Exploring the Relationship Among Lipid Profile Changes, Growth, and Reproduction in Folsomia candida Exposed to Teflubenzuron Over Time

The integration of untargeted lipidomics approaches in ecotoxicology has emerged as a strategy to enhance the comprehensiveness of environmental risk assessment. Although current toxicity tests with soil microarthropods focus on species performance, that is, growth, reproduction, and survival, understanding the mechanisms of toxicity across all levels of biological organization, from molecule to community is essential for informed decision‐making. Our study focused on the impacts of sublethal concentrations of the insecticide teflubenzuron on the springtail Folsomia candida. Untargeted lipidomics was applied to link changes in growth, reproduction, and the overall stress response with lipid profile changes over various exposure durations. The accumulation of teflubenzuron in organisms exposed to the highest test concentration (0.035 mg a.s. kg–1 soil dry wt) significantly impacted reproductive output without compromising growth. The results suggested a resource allocation shift from reproduction to size maintenance. This hypothesis was supported by lipid shifts on day 7, at which point reductions in triacylglycerol and diacylglycerol content corresponded with decreased offspring production on day 21. The hypermetabolism of fatty acids and N‐acylethanolamines on days 2 and 7 of exposure indicated oxidative stress and inflammation in the animals in response to teflubenzuron bioaccumulation, as measured using high‐performance liquid chromatography–tandem mass spectrometry. Overall, the changes in lipid profiles in comparison with phenotypic adverse outcomes highlight the potential of lipid analysis as an early‐warning tool for reproductive disturbances caused by pesticides in F. candida. Environ Toxicol Chem 2024;43:1149–1160. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.


INTRODUCTION
A major fraction of the pesticides used in agriculture end up in the soil compartment, where they potentially pose a threat to many below-ground organisms.Evaluating the harmful effects of these pesticides on soil invertebrates that are not the intended target is a crucial component of environmental risk assessment.Undesirable effects on beneficial soil animals can have major implications for ecosystem functioning.The toxicity tests on such nontargeted species are usually limited to the effects on their fitness, that is, growth, reproduction, and/or survival.
The standard pesticide toxicity tests with springtails (Collembola), a major representative of soil microarthropods, focuses exclusively on reproduction and survival (Organisation for Economic Co-operation and Development [OECD], 2016).This type of assessment restricts the extrapolation of the data to other members of soil fauna, which limits informed decisions about the safety of pesticides to soil functioning.However, expanding toxicity testing to additional soil fauna groups is complex due to the high species diversity in soil.Understanding the mechanisms of the toxicity of pesticides across all levels of organization, from molecules to communities (Zhang et al., 2018), could facilitate the extrapolation across taxa, and improve regulatory decision-making (Bundy et al., 2008;Dreier et al., 2020).In this context, the application of omics techniques to toxicological studies may provide an early understanding of the toxicological pathways and help us to develop a mechanism-based chemical classification and extrapolation.
Metabolomics is a sensitive tool to study small molecules (up to 1.5 kDa) in living organisms; it provides information on molecular events and biochemical pathways (van Ravenzwaay et al., 2016).In the context of untargeted metabolomics, lipids are one of the main molecular classes that are involved in many metabolic pathways and are closely associated with development, growth, and energy storage (Olzmann & Carvalho, 2019).Therefore, metabolomics can be used in risk assessment to detect alteration in organisms exposed to sublethal doses of chemicals in a short period of time, from several hours to a week of exposure (Jeong & Simpson, 2019;Kariuki et al., 2017;Sun et al., 2023).For instance, Jeong and Simpson (2019) demonstrate that alterations in the metabolome of Daphnia magna, exposed to sublethal concentrations of propranolol (18 μg L −1 water) for 6 h, serve as an early warning sign for observed changes in reproduction, decreased growth, and an increase in neonate abnormality after 2 days of exposure.
Because lipid molecules are highly conserved between organisms, untargeted lipidomics is widely applied across different fields, such as biomedical sciences, biology, human health, and toxicology (Davis et al., 2017;van Ravenzwaay et al., 2016;Viant, 2009).There is also a growing interest in their use as bioindicators of effects caused by chemical stress (e.g., pesticides; Aristizabal-Henao et al., 2020), with potential use for early warning systems.Despite the potential benefits of the approach, the use of untargeted lipidomics on soil invertebrates remains limited within ecotoxicological studies (Brunelle et al., 2023;Sun et al., 2023).For instance, Lin et al. (2021) used untargeted metabolomics on the springtail Folsomia candida exposed to chemicals.In their study, F. candida exposed to antimony for 2 and 7 days showed significant changes in fatty acid composition even though reproduction and survival were not affected, indicating oxidative stress, as well as alterations in energy metabolism mediation and enzymatic regulation (Lin et al., 2021).However, there are currently no other studies using untargeted metabolomics on the assessment of ecotoxicological impacts of chemical stressors on F. candida.
The aim of the present study was to assess the effects of sublethal concentrations of the insecticide teflubenzuron on the growth, reproduction, and lipid changes of F. candida.Teflubenzuron is an insect growth regulator that disrupts hormonal regulation and inhibits chitin synthesis in arthropods, leading to alteration in the efficiency or rate of moulting, affecting growth, reproduction, and survival (Schmid et al., 2021).Although the exact mechanism of action is not described for collembolans, it has been shown to reduce body size, delay sexual maturation, and reduce egg laying and hatchability (Campiche et al., 2006;Lee et al., 2019).Previous studies have shown that teflubenzuron negatively affects reproduction of F. candida, with 10% effective concentration (EC10) values as low as 0.006 mg active substance kg −1 soil dw (Fernandes et al., 2023).In the present study, we assessed the effects of teflubenzuron over varying exposure periods-1, 2, 7, 14, and 21 days-to address the temporal patterns in response.We used the results to explore the links between early changes in main lipid classes and changes in individual fitness of the springtails.

Test soil
The toxicity tests were performed using the standard natural Lufa 2.2 soil (LUFA Speyer), which was air-dried and sieved to <4 mm.This is a sandy loam soil with approximately 1.7% organic carbon, 46% water holding capacity (WHC), and soil pH (0.01 M CaCl 2 ) ranging between 5.0 and 6.0.The maximum water holding capacity (WHCmax) and the pH (0.01 M CaCl 2 ) were determined following guidelines of the International Organization for Standardization (ISO, 2019).

Test animals
We used age-synchronized 20 to 22-day-old adult springtails of the species F. candida.This age group was selected to reduce the extent of the experiment compared with the standard toxicity test and ensure enough biomass while using a minimal number of animals (OECD, 2016).The animals were obtained from cultures kept on plaster of Paris at 20 °C and fed with dry baker's yeast (AB Mauri Netherlands) ad libitum.Before selection of the animals for each replicate, the total number of animals required for the test were placed in a container with a moist layer of plaster of Paris and collected randomly to ensure homogeneous samples.

Experimental design
Figure 1 illustrates the experimental design.Teflubenzuron was applied in three concentrations: 0.006, 0.014, and 0.035 mg a.s.kg −1 soil dry weight.This concentration range is below the predicted environmental concentration in soil of 0.06 mg a.s.kg −1 soil dw , based on worst-case-scenarios of application (Campiche et al., 2006) and was selected to ensure that only sublethal effects were recorded during our study.
Teflubenzuron was prepared in acetone at the required concentrations by dilution of stock solution (prepared in pure acetone).These solutions were mixed with 10% of the total amount of soil required to prepare the technical replicates of each treatment.The soil was fully saturated with the acetone solution, and the mixture was sealed in glass jars and left overnight at room temperature.After that, the containers with the soil mixture were opened for a few hours to ensure complete evaporation of the acetone.This dry soil was then homogenized with the remaining amount of soil and moistened with demineralized water to 50% of the maximum WHC.The soil moisture content was maintained at 23% on a dry weight basis (w/w).Controls with demineralized water and solvent control with pure acetone were prepared following the same procedure.Six replicates of solvent controls were prepared for each time point.Water controls were prepared only for 14 and 21 days of exposure (six replicates/time point), to check whether the validation criteria for the percentage of survival, number of offspring, and variance in number of offspring, according to OECD (2016) test guideline 232 were met and no changes were caused by the solvent used.The animals were exposed for 1, 2, 7, 14, and 21 days.At the beginning of the test, 30 technical replicates of each treatment were prepared (6/time point).Each replicate was prepared in 100-mL glass jars containing 30 g dw of soil (approximately 3-4 cm of soil).To ensure enough biomass of animals for lipidomics analysis, 30 age-synchronized individuals were added to each replicate.At each time point, five replicates were selected for analysis of lipidomics, growth, reproduction, and survival, and one replicate was used for physicochemical analysis of the soil.

Toxicity endpoints
Growth and reproduction.At the end of the exposures, the surviving animals and offspring produced were extracted from the soil by flotation in demineralized water.Samples were randomly selected, and the soil was washed with approximately 200 mL of demineralized water into another glass container.This mixture was carefully stirred with a spatula to float the surviving adults and juveniles produced during the experiment.After the flotation, the surviving adults were immediately frozen at -80 °C.Body mass was assessed by weighing the group of frozen surviving animals.For initial body mass determination, 5 replicates of 30 animals each were randomly picked from the age-synchronized culture and processed the same way.The offspring on the surface of the water were photographed and counted using Image J.
Lipidome profiling.Extraction of lipids from the springtail samples was performed according to Xu et al. (2020).Briefly, solid-solvent extraction with ice-cold Milli-Q water: methanol:chloroform (150:150:300 μL, v/v/v) was performed with homogenization steps using a Precellys 24 Dual device (Bertin Technologies) at 6500 rpm for two cycles with 10-and 15-s breaks in between.A volume of 10 μL of internal standard teflubenzuron-d3 (100 ng mL −1 ) was added together with chloroform.The samples were further kept on ice to precipitate the proteins, followed by centrifugation at 13,000 rpm at 4 °C.Lastly, 200 μL of the chloroform fraction (bottom layer) was taken and evaporated under a nitrogen stream and reconstituted in 100 μL of ACN/IPA/H 2 O (5:4:1, v/v/v).Samples were stored at −80 °C prior to analysis.An internal quality control (iQC) sample was prepared using pooled 10-μL samples.The iQC samples were analyzed after every nine samples.
Untargeted lipidomics was performed using High-Performance Liquid Chromatography (HPLC; Agilent 1290 Infinity HPLC system) coupled with a Quadrupole Time-of-Flight (QTOF) mass spectrometer (Compact Bruker).Lipids were detected with electrospray ionization (ESI) in positive and negative modes.The column and chromatographic conditions were set up according to Xu et al. (2022).The QTOF settings were as follows: capillary voltages of ±5000 V (ESI+) and ±4000 V (ESI−); end set plates of ±500 V; nebulizer gas (N 2 ) pressure of 4 bar; drying gas flow rate of 6 L/min; and drying gas temperatures of 200 °C (ESI+) and 250 °C (ESI−).The scan range was 60 to 2000 m/z (mass to charge), with a resolution of 25,000 full width at half-maximum.For the present study, the scan rate was 8 Hz.Mass calibration was performed before every injection with a solution of sodium formate.
Concentration of teflubenzuron in springtails and soil.The internal concentrations of teflubenzuron in springtails were determined using the extracted chloroform fraction right after the lipidomics measurement were finalized.First, samples were diluted in 1 mL of ACN, and Florisil sorbent (50 mg/sample) was added as a clean-up step followed by vortexing for 3 min and centrifugation for 5 min at 5000 rpm at room temperature.Next, the supernatant was evaporated under nitrogen flow until dryness.Lastly, the samples were reconstituted with 50 μL of Milli-Q water:ACN (50:50, v/v), and stored at −20 °C until analysis.Quantitative analysis of teflubenzuron was performed using an HPLC Elute system coupled with an Evoq Triple Quadrupole mass selective detector (Bruker).An Omega Luna 1.6-µm PS C18 (50 × 2.1 mm) column was used for separation.All HPLC and mass spectrometer (MS) conditions were according to Fernandes et al. (2023).Limits of detection (LOD) and quantification (LOQ) were 3.55 and 11.85 ng teflubenzuron g dry weight −1 , respectively.Extraction recovery was 107.0 ± 5.6% (average ± SD; n = 3).Teflubenzuron quantification in soil samples was performed according to the modified QuEChERS method with adaptations described in Fernandes et al. (2023).The extraction protocol is described in Section 1 of the Supporting Information S1.Samples were analyzed with liquid chromatography (LC)-MS using the Elute and Evoq system (Bruker).The mobile phases used for the analysis were a combination of Milli-Q water containing 0.4 mM NH 4 F and ACN.Validation data, extraction efficiency, and instrument precision are detailed in the Supporting Information S1, Table S1.The LOD and LOQ were 0.012 and 0.04 ng teflubenzuron g −1 , respectively, and average (±SD; n = 8) extraction recoveries were 89.7 ± 0.9%.At the end of each time point, the pH (0.01 M CaCl 2 ) of the soil from each treatment was determined following ISO (2019) guidelines.

Data processing and statistical analysis
Lipidomics.The raw MS data were converted to MZML format and used for feature identification via MS-DIAL Ver.4.80 (Tsugawa et al., 2015).Identification and peak alignment settings are described in the Supporting Information S1, Table S2.All the features were identified using LipidBlast library based on m/z, isotope pattern, fragmentation pattern (MS/MS), and retention time, with a total score cut-off of 80% for annotated lipids.Annotated lipids were normalized by sample weight (wet wt of frozen springtails).Data filtering of features was performed using iQC samples, and features with a coefficient of variation (CV) > 30% were removed from further analysis.Feature drift correction of the data was based on the local polynomial regression model using iQC via the open source software NOREVA Ver.2.0 (Yang et al., 2020).After processing, the median iQC CV was 10.7%.Next, the normalized peak areas of annotated lipid features were summed within each class.For instance, the cumulative peak area for the diacylglycerols (DG) class was obtained by summing the area of all features identified as DG lipids.The summed values for each lipid class represent relative abundance (referred to as total content) of lipid classes in each replicate.The results were interpreted as relative changes in lipid composition.Then log 10 transformations were performed to normalize the data of lipid class values.
Data were assessed using heatmaps, hierarchical cluster analysis (clustering method: Ward), t-test/analysis of variance (ANOVA), and Random Forest analysis (for the outlier detection) via MetaboAnalyst Ver.5.0 (Pang et al., 2022).A bar plot was used to compare the lipid class content between exposed groups and controls.For determining the significance of differences, a threshold p value was applied at two levels: the first level used a p value < 0.1 to indicate the major trends of lipid alterations observed with less significant power, and a second-level p value < 0.05 was used to reveal the significant changes of lipid classes compared with the controls.Such an approach was chosen to account for the biological variation in the pooled samples (30 animals) and the low number of replicates.In addition, the applied compromise may reveal additional insights into affected pathways, determine the metabolic perturbations.and at the same time account for the low levels of exposure.The bar plots and two-way ANOVA tests were prepared using GraphPad Prism (Ver.8).
Reproduction and growth effects.Statistically significant differences in growth and reproduction compared with the controls were determined by applying a one-way ANOVA followed by a one-sided Dunnett's post hoc test at a significance level of p < 0.05.The effects of the different concentrations of teflubenzuron on the growth of F. candida over time were described by the following growth and decay equation: Within this equation, W is the biomass at time X (in days), Wm represents the average measured biomass at the onset of exposure, Te indicates the time (in days) when the weight reaches its maximum, and Tm corresponds to the time (in days) at which the growth curve reaches its inflection.Growth was quantified for each concentration and control individually by employing a least-square fitting approach to the average fresh biomass of the surviving organisms within each replication.Significant differences between the parameters (p < 0.05) of the models estimated from controls and the different teflubenzuron concentrations were compared using a generalized likelihood ratio test (LRT).All statistical analyses and graphical presentations were executed using GraphPad Prism (Ver.8).
Bioaccumulation in springtails.Bioaccumulation of teflubenzuron in the springtails was normalized by sample weight and calculated as ng of teflubenzuron per gram of animal (dry wt).A bioaccumulation factor (BAF) was calculated as where C springtails is the concentration of teflubenzuron measured in the springtails (ng g soil dw −1 ) and C soil the concentration of teflubenzuron measured at each time point in the soil (ng g soil dw −1 ).Statistically significant differences between different points of each treatment were determined using a t-test (p < 0.05).Plots were created using GraphPad Prism (Ver.8).

Bioaccumulation of teflubenzuron in springtails
No traces of teflubenzuron were detectable in the control soil samples or the control animals (Supporting Information S1, Table S1).Throughout the experiment, no significant degradation of the pesticide in the soil was observed (Supporting Information S1, Table S1).Bioaccumulation factors for the uptake of teflubenzuron in F. candida are shown in Figure 2A.The accumulation of teflubenzuron increased during the initial 7 days of exposure (Figure 2A).After this period, the concentrations in the animals decreased to levels observed on day 1.This trend was consistent across all three exposure concentrations tested.The lowest concentration resulted in a BAF approximately twofold higher than for the other teflubenzuron concentrations, peaking within the first 2 days of exposure.At the other teflubenzuron exposure concentrations, BAF peaked on day 7. From day 7 onward, the BAF gradually declined with time, despite the absence of any significant fluctuations (p > 0.05) in the pesticide concentrations measured in the soil (Supporting Information S1, Table S1).

Growth, reproduction, and survival
The tests performed met the validity criteria of OECD (2016) test guideline 232 for springtail reproduction and survival.Survival in the water and solvent controls was 94% to 98%, and over 850 juveniles had already been produced after 14 days; many more were produced after 21 days of exposure (Supporting Information S1, Table S3).The CV criteria for control juvenile numbers was met in both cases, with CVs ranging from 15% to 27% (Supporting Information S1, Table S3).The survival of the adults was not affected by tefllubenzuron (Figure 2C).On day 14, the number of juveniles had not been affected by teflubenzuron, but on day 21 a doserelated decrease was seen that was significant compared with the control only at the highest concentration, with a survival reduction of approximately 75% (p < 0.05; Figure 2B).The biomass of the control animals increased in the first 14 days (Figure 3).In fact, all the treatments reached the maximum growth at approximately day 14, except for the concentration 0.014 mg a.s.kg −1 soil dw , which resulted in a significant time delay to reach maximum growth (LRT; p < 0.01, Table 1).On day 14, the biomass of the animals exposed to 0.014 mg a.s.kg −1 soil dw was significantly lower than that of the control (p < 0.01), supporting the delay in growth observed (Figure 3B).

Effect of teflubenzuron on adult F. candida main lipid classes
Changes in lipid profiles of the adult F. candida extracted from the soil were analyzed from days 2, 7, and 14 of exposure to teflubenzuron.All analyzed lipid classes were found in both control and exposure groups.In total, 26 lipid classes were annotated.The major groups were the glycerophosphocholines (lysophosphatidylcholine [LPC] and phosphatidylcholine [PC]), DGs, triacylglycerols (TGs), glycerophosphoethanolamines (lysophosphatidylethanolamine [LPE] and phosphatidylethanolamine [PE]), ceramides (Cers), and fatty acids and conjugates (FAs).The complete list of annotated lipid classes in springtails, with abbreviations, is shown in the Supporting Information S1, Table S4.Cluster analysis (Ward) combined with a heatmap of the lipid classes showed a number of trends related to teflubenzuron exposure (Figure 4).The trends of the lipid classes in the treatments and control are shown with a color gradient (Figure 4).On day 2, the control and the highest concentration of teflubenzuron (0.035 mg a.s.kg −1 soil dw ) showed similar lipid patterns (Figure 4A), whereas a strong upward trend of the relative abundances of most lipid classes ) decreased.On day 14, the top cluster included the lysoglycerophospholipids (LPC, LPE, and LPS) and the fatty acyls (DG and MG), which showed a downward trend across all concentrations of teflubenzuron (Figure 4C).The second cluster showed a prominent upward trend at 0.006 and 0.0014 mg a.s.kg −1 soil dw .This cluster included the sphingolipids (sphinganine, SM, and sufonolipid) and glycerophospholipids (PC, PE, PI, and PS).
Furthermore, significantly affected lipid classes (two-way ANOVA, p < 0.05 and p = 0.05 and 0.1 [both were considered]) were selected and are shown in Figure 5 as log 10 -transformed relative abundances based on the sum of all peak areas of identified lipid features for each lipid class.On day 2, the TG content showed no significant difference between the control group and the highest teflubenzuron concentration (0.035 mg a.s.kg −1 soil dw ) but was significantly upregulated at 0.006 and 0.014 mg a.s.kg −1 soil dw (Figure 5A).The relative abundance of Fas increased at all concentrations tested (p < 0.05; 0.006 and 0.035 mg a.s.kg −1 soil dw ).Furthermore, the DG, MG, PG, CL, and PI contents showed an upward trend, with statistically significant changes at 0.006 mg a.s.kg −1 soil dw (p < 0.05; Figure 5A).
The content of TG on day 7 decreased at a teflubenzuron concentration of 0.035 mg a.s.kg −1 soil dw (p < 0.05; Figure 5B).In contrast, Fas increased significantly at all concentrations of teflubenzuron tested (p < 0.05; 0.006 and 0.035 mg a.s.kg −1 soil dw ).In addition, at the middle and the highest exposure concentrations, upregulation was also observed for NAE, whereas the levels of phospholipids (PG, LPS, and PS) significantly decreased (p < 0.05; 0.014 and 0.035 mg a.s.kg −1 soil dw ).A significant downward trend was also observed for PI at 0.035 mg a.s.kg −1 soil dw (p < 0.05, Figure 5B).
On day 14, the relative abundance of TG exhibited a significant increase at 0.006 mg a.s.kg −1 soil dw (Figure 5C).The FA contents decreased across all teflubenzuron concentrations (p < 0.05), and NAE decreased at the highest concentrations (0.035 mg a.s.kg −1 soil dw , p < 0.05).In addition, CAR and FAHFA were significantly downregulated at both 0.006 and 0.035 mg a.s.kg -1 soil dw (p < 0.05).The level of MG significantly decreased at the concentration of teflubenzuron (p < 0.05).

Effect of teflubenzuron on the investment between growth and reproduction
The results of this study showed that F. candida is capable of tolerating low concentrations of teflubenzuron, despite clear indications of significant bioaccumulation.At the lowest concentration tested, both reproduction and growth remained unaffected.This lack of change has been described in previous studies with the same species under similar conditions of exposure to this insecticide (Fernandes et al., 2023).However, previous studies also show that exposing younger animals of  this species (less than 20 days old) to teflubenzuron results in a delay in sexual maturation and a reduction in egg production (Lee et al., 2019;Xie et al., 2023).Nevertheless, a delay in maturation cannot be the primary factor influencing reproductive inhibition within the context of this study, because the experiments began with 20-day old adult springtails.These individuals reached sexual maturity and initiated egg deposition within the first 2 days of the experiment (Snider, 1973).On the last day of the exposure, even though there were no discernible changes in growth at the highest teflubenzuron treatment, there was a marked reduction in reproduction.These results show a contrasting trend in growth and reproduction, with a greater allocation of resources directed toward size maintenance rather than egglaying or egg hatchability.In contrast, on day 14, an opposite pattern was observed in the middle concentration, with a greater investment in reproductive maintenance alongside evident effects on body size.It is important to mention that the use of fresh weight in our study was required for the lipid analysis.Using fresh mass instead of dry mass may introduce a confounding factor due to fluctuating water content in response to teflubenzuron.Future studies therefore should address the potential effects of teflubenzuron on the water content of F. candida.The observed decline in biomass from day 14 to day 21, irrespective of treatment, could potentially be linked to the substantial population of organisms within each test container (with an average of up to 4000 juveniles in the control group).This elevated number of animals is associated with the use of 30 adult animals/jar, which deviated from the standard procedure of OECD (2016) test guideline 232, in which 10 adults of F. candida are placed in each jar.This likely leads to intensified competition for finite resources, thereby resulting in a diminished biomass.It is worth noting that the selection of specimens for later measurements was randomized, excluding the likelihood of preferentially smaller individuals skewing the results.Additionally, fluctuations in water content of the soil are an improbable contributor to this trend, given that soil moisture content was kept constant throughout the experiment.Similarly, the consistency in sample processing-whereby all samples were uniformly treated during freezing at each time point, and measurements were conducted randomly-mitigates the possibility of methodological discrepancies influencing the observed biomass reduction.Accordingly, the lipid profiling results on days 14 and 21 were very different from the other days across all treatments.This confirms that the high number of animals in the jars introduces an additional confounding factor in the interpretation of the results.Hence, to establish a connection between molecular changes and phenotypic outcomes such as growth and reproduction, without interference from the elevated number of animals in the jar, the most suitable time points for analysis would be the initial 10 days, when no offspring are yet present.For that reason, interpretation of the results in the present study focused on days 2 and 7.It should be noted, however, that transferring the animals from the culture in plaster of Paris to the soil the test jars may have changed their water content.This is, however, accounted for by treating control and exposed animals in the same way.

Changes in lipids in relation to growth and reproduction
The effects of teflubenzuron in F. candida and the link between lipid class alterations observed in early stages of exposure and adverse outcomes such as oxidative stress, growth, and reproduction are shown in Figure 6.The hatching time of the eggs of F. candida is approximately 10 days, so effects on reproduction should be interpreted based on changes at the individual level, such as biomass, and lipid changes observed approximately 10 days before.In this way, the offspring counted on day 14 would be from eggs laid in the first 4 days of exposure.In the same way, the juvenile numbers on day 21 should be related to changes observed in the animals exposed up to day 11 (Figure 6A).Between days 14 and 21, the number of offspring at the highest concentration (0.035 mg a.s.kg −1 soil dw ) remained the same, approximately 1000 individuals (Figure 2).The decrease in reproduction occurred only on day 21, suggesting a delayed effect of the insecticide of approximately 4 days after the initial exposure.This could be caused by an effect of teflubenzuron on egg formation, egg-laying, and hatching success after 4 days of exposure.The same negative effect on both the number of eggs laid by the springtails and the hatching success of these eggs was observed in previous studies (Lee et al., 2019).
The regulation and maintenance of lipid synthesis and homeostasis play vital roles in energy storage, growth, and reproduction of animals (Arrese et al., 2001;Guimaraes et al., 2015;Han, 2016;Olzmann & Carvalho, 2019).In particular, the glycerophospholipid classes (PC, PE, LPC, and LPE) contribute most to the cell membrane constitution and are associated with body growth (Han, 2016).None of the glycerophospholipids mentioned showed significant alterations on any day of the exposure.This lack of changes is in accordance with the overall maintenance of growth observed over time but does not explain the delay in time for reaching peak body mass at the middle teflubenzuron concentration.Recently, positive correlations among growth rate, development of animals, and glycerophospholipid abundance were shown in zebrafish development studies (Xu et al., 2022).The nonmonotonic decrease in PC lipids was shown to affect defence mechanisms and inflammation rather than closely reflecting effects on growth and development (Xu et al., 2022).Hence, further investigation of the glycerophospholipid molecular network in arthropods is necessary to gain a complete understanding of the growth delay observed in the present study.
The glycerolipids (TG and DG) play a vital role in egg formation, egg synthesis, and transportation of vitellogenin, the main protein of the eggs (Arrese & Soulages, 2010;Arrese et al., 2001;Olzmann & Carvalho, 2019;Toprak et al., 2020).The majority of TG is stored in fat bodies and oocytes of invertebrates, so the balance between TG and DG depends on the lipolysis/lipogenesis pathways (Arrese & Soulages, 2010;Silva-Oliveira et al., 2021;Toprak et al., 2020).The phosphatidylserines (LPS and PS) are essential cofactors that bind amino acids and proteins and further interact with sterol lipids (CE, SE, bile acids [BA], sterols [ST]), and participate in cholesterol transport (Rawson, 2003;Schroeder et al., 1990;Vance & Steenbergen, 2005).Therefore, phosphatidylserine and sterol lipids are crucial in cholesterol homeostasis (Rawson, 2003;Schroeder et al., 1990).Cholesterol and sterol lipids act as precursors in the synthesis of vital reproductive hormones, reinforcing their role in the reproductive system (Kumar et al., 2018;Sperfeld & Wacker, 2009).On day 7, TG and DG were both reduced in the teflubenzuron-exposed animals compared with the control, along with a downward trend in LPS and PS.Based on biological factors and phenotypic assessments, the functions of glycerolipids (TG and DG) in reproduction can be understood from various perspectives.The imbalance of TG lipids in aquatic arthropods in response to chemical stress is linked to reproductive changes (Fuertes et al., 2020;Jordão et al., 2015).In particular, complex chemical mixtures in waste waters prevents the transfer of TG to the eggs of D. magna during reproductive stages.In the present study, increased TG levels were observed that are associated with an investment in energy storage for survival, but lower hatchability of the eggs and reduced number of offspring (Brunelle et al., 2023).On the other hand, in a recent lipidomics study of the hepatopancreas of the crustacean Scylla paramamosain exposed to four different juvenoids, a positive correlation was identified between elevated TG levels and ovarian maturation (Fu et al., 2022).Specifically, methyl farnesoate played an extensive role in TG accumulation and at the same time induced reproductive maturity, therefore influencing further fecundity of S. paramamosain (Fu et al., 2022).Considering the biological roles attributed to TG, DG, and PS, it is hypothesized that the egg production and cholesterol homeostasis of F. candida were affected.Therefore, the observed effect of teflubenzuron (0.035 mg a.s.kg −1 soil dw ) on the lipid classes mentioned, together with its significant bioaccumulation on day 7, support the decrease in reproduction observed on day 21 (Figure 6B).
On day 2, TG and DG increased at the lowest and middle concentrations of teflubenzuron, but there was no difference in reproduction between control and treatments on day 14.This disconnection between lipid alteration and reproductive outcomes suggests that TG and DG are involved in alternative molecular pathways (Arrese & Soulages, 2010;Han, 2016;Toprak, 2020).Further investigation is needed to clarify the mechanisms behind the imbalance in glycerolipids observed in the springtails during the early stages of exposure to low levels of teflubenzuron.
Apart from involvement in growth and reproduction, lipid changes can indicate alterations in molecular pathways.The process of synthesis and degradation of TG and DG depends on FAs (Figure 6B).These three lipid classes are essential in energy metabolism, cell signaling, and oxidative stress (Fraher et al., 2016;Olzmann & Carvalho, 2019;Xu et al., 2022).Furthermore, Fas participate in processes such as fatty acid beta-oxidation and lipid peroxidation and can undergo conversion into acetyl-CoA within the mitochondria (Alves-Bezerra et al., 2016;Dean & Lodhi, 2018;Han, 2016).Disruption in Fas indicates an oxidative stress response and the occurrence of inflammation processes (Dunning et al., 2014;Gonçalves et al., 2021;Xu et al., 2022).Teflubenzuron significantly increased Fas on day 2 and day 7, confirming this elevated demand for energy, lipid peroxidation, and oxidative stress.We hypothesize that the decrease in TG on day 7 indicates a higher demand for energy to cope with chemical stress and/or starvation, involving transformation of energyrich TG into free Fas.
Another cluster of glycerophospholipids (PG, PI, and CL) is also associated with mitochondrial activity and energy homeostasis (Han, 2016;Kagan et al., 2005;Lewis & McElhaney, 2009;Morita & Terada, 2015).Moreover, CL and PS play a vital role in oxidation mechanisms in mitochondria; hence CL oxidation may further induce the execution of a proapoptotic program (Kagan et al., 2005).On day 2, PG, PI, and CL exhibited a pattern similar to that of TG and DG, reinforcing the hypothesis that the animals have different molecular pathways to cope with stress depending on the concentration of teflubenzuron.On day 7, PG and PI decreased but the class CL increased.The CL lipids are synthesized from PG and Fas, and the different patterns between PG and PI (decrease) and CL and FA (elevation) might occur to sustain mitochondrial membrane homeostasis and maintain balance in respiratory functions of the organism under chemical stress (Lewis & McElhaney, 2009;Morita & Terada, 2015).

Environmental implications, limitations, and future directions
The present study is the first step in the integration of untargeted metabolomics with traditional ecotoxicological endpoints in Collembola.The results we describe show that early alterations in lipid composition of F. candida exposed to low concentrations of the insecticide teflubenzuron in soil can be related to chronic effects on growth and reproduction of the animals.Our results reveal alterations in several lipid classes such as TGs, DGs, Fas, and PS after 7 days of exposure to the insecticide.Such lipidomic shifts, observed at low concentrations of the pesticide, could indicate broader ecological impacts, particularly because they are associated with effects on reproduction and growth.
The major limitation in the field at this moment is the lack of omics data on soil invertebrates.Thus, interpretation of the function of lipid classes in soil microarthropods is currently highly speculative.Our study presents a nontargeted molecular screening focused on semiquantification and relative abundance measurements.This method implies a level of assumption in interpreting the mechanisms described, warranting prudence in extrapolating these findings to ecological contexts.Future research should investigate the roles of less studied lipid classes in the physiological adaptability of Collembola to environmental contaminants.Additionally, extending this knowledge to other species and taxonomic groups will facilitate extrapolation to ecosystem-level impacts of chemical pollution.This step forward is essential for our comprehension of ecological impacts from chemical exposures, thereby informing more robust and ecologically sensitive risk management strategies.

CONCLUSIONS
The present study emphasizes the value of metabolomics approaches in environmental risk assessment.A combination of conventional endpoints such as growth, reproduction, and survival with metabolomics enhances our mechanistic understanding of teflubenzuron toxicity.By integrating the lipid changes of F. candida exposed to sublethal concentrations of teflubenzuron, we have improved our understanding of the molecular mechanisms driving the effects of this insecticide on individual fitness parameters such as growth and reproduction.The lipid changes observed during the early stages of teflubenzuron exposure explained the observed increase in maintenance and the reduced reproduction effects after chronic periods of exposure.The bioaccumulation of teflubenzuron in the springtails exposed to 0.035 mg a.s.kg −1 soil dw impacted their reproductive cycle but not their growth, which was confirmed by both the lipid profile on day 7 (TG and DG decrease in relative abundance) and offspring produced on day 21 (significant decrease).The overall lipid trends observed after exposure support the different patterns between growth and reproduction observed at different teflubenzuron exposure concentrations.Because metabolite alterations happen much faster than phenotypic adverse effects, lipid analysis in F. candida may indicate changes in reproduction activity at relatively early stages of exposure compared with standard toxicity testing.In addition, our study demonstrates that lipidomics is a valuable tool in the risk assessment of pesticides in soil invertebrates, enabling us to link molecular and phenotypic adverse outcomes.
Supporting Information-The Supporting Information is available on the Wiley Online Library at https://doi.org/10.1002/etc.5851.

FIGURE 1 :
FIGURE 1: Experimental design.Exposure of Folsomia candida to three concentrations of teflubenzuron (0.006, 0.014, and 0.035 mg a.s.kg −1 soil dw ).For each time point, 30 age-synchronized animals at 20-22 days of age were used.The exposure of all the replicates started on the same day, and at each time point, replicates were randomly picked for sampling.

FIGURE 2 :
FIGURE 2: Bioaccumulation factors, reproduction, and survival of Folsomia candida exposed to teflubenzuron (Tef).Bioaccumulation factors (A) for the uptake of teflubenzuron by the adults for different periods of time to three different concentrations (in mg kg −1 soil dw ) of the pesticide in Lufa 2.2 soil.Bioaccumulation factors are shown with standard deviation (n = 5).* shows differences (p < 0.05) compared with day 1 of exposure.Plots for reproduction (B) and survival (C) of F. candida exposed for 14 or 21 days to teflubenzuron show the average with standard deviation (n = 5).* shows differences (p < 0.05) compared with the control at each time point.

FIGURE 3 :
FIGURE 3: Growth of Folsomia candida exposed to teflubenzuron (Tef) in Lufa 2.2soil.The plots show the results for the control and 0.006 (A), 0.014 (B), and 0.035 (C) mg Tef kg −1 soil dw .The data points show the average biomass of the total number of adults in each replicate.The lines show the fit of growth models (Equation1) to the data.* shows differences (p < 0.05) compared with the control at each time point.

FIGURE 5 :
FIGURE 5: The effect of teflubenzuron (Tef) exposure on the lipid profile of Folsomia candida at different days of exposure to different concentrations of teflubenzuron in Lufa 2.2 soil.The different plots show the results for day 2 (A), 7 (B), and 14 (C).The points show the average relative abundance of each lipid class (log 10 -transformed sum of all peak areas of identified lipid features for each lipid class) with the standard deviation (n = 5).*p < 0.1 compared with the control; ** indicates a significant difference (p < 0.05) compared with the control and between exposure groups at each time point.See text for further information and Figure 4 legend for lipid names.

FIGURE 6 :
FIGURE 6: Conceptual relationship between the life cycle of Folsomia candida and lipid alterations caused by teflubenzuron.(A) Details on the reproductive cycle of F. candida during the experiment and effects of teflubenzuron on egg-laying and number of offspring produced.The different colors represent different generations of offspring.(B) Network of biosynthesis and conversion of lipid classes affected by teflubenzuron (p < 0.05) and their roles in molecular processes in the organism, with further projection on adverse outcomes such as oxidative stress, reproduction, and growth.See text for further information and Figure 4 legend for the lipid names.

TABLE 1 :
Parameters extracted from growth curves of Folsomia candida exposed to different concentrations of teflubenzuron (in mg teflubenzuron kg −1 soil dw ) in Lufa 2.2 soil (see Figure4) a Corresponds to the limit of detection of teflubenzuron in the present study.*Significant difference compared with the control (likelihood ratio test, p < 0.01).Values are given with 95% confidence intervals.df = degrees of freedom.