Mónica J.B. Amorim

Assessing joint toxicity of chemicals in Enchytraeus albidus (Enchytraeidae) and Porcellionides pruinosus (Isopoda) using avoidance behaviour as an endpoint.

Contamination problems are often characterized by complex mixtures of chemicals. There are two conceptual models usually used to evaluate patterns of mixture toxicity: Concentration Addition (CA) and Independent Action (IA). Deviations from these models as synergism, antagonism and dose dependency also occur. In the present study, single and mixture toxicity of atrazine, dimethoate, lindane, zinc and cadmium were tested in Porcellionides pruinosus and Enchytraeus albidus, using avoidance as test parameter. For both species patterns of antagonism were found when exposed to dimethoate and atrazine, synergism for lindane and dimethoate exposures (with the exception of lower doses in the isopod case study) and concentration addition for cadmium and zinc occurred, while the exposure to cadmium and dimethoate showed dissimilar patterns. This study highlights the importance of dose dependencies when testing chemical mixtures and that avoidance tests can also be used to asses the effects of mixture toxicity.

Effect of soil properties and aging on the toxicity of copper for enchytraeus albidus, enchytraeus luxuriosus, and folsomia candida

In the present study, the effect of the heavy-metal salt copper chloride (CuCl2.2H2O) in soils freshly spiked (3 d) and aged (70 +/- 10 d; mean +/- SD) was studied in the test species Enchytraeus albidus, E. luxuriosus, and Folsomia candida. Up to nine soils were used: Besides the Organisation for Economic Co-operation and Development (OECD) artificial soil and the Agricultural Testing and Research Agency (Landwirtschaftliche Untersuchungs- und Forschungsanstalt, Speyer, Germany) 2.2 natural standard soils, the others were selected based on the EURO Soil approach, taking into account the effect of different soil parameters (pH, organic matter, grain size distribution, and carbon to nitrogen ratio). Additionally, the effect of the chloride ions was studied separately. The results revealed the following: First, a soil effect was observed; for example, in F. candida, median effective concentrations (EC50s) varied between 262 mg/kg in a sample from the same site as the original EURO Soil 5 soil and greater than 1,000 mg/kg in OECD soil. Second, an aging effect was observed, mainly in F. candida. For example, toxicity of offspring survival was increased twofold in the OECD soil and approximately eightfold with aging in the EURO Soil 7 soil, whereas the enchytraeid species did not react differently after aging. Third, an effect of chloride ions on reproduction of the animals was found; however, this effect was independent of the aging period. Fourth, species variation was seen in terms of sensitivity (EC50), decreasing in the following order: E. luxuriosus > E. albidus >> F. candida. Differences in toxicity of offspring survival between enchytraeids and F. candida might be explained by the different routes of uptake.

Mechanisms of response to silver nanoparticles on Enchytraeus albidus (Oligochaeta): survival, reproduction and gene expression profile.

Silver has antimicrobial properties and silver nanoparticles (Ag-NPs) have been some of the most widely used NPs. Information regarding their effects is still insufficient, in particular for soil dwelling organisms. The standard soil Oligochaete Enchytraeus albidus was used to study the effects of Ag in soils, using differential gene expression (microarray) and population (survival, reproduction) response to Ag-NPs (PVP coated) and AgNO₃. Results showed higher toxicity of AgNO₃ (EC₅₀<50 mg/kg) compared to toxicity of Ag-NPs (EC₅₀=225 mg/kg). Based on the biological and material identity, the difference in toxicity between Ag-NPs and AgNO₃ could possibly be explained by a release of Ag(+) ions from the particles or by a slower uptake of Ag-NPs. The indications were that the responses to Ag-NPs reflect an effect of Ag ions and Ag-NPs given the extent of similar/dissimilar genes activated. The particles characterization supports this deduction as there were limited free ions measured in soil extracts, maybe related to little oxidation and/or complexation in the soil matrix. The possibility that gene differences were due to different levels of biological impact (i.e. physiological responses) should not be excluded. Testing of Ag-NPs seem to require longer exposure period to be comparable in terms of effect/risk assessment with other chemicals.

Effect of Cu-nanoparticles versus one Cu-salt: Analysis of stress biomarkers response in Enchytraeus albidus (Oligochaeta)

Abstract In the present study, the main goal was to compare the effects of ionic copper versus copper nanoparticles in Enchytraeus albidus assessing the effect at the biomarker level, testing different concentrations and exposure times. Measured parameters were lipid peroxidation (LPO), total, reduced and oxidized glutathione content (TG, GSH and GSSG), the enzymatic activity of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), glutathione S-transferases (GSTs) and cholinesterases (ChEs). Results showed that both salt- and nano-copper caused oxidative stress and damage to E. albidus, as confirmed by LPO levels, and effects could be discriminated between the copper forms. Nevertheless and despite the visible discrimination between nano and the salt form (time and exposure dependent), there was no single or a set of biomarkers that provided the best discrimination.

Toxicity of copper nanoparticles and CuCl2 salt to Enchytraeus albidus worms: survival, reproduction and avoidance responses.

Environmental effects of copper nanoparticles are little studied in terrestrial ecosystems. In the present article, the toxicity of copper nanoparticles (Cu-NP) on the enchytraeid Enchytraeus albidus is compared to the toxicity of a copper-salt (CuCl(2)). The effect parameters studied were survival, reproductive output and avoidance behaviour. The results show that Cu-NP were more toxic to E. albidus than the same concentrations of the CuCl(2)-salt. The physic-chemical analysis of the particles indicated that only a small fraction was released as ions. Hence, the results indicated a nanoparticle-specific effect - lower reproductive output and higher avoidance. This was observed as 2-8 fold (significant) lower ECx values for Cu-NP (EC(50-reprod) = 95 mg Cu/kg; EC(50-avoid) = 241 mg Cu/kg) exposed organisms compared to CuCl(2) (EC(50)(-)(reprod) = 251 mg Cu/kg; EC(50-avoid) = 475 mg Cu/kg) exposed organisms. These results corroborate with a nanoparticle-specific effect.

Enchytraeus crypticus as model species in soil ecotoxicology

Enchytraeids are ecologically relevant soil organisms, due to their activity in decomposition and bioturbation in many soil types worldwide. The enchytraeid reproduction test (ERT) guidelines ISO 16387 and OECD 220 are exclusive to the genus Enchytraeus and recommend using the species E. albidus with a 6-week test period. The suggested alternative, E. crypticus has a shorter generation time which may enable the ERT to be twice as fast. To confirm the suitability of a 3-week test period for E. crypticus, the toxicity of five chemicals, with distinct properties and modes of action, was assessed in LUFA 2.2 soil. In all controls the validity criteria were met, as survival of E. crypticus was above 92% and more than 772 juveniles were produced. The good performance supports its appropriateness as model species. Reproduction was more sensitive than survival, with only cadmium and 3,5-dichloroaniline causing significant lethal effects in the tested concentration ranges. The effect concentration causing 50% reduction in the number of juveniles (EC50) was 35 mg kg(-1) for cadmium, <1.0 mg kg(-1) for carbendazim, 145 mg kg(-1) for phenanthrene, 275 mg kg(-1) for pentachloroaniline and 102 mg kg(-1) for 3,5-dichloroaniline. To evaluate the sensitivity of E. crypticus, the present results were compared to literature data for E. albidus. In conclusion, E. crypticus is a suitable model species in soil ecotoxicology, with advantages such as good control performance and speed, leading to a reliable and faster ERT.

Bioaccumulation and elimination of 14C-lindane by Enchytraeus albidus in artificial (OECD) and a natural soil

Bioaccumulation and elimination of 14C-lindane in Enchytraeus albidus was studied in artificial OECD soil and a silty loam from an agricultural field in Central West Portugal. Results showed that enchytraeids were able to bioaccumulate the chemical with a kinetic pattern similar to that of earthworms: fast uptake within a few days and a biphasic elimination pattern. A 10 day period to study uptake was sufficient, but a few more days were probably necessary for elimination. Bioaccumulation was influenced by soil type. The authors suggest that higher organic matter (OM) content and also the higher content on sand particles in the OECD soil may have led to a faster elimination: hydrophobic chemicals tend to adsorb to OM being in this way less bioavailable and therefore less bioaccumulated having bioaccumulation factor value around 6 while in natural soil is 10; the sand could act as abrasive particles (helpers) in the elimination process leading to an elimination of 90% of the chemical in two days while in natural soil 67% was eliminated in the same period of time.

Effects of natural and chemical stressors on Enchytraeus albidus: can oxidative stress parameters be used as fast screening tools for the assessment of different stress impacts in soils?

Enchytraeids are important organisms of the soil biocenosis. They improve the soil pore structure and the degradation of organic matter. These organisms are used in standardized testing, using survival and reproduction (6 weeks) as endpoints. The use of biomarkers, linked to ecologically relevant alterations at higher levels of biological organization, is a promising tool for Environmental Risk Assessment. Here, enchytraeids were exposed for different time periods (two days and three weeks) to different soils (OECD artificial soil, different compositions in its organic matter, clay or pH value, and LUFA 2.2 natural soil) and different chemicals (Phenmedipham and copper). The main question addressed in the present study was if the effects of chemicals and different soil properties are preceded by alterations at the sub-cellular level, and if these endpoints may be used reliantly as faster screening tools for the assessment of different stress conditions in soils. The parameters measured in E. albidus whole body were: lipid peroxidation (LPO), total glutathione (TG), as well as the enzymatic activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione S-transferase (GST). The results showed that biomarker responses in E. albidus were significantly affected by the soil type (GST, CAT, GPx, GR and LPO) and the duration of exposure in OECD artificial soil (GST, GPx, GR, CAT and LPO) but not in LUFA 2.2 natural soil. For the abiotic factors studied, after 2 days, low pH decreased significantly the TG levels and the activities of CAT and GR,and low OM also significantly decreased CAT and GR activities. After 3 weeks, differences in soil properties caused a decrease in GR and GPx activities, whereas increased GST activity was observed due to low organic matter and pH. Copper significantly increased the activities of CAT, GPx and GR, and decreased the activity of GST after 2 days as well as inscreasing LPO levels after 3 weeks. Phenmedipham increased LPO levels, associated with increased levels of TG as well as increased activities of CAT and GPx and decreased GST activity after 3 weeks exposure. This study shows that both abiotic and chemical stresses could be followed through biomarker analysis and that some of these determinations are potential endpoints in a quick soil contamination assessment procedure.

Enchytraeid Reproduction Tests

The Enchytraeid Reproduction Test was added to the test set for the ecotoxicological characterization of wastes as a method for assessing the hazard potential of wastes to terrestrial invertebrates. The method used was an adaptation of the ISO 16387 Guideline. This test was performed with three waste materials identified as INC, SOI, and WOO, diluted with OECD artificial soil to prepare the exposure treatments. Eleven of the twelve tests conducted were classified as acceptable for evaluation of the results. The ecotoxicological data established by the participating groups through these tests were consistent. The minimum-maximum factors for individual EC 50 values were small (2.8 and 3.3), confirming that the Enchytraeid Reproduction Test is a robust method. The Enchytraeid Reproduction Tests showed that the toxic-ity order for waste materials was (from greatest to least) WOO > INC > SOI, based on the respective mean EC 50 values for juvenile production of 13.2, 28.7, and >100% (not toxic). While the EC50 values were about 37% lower than the earthworm acute test, the sensitivity of the two tests to the three materials was statistically similar; the enchytraeid reproduction test can therefore be considered for inclusion in the future test set. Guidelines for determining the appropriate hydration levels for mixtures of test materials with soil will be needed to further improve data quality in future tests.

Biochemical characterization of cholinesterases in Enchytraeus albidus and assessment of in vivo and in vitro effects of different soil properties, copper and phenmedipham

Enchytraeus albidus are important organisms of the soil biocenosis, used as standard test species in environmental risk assessment. The inhibition of cholinesterases (ChE) activity of several species has been widely used to assess the exposure and effects of anti-cholinesterase environmental contaminants. Several studies have shown the association between ChE activity inhibition and adverse effects on behaviour and survival. Extensive studies addressing survival and behavioural endpoints, as well as other biomarkers, have been done in E. albidus with different types of soil contaminants. The main objectives of this study were: (1) to characterize biochemically the ChE present in the soluble post-mitochondrial fraction of E. albidus whole body homogenates, using different substrates and selective inhibitors; (2) to assess the in vivo effects of copper, phenmedipham and different soil properties (pH, organic matter, clay) on the ChE activity; (3) to assess the in vitro effects of copper and phenmedipham on the ChE activity. The results suggest the presence of one ChE in the soluble post-mitochondrial fraction of E. albidus whole body homogenates, which displays properties of both acetylcholinesterase and pseudocholinesterase considering the typical mammalian enzymes. It is also shown that ChE activity is not inhibited by exposure to different soil properties and that copper and phenmedipham inhibited ChE activity both in in vivo and in in vitro conditions and therefore ChE inhibition seems to be a robust biomarker for this herbicide and this heavy metal. This study showed that ChE activity in E. albidus might be correlated to previously determined higher level effects like survival and reproduction, as well as avoidance behaviour.