Soil Toxicity Assessment in Home Gardens within Agro- Ecosystems of an Industrial Zone (Eloor, Kerala, India): Comparative Analysis of Spiked and Field-Contaminated Soils using the Bioindicator Eisenia andrei

doi:10.21203/rs.3.rs-4878029/v1

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Soil Toxicity Assessment in Home Gardens within Agro- Ecosystems of an Industrial Zone (Eloor, Kerala, India): Comparative Analysis of Spiked and Field-Contaminated Soils using the Bioindicator Eisenia andrei

https://doi.org/10.21203/rs.3.rs-4878029/v1

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The toxicity studies using soil invertebrates are good tools for assessing the soil quality of a region. In this study the toxicity assessment of industrial soils using Eisenia andrei was done using field soils and artificial soils spiked with zinc (Zn) and cadmium (Cd). Considering field soils, the soils near the industrial region showed higher toxicity than those away from the region and in spiked soils, the toxicity followed the order Zn < Cd < Zn + Cd. The Mixed Toxicity Index (MTI) revealed an antagonistic response between Zn and Cd (MTI < 0) which points out the influence of one element in reducing the toxicity of the other. The biomass showed the highest reduction in zone 1 soils and Zn + Cd treated soils. A similar trend was noted for the hatchability of cocoons. The bioconcentration studies showed that bioaccumulation has not occurred in field soils and spiked soils. However, despite the bioaccumulation, there were certain responses at short-term (acute) and long-term (chronic) treatments. The study points out the single and combined impact of soil toxicants in the home garden soils on the overall health and survival of invertebrates and also the possibility of reducing those effects using various nutrients in the soil ecosystems.

acute toxicity

bioaccumulation

chronic toxicity

Eisenia andrei

home gardens

Soil pollution is a major issue affecting soil functions and ultimately global soil health. Soil pollutants, which accumulate in the soil and change soil equilibrium (Bailey, 1997) have been found to affect soil organisms, due to the continuous interaction between soil organisms and the non-living soil components (Bailey, 1997; Tresch et al., 2019). Hence assessing the toxicity of various natural and anthropogenic soil components on soil organisms is an inevitable part of ecosystem research as this will help to analyse the negative effect of soil pollutants on various organisms, ultimately helping in the effective management of soil ecosystems.

Home garden agroecosystems are related to human survival, through the provision of food, fodder and economically valuable components like spices and condiments. Home gardens are generally considered sustainable ecosystems (Galhena et al., 2013; Santos et al., 2022; Torquebiau, 1992). However many toxicants found in the home garden agroecosystems like increased amounts of nutrients, pesticides, and other chemicals used as agricultural inputs like fertilisers reduce soil health in the home gardens. In industrial areas, trace elements, petroleum products, recalcitrant chemicals, and radionuclides may also be present in home gardens which in turn can hurt the local soil faunal population, and the effects will be visible in the form of reduced faunal diversity/ abundance and floral health. As humans depend on the home gardens for food to some extent, the effects of these chemicals especially those with a bioaccumulative nature will impact human health through alteration of biological and physiological functions of the body. To ascertain these negative impacts and find viable solutions, experimental studies using model species have become a necessity.

The toxicity of soils on invertebrates can be assessed using single-species and multiple-species assessments. Multiple species assessments indeed give a complete and exact picture of soil toxicity but single toxicity tests are used to get the correct experimental results. For soil pollution-related studies, a relative ranking of contaminated sites, information on the affected groups of species, and identification of the most hazardous compounds are important to making proper management decisions ( De Zwart & Posthuma, 2005; Angerer et al., 2011;). Chemical analysis can indeed give information related to soil composition but the response of soil organisms to changes in soils gives information regarding soil function and soil quality changes. Hence the role of invertebrates in assessing soil pollution has gained importance for soil quality-related research. Earthworms, the invertebrates used as bioindicators of soil toxicity are considered to be models of soil pollution. These organisms are small in size and have some unique features which make them good tools for soil toxicity studies which include their close association with soil habitat, cutaneous respiration, intake of soil particles and contribution to biogeochemical cycles. Among earthworm species, the model earthworm species Eisenia andrei is used as one of the recommended species to study soil pollution by OECD. In this research, we tried to study the toxicity effects of home garden soils in an industrial area Eloor to Eisenia andrei.

Several studies have been conducted using earthworms to study the effects of various toxicants on organisms. These include studies on avoidance behaviour, physiological changes, growth, reproduction etc. Studies on acute toxicity have been conducted using different chemicals (Lukkari et al., 2005; Hackenberger et al., 2012; Liu et al., 2024; Zhang et al., 2014; Tang et al., 2024; Zhu et al., 2020). The results of these tests gave variable results based on the nature of the chemical and its concentration. In addition, many tests have also been conducted with more than one chemical (Khalil et al., 1996; Duque et al., 2023) which reflects the combined effects of toxicants and their bioaccumulation ability in the organisms. However such studies are very rare, especially in home garden agroecosystems. Home garden soils which are usually considered healthy ecosystems are also facing various forms of stress in the form of soil pollution and nutrient imbalance. Our study aims to highlight this fact and the corresponding impacts on the soil invertebrate fauna as a whole. We hypothesised that the toxicity of home garden soils can have direct effects on the growth, reproduction and survival of Eisenia andrei at the organism level and the varied responses are visibly seen at the whole organism level in terms of acute and chronic toxicity, reproduction and bioaccumulation.

2.1. The study area

The study was conducted in Eloor region (10^o4’N-76^o18’E), Kerala, India- a low-lying area (11.21 sq km) located within proximity of the river Periyar. The region has several industries of various kinds (chemical industries, electroplating industries) representing 14% of the total area and is reported to be one of the major toxic hotspots in India (Kumar et al., 2010). Eloor is a low-lying area encircled by river Periyar and located nearly 3 m above mean sea level. The region has a tropical climate with two monsoon seasons, South West Monsoon (June to September), North East monsoon (October to December), and summer (January to May). The soils are sandy loam classified as Entisols (USDA, 1975). Several research works have been done in Eloor region for studying the pollution (Sobha and Anish., 2003; Umadevi et al., 2010; Anjaly et al., 2023). The study was divided into many stages namely collection of soil samples, study of nutrients and trace elements in the soils in the home gardens, toxicity assays of soils using Eisenia andrei, reproductive success, bioaccumulation studies and estimation of toxic responses in the organism. The statistical analysis was done using SPSS version 27.

2.2. Collection of soil samples from the home gardens

The soil samples were collected from three home gardens in the Eloor region. For the collection of soil samples, soil cores of 10 × 10 × 10 cm were made and the samples were collected in triplicates. The bigger particles and plant remnants were removed and the samples were air-dried, sieved through a 2 mm sieve and transported to the laboratory. Among the three home gardens, the first one was in proximity to the industrial centre, the second 3 km West of the industrial region and the third 3.5 km distance from the industrial centre. The soil samples collected from three zones of the home gardens were marked as zone 1, zone 2 and zone 3 respectively for use in toxicity tests (Fig. 1).

2.3. Analysis of nutrients and trace elements in the soil

The nutrients (available nitrogen, phosphorus, potassium, calcium, magnesium, sulphur, iron, copper, zinc, and manganese) and trace elements (lead, cadmium, chromium) in the soils were estimated for the home gardens. The nitrogen was quantified using the Kjeldahl method (Subbiah and Asija (1956), phosphorus using ascorbic method (Watanabe et al., 1965), potassium using a flame photometer (Jackson, 1958). Sulphur was also estimated (DAC, 2011). For the estimation of zinc, copper, iron, and manganese, the soil samples were extracted using 0.005 M DTPA extractant and estimated using an atomic absorption spectrophotometer (AAS, Varian AA 240) ( (Lindsay & Norvell, 1978). For estimating calcium, ammonium acetate extraction followed by EDTA titration was done (Nathan et al., 2012) and magnesium values were calculated from the calcium results. The trace elements lead, cadmium, and chromium were estimated following digestion of the samples in aqua regia followed by quantification using AAS. The soil samples were air-dried, sieved using a 2 mm sieve and ground using a mortar and pestle. One gram of the soil sample was digested in 30 ml aqua regia, evaporated, residue dissolved in distilled water, filtered using Whatman 42 filter paper and made up to 100 ml with distilled water. The analysis of trace elements in the solution was done using an Atomic Absorption Spectrometer (AAS, Varian AA 240), (Davies et al., 1999).

2.4. Soil toxicity assays

Soil toxicity tests were done to estimate the degree of toxicity of the soils to test organisms. The test soils were mixed with control soil in varying concentrations and the organisms were introduced into the test containers. The control used was modified artificial soil in which sphagnum peat was substituted with cocopeat (Shanmugasundaram et al., 2014). The elements which were present in the highest concentrations in the home gardens (zn, Cd) and their combination (zn + Cd) were used for the tests. The tests were done with both field soils and spiked soils. The organisms were first subjected to avoidance tests followed by acute toxicity and chronic toxicity tests. The reproductive success and bioaccumulation studies were also done. The reproduction was quantified in terms of the number of juveniles produced from cocoons and the hatchability of cocoons produced by the earthworms introduced into various concentrations of soils. For the estimation of reproduction, the organisms were introduced to test soils for 28 days and accumulation of elements in the organisms was quantified. The bioconcentration factor was determined to confirm whether the elements were accumulated in the body of the organisms.

2.4.1. Avoidance tests

A short-term behavioural test called "soil avoidance" is used to determine the preference of a particular soil medium over another based on the test organism's avoidance response (Hund-Rinke & Wiechering, 2001). Here, a two-chamber avoidance test was employed, with 750 g test soils from zones 1, 2, and 3 mixed in different proportions with control soils on one side and 750 g modified artificial soil on the other side (the control chamber)(De Silva and van Gestel, 2009; Shanmugasundaram et al., 2014) of a container with dimensions 170 mm x 120 mm x 60 mm. Ten fully grown earthworms were added to each container by setting them on the middle region of the two soil samples. To stop the worms from escaping the containers and to ensure continuous aeration, perforated plastic sheets were placed over the test trays. Following a 48-h test period, the percentage of avoidance was computed by counting the worms in the two chambers on either side of the test and control vessels (Hund-Rinke & Wiechering, 2001). Control soil was added to the soils in zones 1 and 2 in proportions of 10–70% and 25–75%, respectively, based on the avoidance results. The degree of avoidance for field soils at various proportions was ascertained by repeating the experiment in triplicates. To evaluate a single pollutant, single compounds of Zn and Cd were introduced into the soil as ZnCl₂ (Catalogue no 429430, Merck, 95%)and CdCl₂ (Catalogue no 529575, Merck, 99.99%) respectively (Oorts et al., 2021). A range of concentrations of Zn and Cd alone, as well as various combinations of a mixture of Zn and Cd, were used in the tests. The highest Zn and Cd concentrations found in Eloor soils (zone 1, zone 2) were used to spike soils for the initial tests and further, the concentrations were lowered by the earthworms' avoidance response. For the range finding test, the Zn test levels ranged from 250 µg/g to 2000 µg/g, and for the confirmation test, they were 62.5 µg/g to 1000 µg/g. For the range finding test, the Cd test levels ranged from 250 µg/g to 1000 µg/g, and for the confirmation test, the concentrations were from 125 µg/g to 500 µg/g. The avoidance test was done following 48 hours of exposure and the percentage avoidance was calculated.

2.4.2. Acute toxicity tests

E. andrei was used in the acute toxicity test in accordance with OECD guidelines (OECD, 1984). Twelve square transparent plastic trays (170 mm x 120 mm x 60 mm) were covered with black paper and divided into four sets of three in which the first set of three (control) received artificial soil (750 g), while the other three sets received equal amounts (750 g each) of soil from Eloor zones 1, 2, and 3. After being acclimatised for a full day, ten earthworms were cleaned, dried on filter paper, and placed on the test medium's surface in each test tray. The containers were covered with perforated plastic film, the water content was adjusted to 35% of the dry soil weight, and were maintained in a continuous light source of 400–800 lux.

Earthworms were fed autoclaved powdered sieved dry cow dung (2 mm pore size) during the test period and the percentage of mortality in the test and control samples was determined after 14 days (Finney, 1952). As for the avoidance test, the artificial soil spiked at various concentrations of each metal, both separately and in combination, and the toxicity of Zn and Cd on E. andrei was evaluated. The worm mortality was tallied, and LC₅₀ values were computed for each sample. By dividing the LC₅₀ concentration of each element in the mixture by its LC₅₀ values and adding the two values, the combined toxicity of zinc and cadmium was determined (Qiu et al., 2011). The Toxic Unit (TU) (Renaud et al., 2021) was computed as

$$\:\text{T}\text{U}\:\left(\text{M}\right)\:=\:\frac{\text{L}\text{C}\:\:50\:\text{Z}\text{n}\:\text{i}\text{n}\:\text{t}\text{h}\text{e}\:\text{m}\text{i}\text{x}\text{t}\text{u}\text{r}\text{e}\:\:}{\:\text{L}\text{C}\:50\:\text{Z}\text{n}\:\text{i}\text{n}\:\text{i}\text{n}\text{d}\text{i}\text{v}\text{i}\text{d}\text{u}\text{a}\text{l}\:\text{t}\text{e}\text{s}\text{t}\:\:\:}\:+\:\frac{\text{L}\text{C}\:50\:\:\text{C}\text{d}\:\text{i}\text{n}\:\text{t}\text{h}\text{e}\:\text{m}\text{i}\text{x}\text{t}\text{u}\text{r}\text{e}\:\:\:}{\text{L}\text{C}\:50\:\text{C}\text{d}\:\text{i}\text{n}\:\text{i}\text{n}\text{d}\text{i}\text{v}\text{i}\text{d}\text{u}\text{a}\text{l}\:\text{t}\text{e}\text{s}\text{t}\:\:\:}$$

The maximum toxic unit (M₀) is estimated as the maximum toxic unit of the mixture. From M₀ and M, the Mixture Toxic Index (MTI) was estimated as:

Mixture toxic index (MTI) = 1- (Log M/Log M₀)

The interaction between the test elements was categorised as antagonistic (MTI < 0), synergistic (MTI > 1 ) or additive (0 < MTI ≤ 1) (Zeb et al., 2017).

2.4.3. Chronic toxicity tests

Chronic toxicity experiments were conducted to calculate long-term toxicity effects (Greig-Smith, 1992; Robidoux et al., 2000; Zhou et al., 2011) and to detect physiological abnormalities at sublethal dosages comparable to those in the field. Based on the acute toxicity test results, a chronic toxicity assay was conducted at sublethal levels in field soils and soils modified with Zn and Cd. After acclimatising in artificial soil for 24 h, ten mature clitellate earthworms were removed, cleaned, weighed, and depurated on filter paper before placing them back into the test trays. The test containers were weighed, covered with perforated plastic coverings, maintained under light and moistened once a week. The earthworms were fed with autoclaved sieved, powdered dry cow dung (2 mm sieve) during the test period. The worms were removed after 28 days and their biomass, reproduction and bioaccumulation were evaluated. Additionally, a list of the behavioural responses was noted. The alterations in colour, aggregation tendency, constriction of the body, excessive secretion of mucus, and formation of casts were noted. The number of organisms displaying the responses was counted and expressed as percentage. The cast production was estimated in terms of weight/worm/day.

2.4.4. Estimation of biomass

Estimation of an organism's biomass is a good method to determine its health status. In a healthy organism, biomass tends to rise as it grows from a juvenile to an adult as long as it is not under stress. Stress can lead to a reduction in biomass during this growth stage and any alterations in the biomass can point to the altered health status of the organism. The wet weight of the organisms at the beginning of the experiment and after the 28-day experimentation period was used to calculate the biomass of the adult organisms. The biomass difference was expressed as a percentage increase or decrease (Lukkari et al., 2005).

2.4.5. Reproduction tests

The juvenile and adult earthworms were taken out of the test trays after 28 days of exposure, divided and tallied. The cocoons were removed and put in a 1 mm sieve after going through a 2 mm sieve. After cleaning the soil and gathering the cocoons, the unhatched cocoons were put into Petri plates lined with wet filter paper, counted, covered with perforated plastic films, and kept in the dark at 20^o C. After several (28) days, the number of juveniles emerged was counted and hatchability percentage was calculated by counting the number of juveniles out of the total number of cocoons.

2.4.6. Bioaccumulation tests

One of the most important and long-lasting effects of toxicity on soil organisms is the bioaccumulation of toxicants. Toxins in soil can enter an organism's body through a variety of pathways, including the mouth, respiratory system, or skin. Depending on the nature of the chemical, the bioavailable forms of the toxicant may be metabolised, removed by excretion, or a portion may be retained inside the organism. To estimate the bioaccumulation in the test organism the test soils and the earthworms from the corresponding trays were removed individually after 28 day experiment. The samples were weighed, dried for 24 h at 105^o C in an oven (MHOA1, Labline instruments), and digested with 70% nitric acid. Atomic Absorption Spectroscopy (AAS) was used to measure Zn and Cd after diluting the digestate with deionized water. The bioaccumulation factor was estimated by dividing the amount of trace element in the worm by the amount of trace element in the soil.

3.1 Trace elements in the soil

The nutrients and trace elements in the soils were estimated for the home gardens in the three zones (Table 1) and compared with the standard values.

Table 1

Soil nutrients and trace elements in different home garden soils in Eloor, Kerala, India
Zone	Trace elements	Desirable limits	Range	Mean ± SD
Z 1	N	> 280 kg/ha	83.05-408.66	217.21 ± 82.84
	P	> 10 kg/ha	18.43-251.69	94.4 ± 49.25
	K	> 108 kg/ha	45.17-1205.39	159.07 ± 215.41
	Ca	> 30 mg/kg	982.30-3228.85	2354.51 ± 489.62
	Mg	> 120 mg/kg	28.72-1491.57	312.65 ± 329.95
	S	8–30 mg/kg	23.35-112.72	62.27 ± 27.05
	Fe	> 5 mg/kg	52.29–86.88	74.68 ± 10.49
	Cu	> 1 mg/kg	0.20–0.81	0.5 ± 0.16
	Zn	< 40 mg/kg	7.91-135.67	43.76 ± 26.88
	Mn	> 1 mg/kg	15.43–60.14	38.43 ± 10.89
	Pb	< 70	12.34–64.81	37.84 ± 14.57
	Cd	< 1.4	105.22–503.4	183.07 ± 141.13
	Cr	< 64	0.70–8.21	5.87 ± 3.51
Z2	N	> 280 kg/ha	51.02-397.68	213.68 ± 84.56
	P	> 10 kg/ha	24.59-354.45	179.07 ± 76.16
	K	> 108 kg/ha	94.03-1480.55	226.59 ± 241.67
	Ca	> 30 mg/kg	1122.04-3298.84	1882.21 ± 595.59
	Mg	> 120 mg/kg	75.62-767.92	286.53 ± 207.38
	S	8–30 mg/kg	11.01–69.36	31.01 ± 10.51
	Fe	> 5 mg/kg	37.45–62.84	46.46 ± 6.7
	Cu	> 1 mg/kg	0.25–0.91	0.51 ± 0.16
	Zn	< 40 mg/kg	17.64-123.55	58.92 ± 28.81
	Mn	> 1 mg/kg	29.62-124.32	79.2 ± 22.98
	Pb	< 70 mg/kg	11.28–27.34	17.56 ± 3.77
	Cd	< 1.4 mg/kg	11.81–207.44	74.32 ± 58.49
	Cr	< 64 mg/kg	BDL	BDL
Z3	N	> 280 kg/ha	216.98-521.35	334.48 ± 65.86
	P	> 10 kg/ha	55.06-304.46	123.1 ± 77.13
	K	> 108 kg/ha	46.95-308.04	169.99 ± 61.57
	Ca	> 30 mg/kg	1367.22-3476.94	2472.87 ± 614.82
	Mg	> 120 mg/kg	62.43-246.46	161.45 ± 42.13
	S	8–30 mg/kg	11.01–42.81	27.52 ± 6.66
	Fe	> 5 mg/kg	52.29–86.88	60.04 ± 10.17
	Cu	> 1 mg/kg	0.28–0.96	0.55 ± 0.17
	Zn	< 40 mg/kg	39.41–73.72	58.16 ± 9.71
	Mn	> 1 mg/kg	30.85–97.84	64 ± 19.31
	Pb	< 70 mg/kg	4.24–20.53	13.28 ± 3.8
	Cd	< 1.4 mg/kg	1.06–4.99	2.49 ± 0.74
	Cr	< 64 mg/kg	BDL	BDL

3.2. Soil toxicity

The soil toxicity to earthworms were determined with the help of results obtained from earthworm’s avoidance behaviour, acute toxicity, chronic toxicity, biomass estimation, reproduction tests and estimation of bioconcentration factor .

3.2.1 Avoidance tests

The avoidance response was quantified for the earthworms exposed to field soils and spiked soils (Fig. 2). In field soils, avoidance response above 80% denotes poor habitat suitability (Hund Rinke et al., 2003), zone 1 soils are not ecologically suitable, zone 2 is partially suitable, and zone 3 soils are ecologically suitable for the survival of the test organisms. In spiked soils, the organism showed 100% avoidance at 1000 µg/g Cd and above. The 80% avoidance at 500µg/g was reduced to 13.33% at 125 µg/g. In spiked soils with the highest concentrations of Zn and Cd (1500 µg/g Zn + 1000 µg/g Cd) the avoidance was 100%. The percent avoidance reduced to 80% at 750 Zn + 500 Cd, and further to 33% at 375 Zn + 250 Cd. The acute toxicity tests were done using natural soils and spiked soils and the mortality exhibited after 14 days by the earthworms was considered the endpoint of the test (Fig. 3).

3.2.2 Acute toxicity tests

In zone 1 soils, the organisms showed 100% mortality in 75% soil mixture. The mortality was further reduced and was similar to control only at 2.34%. The LC₅₀ was at 18.36% soil. At this test level, Zn content was 342.75 µg/g and Cd was 110.78 µg/g. The soil from zone 2 showed 73.33% mortality in 100% soil. The mortality was further reduced and was similar to control at 6.25%. The LC₅₀ was at 60.72% of soil and at this test level Zn content was 459.09 µg/g and Cd was 125.96 µg/g.

The soil spiked with Zn alone had 100% mortality at ≥ 1500 µg/g of Zn. The LC₅₀ was at 656.55 µg/g of Zn. The soil spiked with Cd alone had 100% mortality at ≥ 1000 µg/g with LC₅₀ at 107.65 µg/g of Cd. The test soils spiked with Zn + Cd mixture exhibited 100% mortality at Zn and Cd concentrations of 1500 µg/g and 1000 µg/g respectively. The lowest mortality was observed at 23.4 µg/g Zn and 15.6 µg/g Cd where the mortality was 16.67%. No mortality was noticed in the mixture with 11.7 µg/g Zn and 7.8 µg/g Cd. The LC 50 for Zn and Cd respectively in Zn-Cd mixture were 199.13 µg/g and 132.75 µg/g. The combined toxicity of Zn and Cd expressed in toxic units was 1.30 and 1.23 respectively with the sum of toxic units (M) 1.53 and M0 value 1.23. The Mixture Toxicity (MTI) index gave a negative value (-1.05) and hence the combined effect of Zn and Cd was antagonistic (Fig. 3).

The worms exposed to soils in zones 1 and 2 did not exhibit any aggregation behaviour, whereas the worms' behaviour in zone 3 did not change. In zone 1, 100% of the worms in 4.95–37.6% of the amended field soils showed colour change; in zones 2 and 3, however, no colour change was detected. Every worm in zone 1 at amended field soils from 8–37.5% of field soils, secreted excessive amounts of mucus. Mucus secretion was not present in soils in zone 2 or zone 3. Earthworms in zone 1 soils had constrictions in 4.95–37.6% of field soils that had been amended. No constrictions were observed in zones 2 or 3. In soils containing Zn spikes, all worms at 750 µg/g and 1500 µg/g exhibited colour changes and constrictions. In 187.5 µg/g of Zn, 100% of the worms showed loss of aggregation. All the worms exhibited colour changes and constrictions when exposed to cadmium levels ranging from 125 µg/g to 500 µg/g of soil (Fig. 4). All the worms showed a loss of aggregation at higher concentrations of Cd (62.5 µg/g). A colour change was observed in 100% of the worms at higher concentrations in soils that had been spiked with combinations of Zn and Cd. Loss of aggregation was present in all concentrations in 100% of the worms. The two main behavioural responses observed in all the soils were the absence of aggregation and the presence of constrictions.

3.2.3. Chronic toxicity tests

In zone 1 the cast production reduced with an increase in test soil concentration (Fig. 4). In test soils of zone 1, the highest amount of cast was produced at 2.34% (0.57) and the lowest at 37.5% (0.45). Compared to the control, the amount of cast was reduced to less than half at the highest concentration. The ANOVA results showed significant differences in the cast production of earthworms treated in zone 1 soils in Eloor region (P < 0.05). The cast production in soils of zone 1 and 2 was less compared to respective controls (Fig. 5). The highest reduction was observed in Zone 1 which may be due to the increased toxicity of soils as observed from acute toxicity tests resulting in increased stress on the organism. In Zone 2 the reduction in cast production was less compared to zone 1 soils. In zone 3 cast production was the same as control probably due to the absence of toxicity in soils. The cast production following exposure of E. andrei to soils spiked with Zn and Cd individually and in combination also showed significant reduction.

3.2.4.Biomass

The biomass showed a reduction with exposure of E. andrei to soils from zone 1 and zone 2 (Fig. 5). The reduction in biomass was most prominent in soils of Zone 1 which may be due to increased levels of toxicants in zone 1. As the toxicity zone 2 soils was less, biomass was not much affected; but there was a concentration-dependent reduction. In zone 3 there was an increase in biomass irrespective of control and no significant difference between control and treatments. Hence it is reasoned that there is no toxicity in the soils of zone 3. In spiked soils, the reduction of biomass was more in Cd spiked soils than Zn spiked soils. In combined mixture also the biomass was very much reduced. The lowest reduction in biomass at low concentrations can be due to the stress in the organism developed due to soil toxicity (Maleri et al., 2007). Studies have shown a correlation with stress-induced reduction in reproductive success and biomass of organisms (Sivakumar, 2015) and the same response is observed in this study (Fig. 6).

3.2.5. Reproduction tests

In Zone 1 the number of cocoons and hatchability decreased with the proportion of field soil (Fig. 7). The hatchability was less than 50% at 37.5% of field soil. In Zone 2 the cocoons had 60.74% hatchability at 50% of field soil. In Zone 3 even in non-amended soil the number of cocoons and their hatchability were similar to control. The ANOVA results showed that there is a significant difference in hatchability between various proportions of field soils (P < 0.05) of zone 1. The number of cocoons and hatchability decreased with the addition of Zn and Cd alone and in combination depending on the metal concentration. The hatchability of cocoons following the addition of Zn reduced to 83% compared to 97% in control. Cd reduced the hatchability to 34% at 500 µg/g. In combination the presence of these metals reduced the hatchability to 24% at a concentration of 750 µg/g Zn + 500 µg/g Cd.

3.2.6. Bioconcentration factor (BCF)

The bioconcentration factor for Zn in zone 1 ranged from 0.21 to 0.31 and that of Cd from 0.18 to 0.8. The bioconcentration factor for Zn in zone 2 ranged from 0.17 to 0.29 and that of Cd from 0.14 to 0.2 (Fig. 8). The ANOVA results showed that the mean difference among various proportions of field soils was significant (P < 0.05). When the control soil was spiked with Zn in the range of 23 µg/g to 750 µg/g the bioconcentration factor ranged from 0.33 to 0.55. The BCF increased from the lower to the higher concentration. A similar trend was observed on exposure of the organism to Cd spiked soil with the range of BCF from 0.24 to 0.66.

The industrial area of Eloor is regarded as a toxic hotspot. Numerous studies have already looked into the pollution of the Periyar River around Eloor (Mohapatra et al., 2011; Charuvilayil, 2013; Jyothi et al., 2021; Dey and Majumdar, 2024; Priya and Varghese., 2024), and the findings from the soil studies also indicate that the region's soils vary in their degree of pollution, which has an impact on the invertebrate fauna.

4.1. Soil toxicity

Lead was found in all three zones of the trace element analysis of the home gardens within permissible limits, suggesting that Lead (Pb) may not be a major trace metal causing soil pollution in the area. However, high Cd concentration was noted in each of the three zones. This confirms the findings of the study (Thomas et al., 2006) that industrial activity had caused elevated levels of in the groundwater of the Eloor region. Since Cd is a trace element that is not necessary for life, its presence in the three zones may increase the risk of both direct and indirect toxicity to higher organisms as well as soil fauna. For example, cattle in Eloor region have demonstrated effects on their red blood cells due to the presence of Cd (Nisha et al., 2009; Varghese et al., 2012) reported that Pseudomonas bacteria in the Eloor industrial region were also found to be bioaccumulating Cd. Chromium (Cr) was also found below permissible limits in Eloor soils. zone 1's proximity to industries may also contribute to the area's Cr content. Additionally, the results for Cr concentrations below the detection limit in zones 2 and 3 showed that there is no Cr pollution in these zones' home gardens.

3.1.1. Avoidance tests

Clear findings were obtained from the toxicity assessment of field soils and spiked soils towards E. andrei. The greater avoidance response observed in zone 1 field soils compared to zones 2 and 3 indicates that, in reaction to soil elements, the organisms in zone 1 were under more stress than those in zones 2 and 3. zone 2 soil’s avoidance response was lower than Zone 1, but Zone 3 soils showed no avoidance response, indicating that zone 2 soils contained relatively few unfavourable components, whereas zone 3 soils contained either none or very few components that could cause stress to soil invertebrates. When it came to spiked soils, Cd was avoided by the earthworms more than Zn. At a concentration of 250 µg/g of test soil, there was a 53.33 per cent avoidance of Cd; in contrast, at the same test dose of zinc, the avoidance percentage was 26.67%. At 33.33% of soils, the avoidance rate was achieved by applying 250 µg/g of Cd and 375 µg/g of Zn together. This demonstrates that when Zn is present, the effects of Cd are reduced. According to research, various invertebrates have been observed to exhibit avoidance behaviour towards Zn and Cd (Morley et al., 2001). This avoidance behaviour can serve as a sensitive test for assessing the toxicity of trace elements to invertebrates found in soil (Amorim et al., 2008). Studies have shown that particular traits in chemoreceptors can affect earthworm avoidance behaviour (Edwards & Bohlen,1996; Stephenson et al., 1998). In a study that used E.fetida, the avoidance response to Zn might have been caused by the binding of free Zn to the chemoreceptor membranes of the earthworms(Ma & Bonten, 2011). It is known that the field soils include additional trace elements in addition to zinc and cadmium. The varied response levels in the various types of soils investigated in the study may be caused by species with different ecological characteristics (Lee, 1985) responding to contaminated soils in different ways (Morgan and Morgan, 1993; Morgan and Morgan, 1999). Additionally, it has been noted that the avoidance response and habitat suitability may be connected. Therefore, the reported > 80% avoidance for soils collected from zone 1 suggests that these soils may not be much suitable to the invertebrates to survive (Hund-Rinke & Wiechering, 2001).

4.1.2 Acute toxicity tests

According to the acute toxicity studies, E. andrei was not found in zone 1 soil, and its mortality rate was 73.33% in zone 2. Zone 3 soils did not exhibit any mortality, indicating that these soils were not lethal to invertebrates. Additionally, zone 1 soil’s LC₅₀ was lower than zone 2. The findings unequivocally show that zone 1 soils were more toxic to living things. The fact that zone 1 is closer to industries than the other two zones lends credence to this. The Cd-spiked soils had low LC₅₀ value compared to Zn which shows that Cd is more toxic than Zn in E. andrei. Even though Zn is an essential element to organisms, an excess level of Zn can cause toxicity and this may result in mortality of organisms at higher concentrations of Zn (Li et al., 2011; Qiu et al., 2011). Cd is found to displace many elements in the organism’s body resulting in toxicity. It is also seen in studies that increased Zn levels can cause a reduction in Cd sorption in organisms (Brzoska and Moniuszko-Jakoniuk, 2001). In soils spiked with Zn and Cd in combination LC₅₀ was attained at a lower concentration of Zn, but at a higher concentration of Cd compared to the LC₅₀ of individual assays. This implies that when applied in combination with Cd, the toxicity of Zn has increased. The higher LC₅₀ level of Cd in the mixture compared to the individual test shows a reduction in toxicity in the mixture. The antagonistic interaction between Zn and Cd is supported by the studies of Brzoska and Moniuszko-Jakoniuk (2001) in earthworms. The different levels of LC₅₀ may be also associated with the differential uptake of Zn and Cd by earthworms. For example, it is understood that cadmium uptake occurs through Calcium channels and Zn uptake through carrier-mediated proteins (Li et al., 2010). Authors also suggest that the internal pathways of these metals may be different (Qiu et al., 2011), and very complex and more research is needed to study them.

4.1.3. Chronic toxicity tests

The behavioural changes varied in different soils used in the study. The organisms exposed to zone 1 soils showed prominent changes in their normal morphology and behaviour. These changes included the presence of constrictions, colour change and loss of aggregation. The loss of mucus usually occurs when an animal comes into contact with any stressor/ toxicant and the same response is noted in the study. Curling and body constrictions were also recorded in certain studies (Sivakumar, 2015). There is ample evidence based on these studies that these responses can be produced as a result of stress from trace elements and that these responses are concentration-dependent. Hence the findings in this study on behavioral responses prove that trace elements do have observable impacts on the physiology/ morphology of earthworms.

The cast production in soils of zone 1 and 2 was less compared to respective controls. The highest reduction observed in zone 1 may be due to the increased toxicity of soils as observed from acute toxicity tests resulting in increased stress on the organism. In zone 2 the reduction in cast production was less compared to zone 1 soils. In zone 3 cast production was the same as control probably due to the absence of toxicity in soils. The cast production following exposure of E. andrei to soils spiked with Zn and Cd individually and in combination also showed significant reduction. Studies show that the earthworms treated in trace element-containing soils produce casts containing a part of the trace elements, as the trace elements in soils are ingested by the organism (Lee et al., 2011; Sizmur & Richardson, 2020). Thus cast production can be associated with the physiological/ digestive activity of the organism and is a part of the toxicity elimination mechanism in an organism.

4.1.4.Biomass

The biomass showed a reduction with exposure of E. andrei to soils from zone 1 and zone 2. The reduction in biomass was most prominent in soils of zone 1 which may be due to an increased level of pollution in zone 1. As the toxicity in soils of zone 2 was less, biomass was not much affected; but there was a concentration-dependent reduction. In zone 3 there was an increase in biomass irrespective of control and there was no significant difference between control and treatments. Hence it is reasoned that there is no toxicity in the soils of zone 3. In spiked soils, the reduction of biomass was more in Cd-spiked soils than in Zn-spiked soils. In the combined mixture also the biomass was very much reduced. The lowest reduction in biomass at low concentrations can be due to the stress in the organism developed due to the toxicity of soils (Maleri et al., 2007). Studies have shown a correlation between stress-induced reduction in reproductive success and biomass of organisms (Sivakumar, 2015)and the same response is seen in this study.

4.1.5. Reproduction tests

The number of cocoons and juveniles in field soils of zone 1 and zone 2 showed a reduction. The highest reduction was in zone 1 probably due to the increased toxicity and the consequent reduction in growth. This may have affected the reproductive ability of the organisms. In zone 3 soils, significant changes in cocoons and juveniles were not observed which implies that the soils did not affect the reproductive ability of the earthworms. In spiked soils, the impact of trace elements was highly evident in Cd-spiked soils than Zn-spiked soils. Compared to individually spiked soils, the reduction of cocoons and juveniles in Zn + Cd soils suggests that mixture toxicities may be more prominent than individual toxicity of trace elements. The study supports other findings ( Robidoux et al., 2000; Qiu et al., 2011; Xie et al., 2013; Kilpi-Koski et al., 2020).

4.1.6. Bioconcentration factor

The estimation of bioaccumulation in earthworms indicates that the amount of trace elements in soils was higher than the amount of trace elements in earthworms both in field soils and spiked soils. This implies that bioaccumulation has not occurred in the exposure period of 28 days. Probably 28 days is too short a period for bioconcentration to occur. However as seen from the laboratory studies, the toxicants in the soil habitat can reduce the overall health of the organisms (evident from the reduction of biomass) and the abundance of the organisms (evident from the concentration-dependent reduction in the number of juveniles and hatchability). The indirect effect of the toxicants at higher trophic levels is more prominent than the direct effect on earthworms, as it affects the ecosystem balance of the region (Sivakumar, 2015). The soils in industrial regions act as a cocktail containing different trace elements in major/ minor proportions, which may have unpredictable effects on the various life cycle parameters and mortality, thus affecting the biodiversity of the invertebrates in the soils. The reduction in invertebrates can have a corresponding effect on associated food webs and the higher trophic levels by reducing the number of vertebrates which solely depend on these invertebrates for food. In parallel, the reduction in invertebrates can affect soil properties and plant growth, as soil aeration and nutrient mixing through bioturbation is an activity which is dependent on the health of earthworms. In short, the toxicity of trace elements in home gardens can have a negative effect on the ecosystem balance of the specified area, in terms of its effects on plants and higher animals.

The study shows the impact of soil pollution on soil invertebrate fauna and brings about the difference between the toxicities of field soils and spiked soils. The acute and chronic toxicity records the highest toxicity for zone 1 soils (in the case of field soils) and Zn + Cd combination in the case of spiked soils. The results are significant because they bring out the short-term effect of soil toxicants in an industrial area through avoidance and acute toxicity tests and long-term effects through life cycle responses and physiological processes like cast production, growth and reproduction. The study is highly significant as it addresses the toxicity of industrial soils at the whole organism level and points out the use of animal models for the estimation of the toxicity of soils, and its quantification and acts as a prerequisite for the management of home garden agroecosystems in the polluted areas. Additionally, it offers the scope for managing soil toxicity using soil nutrients/ amendments.

Funding

The first author acknowledges funding from the University Grants Commission (UGC), Government of India (Grant No. F.15 − 6(DEC 2013)/2014(NET)).

Author Contribution

LG : conceptualized the work, carried out the formal analysis and investigation, writing the original draftAJ, ISB and JP: conceptualized the work, supervised all the experiments, reviewed, edited the manuscript and provided resources

Acknowledgements

The first author thanks the School of Environmental Studies, CUSAT and the National Centre for Aquatic Animal Health (NCAAH), CUSAT for providing the research facilities.

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Soil Toxicity Assessment in Home Gardens within Agro- Ecosystems of an Industrial Zone (Eloor, Kerala, India): Comparative Analysis of Spiked and Field-Contaminated Soils using the Bioindicator Eisenia andrei

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Abstract

Figures

Introduction

Materials and methods

2.1. The study area

2.2. Collection of soil samples from the home gardens

2.3. Analysis of nutrients and trace elements in the soil

2.4. Soil toxicity assays

2.4.1. Avoidance tests

2.4.2. Acute toxicity tests

2.4.3. Chronic toxicity tests

2.4.4. Estimation of biomass

2.4.5. Reproduction tests

2.4.6. Bioaccumulation tests

Results

3.1 Trace elements in the soil

3.2. Soil toxicity

3.2.1 Avoidance tests

3.2.2 Acute toxicity tests

3.2.3. Chronic toxicity tests

3.2.4.Biomass

3.2.5. Reproduction tests

3.2.6. Bioconcentration factor (BCF)

Discussion

4.1. Soil toxicity

3.1.1. Avoidance tests

4.1.2 Acute toxicity tests

4.1.3. Chronic toxicity tests

4.1.4.Biomass

4.1.5. Reproduction tests

4.1.6. Bioconcentration factor

Conclusion

Declarations

Funding

Author Contribution

Acknowledgements

References

Additional Declarations

Supplementary Files

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