Short-term effects of cadmium and mercury on soil nematode communities in a pot experiment

Anthropic activities such as mining, manufacturing, transporting and fertilizing have caused heavy metal contamination in urban and agricultural soils. The heavy metal contamination has resulted in severe threats to human and environment health (Chen et al., 2005; Xia et al., 2004). Cadmium (Cd) and mercury (Hg) are the two most predominant metals in sewage irrigation region of Liaoning province (Li & Tong 2008). How to deal with the toxic pollutants efficiently and safely is becoming one of the most popular environmental-related themes. Phytoremediation has been developed in recent years (Yoon 2007), and has become a new economical method which can reduce some toxic pollutants in an environmentally friendly way (Pilon-Smits, 2005). Morning glory (Pharhiris nil) is a remediation plant which absorbs pollutants from soil and accumulates them in stalk, leaf, and root, in this way, contaminant could be reduced through harvesting the plant body. Xu et al. (2012) reported that petroleum degrading rate was increased by 13 % after 2 months of planting Morning glory. With respect to metal bioaccumulation, plants accumulated higher amounts of metal in root than stem in previous studies (Ali et al., 2004), so the toxicity created by heavy metals impacts roots first, which means soil quality and plant-microbe interactions around the rhizosphere can influence the efficiency of metal phytoremediation (Cherian & Oliveira 2005). However, most investigations about accumulating plants were focused on the aboveground parts, the structure and assemblage of soil fauna around rhizosphere are not well known. Nematodes are one of the most abundant groups of soil invertebrates (Fu et al., 2000) and occupy a central position in the soil food web. Soil nematode communities can provide unique insights into soil processes and supply useful information about soil environment. Soil nematode as an indicator for heavy metal pollution has been widely studied in recent years. (Zhang et al., 2011; Šalamún, 2011; Martinez et al., 2018). Most of previous studies took place in an open environment such as mining area, however, under laboratory condition, the characteristics of rhizosphere nematode communities affected by specific concentrations of single and mixed heavy metals are not well studied. The present study investigated the changes of Cd, Hg and Cd+Hg concentrations after 90 days remediation and characterized the nematode community structure and assemblage around rhizosphere of Morning glory. The objectives of this study were: 1) to evaluate soil quality after phytoremediation experiment; 2) to represent the responses of nematodes to different heavy metal treatments; 3) to provide basic data for using soil nematodes to assess the level of phytoremediation. We hypothesize that only small part of heavy metals could be absorbed by plants during short-term remediation, hence the remaining part still has adverse effects on nematode communities, furthermore, the degree of influence may depend on feeding groups and life-history strategies.

The seeds of Morning glory were planted in non-contaminated garden soil for 7 days before experiment, then, five seedlings were subsequently transplanted into one pot. Each pot (15cm – diam×12cm – -depth) was loaded by 600g soil collected from Dalian Xishan National Forest Park, soil was loamy with 13.1 % clay, 50.4 % silt and 36.5 % sand and was completely mixed. Based on the concentrations of heavy metals obtained from the wastewater irrigation area (max concentration of Cd = 5 mg/kg) (Liao, 1993) and mercury mining area (Hg = 20.4 mg/kg) (Yu et al., 2017), the concentrations of heavy metal were selected at Cd-5mg/kg, Hg-20mg/kg and Cd-Hg5+20mg/kg. Heave metal solutions were applied as Cd(NO₃)₂·4H₂O and HgCl₂, three replicates at each level were established, concentrations of heavy metals before and after treatment were shown in Table 1. The seedlings were grown for three months in a climate chamber at 20±1 °C. Intensity of light was 5000LX with 16 hour of light alternating with 8 hour of darkness. Each pot was watered with 250 ml deionized water every 2 days.

Nematodes were extracted from 100g (fresh weight) soil per pot using elutriate-sieving-flotation and centrifugation method (Barker et al., 1985). Extracted nematodes were heat killed at 60 °C, counted and preserved in 4 % formalin aqueous solution (Steinberger & Sarig, 1993). One hundred nematodes randomly selected specimens per sample were identified to genus level using Olympus inverted compound microscope based on stoma and esophageal morphology (Liang et al., 2001, 2003). The genus was identified according to Bongers (1988) and Li et al. (2017).

Soil moisture of each sample was gravimetric determined by weight loss at 105 °C for 8 hours and expressed as percent dry weight. Another 100g fresh soil per pot was air-dried for 14 days at room temperature to text soil organic matter and heavy metals. Electronic pH meter (model SevenGo™ pH-SG2 ), the potassium dichromate external heating method, graphite furnace atomic absorption spectrometry and microwave dissolution/atomic fluorescence spectrometry were used to determined soil pH, organic matter, Cd and Hg, respectively (Ru, 1999; China National Environmental Monitoring Centre,1997; Ministry of Ecology and Environment of the People’s Republic of China,2013).

The assemblage and characteristics of nematode community were investigated by following approaches: (1) feeding groups (bacterivores-BF, fungivores-FF, plant parasites-PP, omnivores-predators OP) (Yeates et al., 1993; Pen-Mouratov et al., 2004); (2) life-history groups (c–p values range from 1 to 5 represent colonizers to persistors (Bongers, 1990, 1999). (3) ratio of f/b (fungivores/bacterivores); (4) diversity index H’=–Σni (lnni) where ni is the proportion of individuals in the i-th taxon (Shannon & Weaver, 1949) , (5) trophic diversity index TD = TD = 1/Σpi2, where pi is the proportion of each trophic group; (6) evenness J’= H’/ln(S), where S is the number of taxa; (7) maturity indices MI=Σvifi, where vi is the c–p value of the i-th taxa and fi is the frequency of the i-th taxa in the sample (excluding PP) (Bongers, 1990); (8) enrichment index (EI) and structure index (SI), EI = 100(e/e + b), SI = 100(s/s + b), where e =Σkene, s =Σksns, and b =Σkbnb (Ferris et al., 2001).

All nematode data were 1n (x +1) transformed prior to statistical analysis. The significance of the effects of heavy metals on nematode communities was tested by one-way analysis of variance (ANOVA), SPSS 18.0 statistical software, and means compared by LSD’s Test (Least Significant Difference). Differences with P < 0.05 were considered significant. Principal component analysis (PCA) was applied to represent the composition of the soil nematode community using CANOCO software.

Building on previous researches, population structure that was measured by abundance and genus number (Bakonyi et al., 2003), function indexes (MI, SI and EI) (Bongers, 1990; Korthals et al., 1996; Wang & McSorley, 2005) and functional variables such as feeding groups (Yeates et al., 1993) and c-p groups (Bongers, 1990, 1999) were always applied to analyze the responses of nematode communities to disturbance.

The population of nematodes may increase or decrease with heavy metal concentration (Sánchez-Moreno & Navas, 2007). In the present case the pronounced drop of nematode abundance already sent an alert on the heavy metal pollution (Table 4). Our result is different from previous findings by Bakonyi et al. (2003) who reported that Cd had a moderate influence on nematodes. This difference might be contributed by the short-term and high level of pollution. Cadmium could negatively impact on soil enzymatic activities and microbial community structure (Wang et al., 2019). We expect that the abundance of nematode was inhibited by the degradation of their feeding sources in contamination soil. In addition, Hg exposure can lead to multitoxicity, and most of these harmful effects on nematode can be transferred to progeny (Wu et al., 2010). Nematode diversity (H’) and evenness (J’) were lower at Cd-5 and Cd-Hg 5+20 than the control and Hg-20 (Table 3), which suggested single Cd and mixing of Cd and Hg create worse effects on nematode communities than single Hg. Martinez et al. (2019) proposed that nematode-based environmental evaluations should be interpreted in a context-dependent way. Acidic soils can inhibit the diffusion of cadmium (Lu et al., 2005) and enhance its bioavailability (Kim et al., 2009), but the bioavailability of Hg tends to be lower in acidic soils (Mahbub et al., 2016). So the lower values of pH at Cd-5 and Cd-Hg 5+20 seem to be the reason for the lower values of H’ and J’. Xie et al. (2011) found that the mixing of Cd and Hg had stronger toxic effects on soil microbial community than the single Cd or Hg. Similarly, nematode trophic diversity index (TD) was lowest at Cd-Hg 5+20, which indicated the combination of Cd and Hg exert more adverse impacts on nematode trophic groups than individual Cd or Hg in this study.

Nematode assemblage and function level structure presented by MI, SI and EI was found to be skewed away from the theory of these indices which predicts a reduction of the MI and SI as a consequence of heavy metal pollution. The values of MI, SI and EI were relatively uniform throughout all treatments. Our result was in line with Martinez et al. (2018) who discovered a short range for maturity index from different levels of disturbance. The reason for this phenomenon is the rarity and stabilization of relative abundance of high c-p value groups among all treatments. Therefore, we propose that changes in nematode communities could be represented better from abundance and diversity point of view. Since MI, SI and EI are expanded indices derive from proportions of feeding groups and life-history strategies, current findings highlight that more detailed analysis about trophic and c-p groups are needed for a correct interpretation of short-term high level pollution effects on soil nematodes.

After 3 months application, morning glories absorbed small part of heavy metals. Heavy metals had a deleterious effect on soil nematode assemblage, decreased nematode abundance, changed the structure of feeding groups and c-p groups. Single Hg seemed to have smaller impacts than Cd, and single Cd and Hg had fewer side effects than Cd-Hg on nematode communities. Direct analysis of nematode abundance, diversity, trophic and c-p groups could be more useful tools than some indices to assess the degree of soil disturbance in the short-term high level pollution experiment.