Collembola growth in heavy metal-contaminated soils

Abstract

Collembola play a key role in soil ecosystems by decomposing organic matter. Most of them inhabit the upper layers of the soil and are susceptible to contamination present in the pore water. These arthropods serve as model organisms in ecotoxicology for short and long-term exposure. This study aimed to assess the toxicity of three heavy metals (lead [Pb], cadmium [Cd], and copper [Cu]) using the springtail species Folsomia candida as the test organism, with mortality and growth inhibition as the measure of toxicity. We hypothesised that increasing metal concentrations in the soil would correspond to a growth reduction of Collembola. Each heavy metal was tested at a minimum of eight increasing concentrations in six replications. Twenty 10-12-day-old individuals were introduced into each test container filled with contaminated or control soil and incubated for 14 days. The test endpoints included growth inhibition determined by comparing F. candida growth rates in contaminated soil with those in control soil, as well as mortality rates. The EC₅₀ values (mg/kg) for heavy metals were as follows: Cd = 66.89, Cu = 791.01, Pb = 10075.48. Our findings suggest that growth inhibition is a reliable indicator of Collembola toxicity to heavy metals.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-79766-5.

Introduction

The soil is a key habitat for biodiversity, and most soil-dwelling organisms participate directly or indirectly in important processes, such as decomposition and nutrient release¹. However, many organisms are sensitive to contamination, and their health serves as crucial bioindicators of soil health². Contaminants have direct or indirect negative effects on these organisms and may further adversely affect soil functionality³. An important and abundant group of soil organisms are springtails (Collembola), which show a high sensitivity to many environmental factors including anthropogenic contamination. These arthropods occur naturally in soils and some species are also used in ecotoxicological tests⁴. Ecotoxicological tests enable an accurate evaluation of environmental risks associated with soil contamination, without the interference of natural environmental factors⁵.

Collembola are primarily invertebrates that inhabit the soil and are closely linked to soil quality⁶. In field conditions, their population size and community structure serve as indicators of soil disturbance^6,7. Collembola live mainly in soil pores and are typically affected by pollutants in the soil solution, such as pesticides and heavy metals⁸. In soil quality assessment, Collembola’s functional characteristics, such as body size or pigmentation, serve as valuable indicators⁹. Ecotoxicological studies typically use two main test species: Folsomia candida (a parthenogenetic species) and Folsomia fimetaria(a sexual species); however, the use of other species with well-documented biology is also feasible¹⁰. F. candida and F. fimetariaare easy to rear under laboratory conditions, and their responses to different contaminants are widely studied worldwide. Additionally, these species occur naturally in soils in Europe and other areas of the world¹⁰.

The body size of an organism is integral to shaping its biology and interactions within its ecological environment, however, there are currently limited studies assessing the relationship between Collembola body size and soil contamination¹¹. Decreases in body size often coincide with exposure to environmental stressors, illustrating the adaptive capabilities of organisms¹². Specifically, alterations in body size and associated biomass of invertebrates are indicative of responses to habitat disturbances such as contamination or limited food resources¹³. Consequently, this characteristic serves as a crucial endpoint in various standard bioassays, including those that employ the earthworms Eisenia fetida¹⁴ or the crustacean ostracod Heterocypris incongruens¹⁵. Numerous studies have shown that heavy metal contamination significantly inhibits Collembola growth, underscoring its value as a sensitive ecotoxicological endpoint. For example, arsenic contamination in soils has been linked to reduced Collembola body size, making growth inhibition an early indicator of pollution⁶. Other research^16,17has demonstrated that cadmium inhibits growth even at low concentrations, with growth proving to be more sensitive to contaminants than reproduction. Additionally, metals such as cadmium and lead have been confirmed to significantly inhibit growth^18,19. Growth reduction has also been shown to produce a toxicity response comparable to reproductive inhibition^19,20. This suggests that growth inhibition may strongly correlate with reproductive success, as faster growth rates likely enable individuals to reach sexual maturity sooner⁶. However, further research is required to fully understand the mechanisms underlying these effects.

This study aimed to assess the growth response of the springtail species F. candidato soil contamination by cadmium (Cd), copper (Cu), and lead (Pb). These metals were selected due to their common presence in contaminated soils and distinct toxicological profiles. Cadmium, recognized for its high toxicity and environmental persistence, often originates from industrial activities and phosphate fertilizers²¹. Copper, although essential in trace amounts, becomes toxic at higher concentrations typically found in agricultural soils due to its use in pesticides²². Lead, a persistent pollutant from various industrial and historical sources, poses significant risks to soil health and biodiversity²³. Collembola toxicity, in terms of mortality and reduced reproduction and growth, has been established with the metals cadmium, copper, and lead^17,20. It was found that cadmium poses the greatest immediate threat to the Collembola species F. candida and Proisotoma minuta, with both low reproductive and survival thresholds (24–26). Copper presents a moderate risk to the same species, affecting reproduction significantly while exhibiting higher survival thresholds²⁴, ²⁶,²⁷. Lead, while less acutely toxic to F. candida, still has significant effects on reproduction, emphasizing the need for careful assessment of its long-term ecological implications^27,28. Understanding these differences is critical for developing environmental policies and regulations aimed at mitigating metal toxicity in soil ecosystems. Given the varying toxicity levels of these contaminants, we expect different impacts on F. candida.

We hypothesize that increasing concentrations of these heavy metals in the soil will result in growth inhibition of F. candida due to its susceptibility to metal toxicity.

Materials and methodology

Soils and contaminants

For all tests, we used soil sampled from the agricultural field in Wrocław (Poland) 51.169341 N, 17.161579 E. Its physical and chemical properties are as follows: granulometric type: sandy loam, Corg = 5.30 g/kg; pH in H2O = 6.81. The soil was not contaminated with heavy metals or plant protection products. Before the experiment, the soil was sieved and sterilised by autoclaving. The soils were pre-moist 7 days before the tests with distilled water or a solution of heavy metals up to 50% WHC (5 ml for 3 g of dry soil), determined according to the standard OECD 232²⁹.

The concentration ranges of the three metals were determined based on the EC₅₀ and LC₅₀ values by the studies (24–28) of other authors (on F. candida with the same trace materials (Supplementary Table 1). The minimum number of concentrations of each heavy metal was 8 and the control (water) (Supplementary Table 2). The following heavy metal formulations were used: Pb: lead nitrate, (Pb(NO₃)₂ in H₂O), 331.2 g/mol, Cu: copper nitrate (Cu(NO3)2·3H2O), 241.6 g/mol; Cd: Cadmium nitrate (Cd(NO3)2·3H2O); 236.5 g/mol. All reagents were produced by Sigma-Aldrich. The tested solutions were obtained by dissolving the reagents in deionised water. The concentrations of reagents in the soil were transformed into the concentrations of pure heavy metals in the dry mass of the soil [mg/kg]. All concentrations reported in our study are nominal, we have not verified the estimated soil concentrations with strong acid or calcium chloride extraction.

Tested organisms

Collembola species F. candida originated in the culture of the laboratory of the Department of Plant Protection. The springtails were cultured in Paris-charcoal (9:1) plates and fed with baker yeast. For the test, 10–12 days were used. Their body length was measured directly before the test to determine their initial length. Length measurements were taken for the group of individuals captured in photographs measured using a microscope camera (Carl Zeiss Axiocam Erc 5 s camera (Carl Zeiss, Zaventem, Belgium) and the ZEN 2 Core programme. Their average initial size was in the range of 400–500 μm. The length was measured from the end of the posterior abdominal segment to the anterior margin of the head.

Tests details

The test was performed based on the methodology OECD 232²⁹with some modifications and according to Gruss [2022]⁶. One replication was the 100 ml test container filled with 30 g of moist dry soil with distilled water or the tested solution. Each of the tested concentrations was tested in six replications. 20 individuals of Collembola were placed on the soil surface of each container, and a small amount of bakery yeast (2 mg) was added as food. The experimental units were incubated for 2 weeks in climatic chambers at the temperature of 21 ° C and 12/12 h of light / dark conditions. Food and water were supplemented after one week from the beginning of the test. The amount of water released after one week was estimated using the weight method. After 14 days, the organisms were extracted from the soil by flooding the soil with water. Living organisms were extracted from each replication and measured from pictures captured using a microscope camera (Carl Zeiss Axiocam Erc 5 s camera (Carl Zeiss, Zaventem, Belgium) and the ZEN 2 Core programme.

Data analysis

The Kolmogorov-Smirnov test was used to verify the normality of the Collembola body size and mortality data. The normality of residuals was tested using the Shapiro-Wilk Test. The effect of metals on Collembola growth was expressed using the effective concentrations (EC₅₀). As a response variable, growth inhibition was used, calculated according to the formula:

This formula for growth inhibition was proposed in the OECD standard 221 for Lemnasp³⁰. and utilized in this study. Previously the formula used in the study of Gruss et al. 2022⁶.

To estimate the EC₅₀, the non-linear regression method was applied for cadmium, copper, and lead in the soil. To ensure the robustness and reliability of the fitted models, several diagnostic tests were performed: normality of Residuals (Shapiro-Wilk test and Q-Q plots), homoscedasticity (Breusch-Pagan test), independence of residuals (Durbin-Watson test).

For the data analysis, the following formula was used:

, where A1= bottom asymptoml A2 = top asymptomel logx0= centre, p = slope of the slope.

Furthermore, mortality was calculated using the formula.

The growth inhibition and morality were calculated for each replication.

Additionally, the mortality and body sizes of Collembola in response to increasing concentrations of heavy metals and the control group were compared using analysis of variance (ANOVA), and significant differences were identified using a post hoc Tukey test. Furthermore, mortality was compared to growth inhibition using Pearson’s correlation. All analyses were performed in Origin software.

Results

The findings reveal a significant impact of three heavy metals, cadmium (Cd), copper (Cu), and lead (Pb) on growth inhibition after a 14-day incubation in contaminated soil (Table 1; Fig. 1). The ANOVA analysis highlights the statistical significance of growth inhibition responses (p < 0.0001 for Cd, Cu, and Pb), as shown in Table 2 (Fig. 1; Table 2).

Table 1.

Efficient concentration values (mg/kg) for the three heavy metals originated from the concentration-response model of Collembola growth inhibition after soil contamination. Concentrations displayed are nominal.

	Cd	Cu	Pb
EC10	44.15 ± 6.57*	395.96 ± 40.72	9256.25 ± 96.73
EC20	51.47 ± 5.57	511.15 ± 37.06	8885.90 ± 78.48
EC50	66.89 ± 4.23	791.01 ± 40.47	10075.48 ± 97.80
EC80	86.93 ± 8.31	1224.03 ± 110.29	11424.32 ± 183.76
EC90	101.33 ± 13.63	1580.18 ± 194.25	12295.57 ± 253.10

* indicates mean ± standard deviation.

Fig. 1.

Concentration-response curves (growth inhibition) drawn for Collembola in soil contaminated with metals (Cd, Cu, Pb). Concentrations displayed are nominal.

Table 2.

The ANOVA results evaluate the variation in growth inhibition as a function of the presence of the metals cd, Cu, and Pb.

	Df	F value	P value
Cd	4	318.54	< 0.0001
Cu	4	520.31	< 0.0001
Pb	4	1244.58	< 0.0001

Among the metals tested, cadmium (Cd) elicited the most pronounced growth inhibition, with effective concentration values of EC₁₀ = 44.15 mg/kg and EC₅₀ = 66.89 mg/kg, followed by copper (Cu) with EC₁₀ = 395.96 mg/kg and EC₅₀ = 791.01 mg/kg, and lead (Pb) with EC₁₀ = 9256.25 mg/kg and EC₅₀ = 10075.48 mg/kg. In particular, when the levels of toxicity among the metals, a distinct hierarchy emerges with Cd > Cu > Pb. Specifically, cadmium (Cd) shows toxicity levels 12.7 times higher than those of copper (Cu) and 150.6 times higher than those of lead (Pb), highlighting its greater impact on growth inhibition. Furthermore, copper (Cu) exhibits 11.82 times higher than lead (Pb), emphasising its intermediate position in the toxicity hierarchy (Fig. 1; Table 2). Note, all concentrations reported in our study are nominal.

Body size consistently decreased with increasing contamination across all three metals (Cd, Cu, Pb), as shown in Supplementary Table 2. Cadmium (Cd) caused the most pronounced reduction, with significant decreases starting at 10 mg/kg, followed by copper (Cu) at 200 mg/kg, and lead (Pb) at 4400 mg/kg. Each metal showed statistically significant differences compared to the control soil (p = 0.0001 for each metal). Regarding mortality, Cd also led to the greatest increase, with significant effects observed from 10 mg/kg (p = 0.0001). Copper similarly increased mortality, with significant effects from 200 mg/kg (p = 0.0001). In contrast, lead (Pb) had no significant impact on F. candida mortality (p = 0.99).

Furthermore, Collembola mortality was positively correlated with growth inhibition across all metals, with the strongest correlation for Cd (r = 0.76), followed by Cu (r = 0.65), and Pb (r = 0.30) (Table 3; Fig. 2). This indicates that higher mortality was linked to greater reductions in body size, especially in Cd-contaminated soils.

Table 3.

Correlation of growth inhibition and mortality in all experimental series of the three metals (cd, Cu, and pb).

	Pearson coeff (r)	p-value
Cd	0.76	< 0.0001
Cu	0.65	< 0.0001
Pb	0.40	0.0017

Fig. 2.

The correlation between growth inhibition and mortality responses of F. candida under soil contamination with Cd, Cu, and Pb. Concentrations displayed are nominal.

Discussion

In this study, we utilized growth inhibition as the primary toxicity response to evaluate the impact of soil contamination by cadmium (Cd), copper (Cu), and lead (Pb) on the springtail species F. candida. After 14 days of incubation in metal-contaminated soil, our findings revealed a clear correlation between growth inhibition and the survival of F. candida. This relationship is likely attributable to the accumulation of metals within their bodies and the consequent increase in vulnerability due to metabolic disturbances and starvation, aligning with previous observations by Nursita et al²⁴. The mechanisms underlying the toxicity of heavy metals on studied invertebrates are not fully understood. However, cadmium is known to disrupt metal homeostasis, induce oxidative stress, and exert neurotoxic effects on animals³¹. Similarly, excess copper contributes to oxidative stress, inhibits enzymatic activity, and causes tissue damage in invertebrates³². In contrast, lead primarily disrupts enzyme functions and neural systems, resulting in bioaccumulation, oxidative stress, reproductive toxicity, and neurological impairment in earthworms³³.

Our study highlights that growth is more sensitive to heavy metal soil contamination than reproduction. For cadmium, the growth EC₅₀was determined to be 66.89 mg/kg, which is considerably lower than the reproduction inhibition responses reported in other studies, which range from 125 to 351 mg/kg^24–26. This suggests that growth inhibition is a more sensitive endpoint for assessing Cd toxicity in F. candida compared to reproduction inhibition. For copper, the growth inhibition response (EC₅₀ = 791 mg/kg) was similar to the reproduction inhibition value (EC₅₀= 700 mg/kg) reported by Sandifer and Hopkin²⁶. This consistency indicates that both growth and reproduction are equally affected by copper exposure in F. candida, providing a reliable measure of Cu toxicity. Lead toxicity, expressed through growth inhibition (EC₅₀ = 10075 mg/kg), was found to be approximately ten times lower than the values determined for reproduction inhibition (EC₅₀ = 1244 mg/kg). This discrepancy suggests that lead’s impact on F. candida may manifest more significantly in reproductive outcomes than in growth inhibition. However, our study has some limitations. It should be noted, that we used the nominal data, which limits its ability to be compared to other studies.

Our data reveal that cadmium shows toxicity levels (expressed as growth inhibition) 12.7 times higher than copper and 150.6 times higher than lead. This toxicity sequence (Cd > Cu > Pb) corroborates findings from previous studies²⁴ on Proisotoma minuta, emphasizing cadmium’s pronounced toxicity in comparison to copper and lead. While Nursita et al²⁴. observed only slight growth effects at lower doses and with different methodologies (36 days after exposure), our study indicates significant growth inhibition at higher concentrations over a 14-day period.

The results from studies by Bur et al¹⁷. and Crouau and Moïa²⁰provide additional context. Bur et al¹⁷. found no significant differences in the length of juvenile Collembola from the subsequent generation after 50 days of incubation with increasing Cd concentrations. Conversely, Crouau and Moïa²⁰observed slight growth inhibition of Collembola after 15 days of incubation in Cd-contaminated soil, with reproduction being the primary affected response. These variations highlight the importance of exposure duration and methodological differences in interpreting toxicity data. Li et al³⁴. further substantiated that Cd toxicity in F. candidais significantly influenced by soil pH. The decreased growth rates of springtails at higher metal concentrations can be attributed to several factors, including compromised health and reduced food intake, as evidenced by comparisons with control groups²⁴. Studies on earthworms have shown that Cd can form metal-protein complexes within their bodies, leading to adverse effects on survival and reproduction³⁵. Additionally, investigations into natural populations of Orchesella cinctahave indicated increased excretion efficiency in response to Cd-polluted soil, which demands more energy and may result in reduced growth³⁶.

The findings of this study underscore the critical need to consider growth inhibition as a sensitive and relevant endpoint in ecotoxicological assessments of heavy metal contamination in soils. By providing a detailed comparison of the toxic effects of Cd, Cu, and Pb on F. candida, our research contributes valuable data for environmental risk assessments and highlights the varying degrees of sensitivity in different toxicity endpoints. This knowledge is essential for developing effective soil contamination management strategies and protecting soil biodiversity and ecosystem health.

Conclusions

Growth inhibition is an effective toxicity indicator of heavy metal contamination of the collembolan species F. candida. Furthermore, research reveals variations in toxicity levels among heavy metals, with cadmium exerting the highest toxicity followed by copper and lead (Cd > Cu > Pb). Importantly, a negative correlation emerges between growth inhibition and Collembola survival, shedding light on the intricate relationship between metal toxicity and the response of the organism. In further studies we would like to investigate the molecular and biochemical pathways through which cadmium, copper, and lead exert their toxic effects on F. candida.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

The concept of the manuscript was developed by I.G. and J.T.; I.G. and R.L. wrote the main manuscript; the methodology was developed by I.G.; the experiments were conducted by I.G., J.M.-D., and K.T.; the data analysis and visualization were performed by I.G.; the graphs were prepared by I.G.; the review and editing were done by I.G and J.T.

Funding

This APC/BPC financed by the Wroclaw University of Environmental and Life Sciences.

Data availability

The data sets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethics declarations

Experimental research on animals (Collembola) complied with the relevant institutional, national, and international guidelines and legislation.