The mixture of heavy metals and polycyclic aromatic hydrocarbons (PAHs) in classical toxicology studies or in remediation processes is gaining increasing interest in the scientific world [1
]. Indeed, due to anthropogenic activities, namely the application of pesticides in agricultural soil [3
], inorganic fertilizers [4
], industries including metal-plating, mining, tannery, petrochemical, textile, battery and fertilizer production [5
], discharge of waste containing heavy metal [6
] and emission from industries production [7
], agricultural soils contamination with heavy metals and PAHs have increasingly beoame of serious global environmental concern. This pollution poses a huge threat to human beings and natural ecosystems [8
]. Cadmium (Cd), one of the most toxic pollutants because of its non-degradability, persistence in nature, and high toxicity to plants, soil organism and humans [9
] is widely spread in the environment. It negatively affects plant physiological growth [10
], soil organism biomass [11
], and may affect human health through food-chain bioaccumulation [12
]. Even though Cd is indestructible, Liu et al. [13
] reported that it may become less toxic to the environment either by chelating with chelators via chemical or physical remediation, or by shifting the valence by redox reaction by forming interactions, in some specific cases, with PAHs. Phenanthrene, a three-fused ring compound commonly that is present in PAH-contaminated soils [14
], is designed with less toxic effects compared to other degradable PAHs. Although it is not mutagenic or carcinogenic, phenanthrene has been reported to affect nitrifying bacteria in soil [15
], reduce the intraradical colonization of arbuscular mycorrhizal fungi in maize root [16
], inhibit the growth of algae [17
] and, at a given concentration, can inhibit root exudation [18
The joint toxicity of contaminants to organisms in their natural habitat can be complex. It may depend on the chemistries of the individual compounds, environment-specific bioavailability, toxicological mode of action, organism test used, experimental conditions and possible pharmacologic interaction among contaminants once bio-accumulated [19
]. Therefore, the dose combination of two chemicals with a dissimilar toxic mode of action may cause synergistic, independent or antagonistic effects on tested organisms, depending on the end-point investigated and the experimental protocol utilized [20
]. Lu et al. [21
] found that the addition of a moderate dosage of pyrene could promote microbial prosperity in soil and, thus, relieve metal stress. While Gauthier et al. [22
] summarized that the more-than-additive deleterious effects of PAHs–metal mixtures to soil organism were common in metal-PAHs mixtures. Gust and Fleeger [23
] reported that the joint effect of phenanthrene and Cd on Ilyodrilus templetoni
was antagonistic and phenanthrene reduced Cd toxicity on Ilyodrilus templetoni
. In another study, they showed that phenanthrene increased the lethal toxicity of co-occurring Cd in Hyalella Azteca
in sediment exposure [24
]. Thus, it is obvious that the couple “phenanthrene-Cd could as well be more or less toxic to soil or aquatic organisms, compared to their effects in single exposure. Although the main mechanism of their interaction effect is still not deeply understood, studies showed that the interactive effect of phenanthrene and Cd was strongly concentration-dependent rather than being a toxic joint effect [25
]. The combined dose of Cd and phenanthrene at, respectively, 3.2 mg kg−1
and 1.6 mg kg−1
induced high toxicity effect compared to the effect of their combination at 3.2 mg kg−1
and 25.6 mg kg−1
, respectively [26
]. Phenanthrene concentration at 25.6 mg kg−1
reduced significantly Cd toxicity by about 52% [27
]. However, Zhu et al. [26
] reported that the genotoxicity effect of the combined Cd and phenanthrene at, respectively, 50 mg kg−1
and 12.5 mg kg−1
was higher compared to the effect of either Cd or phenanthrene in single exposure. Therefore, there must be a range of concentrations by which phenanthrene could interact with Cd and mitigate its toxic effects on living organisms. Although different organisms reacted differently to an exposure stress situation, earthworms appeared to be excellent candidates for eco-toxicology studies. With their ability to accumulate essential and non-essential heavy metals in their body tissues and their direct or indirect role to modulate the transfer of organic and inorganic pollutants by virtue of their habitation [27
], earthworms are considered as bio-indicators of contaminated soil and key diagnostic indices in eco-toxicology [28
]. They ameliorate soil structure by increasing soil aeration [29
] and enhance the conveyance of soil microorganisms.
Most previous studies on the toxicity of heavy metals and PAHs either in single or combined dose test to earthworms have been concentrated on appraising the concentration of the pollutants in earthworms and their effects on worm growth, biomass and reproduction. However, taking into account the effects of these pollutants, such studies indicated an approximate concentration of pollutants that adult earthworms can tolerate, but provide no indication of the actual toxicity of pollutants to earthworms and how the interaction of organic and inorganic pollutant should be controlled. Exploiting the antagonistic interaction of heavy metal and PAHs in eco-toxicology study would have a significant impact on the control of heavy metals toxicity and could provide clear evidence regarding to the threshold concentration of pollutants in a mixture treatment.
The purpose of this study is to examine the interactive effect of phenanthrene and Cd on two ecologically different species of earthworms; E. fetida (epigeic specie) and A. caliginosa (endogeic specie). The use of ecologically different earthworm species could be promising and effective, since they inhabit the soil at different depth, have different sizes and feeding habits. This paper further determines at a degree of concentration, the relational effect between cadmium and phenanthrene on earthworm life cycles including mortality and body weight variation.
2. Materials and Methods
2.1. Soil Properties
Soil was collected from the test field at Huazhong Agricultural University (HZAU) (30°28′26″ N, 114°20′51″ E). The upper litter was removed, and the soil from the top layer (0–20 cm) was collected. The soil samples were transferred to the greenhouse of Micro-element Research Center at HZAU for grinding and sieving. The used soil presented the following proprieties: pH (soil:H2O 1:2.5) 7.6; organic matter 1.31%; soil moisture content 18.58 ± 0.59%; NH4Cl exchangeable K, 127.99 mg kg−1; total nitrogen N 0.17%; Olsen-P of 39.69 mg kg−1; CEC 11.47 cmolc kg−1; and Ca, 2288.2 mg kg−1.
2.2. Test Soil Preparation
Three kg of air-dried soil was placed into ceramic pots. For cadmium concentration, a desired amount of CdCl2 (98%, purity) was dissolved in aqueous solution. This solution was poured on the soil surface and the soil matrix was mixed thoroughly and incubated at 20 ± 1 °C for three months. Throughout the incubation time, the content of moisture was scrutinized each week and maintained by watering with DI water if needed.
Phenanthrene (97% purity) was dissolved in acetone (analytically pure) and the solution was thoroughly mixed with the soil to produce a final concentration of 5, 10, 15, 20, 25 and 30 mg kg−1 of phenanthrene respectively denoted as P5, P10, P15, P20, P25 and P30. The control treatment was prepared using clean soil spiked acetone only. The fresh spiked soils were stored in open containers in a fume hood until all of the solvent (acetone) evaporated.
2.3. Test Organism
Earthworm species Aporrectodea caliginosa
and Eisenia fetida,
two different ecotypes (endogeic and epigeic respectively) were selected for this study. A litter-dwelling E. fetida
was chosen for its fast growth and its rapid productivity [31
], while the horizontally burrowing mineral soil feeder A. caliginosa
was selected for its ability to transfer nutrients or chemicals elements within a compartment of an ecosystem or between different compartments. [32
]. Both earthworm species were chosen and selected in the earthworms breeding site at HZAU. A sufficient quantity of earthworms (for different species) was initially purchased from a commercial source and transferred to the greenhouse where a controlled earthworm breeding site containing soil filled with household waste (cabbage waste, carrot peelings, banana waste) was installed. The worms used in the present study were selected after 3 months of reproduction in this mentioned site, washed in deionized water, placed on wet filter paper and maintained in the darkness at 20 ± 2 °C for one night in the laboratory [33
] before placing them in the surface of the corresponding experimental pot.
2.4. Acute Toxicity Test
Preliminary studies were carried out to assess the range of Cd concentrations that produced 1–100% mortality. Thus, five concentrations in a geometric series and a control treatment were used to get the concentration value that exhibited 50% mortality (LC50) at 95% confidence interval. Cd concentrations used in the single chemical toxicity experiment were as: 0.5, 1, 3, 5 and 10 mg Cd per kg of soil (dry weight). The choice of the interval of Cd concentration was made to ensure a relatively high effect in the highest concentration and no observable effect in the lowest concentration.
Contrary to Cd, preliminary experiment was realized to evaluate the range of phenanthrene concentration that could at a time produce low observable effect on the tested organism in the highest concentration and have a mitigating effect on Cd toxicity. The concentrations of phenanthrene used were 5, 10, 15, 20, 25 and 30 mg kg−1.
2.5. Toxicity Test with Chemicals Mixture
The effect of Cd in the mixture treatments were based on the LC50 values obtained earlier. Six concentrations of phenanthrene with separation factor of 5 were used to set up the mixture treatment with LC50 of each species. Each treatment was tested in three independent experiments with replicates samples. For each specie, six binary mixtures (CdP5, CdP10, CdP15, CdP20, CdP25 and CdP30) were tested to assess the impact of phenanthrene on Cadmium effect.
2.6. Experimental Monitoring
For each ecotype, 20 adults and healthy individuals with similar fresh weight (1.82 ± 0.06 g and 3.09 ± 0.04 g respectively for E. fetida and A. caliginosa) were randomly selected in the earthworms breeding site at HZAU, regrouped and rinsed with DI water and placed on moist paper for 24 h to void the gut content before placing them on the soil surface of each experimental pot previously mixed with horse dung (3:2) as food. The initial body weight was measured immediately (day-0) and the animals were placed on the pot experiment. The experiment was carried out for 30 days period during which the mortality and the body mass of earthworms was monitored every 5 days. Earthworm was considered dead when it did not show any response on probing.
2.7. Kinetics Parameters
Bioaccumulation factor (BAF), uptake constant rate (k1
) and elimination constant rate (k2
) were monitored to understanding whether there was correlation between earthworm mortality and body mass variation. Every fifth day three individual earthworms for each specie were sorted from each test soil and starved on moist paper for 24 h (elimination phase) to allow them void their gut content. The paper was changed twice during the starvation period (at 6 and 18 h). After starvation, the worms were kept at −28 °C for further analysis [34
]. The gut content was then collect and use to analysis chemical concentration. BAF was calculated as the ratio of the pollutant content in the earthworm tissue to that in the corresponding soil [35
]. Elimination constant rate (k2
) was determine as the concentration eliminated per day and the uptake constant rate (k1
) was calculated by using the formula described by [36
= BAF × k2
2.8. Chemical Analysis
Earthworms were washed in deionized water and placed on wet filter paper to allow depuration for 48 h. Earthworms were then washed again, freeze-dried and ground. A sub-sample of 500 mg was used for chemical analysis.
Earthworm sample was digested with 6 mL of HNO3
mixture (5:1) on a hot plate at 150 °C for 2 h [37
]. The digested solution was evaporated to 1 mL and 1% HNO3
was added to adjust the volume to 25 mL. The diluted solution was filtered, and Cd concentrations were measured by atomic absorption spectrometry (AAS) (Z-2000, HITACHI, Tokyo, Japan).
2.8.2. Phenanthrene Determination
Ultra-sonication extraction method [38
] with slight modification combined the protocol described in [39
] was used to determine phenanthrene concentration. Frozen worms were ground and mixed with 1.5 times its wet weight of Na2
to a fine powder. The mixture was extracted with 10 mL of acetone placed in ultrasonic bath with ice water for 30 min. The solution was shaken for 1 min, resonicated for 30 min and centrifuged at 13,700× g
for 15 min; the whole process was repeated twice. Further chemical treatments which the detail of the method is described in [39
] were performed before determining phenanthrene concentration on High-performance liquid chromatography (HITACHI Chromaster 5300, Hitachi Beijing Tech Information Systems Co., Ltd, Beijing, China.
The accuracy and analysis quality of phenanthrene measurements was checked by using certified standard materials NIST1647 Priority Pollutant Polycyclic Aromatic Hydrocarbons in Acetonitril, purchased from Sigma-Aldrich and the recovery rate was 85 ± 3%.
2.9. Statistical Analysis
All data were subjected to the Analysis of Variance (ANOVA) using Statistical Package for Social Science (SPSS. 20, IBM Company, Chicago, IL, USA) statistical software following by Post hoc Dunnett multiple comparison test and Tukey comparisons test with 95% confidence level to compare the means. Linear regression was used to find out the correlation between concentration of pollutant and different parameters. Cd LC50 mortality and the dose-mortality response were analyzed by probit analysis [41
]. The 30-d LC50 was 2.51 mg kg−1
and 3.47 mg kg−1
, respectively, for E. fetida
and A. caliginosa
. Different graphs were performed using Origin (8 Pro SR4, OriginLab (Guangzhou) Ltd., Guangzhou, China) software.