Based on a microarray (a tool that allows to detect the expression of thousands of genes at the same time) developed for E. albidus, Novais et al. (2012a) showed that the exposure to the organic pesticide phenmedipharm triggered a different set of genes in comparison to the exposure to the metal copper. As a consequence, the two groups of chemicals affected distinct biological functions. For instance, reproduction was only affected by pesticides, and lipid metabolic processes were only affected by metals. Moreover, three pesticides—the insecticide dimethoate, the herbicide atrazine and the fungicide carbendazim—affected biological processes in E. albidus in a dose-related manner, meaning that higher concentrations affected more transcripts than lower ones (Novais et al., 2012b). In this study, changes in gene expression, i.e., translation, regulation of the cell cycle and general response to stress, occurred after 2 days of exposure. Other studies showed that the transcriptional response was time-dependent (Gomes et al., 2011). Transcriptional responses to the herbicide phenmedipham were higher after 2 days compared to 4 and 21 days (Novais et al., 2012c). After 21 days, no more biological responses to pesticide exposure could be detected, perhaps because of biological processes of regulation and stress management.

Most of the studies at the gene level have been carried out with E. albidus. Another microarray is available that allows the study of the expression of targeted genes in E. crypticus in response to a stressor (Ferreira et al., 2010; Castro-Ferreira et al., 2012). Microarrays are so far the only available tools to assess pesticide effects on enchytraeids at this level of organization. However, they have the disadavantage of targeting only certain genes present on the microarray, i.e., they do not allow to screen the genome without an a priori selection of the genes that are expressed. Moreover, microarrays are biased due to signal saturation (Zhao et al., 2014). To screen the genome expression without a priori selection and to quantify the level of expression of the differentially expressed genes, toxicogenomic approaches should be used, i.e., differential transcriptome analysis. However, so far these methods have not been standardized. It seems that there is a potential of using gene expression in risk assessment (Novais et al., 2012b), especially since a database containing genomic information for E. albidus is freely available (Novais et al., 2012a).

Pesticide exposure produces oxidative stress through the generation of free radicals (i.e., reactive oxygen species, ROS) and lipid peroxidation induced in the tissues of mammals and other organisms (Banerjee et al., 2001). All organisms have defense systems including non enzymatic [e.g., vitamines) and enzymatic mechanisms [e.g., production of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase GST] that limit the potentially damaging effects of ROS on cells. Persistent detrimental changes in cell function occur only when all of the detoxification, repair and compensation systems are exceeded. Beyond this “threshold,” cellular homeostasis is no longer ensured, and short and long-term, and often irreversible, negative consequences may occur (Mercurio, 2017).

Oxidative stress represents an imbalance between the production of ROS and the body defenses. Whereas direct measurement of ROS is difficult because of extremely short half-lives (Pryor, 1991), thiobarbituric acid reactive substances (TBARS) are more accessible. TBARS are degradation byproducts of fats formed during lipid peroxidation (Howcroft et al., 2009) that can be detected by the TBARS assay using thiobarbituric acid as a reagent. The measure of TBARS thus ensures the detection of a biochemical response even if some oxidative stress responses have been missed. Regarding cellular responses of enchytraeids to pesticides, several of these biomarkers have already been studied. Novais et al. (2014) assessed the effects of dimethoate, atrazine and carbendazim on the antioxidant defenses of E. albidus at different concentrations known to affect their reproduction (i.e., EC20, EC50, and EC90) and at different timings (i.e., 2, 4, 8, 14, and 21 days). They showed oxidative stress for all tested pesticides at sub-lethal concentrations. Moreover, atrazine induced damage in lipids, measured by lipid peroxidation. Once more, the time of exposure influenced the response of enchytraeids to pesticides since effects were more pronounced after 8 days of exposure than before (i.e., 2 and 4 days). Howcroft et al. (2009) found stronger effects on biomarkers after 3 weeks than after 2 days of exposure to Betanal (i.e., formulation with 157 g/L phenmedipham). This herbicide did not significantly alter the biomarker responses evaluated on E. albidus exposed during 2 days. However, the total glutathione and TBARSlevels increased, associated with an increase in activities of CAT, GPx, and GR and a decrease in GST activity after 3 weeks of exposure.

When neurotoxic pesticides are used, the transmission of the nervous influx can be disrupted (Howcroft et al., 2011). Cholinesterases (ChE) are a family of enzymes that catalyze the breakdown of some choline esters that act as neurotransmitters. It therefore plays a central role in the mechanism of neurotransmission. For instance, when acetylcholinesterase (AChE) activity is inhibited, the cholinergic receptors are overstimulated because of large amounts of acetylcholine accumulate in the synaptic junction. This can lead to behavioral changes and potentially to death (Howcroft et al., 2011). Similarly, gamma-aminobutyric acid (GABA) is the major inhibitory transmitter at neuromuscular synapses and synapses in the central nervous system, being also a biomarker of neurotoxic effects (Bicho et al., 2015). It has been shown that dimethoate caused ChE inhibition in E. albidus, indicating an impairment of the neuronal function, but further work is surely needed before such endpoints can be used in regulatory testing (Novais et al., 2014). Similarly, Howcroft et al. (2011) showed that a commercial formulation of phenmedipham inhibited ChE activity of E. albidus after 3 weeks of exposure, showing that ChE inhibition was a relevant biomarker for the studied pesticide.

Finally, along with the oxidative stress response, the energy reserves can provide information on the health status of the individuals. Because energy is a limiting factor for organisms, presence of pesticides can influence the trade-offs between energy allocated to stress management and life history traits, i.e., survival, growth, or reproduction. Organisms have to allocate their energy not only for maintenance, growth and reproduction but also for stress response (i.e., detoxification processes) while ensuring their basal metabolism and vital functions. Novais and Amorim (2013) studied the effects of three pesticides on cellular energy allocation (CEA) of E. albidus for up to 8 days, using concentrations analogous to the EC10, EC20, EC50, and EC90 values for these chemicals as previously determined in standard laboratory tests. A reduction in CEA was observed but only for atrazine at exposure times longer than 4 days. The authors explained that the low effects on CEA at concentrations known to affect reproduction (ECx) could indicate that the reduction in reproduction was not likely to be caused by a reduction in the total energy budget during the first 8 day of exposure. A complex endpoint such as CEA should thus always be complemented with measurements of the available energy reserves (Ea) and energy consumption (Ec).

Despite the scarce literature, some advice can be given to assess the effects of pesticides on enchytraeids at sub-individual levels. Considering that the molecular and biochemical responses of E. albidus to pesticides appeared to be dose-related and time-dependent, it is recommended to test different concentrations of pesticides and different times of exposure. Experiment durations should be chosen to assess short, medium, and long-term responses, thus allowing to characterize gene expression and biomarker changes in a situation where reproduction occurred. Finally, this literature review makes evident a great need of further research at the sub-individual levels that addresses the effects of multiple stressors on the one hand and more ecologically relevant species of enchytraeids on the other. Moreover, although the reported references allow to infer the cascade of reactions that occurs when potworms are exposed to pesticides, few authors studied the link between different levels of biological organization (Novais et al., 2012b; Bicho et al., 2015) Finally, it is worth noting that to be able to discriminate between stress responses and natural ecological variations of biomarker expression, it is necessary to know the normal operating range (NOR) of a species. The first attempt was done by Novais and Amorim (2014) for E. albidus who provided a naturally varying ecological window for gene expression.

Within the last 10 years, interest in a mechanistic understanding on effects of chemicals in enchytraeids has increased (Spurgeon et al., 2008). Ecotoxicogenomic approaches can be used to analyze initial molecular and cellular effects, and the necessary sequence information is now available for enchytraeids, especially E. albidus. Therefore, it is now possible to address the biochemical basis of species sensitivity, the prevalence of multiple (and unexpected) modes of action, the consequences of chemical-induced change at the population and community level, and to derive a better understanding of the combined effects of pollutants (Spurgeon et al., 2008). While there is certainly an inherent (biological) variability, it seems that it will thus be possible to differentiate between the influence of test conditions and the effects of a stressor. In this context it is important to address metabolic effects of pesticides according to the outcome adverse pathway selection, by which already identified metabolic pathways of individual chemicals can be used as signal of chemical toxicity.

For about 50 years, enchytraeids have been used in laboratory studies with chemicals (e.g., Weuffen, 1968) and pesticides in particular (Way and Scopes, 1968; see also Römbke and Moser, 2002). These old data are difficult to evaluate because often agar or water was used as test substrate (e.g., Römbke and Knacker, 1989; Westheide et al., 1991; Christensen and Jensen, 1995; Kristufek and Ruzicka, 1995). Compared to tests performed with soil they are less useful for the assessment of chemicals since the exposure conditions are too artificial.

From the beginning, almost exclusively species of the genus Enchytraeus were tested, using both acute and chronic endpoints (Purschke et al., 1991) as well as bioaccumulation (Rüther and Greven, 1990). Criteria such as practicability (e.g., short generation times, easy identification, simple breeding) and sensitivity were used to find the most suitable species. For example, Brüggl (1994) compared the biology of E. crypticus and E. minutus (now E. christenseni) under laboratory conditions, measuring cocoon production, number of eggs per cocoon, and population growth. Originally E. albidus was the preferred test organism due to its size (ca. 2 cm), but E. crypticus became more popular due to its broader ecological range and higher practicality (e.g., shorter test duration, higher juvenile numbers, Kuperman et al., 2006). This recommendation has been confirmed several times (Castro-Ferreira et al., 2012; Voua Otomo et al., 2013). Bandow et al. (2013) proposed Enchytraeus bigeminus which reproduces asexually via fragmentation. Its handling and breeding is as easy as that of E. crypticus. In addition, several species from other genera have been proposed as test species, e.g., for forest litter the “typical” species of such habitats, Cognettia sphagnetorum—but testing this fragmenting species is difficult (Augustsson and Rundgren, 1998). Species of the genus Fridericia, being more relevant for agricultural soils, were also investigated in laboratory tests, e.g., in Korea (An and Yang, 2009) or China (Yang et al., 2012a).

For about 15 years, standardized chronic laboratory tests with enchytraeids have been available. The Enchytraeid Reproduction Test (ERT) (Table 2) was standardized in four versions, which differ only slightly: the ISO test (2004) covers retrospective sample testing from contaminated sites, the OECD test (2004) focuses on testing individual chemicals (particularly pesticides), the ASTM test (2004) has a broader approach and includes earthworms, and the ABNT test (2012) covers tropical conditions. Life duration (or the length of the full life-cycle) has been proposed as an additional endpoint more than 10 years ago (Pokarzhevskii et al., 2003). Recently Bicho et al. (2015) have demonstrated that this endpoint could be a worthwhile addition to the ERT.

Enchytraeids can avoid unfavorable environmental conditions, meaning that this behavior can be used as a quick effect endpoint: the organisms could choose between the control and a soil spiked with a pesticide. Such an enchytraeid avoidance test was developed by Achazi et al. (1999). Later on, the design of the standard earthworm avoidance test (International Organization for Standardization, 2008) was used as template, using small two-compartment test vessels. However, enchytraeids did not react more sensitively to the fungicides benomyl and carbendazim in these tests than in chronic tests (Amorim et al., 2005a). The experiments were repeated with different artificial soils (modified in terms of pH, clay or peat content) and different durations in order to improve the test methodology. However, sensitivity remained low and results were highly variable (Amorim et al., 2005b, 2008a,b). In addition, no clear relationship between avoidance behavior and ecologically more relevant endpoints such as reproduction could be established (Novais et al., 2010). When testing mixtures of pesticides with E. albidus Loureiro et al. (2009) found antagonisms for dimethoate and atrazine, while synergisms were detected for lindane and dimethoate.

Some years ago, enchytraeids (especially E. albidus, E. luxuriosus) were included in the oligochaete standard bioaccumulation test (Organisation for Economic Co-operation and Development, 2010; Table 3). Regularly, this test is performed with earthworms, since effect data are usually available only for them. So far, few data from enchytraeid tests have been published (e.g., Bruns et al., 2001; de Amorim et al., 2002).

The effects of insecticides on enchytraeids are compiled in Table S1. In total, 12 active ingredients (plus one PPP metabolite) have been studied in 31 tests. The best known examples are dimethoate and lindane, which were investigated seven and five times, respectively. The former was used as a model chemical in the EU-SECOFASE-project (Løkke and van Gestel, 1998), while both were used in two Ph.D. theses (Amorim et al., 1999; Lock and Janssen, 2002). More than half (i.e., 16) of these tests were performed with E. albidus, 11 with E. crypticus (named E. buchholzi s.l. in three of them), three with E. bigeminus and one with C. sphagnetorum. Twelve tests were performed with OECD artificial soil, seven with the standard field soil LUFA 2.2 and the remaining 12 ones covered a wide geographical and pedological range of field collected soils (including one tropical soil). Fifteen tests were performed according to the OECD guideline No. 220 and four to the ISO guideline (including one draft version). Five avoidance tests were conducted according to an ISO draft guideline. Six tests were performed before the standard guidelines were fixed. With the exception of the avoidance tests, the performance of all tests did not differ much. LC50 values were determined in 17 tests and the NOEC_Reproduction (alternatively, the EC10 is listed rarely) in 19 tests. Thirteen EC50_Reproduction but only five EC50_Avoidance values were found. When several endpoints were measured, mortality and (almost always) avoidance was found to be less sensitive than reproduction. Thus, usually the NOEC_Reproduction was the most sensitive endpoint.

More contrasting results were found when looking at the effects of insecticides on enchytraeids in detail. We firstly discuss the tests in which only small effects were found [i.e., where the most sensitive endpoint is >10 mg a.i. (active ingredient)/kg soil DW (dry weight)], followed by those with effect values <10 mg a.i./kg soil DW—and in particular those, which have been tested several times, often in different soils. The value of 10 mg a.i./kg soil DW was chosen since even in worst case conditions, the exposure in the field will not exceed this concentration.

The first group consists of chlorantraniliprole (no effects on reproduction up to 1,000 mg a.i./kg soil DW, Lavtižar et al., 2016), chlorpyrifos (with just one EC50_Avoidance value of 933 mg a.i./kg soil DW, Amorim et al., 2008b), toxaphene (no effects on survival and reproduction at 620 mg a.i./kg soil DW, Bezchlebová et al., 2007), parathion and its metabolite 4-nitrophenol (all reported effect values >20 mg a.i./kg soil DW, Römbke, 1991; Römbke and Moser, 1999). Natal-da-Luz et al. (2012) reported that spraying the insecticide diazinon on soil samples from Costa Rica did not cause adverse effects on E. crypticus (NOEC_Reproduction of >16 mg ai./kg soil DW). Testing the effects of ethoprophos on E. crypticus in a Mediterranean soil resulted in an EC50 value of 68.5 mg a.i./kg soil DW (Leitão et al., 2014).

Alpha-cypermethrin belongs to the second group (i.e., effect values <10 mg a.i./kg soil DW). Both NOEC and EC50_Reproduction are below 5 mg a.i./kg dry soil (Hartnik et al., 2008). Malathion affects the reproduction of E. albidus at concentrations between 5 and 10 a.i./kg soil DW in two field soils and OECD soil, without any relation to their soil organic matter content (4.3 and 2.3 vs. 10%; Kuperman et al., 1999). This insecticide was more toxic to E. albidus juveniles than to adults in OECD soil.

In the following, some well-studied insecticides will be discussed. Puurtinen and Martikainen (1997) studied the effects of dimethoate on a small Enchytraeus species (probably E. buchholzi s.l.) in uncontaminated field soil at three different moisture levels (40, 55 and 70% of the soil WHC). It is less toxic in dry soil than in moist soil. Martikainen (1996) used the same study design, species and test chemical to investigate the effects of three soils with different texture. High soil organic matter content reduced the toxic effects. This insecticide showed low toxicity for E. buchholzi s.l. A similar result was found in two avoidance tests with E. albidus: the EC50_Avoidance values were 58.3 mg a.i./kg soil DW (Amorim et al., 2008b) and 34 mg a.i./kg soil DW (Loureiro et al., 2009), respectively. Finally, low toxicity of dimethoate was also determined in a test with C. sphagnetorum (Løkke and van Gestel, 1998).

The insecticide lindane has been tested often, especially during the development of the ERT. Early work, e.g., that of Dormidontova (1973) could not be used since almost no information on test conditions or results is available. Loureiro et al. (2009) tested the toxicity of lindane on the mortality and avoidance behavior of E. albidus in LUFA 2.2 soil and found that the effects occurred in a similar range. Using OECD artificial soil, an LC50 of about 200 mg a.i./kg soil DW and a NOEC_Reproduction of about 20 mg a.i./kg soil DW were found (Amorim et al., 1999). Depending on the soil type, Lock et al. (2002) found almost the same result (EC50_Avoidance 172.5 mg a.i./kg soil DW) with E. albidus in OECD soil. Another example is the effect of lambda-cyhalothrin on the reproduction of the fragmenting species E. bigeminus which was examined under three different soil moisture levels (30, 50, and 70% of the soil WHC) (Bandow et al., 2013). A higher toxicity was observed in soil with lower moisture level. For lambda-cyhalothrin, the 21-day EC50Reproduction values at the three levels of soil moisture were calculated to be 1.33, 3.79, and 4.75 mg as/kg soil DW, respectively.

Finally, Chelinho et al. (2012) studied the effects of the insecticide/nematicide carbofuran on E. crypticus under tropical conditions in the laboratory, following basically the ISO standard (International Organization for Standardization, 2004). Actually, a new application method was used, intended to simulate pesticide spraying. The recommended dose of the fungicide carbofuran (1.178 mg/kg soil DW), twice the recommended dose, and a water control were sprayed on plastic trays (1.10 × 0.49 × 0.17 m length width × depth) containing a loamy soil. An EC50 value of 0.739 mg a.i./kg soil DW was determined for reproduction, i.e., lower than the predicted environmental concentration (PEC) after the recommended use of this pesticide. Chelinho et al. (2012) repeated the study with carbofuran and E. crypticus but this time they used soil which was applied in the field on plots varying in size between 3 × 1 and 4 × 2 m. About 18 h after spraying, the soil was collected for enchytraeid tests to be performed similarly as in the previous test. An EC50 value of 0.750 mg/kg soil DW was determined for the reproduction of E. crypticus, again a risk for potworms could not be excluded. In both tests no enchytraeid mortality was observed.

The effects of fungicides on enchytraeids are compiled in Table S2. Only six active ingredients have been studied in 32 tests so far. Twenty-two tests were performed with E. albidus, three with both E. crypticus and E. bigeminus as well as two with E. coronatus. Fridericia ratzeli—a species not cultured but collected from a grassland near Frankfurt—and E. buchholzi were used once. Fifteen and seven tests were conducted with OECD artificial soil or with the standard field soil LUFA 2.2, respectively. Two times LUFA 2.1 and 2.3 soils were used in the early days of the ERT development. Four field soils with varying properties and two forest soils (without and with pH modification (pH = 4.5 and 6.0, respectively) were used in order to evaluate the influence of soil acidity. Nineteen tests were performed according to the ISO guideline 16387, three avoidance tests were tested according to the respective ISO draft and three tests followed the OECD guideline. In seven tests performed during the ERT development no guideline was used. With the exception of the avoidance test, results of these tests were similar, as long as the same soil was used. LC50 values were determined in 17 tests and the NOEC_Reproduction in eight tests. Nine EC50_Reproduction and six EC50_Avoidance values were found.

Azoxystrobin and chlorothalonil were tested just once. Pyrimethanil was studied in three tests, differing only in their soil moisture. The remaining tests were run with pentachlorophenol (PCP) and benomyl (both seven times) and carbendazim (14 times). In the laboratory tests listed in Table S2, PCP was not very toxic to E. albidus (the LC50 values are in a range of 15.5–444 mg a.i./kg soil DW). Mortality is clearly correlated with soil properties, especially with organic matter content, sand and pH.

The high toxicity of benomyl and in particular carbendazim to earthworms has been known for a long time (Stringer and Wright, 1973). Thus, it is used as reference substance in earthworm field tests (International Organization for Standardization, 1998, 1999) and was also selected as a model chemical during the development of the ERT (Römbke and Moser, 1999, 2002). Especially in the international ringtest, a huge data set was compiled (92 tests in total), allowing to assess the variability of this test system (Weyers et al., 2002). Variations were caused by a mixture of factors such as inherent biological variability or the level of experience of the participants. However, the ERT results were robust enough to standardize this method—a view which was supported by the first review on the effects of chemicals on enchytraeids (Didden and Römbke, 2001). Based on these experiences, carbendazim was selected as reference substance for the ERT (Römbke and Moser, 2002). One example from the ERT ringtest shows the effects of carbendazim on the reproduction of E. albidus, indicating that the numbers of juveniles per laboratory were close to the overall mean and that the EC10 values were almost always lower than the NOECs (see Figure 1; Römbke, 2003).

A clear difference in sensitivity between acute and chronic endpoints was found for carbendazim: the acute tests resulted in an LC50 of >10 mg/kg soil DW while the EC50 in the chronic test was 2.8–3.7 mg/kg soil DW. No significant differences were found between test runs following a NOEC design or those performed according to an ECx design, but the latter were less variable. The outcome of this ringtest formed the basis for the OECD, ISO, ASTM, and ABNT guidelines. The high chronic toxicity of carbendazim to enchytraeids has been confirmed several times (e.g., Castro-Ferreira et al., 2012). Avoidance behavior is not more sensitive than reproduction or even mortality (Amorim et al., 2005a). However, when spiking carbendazim in LUFA 2.2 soil and aging it for one, 14 or 28 days before starting the tests, Kobetičová et al. (2009) could show in an avoidance test that E. albidus clearly preferred the soil which was aged for 28 days, i.e., that one with the lowest availability of carbendazim.

Arrate et al. (2002) performed also the ERT with carbendazim, but they used E. coronatus instead of E. albidus—and tested the same compound in parallel in agar. Reduction in the number of juveniles was best explained by reduced hatching from cocoons, leading to a better understanding of the causes of toxic effects on these worms. In addition, the authors could show that the Mode-of-Action (MoA) of carbendazim significantly differs from those determined for other chemicals. Finally, it could be shown that the effects of this fungicide on E. albidus in laboratory tests differed in LUFA 2.2 soil in the absence or presence of 15‰ NaCl₂ (Silva et al., 2015).

Finally, Chelinho et al. (2014) investigated the effects of carbendazim on the reproduction and avoidance behavior of E. crypticus in five to eight Mediterranean soils plus OECD soil. The EC50_Reproduction in the field soils differed between 0.73 and 1.27 (OECD: 0.89) mg a.i./kg soil DW, while in the avoidance tests less effects were determined [1.3–9.4 (OECD: 3.9) mg a.i./kg soil DW]. In both tests there was no difference between the effect size in OECD soil compared to the field soils.

Puurtinen and Martikainen (1997) studied the effects of benomyl on Enchytraeus buchholzi s.l. in uncontaminated field soil at three different moisture levels (40, 55, and 70% of the soil water holding capacity, WHC). The toxicity of benomyl decreased with increasing soil moisture content, but the mechanisms behind this behavior were not clearly understood. In a parallel study, Martikainen (1996) assumed that the high organic matter content of the soil reduced the toxic effects of this pesticide.

The fungicides azoxystrobin and chlorothalonil were tested in the ERT, using E. crypticus, but with a Mediterranean agricultural soil (Leitão et al., 2014). Effects were only observed at high concentrations (ca. 500–1,000 mg a.i./kg soil DW).

Finally, in enchytraeid reproduction tests with the fragmenting species E. bigeminus, pyrimethanil was examined under three different soil moisture levels (30, 50, and 70% of the soil WHC; Bandow et al., 2013). The highest toxicity was observed in soil with the lowest moisture level (i.e., EC50 of 435, 499, and 829 mg a.i./kg soil DW), probably due to synergistic effects of both the fungicide and moisture conditions.

The effects of herbicides on enchytraeids are compiled in Table S3. Only four active ingredients were tested in 23 tests so far. Bromoxynil was tested once, atrazine twice, and 2,4-5-T (banned already 20 years ago because of its carcinogenic properties) four times. All the other 16 tests were performed with phenmedipham, mainly as part of a Ph.D. thesis (Amorim et al., 2005a,b, 2008b). Out of these 23 tests, 17 were performed with E. albidus, five with E. luxuriosus and one with Fridericia bulbosa (an invalid name according to Schmelz, 2003). The lack of tests with E. crypticus indicates that—with two exceptions—most of these tests were performed at least 10 years ago. OECD and LUFA 2.2 soils were used in five and eight tests, respectively. Eight tests were performed with, in total, three field soils. In the remaining test the very sandy LUFA standard soil 2.1 and the slightly humus-richer LUFA standard soil 2.3 were used. Eleven tests were performed according to the ISO guideline 16387, seven avoidance tests were tested according to the respective ISO draft and only one test followed the OECD guideline. In four tests no guideline was used, but they were conducted already in 1988/1989 (Römbke, 1989). LC50 values were determined in 14 tests and the EC50_Reproduction values in 12 tests. Four EC50_Avoidance values were found.

The low number of herbicides tests is probably caused by the fact that, due to their MoA, low effects on enchytraeids are expected. This expectation is fulfilled in the case of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) for which an extremely high LC50 value of 14,150 mg a.i./kg soil DW was found in OECD soil (Römbke, 1989). In the three LUFA soils, the LC50 values were by a factor of more than 10 lower. In contrast, atrazine affected the reproduction of E. albidus already at low concentrations (1–2 mg a.i./kg soil DW), independently from the endpoint (NOEC or EC50). Interestingly, avoidance behavior (EC50: 38 mg a.i./kg soil DW) was found to be an even less sensitive endpoint than mortality (LC50: 12 mg a.i./kg soil DW) (Novais et al., 2010).

Bromoxynil was found to cause quite high mortality of Fridericia bulbosa, even at low concentrations (Yang et al., 2012b). Thus, the authors recommended mortality as “valuable and sensitive” endpoint. This view is not supported by the many studies where reproduction and (sometimes) avoidance is more sensitive than mortality.

Phenmedipham is by far the best studied herbicide (16 tests), covering two species, three endpoints and five soils. Differences in sensitivity between species were low (i.e., within a factor of two in the same soils). There is an influence of soil properties on toxicity: LC50 values in OECD soil were by a factor of two higher than in LUFA 2.2. soil. Results from other field soils are somewhere in between, i.e., there is no clear difference to tests in OECD soil. Mortality and reproduction do not always show the same tendency: in tests with the field soil “Coi3” E. luxuriosus shows almost the same LC50 as in OECD soil, but the EC50_Reproduction differs by almost a factor of 30 (E. luxuriosus reacts much stronger than E. albidus). Surprising differences were also found when performing avoidance tests in different soils but always with E. albidus: the EC50_Avoidance varied between <1 and 252 mg a.i./kg soil DW. This difference might be caused by a combination of different soil properties but also by a lack of experience and/or the higher variability of the results of enchytraeid avoidance tests in general.

In one of the rare tests using soils from the Mediterranean region, Chelinho et al. (2014) investigated the effects of phenmedipham on E. crypticus, in 12 soils from Spain, Italy and Portugal plus OECD soil. Both the ERT as well as the enchytraeid avoidance test were used. The EC50_Reproduction in the field soils differed between 3.8 and 32.8 mg a.i./kg soil DW. In contrast, the EC50_Avoidance could only be determined in seven field soils, showing in general less toxicity (range: 19.1–>81 mg a.i./kg soil DW). Interestingly, the effects in OECD soil in the reproduction tests were almost always lower than in the field soils (EC50_Reproduction: 29.2 mg a.i./kg soil DW), while the opposite was determined in the avoidance tests with OECD soil: EC50_Avoidance: 14.1 mg a.i./kg soil DW). No significant relationships between soil properties and toxicity were found. Probably the range of properties of the selected field soils was too narrow to identify clearly their influence on toxicity.

Scoriza et al. (2015) studied the effects of the herbicide mesotrione which is used in forest restoration in Southern Brazil. Methodically, a combination of field experiments, focusing on soil arthropods, and laboratory tests with E. crypticus was used. Composite samples taken from the field before and one, eight and 22 days after application of 0.4. L/ha mesotrione were studied. Enchytraeid reproduction was severely affected in all samples after application. Thus, the authors recommend to use other herbicides (e.g., Fluazifop-P-butyl or Nicosulfuron), since these compounds did not affect enchytraeid reproduction. Since this herbicide was used in forests, it is not listed in Table S3.

The best example for the use of enchytraeids (E. albidus, E. luxuriosus) in the standard OECD bioaccumulation test (Organisation for Economic Co-operation and Development, 2010) is a study performed with the insecticide lindane, which is, at least in Europe, no longer registered (Bruns et al., 2001; de Amorim et al., 2002). Lindane was quickly accumulated in both species in both soils, but was also quickly eliminated after transfer of the worms into clean soil (Figure 2). The experiment was repeated with both soils but only with E. albidus 1 and 2 months after spiking, i.e., the aging of this insecticide did reduce the bioaccumulation in the enchytraeids. Bioaccumulation factors differed between the two soils, probably because of the lower organic matter content in the latter, natural soil: the BAF was 12.1 in OECD soil and 22.0 (E. albidus)/36.1 (E. luxuriosus) in LUFA St. 2.2 soil. This difference in the bioaccumulation factors (BAF) of lindane in the two species is probably due to size-related differences and the respective volume: surface ratio (E. albidus is larger than E. luxuriosus; Amorim et al., 2002).

First of all, a standard test method (ERT) is available, which has been used in dozens of tests in various laboratories without any difficulties. The ERT allows some flexibility, i.e., various species of the genus Enchytraeus as well as different soils and endpoints can be used. The experiences with enchytraeid laboratory tests can be summarized as follows:

Various types of semi-field methods have been applied in the last 50 years to measure the effects of pesticides on enchytraeids, starting with microcosms. These are small vessels filled with field soil (rarely including plants), kept in the laboratory under controlled conditions. When all components of such a microcosm (including organisms) are selected by the experimenter, they are called gnotobiotic systems (gnotos (gr.) = known) (Mothes-Wagner et al., 1992; Born, 1993; Morgan and Knacker, 1994; Scott-Fordsmand et al., 2008; Schaeffer et al., 2010). The earliest known example is a study in Azalea cultures with various stressors, e.g., the herbicide DBCP (e.g., Heungens, 1968). Some early tests are known from Japan (Kitazawa and Kitazawa, 1980), which already studied combinations of (and thus interactions between) pesticides and ecological factors such as food addition. With few exceptions enchytraeid species were not identified. Most of the pesticides tested have been banned for a long time (e.g., pentachlorophenol, 2,4,5-T, aldicarb), while others such as carbendazim are still used today. Such microcosms are suitable for specific questions, e.g., the influence of pesticides on combinations of standard test soil invertebrates (E. crypticus, together with some springtail species and a predatory mite), but so far no standard guideline is available (Jensen and Scott-Fordsmand, 2012).

Martikainen et al. (1998) were the first to study the effects of an insecticide (dimethoate) and a fungicide (benomyl), used alone or as mixture, in microcosms containing agricultural soil and indigenous soil fauna. They reported no effects on the total number of enchytraeids, but highlighted the added value of microcosm experiments in contrast to laboratory tests when studying complex questions.

The effects of carbendazim were also studied in a gnotobiotic microcosm, i.e., a plastic tube filled with sieved soil from the same site as the one used for the studies described below with Terrestrial Model Ecosystems (TME) (Burrows and Edwards, 2004). Enchytraeids (as well as other organisms) were added, exposed to the same concentrations of carbendazim and studied in the same way as in the TMEs. However, enchytraeids were not affected, meaning that this test system was—at least for this endpoint—not able to predict effects which were found both in the TMEs and in the field. A similar approach has later been used by Jensen and Scott-Fordsmand (2012) who designed a soil multi-species (SMS) test system consisting of one potworm species (E. crypticus), four springtail species and one predatory mite species. With an additional stress factor (here: the predatory mite), the springtails reacted much more sensitively to the insecticide ivermectin (primarily known as a veterinary pharmaceutical) than when exposed alone. Since the mites fed selectively more on enchytraeids than on springtails it is likely but not yet proven that the same phemomenon could happen with potworms.

Sechi et al. (2014) used the same SMS to study the effects of the insecticide alpha-cypermethrin on the same artificial community as described above (including E. crypticus). A community EC50 of 1.26 mg/kg soil DW was determined—a value which is significantly lower as the EC50 value measured in a single-species test with the same enchytraeid (4.91 mg/kg soil DW) (Hartnik et al., 2008).

The best-known example for a “real” semi-field method uses Terrestrial Model Ecosystems (TMEs) (Knacker et al., 2004; Förster et al., 2006; Moser and Römbke, 2007; Moser et al., 2007; Scholz-Starke et al., 2013; Bandow et al., 2016), which was originally called a “terrestrial soil-core microcosm test” (American Society for Testing and Materials, 1993). TMEs are non-disturbed soil cores (diameter 15–45 cm; height 30–60 cm), taken from the field and containing the original soil organism community except, to a certain degree, the macrofauna, especially those species living on or close to the soil surface. TME studies can be performed both in-house, e.g., in temperature–controlled rooms (Figure 3A) as well as out-doors (Figure 3B). A proposal for an OECD test guideline is available (Schaeffer et al., 2010).

In the first TME study with pesticides, Römbke et al. (1994) studied the effects of the insecticide parathion and the herbicide formulation Ustinex (consisting of two active ingredients, amitrole and diuron) on the enchytraeid community of a Central German grassland. Both pesticides were sprayed in two concentrations on top of the intact soil cores. Samples were taken 1 month before application and 1, 2, 3, and 4 months after application. Enchytraeid species number and total abundance were not negatively affected, except in the treatment with the higher parathion concentration. In fact, in the TMEs with the low herbicide concentration their numbers actually increased; maybe because the insecticide eliminated Collembola (food competitors) and predatory mites (main predator).

Similarly, Moser et al. (2007) used intact soil columns collected from three grasslands in Germany, Great Britain and The Netherlands and one arable site in Portugal. They applied six different concentrations of the fungicide carbendazim as formulation Derosal®. At all sites, the genus Fridericia was most negatively affected by the pesticide, mainly 8 and 16 weeks after the application, followed by species of the genus Henlea. Many Achaeta and Enchytraeus species did not decrease or even partly increased (Figure 4A). In general, enchytraeids were not affected by the two lower concentrations (in fact their number increased slightly above control level) but showed a strong decline in the TMEs treated with the two higher concentrations. During the testing period, no indication of recovery could be seen.

At the Flörsheim site, a different effect pattern was found (Figure 4B), meaning that the lowest concentration caused only small and not lasting effects. In the three higher concentrations, the effects were stronger until week eight after application. With the exception of the two highest concentrations, the control level was reached within the study duration. The differences between the enchytraeid effect patterns at the different test sites are probably mainly caused by differences in soil properties. In addition, the different species composition of the enchytraeid communities might have played a role.

The authors explained that these results could be attributed “to the different ecological requirements […] of the different genera.” For example, Fridericia and Henlea species are K-strategists (i.e., long life duration, slow reproduction) whereas Enchytraeus species are r-strategists (i.e., short life duration, rapid reproduction) (Graefe and Schmelz, 1999). This ecological difference may affect recovery of the different species as well. In any case, the low abundance of enchytraeids belonging to the genus Fridericia would indicate a risk of high application rates of carbendazim when using the EU requirements relevant at that time (Weyers et al., 2004). Interestingly, the effects of carbendazim on the total abundance of enchytraeids were correlated with those found when measuring organic matter decomposition (using the filter-paper method) but not with those on the feeding rate as measured in the bait-lamina test (Förster et al., 2004).

Scholz-Starke et al. (2013) found 17 enchytraeid species in 45 TMEs they had collected at a German meadow site. Enchytraeids were sampled after 1, 26, and 149 days after lindane applications. The authors found no significant effects of the pesticide at concentrations ranging from 0.032 to 3.2 mg/kg soil DW on total abundance or that of individual species. Finally, Bandow et al. (2016) found that the fungicide pyrimethanil did not affect the community composition (consisting of Enchytraeus buchholzi, E. bulbosus, E. dichaetus, Fridericia bulboides, F. pretoriana, F. tuberosa, and another Fridericia species) in a TME experiment performed in Portugal, but they reported negative effects from a similar experiment performed in Germany. In the latter one, the strongest effects were found in dry soil, particularly for Fridericia connata after 8 weeks of exposure. It is not clear whether different community composition or soil properties may have caused the different outcome.

In order to survey enchytraeids in the field, soil samples are taken with a corer (diameter usually between 5 and 7.5 cm). Theses samples are separately placed onto sieves hanging in plastic bowls filled with water, and the enchytraeids are driven via wet-extraction from the soil. This procedure has been internationally standardized (International Organization for Standardization, 2007). Species identification is only possible with living specimens, which limits the number of samples that can be handled in parallel. Species determination via genetic methods (DNA barcoding and meta-barcoding) is a promising alternative (Orgiazzi et al., 2015) since the establishment of DNA sequencing as a cheap routine laboratory procedure. However, there is still a problem with the interpretation of the results, because the number of reliable data sets combining genetic and morphological information is small.

The first field study covering the effects of pesticides on enchytraeids, among others, was performed in Northern Germany (Weber, 1953). Edwards et al. (1968) and Edwards and Lofty (1971) described effects of insecticides (i.e., chlorfenvinphos) or nematicides (i.e., methomyl, dazomet or aldicarb) on enchytraeids at agricultural sites. Voronova (1968) studied the effects of the insecticide Sevin on enchytraeids in the Taiga region of Russia. Since hand-sorting was used as extraction method the results of these studies are not reliable. However, Van den Brande and Heungens (1969) used already wet extraction when studying the effects of the nematocide disinfectant DD on enchytraeids in plots with Begonia. However, the outcome is hardly useful since samples were not replicated. Martin (1975) found no effect of the pesticide fenithrothion on enchytraeid abundance in a New Zealand pasture at field-relevant concentrations. The same result was reported by McColl (1984) when studying benomyl (18.6 kg a.i./ha) and phenamiphos (18.6 kg a.i./ha) on grasslands in New Zealand. In contrast, Popovici et al. (1977) studied the effects of atrazine at two concentrations (5 and 8 kg/ha) on enchytraeids–among other soil organisms –, and observed a quick decrease in enchytraeid numbers at both concentrations 1 month after application. However, numbers increased 4 months after application at the lower concentration. In a study using small field plots, Römbke et al. (1994) applied an insecticide (parathion) or a herbicide mixture (Amitrole/Diuron) at the highest recommended application rate or a 5-fold higher concentration rate. A strong increase in enchytraeid abundance and biomass occurred at the lower herbicide rate, but the high rate caused a decrease of 50% for both endpoints. Both application rates of the insecticide did not affect enchytraeid abundance or biomass. However, so far no standardized field test method is available (e.g., Römbke et al., 2009).

Enchytraeids may also avoid chemicals by vertical migration, which has been observed in a field study in a German grassland (Römbke and Federschmidt, 1995). Carbendazim was sprayed on small plots at two concentrations and the abundance, biomass and diversity of the enchytraeid community was studied for two years, divided into an application phase and a recovery phase. Negative reactions on the enchytraeid community were found at concentrations lower than those identified in the laboratory. However, since soil properties were not the same in the laboratory and field tests the results are difficult to compare.

The effects of the fungicide carbendazim (i.e., the formulation Derosal®) on enchytraeids were not only determined in TME tests (Knacker et al., 2004), but also in parallel in the field, using the same concentrations (Moser et al., 2004). This work was done by different partners at one arable site (Coimbra, Portugal) and three grassland sites: Amsterdam (The Netherlands), Bangor (Wales, England), and Flörsheim (Germany). No differences regarding enchytraeid total abundance or number of species were found between the respective TMEs and field sites in the controls. Effects of carbendazim were most pronounced when looking at the abundance of worms of the genus Fridericia (especially 8 and 16 weeks after application), while the abundance of the genera Achaeta and Enchytraeus was not affected. The observed effects did not differ between TME tests and the respective field validation studies (Weyers et al., 2004). Due to high variability of data in both tests, NOEC-values could often not be determined. The EC50-values (based on total abundance) derived from the TME tests and the field validation study indicate that the reproducibility (i.e., the variation between the partners) of the EC50-values was reasonable, although different soils were used at the different sites. The EC50-values, based on total abundance, ranged between 0.7 and 37.8 mg a.i./kg, which is very similar to those values based on the abundance of the most abundant genus Fridericia (i.e., 0.9–24.7 mg a.i./kg soil DW). On the contrary, the EC50-values based on the endpoint number of species was less sensitive (9.5–116.2 mg a.i./kg soil DW). Since no genus was consistently more sensitive than the other genera, it is recommended to include the species level in the assessment of field studies. As in the TME study performed by the same authors, effects on enchytraeids at the four field sites were not correlated with those found in the bait-lamina test but with those from organic matter decomposition tests (Förster et al., 2004).

Potworms have been recommended repeatedly for monitoring programs or assessment schemes, e.g., in the context of post-registration monitoring of pesticides (Schouten et al., 1999; Barth et al., 2000; Jänsch et al., 2005; Bispo et al., 2009). Proposals are available for reference values (diversity, species number, or abundance) of enchytraeids at different sites in the Netherlands and Germany (Rutgers et al., 2008; Beylich and Graefe, 2009).

Recovery of enchytraeids in agro-ecosystems after pesticide exposure has not been studied so far (Kattwinkel et al., 2015). All available information is from forest sites. In a beech forest in Southern Germany, two model pesticides, the fungicide PCP and the herbicide 2,4,5-T were applied bimonthly on small field plots (25 m²) for about 2 years (Römbke, 2001). This study aimed to understand recovery processes after strong stress. Since very high concentrations of these pesticides were used, the enchytraeid populations were strongly affected (especially in a year with a long period of drought) during the application period (Römbke, 1988). After stopping the applications of PCP, enchytraeid abundance started to recover less than half a year later in plots with the lower application rate, while abundance remained significantly lower at the higher application rate for about at least one more year. Thus, it could be shown that such recovery depends strongly on pesticide exposure (here given as applied amount) in interaction with general (mainly climatic) factors (Figure 5)—a scheme which probably is true also for agro-ecosystems. As a side effect, enchytraeid abundance increased to numbers even higher than in the controls, probably because during the application period the litter layer (i.e., food) was not degraded. This picture is mainly caused by r-strategists (i.e., those adapting quickly to changing environments) such as Cognettia sphagnetorum—a potworm which can reproduce by fragmentation (Nielsen and Christensen, 1959). The recovery pattern on the plots treated with 2,4,5-T was very similar.

In 1999, Cortet et al. (1999) firstly summarized the experiences with PPP effects on enchytraeids, listing five papers. Two years later, Didden and Römbke (2001) summarized the information provided in about 30 papers and identified issues deserving further attention. With the improvement of extraction methods and a better availability of taxonomic keys in the past 10 years (Schmelz and Collado, 2010), our knowledge on enchytraeid taxonomy and ecology as well as their reaction to pesticides have certainly increased considerably, not only in Europe but also for example in Brazil (Chelinho et al., 2012; Assis, 2015). On the other side—and despite the fact that standardized methods are available and new ones are in the making (e.g., Bicho et al., 2015)—the use of enchytraeids in regulatory assessment schemes is still very limited. Indeed, enchytraeids played only a minor role in the risk assessment of pesticides in Europe during the past 25 years, mainly because there were no legal requirements for such tests. However, this situation is going to change, since in the “Draft Scientific Opinion addressing the state of the science on risk assessment of plant protection products for in-soil organisms” (EFSA Panel on Plant Protection Products and their Residues (PPR) et al., 2017), Enchytraeidae are one out of seven organisms groups for which Specific Protection Goals (SPGs) are going to be defined, meaning that the relevance of these organisms (and the need to study them) is surely more acknowledged than it has been in the past.

Only few studies addressed the effects of pesticides on enchytraeid communities either under semi-field or field conditions so far. A draft standard method exists for a semi-field method, i.e., TMEs (Schaeffer et al., 2010), but nothing like that is available for field tests. However, the standard earthworm field study (International Organization for Standardization, 1999) could be improved by adding potworm abundance and diversity as additional endpoints. In any case, a central part of practical work (extraction of enchytraeids from soil samples) is already covered in an ISO guideline (International Organization for Standardization, 2007), focusing on monitoring enchytraeids. More difficult is the situation for gnotobiotic semi-field approaches, since several proposals have been made and the amount of information about their pros and cons is still limited. The most promising method is the SMS (a simplified food-web approach, consisting of springtails, enchytraeids and predatory mites; e.g., Menezes-Oliveira et al., 2014). In fact first studies aiming at its standardization have been published (e.g., Sechi et al., 2014).

The experiences with enchytraeid higher-tier testing can be summarized as follows: