ± 1.47
Values are mean ± S.E of three replicates

Bioaccumulation factor (BAF) of various heavy metals viz; As, Zn, Ni, Pb, Cd, Cr, Mn, and Cu in earthworm tissue as shown in Table 3, showed that the BAF was highest for Cadmium in earthworms of Bhogiwal village of Malerkotla (9.19 ± 0.55), followed by Zinc (1.43 ± 0.01 to 2.07 ± 0.12), and lowest for Chromium and Cobalt in village Bhogiwal of Malerkotla. According to Wang et al (2018), Cadmium has the largest bioaccumulation factor (BAF), which was ranked as follows: Cd (10.6–18.8) > Zn (1.15–1.75) > Cu (1.01–1.35) > Pb (0.56–0.95) (Wang et al 2018). There are two main methods that earthworm species might accumulate metals: either by ingesting organic debris and adsorption through the gut tissues, or by absorption following skin contact (Lannan et al, 2004).

Table 3

Bioaccumulation factor (BAF) of Heavy metal in earthworms
METALS	LUDHIANA		MALERKOTLA
	JASSIAN	MANAKWAL	KUPP	BHOGIWAL
ARSENIC	0.34 ± 0.01	0.38 ± 0.04	0.79 ± 0.03	0.70 ± 0.05
CADMIUM	3.48 ± 0.01	3.96 ± 0.18	8.54 ± 0.14	9.19 ± 0.55
CHROMIUM	0.47 ± 0.02	0.51 ± 0.05	0.55 ± 0.04	0.58 ± 0.13
COBALT	0.48 ± 0.02	0.73 ± 0.04	0.54 ± 0.01	0.43 ± 0.02
COPPER	1.30 ± 0.07	1.18 ± 0.05	1.65 ± 0.01	1.33 ± 0.02
MANGANESE	0.36 ± 0.01	0.38 ± 0.02	0.55 ± 0.01	0.36 ± 0.02
LEAD	0.75 ± 0.09	0.44 ± 0.04	0.79 ± 0.07	0.54 ± 0.03
ZINC	1.5 ± 0.02	1.52 ± 0.02	1.80 ± 0.01	1.73 ± 0.03
Values are mean ± S.E of three replicates

The pesticide residues in soil samples and earthworm tissues and the genotoxic effect from Ludhiana and Malerkotla district is shown in Table 5. The soil sample collected from the Jassian village of Ludhiana was contaminated with pesticide chlorpyrifos and the soil sample collected from Bhogiwal village of Malerkotla district was contaminated with chlorpyrifos and Tebuconazole. No pesticide residues were found above limit of quantification (0.05 mg kg^− 1) in earthworm sample of wheat fields from all the locations. Comparable results were found in a study done in soil samples from paddy, wheat and sugarcane fields around Hisar, Haryana, India which detected organophosphates like chlorpyriphos (0.002–0.172 ug/g) and malathion (0.002–0.008 ug/g); organochlorines like DDT, HCH, endosulfan, and chlordane, and synthetic pyrethroids like cypermethrin and fenvalerate (Kumari et al, 2008).

Comet formation due to damaged DNA, as shown in Table 4, was found in earthworms collected from wheat field of village Jassian of Ludhiana in which heavy metals Cd, Co, Cu, Zn, Pb were detected above the permissible limit and pesticide residue of chloropyrifos was detected but below the limit of quantification (LOQ). The average tail length of 13.53 µM and per cent tail DNA 21.04 was observed. In the earthworm samples of village Bhogiwal of Malerkotla, pesticide residue of chloropyrifos and tebuconazole were detected below the limit of quantification (LOQ) and two heavy metals Cd, Pb were detected above permissible limit. The average tail length of 11.81 µM and per cent tail DNA 18.04 was observed.

Table 4

DNA damage observed in coelomocytes of earthworms from central zone of Punjab
District	Village	Pesticide detected in soil	Pesticide detected in earthworm	Heavy metals detected in soil above PL Agarwal 2009	Average tail length µM	per cent tail DNA
Ludhiana	Jassian	Chloropyrifos	ND	Cd, Co, Cu, Zn, Pb	13.53	21.04%
Ludhiana	Manakwal	ND	ND	Cd, Cu, Zn, Pb	-	-
Malerkota	Kupp	ND	ND	Cd, Co, Zn, Pb	-	-
Malerkota	Bhogiwal	Chloropyrifos Tebuconazole	ND	Cd, Pb	11.81	18.04%
Values are mean ± S.E of three replicates
PL- Permissible Limit

Heavy metals also have deleterious impact on earthworms at all levels of organization, including inhibiting enzyme activities, causing DNA damage, reducing survival, growth and cocoon production, altering behaviour and reducing total earthworm community diversity and biomass (Yadav et al, 2023). A study conducted on Pheretima peguana earthworms showed, 8% of tail DNA and tail length of 20 um was observed at 13.66 mg/kg Pb concentration while 11% of tail DNA and 30 um of tail length was detected at 0.75 mg/kg Cd concentration (Muangphra and Gooneratne, 2011).

The observations made from the present study indicate the presence of pesticides and heavy metals in central and undulating plain zones of Punjab directly or indirectly leading to genotoxic effects on earthworms. Our results further provide insights to the variations in the bioaccumulation of different heavy metals by earthworms which can be further studied for differential risk assessment in heavy metal contaminated soils. Therefore, the pesticide and heavy metal contamination must be monitored and mitigated in order to preserve biodiversity.

Author Contribution

Devika did the research part Ragini Sharma , compiled the data and wrote manuscriptRandeep Kaur Aulakh helped in writing manuscriptNeeraj Rani provided research facilities

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No competing interests reported.

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Analysis of pesticide and heavy metal bioaccumulation in earthworms from Central plain zone of Punjab

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Abstract

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Conclusion

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