• Metals;
  • Geraniol;
  • Benzo[a]pyrene;
  • Oppia nitens;
  • Hormesis


  1. Top of page
  2. Abstract
  8. Acknowledgements

The oribatid mite Oppia nitens has been suggested as a test species for ecotoxicological assessment of contaminated boreal soils. Knowledge of the ecotoxicity of pollutants of different modes of action to this species is necessary to assess its relative sensitivity in comparison with other invertebrates. The toxicity of four metals and two organic chemicals to O. nitens was evaluated over a 28- or 35-d period. Mite survival, reproduction, and tissue accumulation were assessed at the end of the test. Reproduction was a more sensitive endpoint than survival for all of the compounds except geraniol. The reproduction median inhibitory concentration (IC50) values for Cu, Zn, Cd, and Pb were 2,896, 1,562, 137, and 1,678 mg/kg, respectively, whereas those for benzo[a]pyrene and geraniol were greater than 1,600 and 283 mg/kg. The median lethal concentration (LC50) values for Cu, Zn, Cd, and Pb were 3,311, 2,291, 603, and 6,761 mg/kg, respectively, whereas those for benzo[a]pyrene and geraniol were greater than 1,600 and 251 mg/kg. When effects on reproduction are compared with those of other soil invertebrates, O. nitens appears less sensitive to Cu and Zn but within the same order of magnitude of sensitivity as that for Cd and Pb. Despite its lower sensitivity to Cu and Zn, O. nitens is a member of a group underrepresented in ecotoxicological evaluations and should therefore be included in test battery for risk assessment of contaminated boreal and other northern soils. Environ. Toxicol. Chem. 2012; 31: 1639–1648. © 2012 SETAC


  1. Top of page
  2. Abstract
  8. Acknowledgements

Soil contamination is often the most common form of anthropogenic stress in ecosystems and can pose threats to human health and the environment. In the soil environment, invertebrates are abundant and good indicators of stress in their environment. Soil organisms are among the major components of soil biomass and play important roles in maintaining the structure and fertility of soil. Invertebrate-mediated processes such as drainage, aeration, and incorporating and degrading organic matter are important in improving soil quality 1. Among the soil invertebrates, mites are an ecologically relevant mesofauna because of their large diversity and abundance compared with other groups of soil invertebrates. They are rich in species diversity and are abundant in the forest soil, especially in the organic layer, where they contribute to litter decomposition. Because of the character of their habitats, they are frequently exposed to high concentrations of pollutants in their environment.

Soil mites are well suited for ecotoxicological assessment of pollutants because they are known accumulators and respond to toxic substances in their environment 2–4. However, fewer studies use mites than use other species such as earthworms, collembolans, and enchytraeids. One of the first mite species to be used in laboratory toxicity tests was the parthenogenetic oribatid species Platynothrus peltifer 5. The testing presented many challenges such as adult mortality, difficulty in establishing laboratory cultures, and test duration, which dissuaded many authors from using this species. Many other species of mites have since been used in laboratory experiments. The most commonly investigated species are Archegozetes longisetosus 3, 4, another parthenogenetic oribatid mite and the predatory mesostigmatid mite Hypoaspis aculeifer 6, 7, which occupies a higher trophic level. Although H. aculeifer, for which a standardized test guideline exists 8, is found worldwide, A. longisetosus is tropical in distribution. For ecotoxicological assessment of northern and boreal systems, the oribatid mite Oppia nitens has been advocated as a likely test species. Recently, a standardized toxicity test procedure has been developed 9. Therefore, a study investigating the toxicity of common contaminants on its survival and reproduction is warranted.

Two soil contaminant groups that have attracted global attention are metals and organic compounds. Metal pollution has become a great source of concern, mostly because metals interact with the soil matrix and persist, posing long-term hazards. Soil contamination with metals results in accumulation and subsequent toxicity to plants, microbes, and invertebrates 10, 11. Apart from metals, several organic compounds cause serious effects on soil organisms and could be transferred into the food chain. Among these, polycyclic aromatic hydrocarbons (PAHs) are often a main concern. Polycyclic aromatic hydrocarbons are widely distributed organic contaminants that have detrimental biological effects, toxicity, mutagenicity, and carcinogenicity. Benzo[a]pyrene (BaP) is the model PAH for human toxicity concerns because of its classification as a known human carcinogen 12. Conflicting results on its toxicity to soil organisms are available in the literature; although some authors have reported high toxicity of BaP 13, others reported low toxicity 6, 7, 14 with respect to environmental contamination level. Another organic compound that is often not well studied is geraniol, a commercially important terpene alcohol occurring in the essential oils of several aromatic plants. It is one of the most important molecules in the flavor and fragrance industries and is a common ingredient in consumer products produced by these industries. In addition to its pleasant odor, it is known to exhibit insecticidal 15 and repellent properties. Thus, geraniol is used as a natural pest control agent with low toxicity to mammals. Whether it will negatively affect the survival and reproduction of oribatid mites is not known.

The specific objectives of the present study were to assess the effects of four heavy metals commonly used in toxicity tests (Cu, Zn, Cd, and Pb) and two organic compounds (BaP and geraniol) on the survival and reproduction of the oribatid mite O. nitens. Another objective was to compare sensitivity of this mite species with those of other species commonly used in toxicity tests. We also assessed whether contaminant accumulation could be used as an indicator of toxicity to the mite.


  1. Top of page
  2. Abstract
  8. Acknowledgements

Test organism

The test organism used for the present study was the oribatid mite Oppia nitens (C.L. Koch, 1836). Its biology and ecology have been previously reviewed 9. Specimens used for the present study were age-synchronized from cultures kept in the laboratory of the Soil Environmental Toxicology Group, Soil Science Department of the University of Saskatchewan, Canada. The culture was obtained from Environment Canada in 2009, with the species' provenance described in Princz et al. 9. The organisms were cultured in the laboratory kept at 20°C with a photoperiod of 16:8 h light:dark. Specimens were reared in 125-ml glass mason jars lined with an 8:1:8 (w/w/v) ratio of plaster of Paris, charcoal substrate, and distilled water thoroughly mixed and added in order to reach the 1-cm mark of the jar. The jars were covered with a perforated lid, and the substrates were moistened once per week with deionized water. Grains of Baker's yeast were added ad libitum as a food source.

Test soil

Experiments were done in Organisation for Economic Co-operation and Development (OECD) soil. The OECD soil was prepared as described by OECD guideline 222 16. It consisted of 70% sand, 20% kaolin clay, and 10% sphagnum peat by dry weight. The pH was adjusted to 6.0 ± 0.5 by CaCO3. The maximum water holding capacity was 65%. Water was added to reach 60% of the maximum water-holding capacity of the soil. The pH-H2O and water-holding capacity of these soils was determined according to standard procedures 17, 18.

Test substances

Test chemicals used in our experiments included four metals: Cu (added as CuSO4.5H2O; Scholar Chemistry), Zn (added as ZnSO4.7H2O; BDH Chemical), Cd (added as CdSO4; Sigma-Aldrich), and Pb (added as Pb[NO3]2; BDH Chemical), as well as two organic compounds: benzo[a]pyrene (Sigma-Aldrich), and geraniol (Sigma-Aldrich). Selected test concentrations for each chemical were based on the results of a range-finding test conducted earlier. The concentrations of contaminants used in the present study (in mg/kg) were the following: 100, 250, 500, 1,000, 2,000, 4,000, and 8,000 (Cu); 100, 250, 500, 1,000, 2,000, 4,000 (Zn); 50, 100, 200, 400, 800, and 1,200 (Cd); 1,000, 2,000, 4,000, 8,000, and 16,000 (Pb); 50,100, 200, 400, and 800 (geraniol); and 50,100, 200, 400, 800, and 1,600 (BaP). For metals, the test chemicals were added as aqueous solution in deionized water to reach 60% of the maximum water-holding capacity of the soil. For organic chemicals, the compounds were introduced with acetone as a carrier, but control soils were also given acetone equivalent to the volume used in the treatment soils. The soils were left in a fume hood overnight to evaporate the solvent before moistening with deionized water to 60% of maximum water-holding capacity of the soil. Prepared soil treatments were introduced into the test vessels and allowed to equilibrate for 7 d and 1 d for metals and organic chemicals, respectively, before they were used in the experiment.

Experimental procedures

Effects on survival and reproduction were performed according to procedures used in a recent study 9. Ten adult mites were introduced in each test vessel containing 30 g of hydrated soil. The mites were fed weekly with Baker's yeast. Moisture changes were assessed weekly by weighing the containers, and the corresponding water loss was replenished. Four replicates per treatment were used. Testing was conducted using the 16:8 h light:dark photoperiod cycle and a temperature of 20°C for four weeks in the test with Zn as used in the earlier study 9. However, we extended the test duration for the other compounds for one more week to allow more juvenile production in control soils, because only a few juveniles were produced after four weeks in the test with Zn. At the end of the test, the number of mites in each container was counted after 48-h extraction with a modified Berlese-Tullgren apparatus. Measurement endpoints included the number of surviving adults and total juveniles (protonymphs, deutonymphs, and tritonymphs) produced after the test end. The juveniles were distinguished from the adults by their pale color. The mites were placed in small vials and frozen at −4°C until needed for chemical analysis.

Determining the metal contents in mites

To determine the internal metal concentration, all mites recovered from each treatment were pooled together into two groups so that all treatments had two replicate samples for metal analysis. For the highest concentrations of metals, and because only a few mites survived in all replicates used for the toxicity tests, replication was not feasible. Therefore, the few surviving mites were pooled together for metal analysis. Preparing the oribatid mites for metal analysis was done at the Toxicology Centre, University of Saskatchewan, Canada. Mites were digested in 6.5 ml of a mixture of concentrated nitric acid (OmniTrace Ultra HNO3 69%) and perchloric acid (Ultrex grade) in a ratio of 7:1 by volume, respectively. Samples were heated until dry, and the pellet was dissolved in 2% HNO3 before metal analysis. For every batch of mites that was digested, a blank was prepared to detect and eliminate possible contamination during the digestion process. Determinations of Cu, Zn, Cd, and Pb concentrations were done using X Series11 inductively coupled plasma mass spectrometer with PlasmaLab software and collision cell (Thermo Fisher Scientific), with optimized protocols for each metal. Detection limits were estimated from the standard deviation and mean of procedural blanks and related to the sensitivity of each metal. Detection limits of Cu, Zn, Cd, and Pb were 0.05, 0.04, 0.02, and 0.01 mg/kg, respectively. Metal concentrations measured in all samples were higher than the detection limits. Metal contents in certified reference material (lobster hepatopancreas) from the National Research Council, Canada were determined with the same procedure used for the samples to assure quality control. The measured values (mg/kg) for Cu, Zn, Cd, and Pb in 0.001-mg samples were the following (means and standard deviations, certified values in parentheses): Cu: 127 ± 21 (106 ± 10); Zn: 145 ± 28 (180 ± 6); Cd: 30 ± 4.5 (26.7 ± 0.6); and Pb: 0.32 ± 0.67 (0.35 ± 0.13). All measurements were within the acceptable range for the certified reference material.

Determining organic compounds contents in mites

Benzo[a]pyrene concentrations in mites were determined by extraction using accelerated solvent extraction with dichloromethane/hexane (80/20%, v/v) solvent at a temperature of 160°C and pressure of 231 psi. For every concentration of each compound, measurements were done in triplicate. For every batch of mites that was digested, a blank was prepared to detect and eliminate possible contamination during the digestion process, as was done in the case of the metals. This was followed by characterization of the extract by high-performance liquid chromatography with fluorescence detection. A 10-µl aliquot of each sample was injected onto a Varian PAH Pursuit (3-µm particle size, 100-mm length, and 4.6-mm inner diameter) column guarded with a Varian MetaGuard 3 µm C18 4.6-mm column. The column was maintained at 28°C using a column heater. The runtime was set to 30 min, and the mobile phase for the high-performance liquid chromatography was 60:40 acetonitrile:water. The water samples were diluted with acetonitrile and injected into the high-performance liquid chromatography system. Samples were quantified by comparing the area under the chromatogram peak with areas of known concentrations of the samples. The correlation (r2) of the test substance standard curve was greater than 0.90 for the test substance. Detection limits were estimated from the standard deviation of procedural blanks and were found to be 0.15 pg for BaP. Geraniol concentrations in mites were not assessed.

Assessing the pH effect

Because the pH of soils receiving high metal salts decreased substantially because the SOmath image and NOmath image increased, quantifying the effect of pH change on mite reproduction was necessary. The pH of the OECD soil was adjusted with either H2SO4 or CaCO3 to pH values ranging from 3.1 to 8.1. Four replicates of the five treatment groups were used, and experimental procedures were as described previously. Survival and reproduction were assessed after 35 d.


Data were presented as mean ± standard error. The LC50 values (lethal concentration at which 50% of the population were killed) were calculated using the trimmed Spearman-Karber method 19. The IC50s (effect concentrations causing 50% inhibition of reproduction) for reproduction were estimated with reparameterized regression models for exponential, logistic (three-parameter), logistic (four-parameter), Gompertz, and hormetic concentration–response curves, which have been described previously 19. The regression model with the highest adjusted r2 value was selected to calculate the inhibitive concentrations at a 50% level. However, in cases in which none of the models showed a good fit or when either the assumption of normality or homogeneity of variance was not met, those data were reanalyzed using linear interpolation, and the expanded confidence limits were used because fewer than seven replicates existed per treatment. The biota–soil accumulation factor (BSAF) for each metal was estimated from the slope of the relationship between concentrations of metals in mites and the concentrations of metals in soil. The relationship between mite metal burden and metal concentrations in soil was done with the Spearman-Rank correlation. Normality and homogeneity of variance for the data of the survival and number of juveniles produced at different pH regimens were assessed with Shapiro-Wilk's and Levene's test, respectively. Because the data did not violate the assumptions of normality and homogeneity of variance, the data were analyzed with analysis of variance, and the least significant difference test was used for post hoc comparison. All statistical analyses were done using SYSTAT 13 software.


  1. Top of page
  2. Abstract
  8. Acknowledgements

pH changes and pH effect

With increased metal concentration, a significant decrease occurred in soil pH in all treatments for all metals. For Cu and Zn, spiking with the highest metal concentration reduced the mean pH from 6.15 in the control to 4.54 and 5.11, respectively. For Cd and Pb, spiking with the highest concentration reduced the mean pH to 5.93 and 4.15, respectively. From these initial values, pH values increased slightly in all treatment groups after a 35-d exposure period. The overall range for pH increase in all treatments was 0.23 to 0.48.

Spiking the soil with either geraniol or BaP did not change the pH of the soil substantially. Soil pH at the highest concentration of geraniol was 6.2, compared with 6.1 for the control soil. Soil pH at the highest concentration of BaP was 6.3. However, pH values increased slightly in all treatment groups after a 35-d exposure period. The overall range for pH increase in all treatments was 0.38 to 0.49.

No significant difference (analysis of variance, p > 0.05) was found in mite survival among the different pH treatments. However, a significant difference (analysis of variance, p < 0.05) in juvenile production was found as the pH values increased (Fig. 1). Soil treatments with pH values above 7.3 decreased (least significant difference, p < 0.05) juvenile production when compared with those of standard soil (pH = 6.1) and those of pH values below that of standard soil.

thumbnail image

Figure 1. Survival and juvenile production in the oribatid mite Oppia nitens after 35-d exposure in Organisation for Economic Co-operation and Development soil of different pH regimens. Symbols represent the average of five replicates, with 10 mites added per replicate test. Acidity was adjusted using H2SO4 or CaCO3. Asterisks indicate juvenile production significantly different from that at pH 6.0 at 0.05 significance.

Download figure to PowerPoint


Effects on survival and reproduction

The proportions of adult survival and those of juveniles produced when the mites were exposed to the four metals are shown in Figure 2A through D. Generally, all four metals showed a clear dose–response curve on survival, but those of Cu and Zn were steeper. The LC50 values and confidence intervals (CI) for Cu, Zn, Cd, and Pb were 3,311 (3,020–3,631); 2,291 (1,995–2,630); 603 (537–691); and 6,761 (5,623–8,128) mg/kg, respectively. For reproduction, a clear dose–response curve was only observed for Cd and Pb. Hormetic effect on juvenile production was observed for Cu and Zn, up to 2,000 mg/kg Cu and 1,000 mg/kg Zn, respectively, after which a sharp decline occurred in juvenile production by the mites (Fig. 2A,B). The IC50 values and confidence intervals for Cu, Zn, Cd, and Pb were 2,896 (2,375–2,938); 1,562 (1,363–1,952); 137 (73–258); and 1,678 (1,066–2,290) mg/kg, respectively. In the case of Cu and Zn, where strong hormetic effects were observed, the control groups used for calculating IC50 were those that received no metal treatment.

thumbnail image

Figure 2. Mean (± standard error) survival and reproduction of four groups, each consisting of 10 mites (Oppia nitens) after exposure for 28 or 35 d in Organisation for Economic Co-operation and Development soil spiked with four metals: (A) Cu, (B) Zn, (C) Cd, and (D) Pb. Test duration was 28 d for Zn and 35 d for the other metals.

Download figure to PowerPoint

Metal concentrations in mites

The metal concentrations in the mites at test end in relation to the nominal metal concentration in the soil are presented in Figure 3. All of the four metals were accumulated to varying degrees in the mites over the test period. The mite O. nitens exhibited a dose-dependent accumulation of Cd, Cu, Pb, and Zn, except at the highest concentration of Zn and Pb. For Zn and Pb, the lower accumulation of metals at the highest concentrations in comparison with the preceding concentration was mostly because only a few mites survived at these concentrations, and they were the only ones digested for the metal analysis. This could have introduced a margin of error in the analysis, considering the small size of the mites. All tested metals showed a strong link between mite body burden and soil concentration (r2 between 0.70 and 0.99, p < 0.05; Fig. 3). Zinc and Cd were the most highly accumulated metals, with mite Zn or Cd concentrations being equal to soil concentrations in some instances. For example, internal Zn concentration was 2,118 mg/kg, similar to the concentration spiked into the soil (2,000 mg/kg), whereas internal Cd concentration was approximately 300 mg/kg when soil Cd concentration was 400 mg/kg. The estimated BSAF was highest for Zn (1.07) followed by Cd (0.71), Pb (0.42), and Cu (0.12).

thumbnail image

Figure 3. Metal (Cu, Zn, Cd, and Pb) concentrations (y axis) in the mite Oppia nitens after 28- or 35-d exposure plotted against nominal soil metal concentration (x axis) spiked into Organisation for Economic Co-operation and Development soil. Test duration was 28 d for Zn and 35 d for the other metals.

Download figure to PowerPoint

Organic chemicals

Effects on survival and reproduction

Geraniol was more toxic than BaP regardless of whether survival or reproduction was considered (Fig. 4A, B). A dose–response relationship was only observed for geraniol and survival of mites. However, the curve was steep, with survival not being affected up to 200 mg/kg geraniol but with total mortality occurring at 400 mg/kg. As in the case for Cu and Zn, hormetic effect on juvenile production was observed for geraniol at up to 200 mg/kg, after which a sharp decline in juvenile production by the mites occurred (Fig. 4A). The LC50 and IC50 values with confidence intervals for geraniol were 234 and 283 (256–283) mg/kg, respectively. Confidence interval could not be computed for the LC50 value. No significant mortality was found at all concentrations of BaP used. Hormetic effect on juvenile production was found up to 800 mg/kg BaP, after which juvenile production became similar to that of control mites at 1,600 mg/kg BaP. Therefore, LC50 and IC50 values could only be estimated as greater than 1,600 mg/kg BaP.

thumbnail image

Figure 4. Mean (± standard error) survival and reproduction of four groups, each consisting of 10 mites (Oppia nitens) after exposure for 35 d in Organisation for Economic Co-operation and Development soil spiked with (A) geraniol and (B) benzo[a]pyrene.

Download figure to PowerPoint


After 35 d of exposure, only trace values (below detection limit) of BaP were found in the mites irrespective of concentrations of B[a]P spiked in soil.


  1. Top of page
  2. Abstract
  8. Acknowledgements

The present study found that the oribatid mite O. nitens showed a clear difference in sensitivity to four metals (Cu, Zn, Cd, and Pb) and two organic compounds (geraniol and BaP) based on survival and reproduction. Spiking of soil with metal salt reduced the pH of soil sometimes to values up to two units below those of unspiked soils with a mean pH of 6.1. However, additional experiments conducted to assess the influence of pH on mite reproduction showed no significant difference in mite reproduction within pH of 3.1 and 6.1. In a recent study, the mite O. nitens was reported to be tolerant to a relatively large soil pH range (3.9–7.5) 9. Previous studies have also shown that micro-arthropods thrive in acidic soils 20. These results suggest that the direct impact of pH changes on the toxicity of chemicals in the present study will be negligible. It is well known, however, that the bioavailability of metals and organic compounds increases with reduced pH 21. The present study was not aimed at assessing the bioavailability of metals; therefore we could not account for any increase in bioavailability of chemicals as a result of pH reduction in the present study.

When the test lasted for 28 d (in the case of Zn), the mean number of juvenile produced in control OECD soil in the present study was 24.5. This is similar to the mean number of juveniles of 19 reported in OECD soil by an earlier study 9. When test duration was extended to 35 d (for other compounds) in the present study, the mean number of juveniles in the control soils increased to 61.5. This suggests that extending the test duration by an additional week might help achieve more reproduction in control soils and therefore might help in getting a better dose–response curve than 28 d as previously assessed.


Cadmium was clearly the most toxic of the metals to the oribatid mite O. nitens, having the lowest LC50 and IC50 values. Lower toxicity of Zn, Cu, and Pb to the mites compared with Cd observed in the present study agrees with reports available for other oribatids in the literature. Cadmium had more detrimental effects than Pb and Cu on reproduction of P. peltifer 22, A. longisetosus 23, or Pergalumna nervosa 24. This is similar to the finding of Khalil et al. 25, who studied the association and presence of mites in metal-contaminated field soil and reported that only Cd among the metals present had an effect on the community structure of mites. Although most of these studies with other mite species used exposure via food whereas we used exposure via soil, the difference in the route of exposure appears to not make a significant impact on the relative toxicity of these metals to mites. These results obtained for mites are similar to findings with collembolan species F. candida 26 and Proisotoma minuta 27, in which Cd was the most toxic of the four metals studied.

Differences in experimental and exposure conditions and test duration hamper a straightforward comparison of data of the present study with those available for other mite species as well as other soil invertebrate groups. However, compared with other mite species, O. nitens appears to have similar sensitivity to Cd as P. peltifer and is probably more sensitive to Cu than H. aculeifer (Table 1). The toxicity of these metals to the mite O. nitens when compared with their toxicity to the collembola, earthworm, enchytraeid, and isopod species commonly used in toxicity tests (Table 1) showed that the mite O. nitens is less sensitive to Cu and Zn but is within the same order of magnitude for Cd and Pb when compared with other soil invertebrates. When reproduction IC50 values for the least sensitive species were ranked to the 100th percentile and the IC50 values for other more sensitive species were relatively ranked with respect to that of the least sensitive species, for each metal, it became very clear that for Cd and Pb the mite O. nitens is within the 40th to 60th percentile of toxicity for the least sensitive species, whereas for Cu and Zn, it is clearly the least sensitive species (Fig. 5). For hydrocarbon- and salt-contaminated soils from the boreal regions in Canada, the mite O. nitens was reported to have intermediate sensitivity when compared with standard and boreal earthworm (Eisenia fetida and Dendrodrilus rubidus) and collembolan (Folsomia candida, F. nivalis, and P. minuta) species 28. Therefore, despite its lower sensitivity to Cd and Pb when compared with other invertebrates, its use as an additional test species representing a group mostly that is underrepresented in ecotoxicological evaluations is recommended.

Table 1. Comparison of LC50 and EC50 values (mg/kg) derived in toxicity of selected metals to different groups of soil invertebrates
Groups/speciesDuration (days)CuZnCdPb
  • a

    Present study.

    IC50 = median inhibitory concentration; EC50 = median effective concentration.

 Oppia nitens353,311a2,896a2,291a1,562a603a137a6,761a1,678a
 Hypoaspis aculeifer14

>1,500 44

; >3,746 45

674 44      
 Platynothrus peltifer49    297 29246 29  
 63    817 46   
 108    357 46   
 Sinella communis28 400.3 20  374 2050.1 20  
 Sinella coeca42    11.8 4727.6 47 490 47
 Sinella umesaoi28     40.9 48  
 Folsomia candida281,810 20

700 49

; 751 20

5,150 50

865 20

; 375 50

; 900 49

2,310 20

315 20

; 590 49


2,560 20

; 2,970 49

 35    854 29>326 29  
 42   6835165.9 47


; 47.4 47

 Proisotoma minuta281,180 20157.5 47      
 42 696 27 283 27 125 27 >2,000 27
 Folsomia fimetaria21 129 52      
 Orchesella cincta63    177 2985.5 29  
 Onychiurus yodai28     154.7 48  
 Eisenia fetida14683 53 1,010 53 >300 53   
 21836 54715 54


; 1,078 54

705 50

; 357 54

>300 54

; 1,260 55

108 55

; 295 54

>10,000 541,629 54
 28764 11316 11      
 56  745 53462 53>300 53   
 Aporrectodea caliginosa56    540 5634.8 56  
 Lumbricus terrestris14    256 57   
 Lumbricus rubellus28  728 58348 58    
 Enchytraeus albidus 799 59305 60566 50267 50476 55158 5511,400 59320 60
 Enchytraeus crypticus42   188 61    
 Porcellio scaber633,755 441,858 44  270 29   
thumbnail image

Figure 5. Comparison of rank percentile obtained from reproduction median effective concentration (EC50) values derived in toxicity of metals (Cu, Zn, Cd, and Pb) to the mite Oppia nitens and other major groups of soil invertebrates commonly used in toxicity tests. Data used were obtained from the present study and previous studies cited in Table 1.

Download figure to PowerPoint

The toxicity of the metals investigated here to O. nitens reflects their essential (Cu and Zn) and nonessential (Cd and Pb) statuses for invertebrates. Copper and Zn had similar IC50 and LC50 values and within the same order of magnitude, whereas Cd and Pb have IC50 values within four to five orders of magnitude of the LC50 values. This is an indication that for the essential metals Cu and Zn, the organisms were not affected at the onset of accumulation, because the metals are essential for normal metabolic activities in these organisms. The mites likely kept accumulating these metals with little effort at depuration and elimination even when the threshold for toxicity was overreached, leading to sudden breakdown in metabolic, depuration, and elimination activities and therefore concurrent sudden effect on survival and reproduction. This assumption was supported by the accumulation data for Zn, which reached a very high value at 2,000 mg/kg Zn, the same concentration at which sudden effect on survival and reproduction was more pronounced. For Cd and Pb, which are known to be nonessential metals, the metabolic instruments of the mites were sensitive to increasing concentrations; therefore, the mites quickly metabolized the compounds, with a progressive toxic effect (first on reproduction and later on survival) at increasing exposure concentrations. Crommentuijn et al. 29 introduced the sublethal sensitivity index as the ratio of the LC50 to the no observed effect concentration for reproduction. A large value for the sublethal sensitivity index indicates that reproduction is inhibited at a level far below the LC50; a low value for the sublethal sensitivity index indicates that reproduction is maintained until the organism's death. Van Straalen 30 argued that the sublethal sensitivity index will depend not only on the life-history strategy of the species, but also on the mode of action of the substance. For example, for chemicals that act on the nervous system, the critical thresholds being exceeded would lead directly to the organism's death without first introducing sublethal effects on reproduction. On this basis, the modes of action of Cu and Zn to O. nitens are similar and in contrast with those of Cd and Pb.

Hormetic effect of low concentrations of the essential metals Cu and Zn but not the nonessential metals Cd and Pb was found on juvenile production by the mites in the present study. Hormesis has been defined as a diphasic dose–response characterized by low-dose stimulation and a high-dose inhibitory or toxic effect 31. It is often associated with, but not limited to, essential elements. For example, apart from Cu, such hormesis has been reported with small doses of Pb on the fecundity of P. peltifer 22 and A. longisetosus 23 and for oribatid mite communities along a gradient of heavy metal contamination 2. Moreover, hormetic effect has been reported at low concentration of Pb for other soil organisms. For example, a low concentration of Pb stimulated juvenile production in the earthworm E. fetida and Perionyx excavatus 32 and the collembolan P. minuta 27. The hormetic effect found for geraniol and BaP further showed that not only essential metals but also organic compounds can induce hormesis in mites.

In the present study, the mite O. nitens accumulated the four metals to varying degrees. The fact that mites accumulate metals to very high internal concentrations is well known 2, 33. With respect to concentrations spiked, the pattern of BSAF in O. nitens is as follows: (Cu < Pb < Cd < Zn). Zaitsev 34 found a slightly similar pattern of BSAF (Cu < Zn < Cd) in nine species of oribatid mites in the vicinity of a metallurgical plant in Russia. This is contrary to a similar study on mites 2 in which BSAF increased along the range (Zn < Cd < Cu). That study reported, however, that species-specific BSAF of metals was evident. The difference between the pattern of BSAF for the metals in the present study and this previous study 2 could be explained by differences in mite species, because their study did not include O. nitens. Bioaccumulation in O. nitens increased as soil concentrations increased for all four metals studied (Fig. 3). This pattern of increased accumulation of metals by O. nitens with increased soil concentration was also observed for three mite species (Oribatula tibialis, Eupelops tardus, and Adoristes ovatus) and Cu 2. For Oppiella nova and Cu, as well as E. tardus and A. ovatus with Zn, the mites seemed to prevent increased accumulation as soil concentration increased 2. These data suggest a complex interaction regarding concentration and accumulation, depending on the mite species and metals involved.

Organic compounds

For BaP, a no significant effect on survival and reproduction was seen within the maximum concentration (1,600 mg/kg BaP) used in the present study. Lack of effect on reproduction of such relatively high concentrations of BaP on the mite is not very surprising. An overview of toxicity data for the effects of BaP on soil organisms has been given by Sverdrup et al. 7. All except one of these studies reported low toxicity of BaP to soil organisms. For example, BaP concentrations of up to 840 mg/kg and 931 mg/kg were found not to be toxic to the collembolans Folsomia fimetaria 35 and F. candida 6, respectively. Similarly, a concentration of up to 1,000 mg/kg BaP had no toxic effect on the survival and reproduction of the enchytraeid Enchytraeus crypticus and the mite H. aculeifer 7. A concentration of 48,000 mg/kg BaP was found to be nontoxic to the earthworm E. fetida 14 in OECD soil. However, Achazi et al. 13 found significant toxicity of BaP to the enchytraeid E. crypticus and earthworm E. fetida at a concentration of 10 mg/kg in a natural soil. The data of Achazi are still being used as the main reference for invertebrate data and for setting soil quality criteria in many countries, including Canada 36, 37. With more recent data indicating low toxicity of BaP to many invertebrates, one could argue that BaP is generally less toxic to soil organisms 7 and might pose low environmental concern on soil invertebrates. This is in view of low environmental concentrations of BaP reported in different countries that were mostly less than 300 µg/kg 38–40.

Benzo[a]pyrene did not accumulate in the mites after a 35-d exposure. Many PAHs are metabolized in insects via the cytochrome P450 pathways 41. Metabolism of PAH often takes place within a few days of uptake, however (J. Princz, Environment Canada, Ontario, Canada, personal communication). Because we did not assess uptake until many weeks later, establishing whether initial accumulation or metabolism of BaP occurred in the mites was difficult. However, BaP could be metabolized to diol-epoxide metabolites, which are considerably more mutagenic than the parent compound 42. Further studies monitoring the fate of BaP and its probable transformation in the mites are warranted.

In the present study, geraniol was found to be very toxic to the mite O. nitens. The high toxicity of this natural product contrasts sharply with the low toxicity of the xenobiotic compound BaP. The lower toxicity of BaP in comparison with geraniol might be related to its lower water solubility of approximately 0.01 mg/L compared with 100 mg/L for geraniol. Geraniol is one of the most important molecules in the flavor and fragrance industries and is a common ingredient in consumer products produced by these industries. In Europe, its production exceeds 1,000 metric tons per annum 43. Its high production and negative effect on O. nitens suggest that it may have a negative consequence for oribatid mites and other soil organisms. When used for industrial purposes, proper disposal methods should be used to avoid negative effects on soil organisms.


  1. Top of page
  2. Abstract
  8. Acknowledgements

The present study investigated the toxicity and accumulation of four metals (Cu, Zn, Cd, and Pb) and two organic compounds (geraniol and benzo[a]pyrene) to the oribatid mite O. nitens. Among the metals, Cd was the most toxic metal, and this agrees with most studies on metal toxicity to soil invertebrates. The mite O. nitens was a good accumulator of all four metals; therefore, subsequent kinetic data might reveal patterns of accumulation. Kinetic studies are of particular importance and may provide more useful toxicological information on time-dependent uptake patterns of metals in O. nitens. The strong hormetic response on mite reproduction observed for benzo[a]pyrene, geraniol, as well as Cu and Zn indicated that metals and organics could elicit hormetic response in this mite. Care should be taken in interpreting results when only a few concentrations of these compounds are used in ecotoxicity studies. Overall, the results of the present study and other studies 28 suggest that the mite O. nitens has intermediate sensitivity to Cd, Pb, hydrocarbon, and salt when compared with other soil invertebrates commonly used in toxicity tests but has lower sensitivity to Cu and Zn. Oribatid mites are the most numerous group of soil invertebrates but generally not well represented in ecotoxicological evaluations of contaminated soils in boreal and other areas. Despite lower sensitivity to some compounds, the use of O. nitens as an additional test species will provide a robust risk assessment of contaminated boreal and other northern soils 28.


  1. Top of page
  2. Abstract
  8. Acknowledgements

The present study received financial support from a Natural Sciences and Engineering Research Council Community Research and Development grant to S.D. Siciliano.


  1. Top of page
  2. Abstract
  8. Acknowledgements
  • 1
    Barber I, Bembridge J, Dohmen P, Edwards P, Heimbach F, Heusel R, Romijn K, Rufli H. 1998. Development and evaluation of triggers for earthworm toxicity testing with plant protection products. In Sheppard S, Bembridge J, Holmstrup M, Posthuma L, eds, Proceedings, Advances in Earthworm Ecotoxicology: 2nd International Workshop on Earthworm Ecotoxicology, April 2-5, Amsterdam, The Netherlands. SETAC, Pensacola, FL, USA, pp 269278.
  • 2
    Skubala P, Kafel A. 2004. Oribatid mite communities and metal bioaccumulation in oribatid species (Acari, Oribatida) along the heavy metal gradient in forest ecosystems. Environ Pollut 132: 5160.
  • 3
    Kohler H, Alberti G, Seniczak S, Seniczak A. 2005. Lead-induced hsp70 and hsp60 pattern transformation and leg malformation during postembryonic development in the oribatid mite, Archegozetes longisetosus Aoki. Comp Biochem Physiol Part C 141: 398405.
  • 4
    Seniczak A, Ligocka A, Seniczak S, Paluszak Z. 2009. The influence of cadmium on life-history parameters and gut microflora of Archegozetes longisetosus (Acari: Oribatida) under laboratory conditions. Exp Appl Acarol 47: 191200.
  • 5
    VanGestel CAM, Doornekamp A. 1998. Tests on the oribatid mite Platynothrus peltifer. In Løkke H, Van Gestel CAM, eds, Handbook of Soil Invertebrate Toxicity Tests. John Wiley & Sons, Chichester, pp. 113130.
  • 6
    Bleeker EAJ, Wiegman S, Droge STJ, Kraak MHS, Van Gestel CAM. 2003. Towards an improvement of the risk assessment of polycyclic (hetero)aromatic hydrocarbons. Report 2003-04 of the Institute of Ecological Science, Vrije Universiteit, Amsterdam, The Netherlands.
  • 7
    Sverdrup LE, Hagen SB, Krogh PH, van Gestel CAM. 2007. Benzo(a)pyrene shows low toxicity to three species of terrestrial plants, two soil invertebrates, and soil-nitrifying bacteria. Ecotoxicol Environ Saf 66: 362368.
  • 8
    Organisation for Economic Co-Operation and Development. 2008. Guideline for testing of chemicals No. 226. Predatory mite reproduction test in soil (Hypoaspis (Geolaelaps) aculeifer). Paris, France.
  • 9
    Princz JI, Behan-Peletier VM, Scroggins RP, Siciliano SD. 2010. Oribatid mites in soil toxicity testing: The use of Oppia nitens (C.L. Koch) as a new test species. Environ Toxicol Chem 29: 971979.
  • 10
    Gimmler H, Carandang J, Boots A, Reisberg E, Woitke M. 2002. Heavy metal content and distribution within a woody plant during and after seven years continuous growth on municipal solid waste (MSW) bottom slag rich in heavy metals. J Appl Bot 76: 203217.
  • 11
    Owojori OJ, Reinecke AJ, Rozanov AB. 2009. The combined stress effects of salinity and copper on the earthworm Eisenia fetida. Appl Soil Ecol 41: 277285.
  • 12
    International Agency for Research on Cancer. 2010. Some non-heterocyclic polycyclic aromatic hydrocarbons and some related exposures. IARC monographs on the evaluation of carcinogenic risk of chemicals to humans, vol 92. Lyon, France, pp 394423.
  • 13
    Achazi RK, Chroszcz G, Duker C, Henneken M, Rothe B, Schaub K, Steudel I. 1995. The effect of fluoranthene (Fla), benzo(a)pyrene (B[a]P) and cadmium (Cd) upon survival rate and life cycle parameters of two terrestrial annelids in laboratory tests systems. Newslett Enchytraeidae 4: 714.
  • 14
    Canadian Council of Ministers of the Environment. 1997. Recommended Canadian Soil Quality Criteria. Report from the Canadian Council of Ministers of the Environment (CCME), ISBN: 1-895-925-92-4, Ottawa, Ontario.
  • 15
    Zahran HEM, Abdelgaleil SAM. 2011. Insecticidal and developmental inhibitory properties of monoterpenes on Culex pipiens L. (Diptera: Culicidae). J Asia-Pacific Entomology 14: 4651.
  • 16
    Organisation for Economic Co-operation and Development. 2004. Earthworm reproduction tests (Eisenia fetida/Eisenia andrei): Guideline for the testing of Chemicals. 222. Paris, France
  • 17
    Soil Conservation Service. 1984. Soil survey laboratory methods and proceedings for collecting soil samples. Soil Survey Investigation, Report 1. U.S. Government Printing Office, Washington, DC.
  • 18
    International Organization for Standardisation. 1996. Soil quality-effects of pollutants on earthworms (Eisenia fetida). Part 2: Determination of effects on reproduction, No. 11268-2. Geneva, Switzerland.
  • 19
    Environment Canada. 2005. Guidance Document. Statistical Methods for Environmental Toxicity tests. ESP 1/RM/46. Environmental Protection Series, Ottawa, Ontario.
  • 20
    Greenslade P, Vaughan G. 2003. A comparison of Collembola species for toxicity testing of Australian soils. Pedobiologia 47: 171179.
  • 21
    Alexander M. 2000. Aging, bioavailability, and overestimation of risk from environmental pollutants. Environ Sci Tech 29: 27132717.
  • 22
    Denneman CAJ, Van Straalen NM. 1991. The toxicity of lead and copper in reproduction tests using the oribatid mite Platynothrus peltifer. Pedobiologia 35: 305311.
  • 23
    Seniczak A, Ignatowicz S, Seniczak S. 2000. Effect of some heavy metals on the bionomy of mite Archegozetes longisetosus (Acari, Oribatida) in the laboratory conditions. Ecol Chem Eng 10: 10851091.
  • 24
    Seniczak A. 2007. Preliminary studies on the toxicity of copper and lead in Pergalumna nervosa (Berlese, 1914) (Acari, Oribatida) in laboratory tests. In Tajovský K, Schlaghamerský J, Piql V, eds, Contributions to Soil Zoology in Central Europe. II. Beské Bud5jovice, Czech Republic, pp 131134.
  • 25
    Khalil MA, Janssens TKS, Berg MP, Van Straalen NM. 2009. Identification of metal-responsive oribatid mites in a comparative survey of polluted soil. Pedobiologia 52: 207221.
  • 26
    Fountain MT, Hopkin SP. 2001. Continuous monitoring of Folsomia candida (Insecta: Collembola) in a metal exposure test. Ecotoxicol Environ Saf 48: 275286.
  • 27
    Nursita AI, Singh B, Lees E. 2003. The effects of cadmium, copper, lead, and zinc on the growth and reproduction of Proisotoma minuta Tullberg (Collembola). Ecotoxicol Environ Saf 60: 306314.
  • 28
    Princz JI, Moody M, Fraser C, Van Der Vliet L, Lemieux H, Scroggins R, Siciliano SD. Evaluation of a new battery of toxicity tests for boreal forest soils: Assessment of the impact of hydrocarbon and salts. Environ Toxicol Chem 31: 766777.
  • 29
    Crommentuijn T, Doodeman CJAM, Van Der Pol JJC, Doornekamp A, Rademaker MCJ, Van Gestel CAM. 1995. Sublethal sensitivity index as an ecotoxicity parameter measuring energy allocation under toxicant stress: Application to cadmium in soil arthropods. Ecotoxicol Environ Saf 31: 192200.
  • 30
    Van Straalen NM. 2000. Physiological and ecological factors determining variability of ecotoxicological responses in animals. In Badejo MA, Van Straalen NM, eds, Pollutants and Their Effects on Terrestrial and Aquatic Ecosystems. College Press Limited, Ibadan, Oyo State, Nigeria.
  • 31
    Calabrese EJ, Baldwin LA. 2003. Toxicology rethinks its central belief. Nature 421: 691692.
  • 32
    Maboeta MS, Reinecke AJ, Reinecke SA. 1999. Effects of low levels of lead on growth and reproduction of the Asian earthworm Perionyx excavatus (Oligochaeta). Ecotoxicol Environ Saf 44: 236240.
  • 33
    Zaitsev AS, Van Straalen NM. 2001. Species diversity and metal accumulation in oribatid mites (Acari, Oribatida) of forests affected by a metallurgical plant. Pedobiologia 45: 467479.
  • 34
    Zaitsev AS. 1999. Metal accumulation by oribatid mites in the surroundings of the Kosogorsky metallurgical plant. In Butovsky RO, Van Straalen NM, eds, Pollution-Induced Changes in Soil Invertebrate Food-Webs, vol 4. Amsterdam and Moscow, Vrije Universiteit, Amsterdam, Netherlands, pp 5170.
  • 35
    Sverdrup LE, Nielsen T, Krogh PH. 2002. Soil ecotoxicity of polycyclic aromatic hydrocarbons in relation to soil sorption, lipophilicity, and water solubility. Environ Sci Tech 36: 24292435.
  • 36
    Kalf DK, Crommentuijn T, van de Plassche EJ. 1997. Environmental quality objectives for 10 polycyclic aromatic hydrocarbons (PAHs). Ecotoxicol Environ Saf 36: 8997.
  • 37
    Canadian Council of Ministers of the Environment. 2008. Canadian Soil Quality Guidelines. Carcinogenic and other polycyclic aromatic hydrocarbon (PAHs) (Environmental and Human Health Effects). Scientific Supporting Document. Winnipeg, MB, Canada.
  • 38
    Wild SR, Jones KC. 1995. Polynuclear aromatic hydrocarbons in the United Kingdom environment: A preliminary source inventory and budget. Environ Pollut 88: 91108.
  • 39
    Nadal AM, Schuhmachera M, Domingo JL. 2004. Levels of PAHs in soil and vegetation samples from Tarragona County, Spain. Environ Pollut 132: 111.
  • 40
    Crnković D, Ristić M, Jovanović A, Antonović D. 2007. Levels of PAHs in the soils of Belgrade and its environs. Environ Monit Assess 125: 7583.
  • 41
    Van Pottelberge S, Van Leeuwen T, Van Amermaet K, Tirry L. 2008. Induction of cytochrome P450 monooxygenase activity in the two-spotted spider mite Tetranychus urticae and its influence on acaricide toxicity. Pesticide Biochem Physiol 91: 128133.
  • 42
    Raszinsky K, Basler A, Rohrborn G. 1979. Mutagenicity of polycyclic hydrocarbons. V. Induction of sister chromatid exchanges in vivo. Mutat Res 66: 65.
  • 43
    Rastogi SC, Heydorn S, Johansen JD, Basketter DA. 2001. Fragrance chemicals in domestic and occupational products. Contact Dermatitis 45: 221225.
  • 44
    Rundgren S, Van Gestel CAM. 1998. Comparison of species sensitivity. In Lokke H, van Gestel CAM, eds, Handbook of Soil Invertebrate Toxicity Tests. John Wiley and Sons, West Sussex, UK.
  • 45
    Scholer VVC. 2006. Optimierrung des raubmilben-reproduktionstestes (Hypoaspis aculeifer, Canestrini [Acari, Laelapidae)] fur im boden persistente substanzen. Diploma thesis. Submitted to Fachhochschule Bingen (in German).
  • 46
    Van Straalen NM, Schobben JHM, De Goede RGM. 1989. Population consequences of cadmium toxicity in soil microarthropods. Ecotoxicol Environ Saf 17: 190204.
  • 47
    Menta C, Maggiani A, Vattuone Z. 2006. Effects of Cd and Pb on the survival and juvenile production of Sinella coeca and Folsomia candida. Eur J Soil Biol 42: 181189.
  • 48
    Nakamori T, Yoshida S, Kubota Y, Ban-nai T, Kaneko N, Hasegawa M, Itoh R. 2008. Sensitivity to cadmium of the standard test species Folsomia candida compared to two other species, Onychiurus yodai and Sinella umesaoi (Collembola). Eur J Soil Biol 44: 266270.
  • 49
    Sandifer RD, Hopkin SP. 1996. Effects of pH on the toxicity of cadmium, copper, lead and zinc to Folsomia candida Willem, 1902 (Collembola) in a standard laboratory test system. Chemosphere 33: 24752486.
  • 50
    Lock K, Janssen CR. 2001. Modeling zinc toxicity for terrestrial invertebrates. Environ Toxicol Chem 20: 19011908.
  • 51
    Van Gestel CAM, Hensbergen PJ. 1997. Interaction of Cd and Zn toxicity for Folsomia candida Willem (Collembola:Isotomidae) in relation to bioavailability in soil. Environ Toxicol Chem 16: 11771186.
  • 52
    Scott-Fordsmand JJ, Krogh PH, Weeks JM. 1997. Sub-lethal toxicity of copper to a soil-dwelling Springtail (Folsomia fimetaria) (Collembola: Isotomidae). Environ Toxicol Chem 16: 25382542.
  • 53
    Spurgeon DJ, Hopkin SP, Jones DT. 1994. Effects of cadmium, copper, lead and zinc on growth, reproduction and survival of the earthworm Eisenia fetida (Savigny): Assessing the environmental impact of point-source metal contamination in terrestrial ecosystems. Environ Pollut 84: 123130.
  • 54
    Spurgeon DJ, Hopkin SP. 1995. Extrapolation of the laboratory-based OECD earthworm test to metal-contaminated field sites. Ecotoxicology 4: 190205.
  • 55
    Lock K, Janssen CR. 2001. Cadmium toxicity for terrestrial invertebrates: Taking soil parameters affecting bioavailability into account. Ecotoxicology 10: 315322.
  • 56
    Khalil MA, Abdel-Lateif HM, Bayoumi BM, Van Straalen NM, Van Gestel CAM. 1996. Effects of metals and metal mixtures on survival and cocoon production of the earthworm Aporrectodea caliginosa. Pedobiologia 40: 548556.
  • 57
    Fitzpatrick LC, Muratti-Ortiz JF, Venables BJ, Goven AJ. 1996. Comparative toxicity in earthworms Eisenia fetida and Lumbricus terrestris exposed to cadmium nitrate using artificial soil and filter paper protocols. Bull Environ Contam Toxicol 57: 6368.
  • 58
    Van Gestel CAM, Dirven-van Breemen EM, Baerselman R. 1993. Accumulation and elimination of cadmium, chromium and zinc and effects on growth and reproduction in Eisenia andrei (Oligochaeta, Annelida). Sci Total Environ (Suppl): 585597.
  • 59
    Lock K, Janssen CR. 2001. Test design to assess the influence of soil characteristics on the toxicity of copper and lead to the oligochaete Enchytraeus albidus. Ecotoxicology 10: 137144.
  • 60
    Lock K, Janssen CR. 2002. Mixture toxicity of zinc, cadmium, copper and lead to the potworm Enchytraeus albidus. Ecotoxicol Environ Saf 52: 17.
  • 61
    Posthuma L, Baerselman R, Van Veen RPM, Dirven-Van Breemen EM. 1997. Single and joint toxic effects of copper and zinc on reproduction of Enchytraeus crypticus in relation to sorption of metals in soils. Ecotoxicol Environ Saf 38: 108121.