Soil Invertebrates as Bio-Monitors of Toxic Metals Pollution in Impacted Soils

The bio-indicator potentials of two invertebrate species, Eudrilus eugeniae (earthworm) and Pachybolus ligulatus (millipede) in toxic metals impacted soils in Akwa Ibom State, in the Niger Delta region of Nigeria were assessed. The study involved collection of Eudrilus eugeniae and Pachybolus ligulatus, and their site soil samples from different impacted and non-impacted soils in ten locations spread over four Local Government Areas. Toxic metals (Pb, Fe, Zn, Ni and Cd) in soils and in the bio-indicator organisms were analysed using Atomic Absorption Spectrometry, after acid digestion. Geo-accumulation Index (Igeo) was used to measure the extent of soil pollution; Bio-Accumulation Factor (BAF) to compute the metal uptake from soil by bio-indicators; and multivariate statistics to identify interrelationships among metals and the pollution sources. Metals in all soils were higher than the background levels, and were classified as unpolluted to very strongly polluted. Pb, Fe, Zn and Cd were higher in Eudrilus eugeniae, and Ni in Pachybolus ligulatus at 0.05 level. The organisms absorbed all metals (BAF<1), but Eudrilus eugeniae accumulated Cd and Fe and Pachybolus ligulatus, Ni and Fe (BAF>1). Metals were clustered into three (Eudrilus eugeniae) and two (Pachybolus ligulatus) dissimilar groups. Two principal components extracted, each for Eudrilus eugeniae and Pachybolus ligulatus accounted for 64% and 72% of total variations, respectively. The invertebrates exhibited varying levels of affinity in metals uptake. Eudrilus eugeniae appeared to accumulate Pb, Fe and Cd more, and could serve as a better bio-indicator for monitoring these metals. Pachybolus ligulatus accumulated 3-folds more Ni, and could provide a monitoring tool especially in agriculture, in the Niger Delta Region of Nigeria, plagued by crude oil pollution of its land and water bodies. key words: Soil bio-indicators, Pollution, Multivariate analysis, Atomic Spectroscopy.


InTrodUCTIon
From the advent of Agricultural to the Industrial revolution, the scale of natural resource exploitation has progressively exerted pressure on the environment, and the signs of 'weakness' have become very obvious.Consequently, 'sustainable development' 1 , a global concept and panacea, is now in the front burner of all development issues.The concept recognizes the fact that human health and life are inextricably linked to a robust and healthy ecosystem-the soil being an important component.Measurement of the different soil properties or processes provides quality index-indicators that can be used to estimate soil quality; these include visual, physical, chemical and biological processes (micro and macro-organisms, their activities or functions) 2-4   .
Terrestrial arthropods (e.g. the diplopods like millipedes) and oligochaetes (e.g.earthworms) are ubiquitous and have great diversity of species.These invertebrates which dwell in the various soil layers and are in contact with the soil and/ or litters 5 , invariably, are affected by deposition of toxic chemicals like metals, organic compounds and other complex substances.Heavy metals have been found to cause reduction in the abundance of soil decomposers, and in the diversity of animal communities [6][7] .
Heavy metals deposition in terrestrial invertebrates is reported to depend on the extent of environmental pollution 5,[8][9][10][11][12] .Consequently, researchers have used various species of invertebrates including earthworms and millipedes to evaluate contaminated and uncontaminated soils 9,[13][14] .Millipedes, which belong to the class of diplopods, are particularly abundant in the tropics; they are herbivores and predominantly nocturnal.Their importance in recycling of nutrients, aeration and fertilization of soil has been well documented 14- 19 .
The use of terrestrial invertebrates as bioindicators of soil pollution has however, focused mainly on the toxic metals aspect 9,13,[18][19][20] .Hopkin 21 and Morgan and Morgan 22 reported a higher uptake of Cu, Zn and Cd by these organisms from contaminated relative to uncontaminated soils; also, olayinka et al 12 recorded higher Pb, Cu, Mn, Zn, Cd and Co around a cement factory compared with the control site.Earthworms from dump site areas in Nigeria have been shown to bio-accumulate high levels of toxic metals 11,20,13 .
Studies have also been conducted on cytotoxic effects of metals 24 ; the detoxification mechanism 25 ; trait-based approach to depict disturbance impact on soil invertebrate communities 26- 28 and ecological remediation 18,[29][30][31] .The histological and histochemical analyses as well as ultrastructural analysis, revealed that chemical substances (both organic and inorganic) are equally toxic to soil invertebrates since the mineralized bodies varied in structural organization within the individual midgut and fat body cells 12,14,17 .
Most authors have used the earthworms to determine soil health, but information on use of millipedes is scarce; and little or no information is available here in Nigeria despite the abundance of different species of millipedes.The aim of the present study was to compare patterns of metals accumulation in these two invertebrates-earthworms (Eudrilus eugeniae) and millipedes (Pachybolus ligulatus), establish which is a better indicator of soil metal pollution, determine influence of location on bio-accumulation of the toxic metals by indicator organisms, and subject the database to factor analysis to determine the possible source(s) of pollution; also, to establish the similarities and dissimilarities among locations and metals.

Sample collection and preparation of earthworms (Eudrilus eugeniae), millipedes (Pachybolus ligulatus) and soil
Soil sample from ten locations in four Local Government Areas (LGAs) in Akwa Ibom State of Nigeria were used for this study.The LGAs were Etinan (around a paint factory, locations 2 and 5), Uyo (the busy state capital, locations 7 [ravine], 6 and 10 [auto-mechanic workshops], 8 [high traffic road] and 9 [agricultural land]), Uruan (an old sea port city, locations 1 and 4) and Ibesikpo (a rural area as control, location 3) (Figure 1).Earthworms (Eudrilus eugeniae) were manually harvested (hand sorted) from soil cores of 100 cm x 100 cm dug to depth of 15 cm with a spade.Each location consisted of four composites in order to obtain enough quantity of Eudrilus eugeniae for analysis.Millipedes (Pachybolus ligulatus) were picked between the leaf litters and surface soil.Eudrilus eugeniae and Pachybolus ligulatus (Figure 2A & B) were thoroughly rinsed with de-ionised water and each composite stored in different transparent rubber bowls lined with wet Whatman No. 1 filter paper, and covered with perforated lid. on reaching the laboratory, Eudrilus eugeniae (soft-bodied) and Pachybolus ligulatus (hard-bodied) samples were kept in the stored condition for 24 hours, and at laboratory temperature (27°C-30°C) to allow for evacuation of gut content; then oven-dried at 105°C to constant weight.The corresponding soil samples were air-dried at ambient temperature.The dried samples were crushed to fine powder using mortar and pestle, and passed through 0.5 mm sieve.

determination of metals in eudrilus eugeniae and Pachybolus ligulatus
To 500 mg each of the dried and sieved samples of Eudrilus eugeniae and Pachybolus ligulatus, 5 ml concentrated HNo 3 (analar grade Aldrich) was added and kept overnight in Teflon beakers; then heated on a hot plate to near dryness.After cooling, the solution was made up to 25 ml with distilled de-ionised water.Blank solutions were also prepared (without the samples).Metal content was determined using UNICAM 939/959 Atomic Absorption Spectrometer (AAS) for Pb, Zn, Fe, Ni and Cd.

Fig. 1: Sample Locations (a) Study area (B) auto-repair workshop determination of metals in soils
Five hundred milligrammes of the soil samples were digested with mixture of 15 ml concentrated HNo 3 :5 ml concentrated HCl mixture in a Teflon beaker.After cooling, the solution was made up to 50 ml with distilled-deionized water.Blank solutions were also prepared and the metal content determined as described above.Soil pH and organic matter were determined using methods by IITA 32 .Where Cn = concentration of metal 'n' in soil; Bn = background concentration of the metal 'n' [33][34] ; 1.5 = correction factor to take care of possible variations in background values for a given environment as well as small anthropogenic influence.

Bio-accumulation Factor (BaF)
The Bio-Accumulation Factor (BAF) in earthworms and millipedes was calculated using the formula 36

Metals accumulation in potential bio-indicators of soil pollution at different locations
Mean metal levels in Eudrilus eugeniae and Pachybolus ligulatus at the individual locations were significantly different from each other at a=0.05 (Table 4) except for locations 3 and 8 for Pb, 1, 3 and 8 for Ni and 7 for Cd.The highest level (mg/kg) of Pb was found in the paint factory (locations 2 and 5).Pb was generally higher in Eudrilus eugeniae than in Pachybolus ligulatus at all the locations except for locations 5 (paint factory), 7 and 9 (Uyo, a busy city) where Pb was more in Pachybolus ligulatus samples.Eudrilus eugeniae accumulated more Fe (2-24-folds) at all locations than Pachybolus ligulatus (Tables 4 and 5).The paint factory (location 5 in Etinan) followed by mechanic village (location 10 in Uyo) had  4 and 5).Seventy percent (70%) of the locations recorded higher values of Zn in Eudrilus eugeniae while Pachybolus ligulatus recorded higher levels of Zn at locations 1, 7 and 8 (30%).Ni accumulated much more (greater than 3-folds) in Pachybolus ligulatus, ranging 0.01-12.6mg/kg, while Eudrilus eugeniae ranged 0.01-3.70mg/kg.Cadmium (Cd) was much higher in Eudrilus eugeniae (6.89 mg/ kg) than in Pachybolus ligulatus (0.10 mg/kg).Lead (Pb), Fe, Zn and Cd levels were higher in Eudrilus eugeniae (feeds on soil), and Ni higher in Pachybolus ligulatus (herbivore, feeds on litters) (Table 5).Moisture content of Eudrilus eugeniae was higher for most locations except location 10 with higher values for Pachybolus ligulatus.However, moisture content in the body of Eudrilus eugeniae (73.25%) though higher than in Pachybolus ligulatus (59.52%), was not significant at a=0.05 (Table 6).The areas of the paint factory, mechanic workshop and Uyo city high vehicular traffic roads had the greatest impact on soil as evidenced in the higher levels of metals in the bio-indicators.ANoVA results indicated a significant influence of location (p<0.0001) on accumulation of metals by the indicator organisms (p<0.0001).Interaction between these two factors were significant at a = 0.05, implying that there are differences in Pb, Fe, Zn, Ni and Cd accumulation

Bio-accumulation Factor (BaF) of metals in Eudrilus eugeniae and Pachybolus ligulatus
Bio-Accumulation Factors (BAFs) are presented in Table 7. Generally, BAFs were <1 for the selected metals in both Eudrilus eugeniae and Pachybolus ligulatus at the different locations,   6B).
Cluster analysis (CA) performed on Eudrilus eugeniae and Pachybolus ligulatus data sets grouped similar sampling sites (spatial variability) in the ten locations across the four Local Government Areas (LGAs) (Figures 7A and B), and the variables (metals) within the bio-indicators (Figures 7C and D).The spatial variability, that is, similarity and dissimilarity between locations where bio-indicators were collected, showed four clusters for Eudrilus eugeniae and three for Pachybolus ligulatus.For Eudrilus eugeniae data set, cluster one was made of locations 1, 3, 6 and 5; cluster two, locations 2 and 10; cluster three, location 9 only; and cluster four contained locations 4, 7 and 8 (Figure 7A).Pachybolus ligulatus data set contained in cluster one, locations 1, 8, 2, 7 and 3; Cluster two, locations 4, 10 and 6; and cluster three, locations 5 and 9 (Figure 7B).Cluster one had more locations in both dendrograms.Metals in bio-indicator organisms showed three clusters for Eudrilus eugeniae: Cluster one with Cd only, cluster two had Fe, Zn and Pb and cluster three Ni only (Figure 7C).For Pachybolus ligulatus, two clusters emerged: Cluster one contained Cd and Zn and cluster two, Fe, Ni and Pb (Figure 7D).

dISCUSSIon
The soil ecosystem is in equilibrium between its abiotic and biotic components; any change in one adversely affects the other 14, 26,39,40.Toxic metals on release into the soil (the ultimate sink of pollutants) disrupt this equilibrium.Their accumulation in soils is as a result of anthropogenic activities-from rapid industrialization, agriculture and urbanization.
The mean soil metal (Zn, Pb, Cd, Ni, and Fe) levels in the study areas were higher than the background levels [33][34] .High levels were especially recorded in the urban city of Uyo (with higher vehicular emissions), the mechanic workshops and the paint factory.This confirms reports by previous researchers 5,[41][42][43] that most areas near busy cities, industries and road systems have high concentration of toxic metals.Heavy metals may persist in the soil for up to a hundred decades; and can directly pollute soil and endanger the ecosystem by availability to flora and fauna, transfer to food chain and contamination of surface and ground water 26,39,40,44,45 .Soil characteristics such as pH and organic matter content affect the heavy metals adsorbed in soil 46 .According to Souza et al. 47 , decreasing the pH made available metals that were strongly adsorbed in organic matter of the soil.Trace elements are generally more solubilized in acid soil conditions.The present study revealed high positive correlations between soil organic matter and/soil pH (r = 0.8800); also, between soil Zn/Fe (r = 0.7580), Cd in Eudrilus eugeniae/soil organic matter (r = 0.6646, P = 0.0360).The total soil Pb and Cd were higher than values reported by olayinka et al 12 and Iorungwa et al 48 from a cement factory area and waste dump site in a mechanic village, respectively both in Nigeria, and Spurgeon and Hopkin 13 in a smelter plant area in the UK; but lower than from a mine in the UK 13 .Fe had values higher than reported by Iorungwa et al 48 ; Cd was higher than values reported in soil from a smelter plant but lower than from a mine, both in the UK 13 (Table 9).
Igeo values for Pb, Fe, Zn, Ni and Cd metals in soil were generally higher in soils around the paint factory and Uyo (a metropolitan city).The study areas soil quality varied from strongly polluted (soils from the paint factory, auto-mechanic village, and high traffic areas) to unpolluted (in the control).Qingjie et al 35 reported soil quality varying from polluted to unpolluted in Beijing Parks, from the center to the outskirts of Beijing City (Table 9).
Regular exposure of soil invertebrates to metals in the superficial layers of soil causes them to accumulate metals in their tissues 49 , hence the organisms can be used for monitoring soil pollution.Heikens et al 49 , further reported that the concentration of metals in terrestrial invertebrates were in the order Pb > Cd > Cu; which is closely similar to the trend observed in this study with Eudrilus eugeniae (Pb > Cd).our results also, are in line with findings that earthworms in impacted areas-near road sides, factories, mechanic villages and recreational areas agro-ecosystem and dump sites areas accumulate much more metals 11- 13,19,23,35,48 (Table 10).The variation in metals levels in the two organisms may be due to factors including pathway of metal uptake-dermal and/or oral, the body composition/metabolism and feeding habit 50 .Diplopods have been reported to metabolize and/or accumulate various types of metal ions in the midgut, and the fat body 8 which is distributed throughout the body cavities.Accumulation of more Pb, Fe, Zn and Cd in Eudrilus eugeniae tissues than Pachybolus ligulatus may be explained in their feeding habits.Pachybolus ligulatus species are herbivores and live between the soil surface and litters, feeding mainly on decayed plants materials very rich in organic matter, while Eudrilus eugeniae species are burrow animals that feed on soil particles with lesser organic matter down the soil profile.Soil being a sink for pollutants, could be responsible for the higher concentrations of metals in earthworms (Eudrilus eugeniae) than in millipedes (Pachybolus ligulatus).
Bio-accumulation in terrestrial invertebrates often has been the sum of the metals adsorbed to the body wall and absorbed into the body.However, Vijver et al 51 , in their work on earthworm and isopod reported that adsorption of Cd and Zn to surface of the organisms were negligible.They further showed that the metal uptake levels in the organisms were mainly the internalised metals with little or no external component; contrary to previous report 52 .Issues of variation and/or affinity of metals therefore could reside in the internal mechanisms/processes of the organism.The highest concentration of Cd (6.89mg/kg) in Eudrilus eugeniae compared with 0.10 mg/kg in Pachybolus ligulatus may result from its localization in the chloragogenous tissue 9,50 which is unique to oligochaetes, and has a function in metal storage and trafficking 53 ; and essential metals e.g.Zn when involved, also function as buffer for the coelomic fluid and blood.on the other hand, for arthropods, the hepatopancreas appears to be the most important storage organ for metals.The hepatopancreas is packed with different types of granules like the chloragogenous tissue in earthworms.Metals are precipitated in different chemical forms in these granules 21,54 .The binding chemicals of the hepatopancreas in millipedes may have more affinity for Ni (12.60mg/l) than the chloragogenous tissue of earthworms (3.70 mg/l), hence the higher concentration of Ni in Pachybolus ligulatus than Eudrilus eugeniae.other factors that could influence uptake of metals in the bioindicators include the solubility of the soil metal bearing mineral 41,55 .The higher the solubility, the higher the accumulation of metals in invertebrates.This study revealed high positive correlations between soil organic matter/soil pH (r = 0.8800); also, between Cd in Eudrilus eugeniae/soil organic matter (r = 0.6646).Another important factor in bioaccumulation of metals in terrestrial invertebrates is their ability to eliminate/excrete excess of metals.For the xenobiotic metals e.g.Cd and Pb, it is reported that the excretion is slow or absent 9 , which could also be responsible for higher accumulation of Pb, Cd in Eudrilus eugeniae and Ni in Pachybolus ligulatus.
Soils, and consequently the indicator organisms are impacted by activities of the paint factory, auto-mechanic workshop and the municipal city.PCA revealed that for Eudrilus eugeniae Eigen values >1 obtained explained more than 64.116% of the total variation of metals.PC1 showed strong positive factor loading on Fe and Zn, and moderate positive factor loading on Cd; and PC2, strong positive factor loading on soil organic matter and soil pH, and moderate positive factor loading on Pb.For metals in Pachybolus ligulatus, PC1 accounted for 37.558% of the total variation and showed strong positive factor loading on Ni, soil organic matter (oM) and soil pH; which could also explain the preferential accumulation of organically bound metal (Ni) by Pachybolus ligulatus while Eudrilus eugeniae appears to be indicator for the inorganic species of metals.on the other hand, PC2 explained 34.304% of the total variation, with strong positive factor loading on Pb and Fe; and strong negative factor loading on Cd.These suggest anthropogenic input of metals from waste from the paint factory, mechanic workshop, agricultural activities, decomposed organic wastes and acid precipitation.

ConCLUSIon
The invertebrates exhibited (preferential) levels of affinity in metals uptake.Eudrilus eugeniae (soil feeder) appears to accumulate the inorganic metals species -Pb, Fe and Cd more, and could serve as bio-indicator of these metals; while millipede (decayed litter feeder) accumulated 3-folds more Ni (organically bound metal), thus presenting the organism as a suitable indicator of Ni-polluted soils.The endowment of Pachybolus ligulatus for accumulation of nickel makes the invertebrate both suitable and choice bio-indicator of crude oil pollution.This could provide a monitoring tool for agriculture in the Niger Delta, a region of Nigeria that is plagued by decades-long oil pollution of its water bodies and land.
We are of the opinion that despite other possible factors, preference in metals uptake/storage in the organisms may reside largely at the level of the chloragogenous and hepatopancreas tissues of the earthworm (Eudrilus eugeniae) and millipede (Pachybolus ligulatus), respectively.A more detailed research on the affinity of the tissues for metals would be required.In the overall, the activities in the areas studied clearly contributed to the pollution of locations.

Table 4 : T-test (LSd) results for metal distribution in Eudrilus eugeniae and Pachybolus ligulatus from different locations
Means with the same letters are not significantly different; Ee= Eudrilus eugeniae; PI= Pachybolus ligulatus ; LSD= Least Significant Level geological index of soil pollution (Igeo)

a: Eudrilus eugeniae B: Pachybolus ligulatus C: Eudrilus eugeniae d: Pachybolus ligulatusTable 8 : Total variance explained by PCa for metals in Eudrilus eugeniae and Pachybolus ligulatus
Eudrilus eugeniae and Pachybolus ligulatus after Varimax rotation with Kaiser normalization resulted in the extraction of two Principal Components (PCs), each of which was used in interpreting the sources of pollution of the indicator organisms and the corresponding soil.PCs with Eigen values >1 are considered significant 38 .Table 8 shows the variance explained by the PCs for metals in Eudrilus eugeniae and Pachybolus ligulatus.Two PCs obtained with Eigen values >1 explained more than 64.116% of the total variance of metals in Eudrilus eugeniae.PC1