Extrapolation of the laboratory-based OECD earthworm toxicity test to metal-contaminated field sites

The effects of cadmium, copper, lead and zinc on survival, growth, cocoon production and cocoon viability of the earthworm Eisenia fetida (Savigny) were determined in three experiments. In experiment 1, worms were exposed to single metals in standard artificial soil. For experiment 2, worms were maintained in contaminated soils collected from sites at different distances from a smelting works situated at Avonmouth, south-west England. In experiment 3, worms were exposed to mixtures of metals in artificial soil at the same concentrations as those present in the field soils. A survey of earthworm populations was carried out also. Population densities and species diversities of earthworms declined with proximity to the smelting works. No earthworms were found within 1 km of the factory. Comparison of toxicity values for the metals determined in the experiments indicated that zinc is most likely to be limiting earthworm populations in the vicinity of the works. Zinc was at least ten times more toxic to E. fetida in artificial soil than in contaminated soils collected from the field. This difference was probably due to the greater bioavailability of zinc in the artificial soil. The results are discussed in the context of setting protection levels for metals in soils based on laboratory toxicity data.     

Soil pH effects on the comparative toxicity of dissolved zinc,non-nano and nano ZnO to the earthworm Eisenia fetida

To determine how soil properties influence nanoparticle (NP) fate, bioavailability and toxicity, this study compared the toxicity of nano zinc oxide (ZnO NPs), non-nano ZnO and ionic ZnCl₂ to the earthworm Eisenia fetida in a natural soil at three pH levels. NP characterisation indicated that reaction with the soil media greatly controls ZnO properties. Three main conclusions were drawn. First that Zn toxicity, especially for reproduction, was influenced by pH for all Zn forms. This can be linked to the influence of pH on Zn dissolution. Secondly, that ZnO fate, toxicity and bioaccumulation were similar (including relationships with pH) for both ZnO forms, indicating the absence of NP-specific effects. Finally, earthworm Zn concentrations were higher in worms exposed to ZnO compared to ZnCl₂, despite the greater toxicity of the ionic form. This observation suggests the importance of considering the relationship between uptake and toxicity in nanotoxicology studies.     

Bioavailability of chemical pollutants in contaminated soils and pitfalls of chemical analyses in hazard assessment

Decision-making for remediation of industrial wastelands are still based on the concentrations of pollutants of concern measured in soils. In this work, two soils polluted by polycyclic aromatic hydrocarbons (PAHs) and metals were investigated for their toxicity on earthworms (Eisenia fetida), collembolae (Folsomia candida), and higher plants (Brassica chinensis, Lactucca sativa and Avena sativa) in order to study the relationships between chemical contamination and biological effects. Although the level of contamination by PAHs was elevated and commensurate in the two soils, their toxicity profile was quite different. Soil A affected survival and reproduction of invertebrates and growth of higher plants. Surprisingly, soil B, heavily contaminated by metals in addition to PAHs, was devoid of toxicity. Our results indicate that toxicity cannot simply be extrapolated from pollutant concentrations in a complex matrix in which bioavailability of pollutants may be reduced by ageing. Moreover, the use of toxicity data obtained from spiked soils characterized by readily bioavailable pollutants can also be called into question for such extrapolations. Predicting biological effects therefore requires biological tools to avoid any erroneous conclusions that can be drawn from sole extrapolation of analytical results.     

The hydroxyl radical generation and oxidative stress for the earthworm Eisenia fetida exposed to tetrabromobisphenol A

Biochemical effects of tetrabromobisphenol A (TBBPA) on the earthworm Eisenia fetida, including superoxide dismutase, catalase, glutathione-S-transferase, reduced glutathione, oxidized glutathione, GSH/GSSG ratio and malondialdehyde (MDA) level, were measured to assess ecological toxicity of TBBPA. With OECD standard filter-paper contact test method, earthworms were exposed to TBBPA of a range of concentrations (0.00, 0.01, 0.05, 0.1, 0.5 and 1.0 mg L⁻¹). According to the electron paramagnetic resonance spectrum, reactive oxygen species (ROS) generated in the earthworm was identified as the hydroxyl radical (^•OH) which was significantly induced at all TBBPA concentrations. With the increasing of TBBPA concentration, the antioxidant enzymes, glutathione and MDA levels varied significantly. The results showed that TBBPA exerts its toxic effects on E. fetida by inducing the generation of ROS and resulting in oxidative damage. The results show that the ^•OH production leads to oxidative stress in the tissues of the earthworm E. fetida.     

Toxicity and transformation of insecticide fenamiphos to the earthworm <Emphasis Type="Italic">Eisenia fetida</Emphasis>

This study was conducted to investigate the toxicity of the organophosphate insecticide fenamiphos to earthworms (Eisenia fetida) under laboratory conditions. Earthworms were exposed to soils differing in their physico-chemical properties spiked with fenamiphos at concentrations ranging from 10 to 200 mg kg⁻¹ for a period of 4 weeks. Residues of fenamiphos and its metabolites were determined in both soils and earthworms after 4 weeks of pesticide exposure. Fenamiphos was degraded faster in the alkaline soil than in the neutral and acidic soils. Median lethal concentration of fenamiphos in the neutral soil was 228 mg kg⁻¹ soil. Residues of fenamiphos caused a significant reduction in the biomass of worms, especially the ones exposed to the pesticide in the acidic soil. In vitro experiments suggested that fenamiphos was biotransformed in the earthworms principally to its oxide metabolite. To our knowledge, this is the first study demonstrating the biotransformation of fenamiphos by E. fetida.     

Biochemical and genotoxic effect of triclosan on earthworms (Eisenia fetida) using contact and soil tests

Triclosan (TCS) is a broad‐spectrum bactericide that is used for a variety of antimicrobial functions. TCS is frequently detected in the terrestrial environment due to application of sewage sludge to agricultural land. In the present study, 48‐h paper contact and 28‐day spiked soil tests were conducted to examine the toxic effects of TCS on the antioxidative and genetic indices of earthworms (Eisenia fetida). The activity of antioxidative enzymes (superoxide dismutase, SOD; catalase, CAT) and the content of the lipid peroxidation product (malondialdehyde, MDA) were determined as biomarkers of oxidative stress in E. fetida. Moreover, single cell gel electrophoresis (SCGE) was used as a biomarker of genotoxicity. The results showed that triclosan induced a significant increase (P < 0.05) in antioxidative enzyme activities and MDA content. Of all of the biomarkers examined, CAT activity was most sensitive to TCS, and the CAT activity increased significantly (P < 0.05) at bactericidal concentrations of 7.86 ng cm^?2 in the contact test and 10 mg kg^?1 in the spiked soil test. The comet assay showed that TCS treatments significantly induced (P < 0.05) DNA damage in E. fetida, and that 78.6 ng cm^?2 caused significant genotoxic effects in the acute test (48 h). Clear dose‐dependent DNA damage to E. fetida was observed both in contact and spiked soil tests. These results imply that TCS may have potential biochemical and genetic toxicity toward earthworms (E. fetida). A battery of biomarkers covering multiple molecular targets of acute toxicity can be combined to better understand the impacts of TCS on E. fetida. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.     

Persistence of the parabens in soil and their potential toxicity to earthworms

Due to their antimicrobial activity, parabens are commonly used as preservatives in a variety of consumer goods including cosmetics, pharmaceuticals, and personal care products. During the production, usage and disposal of these products, parabens are released into the environment. In this study, the persistence of three widely used parabens; methyl-, propyl-, and butyl paraben in soil and their toxic effects on the soil invertebrate, Eisenia fetida was investigated. The results of this study indicate that selected parabens do not negatively affect the survival, growth, and reproduction of Eisenia fetida up to 1000 mg Kg⁻¹ concentration. Further, these parabens (0–1000 mg Kg⁻¹) exhibited a low persistence in soil with more than 90 % disappearing within three days. In contrast, only 16–54 % degradation of parabens occurred in frozen soil suggesting a microbial role in parabens degradation. This study demonstrates that methyl-, propyl-, and butyl parabens degrade rapidly in the terrestrial environment and therefore, are unlikely to pose a threat to species such as Eisenia fetida. To our knowledge, this is the first report on the toxicity of parabens to earthworms.     

Test Designs to Assess the Influence of Soil Characteristics on the Toxicity of Copper and Lead to the Oligochaete Enchytraeus albidus

Statistical test designs were used to identify and quantify the soil parameters important for determining the ecotoxicological effects of copper and lead to the potworm Enchytraeus albidus. The application of a Fractional Factorial Design revealed that the acute toxicity of copper and lead to E. albidus can vary over more than two orders of magnitude depending on the physico-chemical characteristics of the (artificial) soils. The differences in metal ecotoxicity were mainly determined by pH and organic matter content or cation exchange capacity (CEC). Using a Central Composite Design, models were developed describing the ecotoxicity of copper and lead to E. albidus as a function of these parameters. To validate the developed response surface models, two field soils and the standard artificial soil prescribed by OECD were spiked with copper and lead and the acute toxicity to E. albidus was assessed. These validation experiments confirmed that the toxicity of copper and lead could be predicted using information on the total metal concentration, the pH and the CEC of the soil with toxicity decreasing with increasing pH and CEC.     

Zinc,among a ‘cocktail’ of metal pollutants,is responsible for the absence of the terrestrial isopod Porcellio scaber from the vicinity of a primary smelting works

Porcellio scaber Latreille (Crustacea: Isopoda) of one month in age were reared for a year on leaf litter of field maple (Acer campestre) contaminated in the laboratory with a range of concentrations of cadmium, copper, lead or zinc. The metals were applied topically to the leaves as nitrates. Growth and survival, numbers of live offspring produced by females that matured, and concentrations of metals in adult isopods at the end of the experiment were measured.Critical concentrations of metals in food at which all the isopods died before producing offspring were 100 g Cd g^–1, 100 g Cu g^–1, 2000 g Pb g^–1 and 1000 g Zn g^–1 (on a dry weight basis). The relative toxicities of the four metals in the laboratory were compared with concentrations of cadmium, copper, lead and zinc in surface leaf litter in the vicinity of a primary smelting works at Avonmouth, South West England. The results support the hypothesis that the absence of Porcellio scaber from sites in the immediate vicinity of the factory is due to zinc poisoning. Although cadmium is approximately ten times more toxic to isopods than zinc in the laboratory, zinc is most likely to be killing isopods in the field because its concentration is always at least 30 times higher than cadmium in Avonmouth leaf litter, and more than 100 times higher at most sites.Populations of Porcellio scaber survive in field sites where surface leaf litter contains up to 5000 g Zn g^–1. This is at least five times higher than the critical concentration in laboratory experiments. Thus, the methodology for assessing metal toxicity described in this paper, exaggerates the potential effects of metals to isopods in the field. Such differences between laboratory and field toxicities of metals should be taken into account when environmental protection levels for metals are being proposed for soil invertebrates based on ecotoxicological tests conducted in the laboratory.     

Increased cytotoxic and genotoxic tolerance of <Emphasis Type="Italic">Eisenia fetida</Emphasis> (Oligochaeta) to cadmium after long-term exposure

Since life-cycle studies showed that the earthworm species Eisenia fetida can develop increased tolerance after long-term exposure to a sub-lethal concentration of Cd in the laboratory, we assessed both the cytotoxicity and genotoxicity of Cd in a long-term Cd-exposed population. We exposed E. fetida specimens from this population, from a laboratory control population and from a field population to various concentrations of CdSO₄ in artificial soil water. Toxic effects were measured using the MTT test and the comet assay. The group that had been exposed to Cd for more than a decade was found to be more tolerant to the deleterious effects of Cd at both cellular and molecular levels than the laboratory control population. The field population, which came from a severely metal polluted environment, displayed high tolerance at molecular level as well. The results provide novel biomarker evidence of increased Cd tolerance in E. fetida, but the mechanisms supporting the apparent tolerance, still need to be clarified.     

Copper toxicity in a natural reference soil: ecotoxicological data for the derivation of preliminary soil screening values

The risk assessment of contaminated soils is conventionally done with the support of soil screening values (SSVs). Since SSVs are still unavailable for many European countries, including Portugal, standardized toxicity tests are urgently claimed for their derivation. Hence, this work aimed the generation of toxicity values for copper (Cu) in a natural reference soil (PTRS1) targeting different terrestrial species, endpoints and soil functions, as to derive a preliminary Cu SSV. For this, the Assessment Factor approach was applied, which allowed calculating predicted no effect concentrations (PNEC) for Cu that will be the basis for SSV proposal. In order to increase the reliability of the PNEC, and hence of the SSV, a lab/field factor was applied to correct the toxicity values used for PNEC determination. Cu affected urease, cellulase and nitrogen mineralization activities. The EC₅₀ values calculated for the invertebrates reproduction were 130.9, 165.1 and 191.6 mg Cu Kg^?1soil_dw for Eisenia andrei, Enchytraeus crypticus and Folsomia candida, respectively. Cu inhibited seed germination mainly for Lactuca sativa, whilst it was toxic for the growth of different plant species (EC₅₀s between 89 and 290.5 mg Cu Kg^?1soil_dw). Based on the outcomes gathered, we proposed SSVs for Cu ranging between 26.3 and 31.8 mg Kg^?1 soil_dw, which is above the background values reported and below all the EC₂₀s recorded for the species and endpoints herein analyzed. Overall, this work describes a procedure that could be easily followed by other European countries wishing to derive SSVs adjusted to their soils.     

Genotoxic and oxidative responses in coelomocytes of Eisenia fetida and Hediste diversicolor exposed to lipid‐coated CdSe/ZnS quantum dots and CdCl2

The emerging of Quantum Dots utilization in industrial or medicinal fields involved a potentially increase of these nanoparticles in environment. In this work, the genotoxic (comet assay) and oxidative effects (SOD activity, TBARS) of functionalized‐QDs and cadmium chloride were investigated on Hediste diversicolor and Eisenia fetida coelomocytes. Results demonstrated that functionalized‐QDs (QDNs) and cadmium chloride induced DNA damages through different mechanisms that depended on the nano‐ or ionic nature of Cd. The minimal genotoxic concentrations for H. diversicolor (<0.001ng/g for QDNs and CdCl₂) were lower than for E. fetida (between 0.01 and 0.1 ng/g for QDNs, and between 0.001 and 0.01 ng/g for CdCl₂). These results showed that H. diversicolor was more sensitive than E. fetida. The two contaminants had a low impact on the oxidative stress markers. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 918–926, 2015.     

Oxidative damage of naphthenic acids on the Eisenia fetida earthworm

Naphthenic acids (NAs) have been gaining recognition in recent years as potentially harmful environmental contaminants. Few studies have focused on the potential ecotoxicity of NAs to terrestrial environment. In this study, the responses of antioxidant system and lipid peroxidation and DNA damage were investigated after exposing Eisenia fetida to soil contaminated with NAs. The results indicated that NAs induced a significant increase (p < 0.05) in superoxide dismutase and catalase enzyme activities. The glutathione peroxidase enzyme activities were significantly inhibited (p < 0.05) in the medium and high dose treatments. An increase in malondialidehyde indicated that NAs could cause cellular lipid peroxidation in the tested earthworms. The percentage of DNA in the tail of comet assay of coelomocytes as an indication of DNA damage increased after treatment with different doses of NAs, and a dose‐dependent DNA damage of coelomocytes was found. In conclusion, oxidative stress caused by NAs exposure induces physiological responses and genotoxicity on earthworms. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1337–1343, 2016.     

Effects of zinc and cadmium on oxygen consumption and ammonium excretion in pink shrimp (Farfantepenaeus paulensis, Pérez-Farfante, 1967, Crustacea)

In Brazil, pink shrimp (Farfantepenaeus paulensis) is an important commercially exploited species and is an ideal animal for studying the impairment caused by the effects of heavy metals that are often detected in coastal areas. The main purpose of the present study was to detect the acute toxicity of cadmium and zinc to F. paulensis and investigate their effects on oxygen consumption and ammonium excretion, investigations that have not been carried out in this species before. First, the acute toxicity of zinc and cadmium to F. paulensis for 24, 48, 72, and 96 h—medium lethal concentration was examined, which resulted in the following values: 9.39, 6.00, 4.88, and 3.31 mg/l for zinc and 2.35, 1.67, 1.26, and 0.83 mg/l for cadmium. Furthermore, we also found that exposure of shrimp to zinc and cadmium caused an inhibition in oxygen consumption of 25 and 32.4%, respectively, relative to the control. In addition, after separate exposure to cadmium and zinc, elevations in ammonium excretion were obtained, which were 42.85 and 51.85% higher than the control, respectively.     

Effect of silver nanoparticle surface coating on bioaccumulation and reproductive toxicity in earthworms (Eisenia fetida)

Abstract

The purpose of this study was to investigate the effect of surface coating on the toxicity of silver nanoparticles (Ag NPs) soil. Earthworms (Eisenia fetida) were exposed to AgNO₃ and Ag NPs with similar size ranges coated with either polyvinylpyrrolidone (hydrophilic) or oleic acid (amphiphilic) during a standard sub-chronic reproduction toxicity test. No significant effects on growth or mortality were observed within any of the test treatments. Significant decreases in reproduction were seen in earthworms exposed to AgNO3, (94.21 mg kg^-1) as well as earthworms exposed to Ag NPs with either coating (727.6 mg kg^-1 for oleic acid and 773.3 mg kg^-1 for polyvinylpyrrolidone). The concentrations of Ag NPs at which effects were observed are much higher than predicted concentrations of Ag NPs in sewage sludge amended soils; however, the concentrations at which adverse effects of AgNO₃ were observed are similar to the highest concentrations of Ag presently observed in sewage sludge in the United States. Earthworms accumulated Ag in a concentration-dependent manner from all Ag sources, with more Ag accumulating in tissues from AgNO₃ compared to earthorms exposed to equivalent concentrations of Ag NPs. No differences were observed in Ag accumulation or toxicity between earthworms exposed to Ag NPs with polyvinylpyrrolidone or oleic acid coatings.     

Investigation on bioavailability of some essential and toxic elements in medicinal herbs

Trace and major elements were determined in medicinal herbs (Cynara scolymus, Matricaria chamomilla, Artemisia absinthium L., Achillea millefolium, and Inula britannica) as well as in rhizosphere soil samples. Based on the results obtained after microwave-acid-assisted digestion (nitric acid + hydrogen peroxide) and single-step extraction (ammonium acetate), the real and potential acidity and redox potential of the soils, uptake, mobility, and bioavailability of potassium, calcium, magnesium, iron, manganese, copper, zinc, nickel, chromium, lead, and cadmium are discussed. By calculating the bioconcentration factors and their deviation from the recommended values, elevated concentrations, were explained in terms of contamination and pollution. The concentrations measured in both plants and soil samples were below maximum allowable concentration ranges considered for the European Union.     

A Comparative Study of the Effects of Metal Contamination on Collembola in the Field and in the Laboratory

We examined the species diversity and abundance of Collembola at 32 sampling points along a gradient of metal contamination in a rough grassland site (Wolverhampton, England), formerly used for the disposal of metal-rich smelting waste. Differences in the concentrations of Cd, Cu, Pb and Zn between the least and most contaminated part of the 35 metre transect were more than one order of magnitude. A gradient of Zn concentrations from 597 to 9080 microg g(-1) dry soil was found. A comparison between field concentrations of the four metals and previous studies on their relative toxicities to Collembola, suggested that Zn is likely to be responsible for any ecotoxicological effects on springtails at this site. Euedaphic (soil dwelling) Collembola were extracted by placing soil cores into Tullgren funnels and epedaphic (surface dwelling) species were sampled using pitfall traps. There was no obvious relationship between the total abundance, or a range of commonly used diversity indices, and Zn levels in soils. However, individual species showed considerable differences in abundance. Metal "tolerant" (e.g., Ceratophysella denticulata) and metal "sensitive" (e.g., Cryptopygus thermophilus) species could be identified. Epedaphic species appeared to be influenced less by metal contamination than euedaphic species. This difference is probably due to the higher mobility and lower contact with the soil pore water of epedaphic springtails in comparison to euedaphic Collembola. In an experiment exposing the standard test springtail, Folsomia candida, to soils from all 32 sampling points, adult survival and reproduction showed small but significant negative relationships with total Zn concentrations. Nevertheless, juveniles were still produced from eggs laid by females in the most contaminated soils with 9080 microg g(-1) Zn. Folsomia candida is much more sensitive to equivalent concentrations of Zn in the standard OECD soil. Thus, care should be taken in extrapolating the results of laboratory toxicity tests on metals in OECD soil to field soils, in which, the biological availability of contaminants is likely to be lower. Our studies have shown the importance of ecotoxicological effects at the species level. Although there may be no differences in overall abundance, sensitive species that are numerous in contaminated sites, and which may play important roles in decomposition ("keystone species") can be greatly reduced in numbers by pollution.     

<Superscript>1</Superscript>H NMR metabolomics of earthworm responses to polychlorinated biphenyl (PCB) exposure in soil

¹H NMR-based metabolomics was used to examine the metabolic profile of D₂O-buffer extracted tissues of Eisenia fetida earthworms exposed for 2 days to an artificial soil spiked with sub-lethal concentrations of polychlorinated biphenyls (PCBs) (0, 0.5, 1, 5, 10, or 25 mg/kg Aroclor 1254). Univariate statistical analysis of the identified metabolites revealed a significant increase in ATP concentration in earthworms exposed to the highest soil PCB concentration, but detected no significant changes in other metabolites. However, a multivariate approach which considers alterations in multiple metabolites simultaneously, identified a significant linear relationship between earthworm metabolic profiles and PCB concentration (cross-validated PLS-regression with 7 components, R²X = 0.99, R²Y = 0.77, Q²Y = 0.45, P < 0.001). Significant changes in pair-wise metabolic correlations were also detected as PCB concentration increased. For example, lysine and ATP concentrations showed no apparent correlation in control earthworms (r = 0.22, P = 0.54), but were positively correlated in earthworms from the 25 mg/kg treatment (r = 0.87, P = 0.001). Overall, the observed metabolic responses suggest that PCBs disrupted both carbohydrate (energy) metabolism and membrane (osmolytic) function in E. fetida. The ability of ¹H NMR-based metabolomics to detect these responses suggests that this method offers significant potential for direct assessment of sub-lethal PCB toxicity in soil.