Metabolism underpins cellular physiology, whereby the preference for specific substrates and catabolic pathways shapes the production of energy, anabolic substrates, and metabolite signals to address bioenergetic demands. Substrate catabolism can be directly examined by measuring metabolic endpoints. For instance, substrate oxidation can be quantified by the incorporation of carbon from labelled glucose or fatty acids into carbon dioxide, providing a sensitive and specific readout of metabolic flux. However, current platforms require relatively large culture volumes, lacking adaptability for small-scale or complex cell culture formats. Herein, we develop and validate a modular platform that can quantify substrate oxidation in a range of cell culture systems, including two- and three-dimensional cultures grown in 12- and 96-well plate formats. This platform was engineered for precise gas equilibration, minimal gas leakage, and bioinert adapters suitable for smaller-scale cultures, using inexpensive and accessible components. We demonstrate the versatility of this system by showing that: (i) dendritic cells modulate glucose catabolism in response to a tolerance-inducing biologic (anti-inflammatory protein 2), and (ii) human cardiac organoids maintain fatty acid oxidation during acute inflammatory stress. This platform can be performed in parallel with orthogonal metabolomics assays and live-cell imaging, enabling integrated analysis of metabolic and functional readouts. Together, this platform expands access to measuring substrate oxidation across a range of cellular systems.

Microplastic (MP) accumulation and salinization frequently co-occur in terrestrial ecosystems, posing potential risks to soil health. However, their combined toxicity to soil invertebrates remains unclear. This study investigated combined effects of NaCl and polyethylene MPs (< 35, < 125, < 500 μm) on earthworms. The non-saline avoidance EC50 for < 35 μm MPs was 18 wt%, while avoidance only occurred at 50 wt% for < 125 μm MPs and not at all for < 500 μm MPs. Synergistic effects between NaCl and small MPs increased avoidance. High (10 wt%) MP concentrations by themselves did not increase earthworm mortality; mortality was lower with both MPs and 4000 mg/kg NaCl than with NaCl alone. Co-exposure to MPs and 1000 mg/kg NaCl further reduced growth rates by 16 % but increased cocoon production by 259 % compared to NaCl exposure alone. NaCl exposure induced earthworm oxidative and osmotic stress; overall stress levels were unchanged by MP co-exposure. 16S rRNA gene sequencing revealed that both NaCl and MPs significantly altered earthworm gut microbiome, suggesting potential impaired gut health. MPs more strongly impacted gut microbial community and functions when no salt was added. Overall, combined effects of NaCl and MPs on earthworms are mainly additive or antagonistic.

Repeated dietary dose testing is used to assess longer term toxicity of chemicals, such as pesticides, to mammals. However, the internal pesticide concentration varies significantly as feeding rate relative to body size fluctuates over time. Toxicokinetic-toxicodynamic (TK-TD) models can estimate internal toxicant concentration over time and link this directly to observed effects on endpoints such as the growth rate of laboratory rats. Using TK-TD models it is therefore possible to predict the effects that would result from a constant internal concentration of a pesticide. This presents the possibility of comparison with data from in vitro experiments, potentially facilitating quantitative in vitro to in vivo extrapolation (QIVIVE). We used in vivo TK-TD models to identify relevant internal concentrations and then estimated the experimental conditions required to replicate these in cultured cells, using in vitro TK models. Cell population growth was measured, with a view to extrapolating through time and comparing effect sizes with in vivo predictions. However, observed cell proliferation was not significantly affected by the tested concentrations of any of the five pesticides in this study and so extrapolation was not possible. In light of this negative result, we highlight areas for future work towards QIVIVE of graded sublethal effects in mammals. The most pressing objective is improving the accuracy of in vivo TK predictions, which could be achieved with dietary dosing in TK studies.

Biodegradable polylactic acid (PLA) mulch has been developed to replace conventional polyethylene (PE) mulch in agriculture as a response to growing concerns about recalcitrant plastic pollution and the accumulation of microplastics (MPs) in soil. Cadmium is a significant soil pollutant in China. MPs have been shown to adsorb metals. In this study the earthworm Lumbricus terrestris was exposed to either Cd (1.0-100 mg / kg) or MPs (PE and PLA, 0.1-3 % w / w), or a combination of the two, for 28 days. Cd bioavailability significantly decreased in the presence of MPs. In particular, at the end of the experiment, PLA treatments had lower measured Cd concentrations in both earthworms (2.127-29.24 mg / kg) and pore water (below detection limits - 0.1384 mg /L) relative to PE treatments (2.720-33.77 mg / kg and below detection limits - 0.2489 mg / L). In our adsorption experiment PLA MPs adsorbed significantly more Cd than PE MPs with maximum adsorption capacities of 126.0 and 23.2 mg / kg respectively. These results suggest that the PLA MPs reduce earthworm exposure to Cd relative to PE by removing it from solution and reducing its bioavailability.

In plant growth experiments, the presence of microplastics (MPs) often reduces plant growth. We conducted laboratory experiments to investigate the potential of microplastics to adsorb the major soil nutrient phosphate; adsorption to MPs was then compared to adsorption to soil. Adsorption experiments used two contrasting soils, pristine high density polyethylene and artificially weathered material (the same material but exposed to 185 nm UV light for 420 h over 105 days), phosphate solutions (dissolved KH2PO4) ranging from 0.2 to 200 mg L−1 and a solid (g) to liquid (mL) ratio of 1: 150 at different values of pH (2–12) and different concentrations of background electrolyte (0.00–0.10 M NaNO3). The adsorption data were best fitted to linear and Freundlich isotherms. In initial experiments where pH was not fixed and with a background electrolyte of 0.10 M NaNO3, Kd values ranged from 3.37 to 27.65 L kg−1, log Kf from 1.21 to 1.96 and 1/n from 0.36 to 0.84. Exposure of the MP to 185 nm UV radiation led to the appearance of a C=O functional group in the MP; the partition coefficient Kd, calculated from the linear isotherm did not increase but the logKf value derived from fits to the Freundlich isotherm increased by a factor of 1.5. Kd values for soils were 3–7.5 times greater than those for MPs and log Kf values 1.1–1.7 greater. In the experiments in which initial pH and ionic strength were varied, adsorption was similar across all treatments with adsorption parameters for the higher organic content soil sometimes having the highest values and the pristine microplastic the lowest. In the desorption experiments most of the adsorbed phosphate desorbed. Overall our findings indicate that despite their ability to adsorb phosphate, MPs are unlikely to control the fate and behaviour of phosphate in soil.

Existing models for estimating pesticide bioconcentration in earthworms exhibit limited applicability across different chemicals, soils and species which restricts their potential as an alternative, intermediate tier for risk assessment. We used experimental data from uptake and elimination studies using three earthworm species (Lumbricus terrestris, Aporrectodea caliginosa, Eisenia fetida), five pesticides (log Kow 1.69-6.63) and five soils (organic matter content = 0.972-39.9 wt %) to produce a first-order kinetic accumulation model. Model applicability was evaluated against a data set of 402 internal earthworm concentrations reported from the literature including chemical and soil properties outside the data range used to produce the model. Our models accurately predict body load using either porewater or bulk soil concentrations, with at least 93.5 and 84.3% of body load predictions within a factor of 10 and 5 of corresponding observed values, respectively. This suggests that there is no need to distinguish between porewater and soil exposure routes or to consider different uptake and elimination pathways when predicting earthworm bioconcentration. Our new model not only outperformed existing models in characterizing earthworm exposure to pesticides in soil, but it could also be integrated with models that account for earthworm movement and fluctuating soil pesticide concentrations due to degradation and transport.

Modelling approaches to estimate the bioaccumulation of organic chemicals by earthworms are important for improving the realism in risk assessment of chemicals. However, the applicability of existing models is uncertain, partly due to the lack of independent datasets to test them. This study therefore conducted a comprehensive literature review on existing empirical and kinetic models that estimate the bioaccumulation of organic chemicals in earthworms and gathered two independent datasets from published literature to evaluate the predictive performance of these models. The Belfroid et al. (1995a) model is the best-performing empirical model, with 91.2% of earthworm body residue simulations within an order of magnitude of observation. However, this model is limited to the more hydrophobic pesticides and to the earthworm species Eisenia fetida or Eisenia andrei. The kinetic model proposed by Jager et al. (2003b) which out-performs that of Armitage and Gobas (2007), predicted uptake of PCB 153 in the earthworm E. andrei to within a factor of 10. However, the applicability of Jager et al.’s model to other organic compounds and other earthworm species is unknown due to the limited evaluation dataset. The model needs to be parameterised for different chemical, soil, and species types prior to use, which restricts its applicability to risk assessment on a broad scale. Both the empirical and kinetic models leave room for improvement in their ability to reliably predict bioaccumulation in earthworms. Whether they are fit for purpose in environmental risk assessment needs careful consideration on a case by case basis.

The uptake and elimination kinetics of pesticides from soil to earthworms are important in characterising the risk of pesticides to soil organisms and the risk from secondary poisoning. However, the understanding of the relative importance of chemical, soil, and species differences in determining pesticide bioconcentration into earthworms is limited. Furthermore, there is insufficient independent data in the literature to fully evaluate existing predictive bioconcentration models. We conducted kinetic uptake and elimination experiments for three contrasting earthworm species (Lumbricus terrestris, Aporrectodea caliginosa, Eisenia fetida) in five soils using a mixture of five pesticides (log Kow 1.69 - 6.63). Bioconcentration increased with pesticide hydrophobicity and decreased with soil organic matter. Bioconcentration factors were comparable between earthworm species for hydrophilic pesticides due to the similar water content of earthworm species. Inter-species variations in bioconcentration of hydrophobic pesticides were primarily accounted for by earthworm lipid content and specific surface area (SSA). Existing bioconcentration models either failed to perform well across earthworm species and for more hydrophilic compounds (log Kow < 2) or were not parameterised for a wide range of compounds and earthworm species. Refined models should incorporate earthworm properties (lipid content and SSA) to account for inter-species differences in pesticide uptake from soil.

Fe oxyhydroxides are known to adsorb organic carbon in soil, protecting it from degradation. In this presentation we will report the results of experiments in which we added waste ochre (Fe oxy-hydroxide) precipitated by waters draining former UK coal mines to soils and observed decreases in carbon lability and thus increased carbon storage. In batch experiments in which ochre was added to soil at rates of 5 wt % and 10 wt % by mass and shaken in 20 mL 0.01 M CaCl2, organic carbon release into solution was significantly reduced suggesting that the ochre had adsorbed organic carbon. In an initial set of plant growth experiments soil was amended with 5 wt% ochre and wheat plants grown for 6 weeks. The concentration of organic carbon leached from the soil over the course of the experiment and the hot water extractable organic carbon at the end of the experiment were significantly reduced by c. 43 % and 16 % respectively indicating a reduction in carbon lability. However, plant growth was reduced by c. 50 % by the ochre amendments. As Olsen P was reduced by the ochre amendments we ascribe this growth reduction to reduced P availability due to adsorption of P to the ochres. In a follow up set of experiments we added both ochre and KH2PO4 to soils. The KH2PO4 was added at a rate equivalent to levels of recommended P fertiliser application in the UK. In this second plant experiment, there was no difference in plant biomass between the study control and the ochre-treated soils and carbon lability was still significantly reduced by the ochre treatments. In addition to this the ochre amended soils leached less phosphate over the course of the experiment. These results suggest that Fe oxide amendments to soil may be a practical way of changing soil chemistry in order to increase the amount of carbon retained in soils but that care must be taken to ensure that phosphorus availability is not negatively affected. However, rough calculations indicate that the global supply of Fe-rich waste may be insufficient for Fe-oxide waste amendments to generate significant increases in soil carbon at a global scale. So addition of Fe-oxide wastes may represent a local means of waste-generating industries to offset their carbon footprint.

Plastics fragment and threaten soil ecosystems. Degradation of soil structure is one of the risks. Despite this, data on impacts of different sized microplastics (MPs) on soil aggregates is lacking. This study systematically investigated the effects of pristine polyethylene powders of different sizes (< 35, < 125, < 500 µm) and concentrations (0, 0.1, 1.0, 10 wt%) on aggregate formation and their properties for two contrasting soils (woodland soil, WS; agricultural soil, AS). 75 day wet-dry cycles produced newly-formed aggregates in all treatments. MP size and concentration impacted the incorporation of MPs in aggregates and this varied with aggregate size; the size distribution of aggregates also varied with MP size and concentration. Aggregates produced in soil containing 10 wt% < 35 µm MPs had significantly lower MWDs (mean weight diameters) than controls. The wettability of aggregates (> 4 mm) reduced with increasing MP exposure concentration and decreasing MP exposure size. MP incorporation decreased the water stability of aggregates (1-2 mm) in WS but increased it in AS. The particle density of aggregates (> 4 mm) significantly decreased with increasing MP concentration, whereas MP size had no effect. As MPs breakdown, fragment and become smaller over time, their potential risk to the aggregated structure of soil increases.

There is an increasingly urgent need to remove CO2 from the atmosphere. Increasing C storage in soils has been discussed as a possible method of achieving this. Regardless of whether increasing soil C would result in reduced atmospheric C or not, increasing the amount of organic carbon (OC) stored as organic matter in soils is widely regarded as leading to agronomic benefits such as increased water retention and resistance to erosion. Fe oxides adsorb OC in soil, protecting it from degradation. In this study we investigated the use of waste ochre (Fe oxy-hydroxide) that precipitates from the drainage of former coal mines as a soil amendment to increase C storage in soils via reduced C lability. In preliminary batch experiments, ochres reduced the release of OC from soils into solution. In plant growth experiments with wheat (var. Skyfall, RAGT) 5 wt% amendments of ochre to soils significantly reduced the concentration of hot water extractable OC by approximately 16% and OC lost from the soils in leachate by approximately 43%. Above ground plant biomass was significantly reduced by approximately 50% in the amended soils. There was no evidence for increased uptake of potentially toxic elements in the plants from the ochre-amended soils compared to the study controls but Olsen P extractable phosphate was decreased by the ochre amendments. In subsequent experiments with agronomically realistic additions of KH2PO4 there was no difference in plant biomass between the study controls and the ochre-treated soils suggesting that reduced plant growth was due to reduced P availability; C lability was still significantly reduced. Furthermore, the ochre amendments helped retain th added phosphate in the soil. These results indicate that Fe oxide amendments to soil may be a viable way of changing soil chemistry in order to increase the amount of OC retained in soils. Further experiments investigating the impacts of the ochre amendments on greenhouse gas emissions and soil biology are required followed by field trials where the impact of more variable soil moisture and temperature effects can be assessed and crops grown to harvest. A full Life Cycle Analysis could then be performed. Given the relatively limited quantities of former coal mine ochres available in the UK and the carbon footprint of transporting materials prior to adding them to soils, similar experiments to those reported here, taking into account chemical and mineralogical variations, are warranted to determine the global stock pile of Fe-rich wastes that could potentially be used to reduce C lability in soils.

Earthworms enhance plant growth but the precise mechanism by which this occurs is not known. An understanding of the mechanism could potentially support changes in agricultural management reducing fertiliser usage and therefore costs and the carbon footprint of agriculture. We conducted a factorial experiment in which 5 strains of wheat were grown in the presence and absence of earthworms under regular watering and droughted conditions. The different wheat strains all responded in a similar fashion. Plant biomass was greater in the presence of earthworms and under regular watering. The presence of earthworms reduced the impact of drought on plant biomass and also slowed down the rate of drying of the droughted soils. Plant nutrient content (N, P, Si) showed no consistent pattern with treatments but plant total N, P and Si mirrored plant biomass and decreased in the order earthworm-present watered > earthworm-present droughted > earthworm-absent watered > earthworm-absent droughted. Nutrient availability in the soil, as assessed by chemical extractions showed no consistent pattern with treatments. Differential gene expression of plants was greater between watering treatments than between earthworm treatments. Genes that were differentially expressed between the earthworm treatments predominantly related to plant defences, abiotic stress and control of plant growth though a couple were linked to both nitrogen cycling and stress responses. The soil microbiome of the earthworm-present treatments was more associated with nutrient-rich environments, the promotion of plant growth and the suppression of plant pathogens whilst that of the earthworm-absent treatments included a variety of plant pathogens. Our data are consistent with enhanced plant growth being due to changes in the microbiome brought about by earthworm processing of the soil rather than changes in nutrient availability directly due to earthworm activity.

The European environmental risk assessment (ERA) of plant protection products follows a tiered approach. The approach for soil invertebrates currently consists of two steps, starting with a Tier 1 assessment based on reproduction toxicity tests with earthworms, springtails, and predatory mites. In case an unacceptable risk is identified at Tier 1, field studies can be conducted as a higher-tier option. For soil invertebrates, intermediate tiers are not implemented. Hence, there is limited possibility to include additional information for the ERA to address specific concerns when the Tier 1 fails, as an alternative to, for example, a field study. Calibrated intermediate-tier approaches could help to address risks for soil invertebrates with less time and resources but also with sufficient certainty. A multistakeholder workshop was held on 2-4 March 2022 to discuss potential intermediate-tier options, focusing on four possible areas: (1) natural soil testing, (2) single-species tests (other than standard species), (3) assessing recovery in laboratory tests, and (4) the use of assembled soil multispecies test systems. The participants acknowledged a large potential in the intermediate-tier options but concluded that some issues need to be clarified before routine application of these approaches in the ERA is possible, that is, sensitivity, reproducibility, reliability, and standardization of potential new test systems. The definition of suitable assessment factors needed to calibrate the approaches to the protection goals was acknowledged. The aims of the workshop were to foster scientific exchange and a data-driven dialog, to discuss how the different approaches could be used in the risk assessment, and to identify research priorities for future work to address uncertainties and strengthen the tiered approach in the ERA for soil invertebrates. This article outlines the background, proposed methods, technical challenges, difficulties and opportunities in the ERA, and conclusions of the workshop. Integr Environ Assess Manag 2024;20:780-793. © 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Size distribution is a crucial characteristic of microplastics (MPs). A typical method for measuring this property is wet laser diffraction. However, when measuring size distributions of MPs, despite it being a poor dispersant for many MPs, water is commonly selected, potentially limiting the reliability of reported measurements. To evaluate dispersant suitability, different aqueous concentrations of ethanol (0, 10, 20, 30, 40, 50, 75, 100 wt%) and aqueous solutions of 0.001 wt% Triton X-100 and a mixture comprising 10 wt% sodium pyrophosphate and 10 wt% methanol were used as dispersants in a laser granulometer (Mastersizer 2000) to determine particle size distributions (PSDs) of granular polyethylene MP35, MP125 and MP500 particles (nominally <35, <125 and, < 500 μm in size). The reliability of the PSDs depended on the dispersant used and size of primary MPs. With increasing ethanol concentrations, PSD curves of MP35 particles shifted from multi-modal to mono-modal distributions. The measured size distribution reduced from 1588.7 to 4.5 μm in water to 39.9 to 0.1 μm in 100 wt% ethanol. Generally, as ethanol concentration increased, uncertainty associated with the PSD parameters decreased. Although Triton X-100 and the mixed solution also showed better dispersion than water, measured particle sizes and coefficient of variation (COV, %) were notably larger than those for 100 wt% ethanol. Similar trends were observed for larger-sized MP125 and MP500 particles, but differences in PSD curves, PSD parameters, and COV (%) among dispersants were less pronounced. In all dispersants, the volume weighted mean diameters (VWMD) in 100 wt% ethanol (MP35: 14.1 μm, MP125: 102.5 μm, MP500: 300.0 μm) were smallest and close to diameters determined from microscope observations (MP35: 14.6 μm, MP125: 109.0 μm, MP500: 310.6 μm). Therefore, for accurate determinations of the PSDs of MP by wet laser diffraction, ethanol rather than water should be used as the dispersant.

Earthworms have a positive effect on plant growth; this is typically attributed to increased N availability. Earthworms can also increase Si availability; plant Si plays a role in drought tolerance. Finally, earthworms effect microfaunal diversity. We conducted a glasshouse experiment to investigate interactions between these factors. Wheat varieties (Skyfall and four ancient strains) were grown in a greenhouse under presence / absence of adult Allolobophora chlorotica and no-drought / drought watering regimes (39 days watering then a further 17 days of watering or no watering). Despite earthworm aestivation in drought treatments, plant growth was greater in both the no-drought and drought treatments. Plant %N was greater in the watered treatments; for the drought treatments, it was greater in the earthworm-present treatments. Plant %C was greater in the earthworm-present treatments. In the earthworm-absent treatments Plant %P and %Si was greater in the watered treatments. In the watered treatments %P and %Si was greater in the absence of earthworms. Soil pH (c. 8.1) was slightly decreased in the earthworm-present and in the watered treatments. The watered treatments contained more extractable nitrate. Extractable P showed no difference across treatments. The droughted earthworm-present treatments contained more extractable Si than both the earthworm-present watered treatments and earthworm-absent drought treatments. Different wheat strains behaved similarly with Skyfall showing greater biomass and, sometimes, elemental concentrations. Bacterial and fungal beta-diversity varied with both watering and earthworm treatments; fungal diversity also varied between wheat strains. Plants showed significant differences in RNA expression between both watering and earthworm treatments including for genes linked to N uptake. Earthworms promoted plant growth under both watered and drought conditions. Under drought conditions this does not appear to be related to Si availability or uptake. Similarly we observed no simple relationship between earthworm-presence, N (or P) and plant growth though under drought conditions the presence of earthworms promoted growth and %N in the plants despite lower extractable nitrate. The RNA response of plants suggests a N-related effect perhaps mediated by changes in microbial diversity.

A major limitation of dietary toxicity studies on rodents is that food consumption often differs between treatments. The control treatment serves as a reference of how animals would have grown if not for the toxicant in their diet, but this comparison unavoidably conflates the effects of toxicity and feeding rate on body weight over time. A key advantage of toxicity models based on dynamic energy budget theory (DEB) is that chemical stress and food consumption are separate model inputs, so their effects on growth rate can be separated. To reduce data requirements, DEB convention is to derive a simplified feeding input, f, from food availability; its value ranges from zero (starvation) to one (food available ad libitum). Observed food consumption in dietary toxicity studies shows that, even in the control treatment, rats limit their food consumption, contradicting DEB assumptions regarding feeding rate. Relatively little work has focused on addressing this mismatch, but accurately modelling the effects of food intake on growth rate is essential for the effects of toxicity to be isolated. This can provide greater insight into the results of chronic toxicity studies and allows accurate extrapolation of toxic effects from laboratory data. Here we trial a new method for calculating f, based on the observed relationships between food consumption and body size in laboratory rats. We compare model results with those of the conventional DEB method and a previous effort to calculate f using observed food consumption data. Our results showed that the new method improved model accuracy while modelled reserve dynamics closely followed observed body fat percentage over time. The new method assumes that digestive efficiency increases with body size. Verifying this relationship through data collection would strengthen the basis of DEB theory and support the case for its use in ecological risk assessment.

To assess the extent of soil contamination with a potentially toxic element, it is important to determine the background concentration of that element in the soil. In this study we decided to investigate the relationship between trace element concentrations in soils and the lithology of their parent rocks. We collected 85 topsoils (0-20 cm) samples from the Petorca, La Ligua, Aconcagua, and Casablanca River basins (Valparaiso region, central Chile). For soil sampling, we chose 11 tributary watersheds free of anthropogenic influence. The areas that contributed to the formation of each alluvial soil were delineated and the parent lithologies were acquired from 1:250,000 geological maps. The results show that the total concentrations of arsenic (As), copper (Cu), lead (Pb), and zinc (Zn) in soil vary considerably among the four river basins studied. Soil concentrations of all el-ements under study are positively correlated with the presence of andesitic volcanic rocks. Furthermore, soils formed from marine sediments and felsic volcanic rocks had the highest concentrations of Pb and As. The carbonate-bearing marine sediments may have adsorbed As and Pb released by weathering of felsic volcanic rocks found upstream of the study area. In contrast, lithologies dominated by continental sedimentary and plutonic rocks showed negative correlations with the concentrations of As, Cu, Pb, and Zn in the soil. Thus, the differences in soil element concentrations in the watersheds under study were due to the different rock types present in the area. We argue that absolute "one-limit-fits-all" background trace element values may be inade-quate to assess the extent of soil contamination. Therefore, it is important to determine background concen-trations of soil elements in each river basin in the anthropogenically impacted areas. In contrast, erroneous conclusions can be drawn when background concentrations are determined in one region and then applied in another region that is lithologically different.

For a long time, soils were considered an inert substrate, however, now it is widely acknowledged that soils are a dynamic system comprising considerable biodiversity. Earthworms are an essential component of the biological activity in soils. These organisms contribute to plant productivity, plant and soil health and many other ecosystem services. Here, we review the contribution of earthworms to soil water regulation. We particularly synthesize our understanding of how earthworms affect soil water infiltration and movement and soil water retention and storage dynamics. We briefly review factors that impact soil water regulation, and we show a substantial knowledge gap related, in particular, to difficulties in identifying processes by which earthworms impact on soil water storage and how interactions of earthworms of different species with different plant rooting strategies impact water flow and storage. This review aims to provide guidance to obtain a general emerging framework of earthworm effects on soil water regulation.