Lysosomal neutral red retention time as a biomarker of organophosphate exposure in the earthworm Aporrectodea caliginosa: laboratory and semi-field experiments

Aporrectodea caliginosa is the most common endogeic (topsoil) earthworm in New Zealand and, because of its habitat, is potentially vulnerable to surface-applied pesticides. Lysosomal damage to earthworms, which can be visualized by the use of the neutral red retention assay (NRRA) has been evaluated in this species as a biomarker of organophosphate exposure. Earthworms were exposed in the laboratory to sub-lethal concentrations of chlorpyrifos and diazinon. In a semi-field experiment, earthworms were placed in mesocosms in a field sprayed with these pesticides at the rate recommended for a vegetable crop. In the laboratory, the neutral red retention time (NRRT) was significantly reduced following exposure to both pesticides compared with controls. In the semi-field experiment, earthworm NRRT was significantly reduced by both pesticides. These experiments have shown that the NRRA is very sensitive to exposure to chlorpyrifos and diazinon even at field rates. It therefore shows promise as a potential biomarker of contamination of soil by organophosphates.     

Sensitive biomarker of polycyclic aromatic hydrocarbons (PAHs): urinary 1-hydroxyprene glucuronide in relation to smoking and low ambient levels of exposure

The lysosomal neutral red retention time (NRRT) assay, a biomarker for lysosomal membrane stability, and the total immune activity (TIA) assay, a measure of non-specific immune system activity, were used in laboratory studies to assess the toxic effects of 2,4,6-trinitrotoluene (TNT) on earthworms (Eisenia andrei) in vivo. The results were compared with the concentration of TNT and its metabolites in earthworm tissue, as well as standard sublethal toxicity endpoints including growth (i.e. weight change) and reproduction effects from previously published studies. Filter paper experiments indicated a significant decrease in NRRT at ≥1.8 μg TNT cm^-2, whereas sublethal (weight loss) and lethal effects to earthworms were detected at ≥3.5 and 7.1 μg TNT cm^-2, respectively. Experiments in artificial soil showed that NRRT effects could be detected at lower TNT concentrations (≥55 mg TNT kg^-1 soil dry weight) compared with other sublethal endpoints (effects on growth and reproduction). The TIA biomarker did not significantly respond to TNT. Copper (as CuSO₄, filter paper contact tests) and 2-chloroacetamide (soil tests), which were used as reference toxicants, also decreased the NRRT. The use of the NRRT assay linked with tissue concentrations of TNT metabolites in earthworms was identified as a potentially appropriate biomarker approach for TNT exposure assessment under laboratory conditions and a novel tool for effects-based risk assessment.     

Evaluation of tissue and cellular biomarkers to assess 2,4,6-trinitrotoluene (TNT) exposure in earthworms: effects-based assessment in laboratory studies using Eisenia andrei

The lysosomal neutral red retention time (NRRT) assay, a biomarker for lysosomal membrane stability, and the total immune activity (TIA) assay, a measure of non-specific immune system activity, were used in laboratory studies to assess the toxic effects of 2,4,6-trinitrotoluene (TNT) on earthworms (Eisenia andrei) in vivo. The results were compared with the concentration of TNT and its metabolites in earthworm tissue, as well as standard sublethal toxicity endpoints including growth (i.e. weight change) and reproduction effects from previously published studies. Filter paper experiments indicated a significant decrease in NRRT at ≥1.8 µg TNT cm^-2, whereas sublethal (weight loss) and lethal effects to earthworms were detected at ≥3.5 and 7.1 µg TNT cm^-2, respectively. Experiments in artificial soil showed that NRRT effects could be detected at lower TNT concentrations ( ≥55 mg TNT kg^-1 soil dry weight) compared with other sublethal endpoints (effects on growth and reproduction). The TIA biomarker did not significantly respond to TNT. Copper (as CuSO₄, filter paper contact tests) and 2-chloroacetamide (soil tests), which were used as reference toxicants, also decreased the NRRT. The use of the NRRT assay linked with tissue concentrations of TNT metabolites in earthworms was identified as a potentially appropriate biomarker approach for TNT exposure assessment under laboratory conditions and a novel tool for effects-based risk assessment.     

用蚯蚓溶酶体作为检测土壤污染的生物标志物

蚯蚓体腔细胞内的溶酶体能积累中性红染料.当受到环境胁迫时,溶酶体膜容易破裂,染料就逐步泄露到细胞质中,使细胞染成红色.中性红的保持时间(neutral red retention time,NRRT)可反映溶酶体受损情况.用不同浓度乙草胺和不同浓度Se处理赤子爱胜蚓(Eisenia fetida),观察体腔细胞溶酶体中性红染色的保持时间.结果表明,随乙草胺和Se处理浓度的增高和处理时间的延长,NRRT缩短;NRRT与土壤中乙草胺残留的浓度呈明显的负相关,与蚯蚓体内富集的Se也有明显的负相关.可见,蚯蚓溶酶体中性红染色保持时间是测定土壤污染物的生物标志物,在环境风险评价和土壤监测中具有稳定性和准确性,可作为早期预警生物标志物.     

Drought stress in rice (Oryza sativa L.) is enhanced in the presence of the compacting earthworm Millsonia anomala

Earthworms increase growth of most plant species through a number of poorly investigated mechanisms. We tested the hypothesis that earthworm modifications of soil structure and the resulting changes in water availability to plants explain this positive effect. Addition of endogeic earthworms Millsonia anomala induced a 40% increase in shoot biomass production and a 13% increase in CO₂ assimilation rate of well watered rice plants grown in pots. Conversely, when plants were subjected to water deficit, presence of earthworms had no effect on shoot biomass production and a negative impact on CO₂ assimilation rate (−21%). Early stomatal closure in presence of earthworms indicated lower water availability. The hypothesis that earthworms improve plant biomass production through soil physical structure modification was thus rejected. Three hypotheses were tested to explain this decrease in water availability: (i) a decrease in soil water retention capacity, (ii) an increase in evaporation from the soil or/and (iii) an increase in plant transpiration. Results showed that earthworms significantly reduced soil water retention capacity by more than 6%, but had no effect on evaporation rate. Water losses through transpiration were greater in the presence of earthworms when the soil was maintained at field capacity, but this was not the case under drought conditions. This experiment showed that the endogeic compacting earthworm M. anomala significantly increased plant photosynthesis by an undetermined mechanism under well-watered conditions. However, photosynthesis was reduced under drought conditions due to reduced soil water retention capacity.     

蚯蚓生物标记物在土壤生态风险评价中的应用

蚯蚓在土壤中行使了很多重要的生态功能,蚯蚓生物标记物常用作土壤污染风险评价研究。这篇综述的目的是探讨当前蚯蚓生物标记物研究是否可以应用到实际的土壤污染风险评价。1)讨论了蚯蚓生物标记物在土壤污染风险评价体系中的重要性,认为它是化学分析方法的有益补充,可以提供更为全面和客观的土壤污染信息;2)综述了相关研究中所使用的蚯蚓类型,土壤类型和生物标记物类型,及其它试验设计要素和最后结果的变异,认为目前蚯蚓生物标记物研究以实验室基础研究为主,筛选出了大量的生物标记物,一定程度上揭示了生物标记物的对各类典型污染物及其组合的应答机制;同时也认为,未来的蚯蚓生物标记物研究应该重点探讨将其应用到实际的土壤污染风险评价中的可行性及如何应用;3)目前不同研究之间从试验设计到结果都具有很大的变异,难以通过综合比较获得完全可靠的具有实践意义的结论和成果,因此,有必要通过建立标准化的蚯蚓生物标记物研究方法,推动生物标记物的研究工作;4)提出了蚯蚓生物标记物研究方法标准化的具体建议,推荐了蚯蚓生物标记物走向实际应用所需要解决的问题。     

Acetylcholinesterase activity in the earthworm Eisenia andrei at different conditions of carbaryl exposure

Recent reports have stressed the need for a better understanding of earthworm biomarker responses. We aimed at investigating acethylcholinesterase (AChE) activity in the earthworm Eisenia andrei after exposure to carbaryl or its commercial formulation Zoril 5 under different in vitro and in vivo experiments. In addition, lysosome membrane stability was assessed by neutral red retention assay in the same experimental conditions. AChE basal Km and Vm values were about 0.16 mM and 41 nmol min(-1) mg protein(-1), respectively. Carbaryl dose-dependently decreased Vmax, while not affecting Km values. Carbaryl reduced earthworm AChE activity within 1 day of in vivo exposure to contaminated filter paper. Tested on soil, carbaryl inhibited AChE with the maximum effect after 3 days; in contrast, lysosome membrane stability of coelomocytes indicated a maximum toxicity after one day, followed by a recovery. AChE inhibition by Zoril 5 was highest after one day, while lysosome membrane stability declined progressively. In all cases, carbaryl dose-dependently decreased Vmax while not affecting Km values. In conclusion, E. andrei AChE activity assessed in vitro is dose-dependently inhibited by the carbamate compound carbaryl, which acts as a pure competitive inhibitor. In vivo experiments suggested that pure and co-formulated carbaryl have different time and/or dose dependent effects on earthworms. Our results further support the use of AChE inhibition as an indicator of pesticide contamination, to be included in a battery of biomarkers for monitoring soil toxicity.     

Does pollen availability mitigate the impact of pesticides on generalist predatory mites?

The effect of the provision of pollen on the impact of pesticides on the predatory mite Kampimodromus aberrans was assessed at individual and population levels. In the laboratory we evaluated the influence of pollen amount and pollen application frequency on lethal and sub-lethal effects of chlorpyrifos and spinosad. In a potted plant experiment, the effects of pesticides and pollen were assessed on predatory mite population abundance. In the laboratory, survival and fecundity of predatory mites were reduced by insecticides, and spinosad was more toxic than chlorpyrifos. In the same experiment, high pollen application frequency alleviated the sub-lethal effect induced by chlorpyrifos. On potted plants, pollen applications reduced the impact of chlorpyrifos on K. aberrans, whereas without pollen applications the impact of spinosad and chlorpyrifos on the predatory mite population was similar. Results obtained here highlight that the provision of fresh pollen is of particular importance for predatory mites when pesticides are applied.     

Gas Chromatography- Mass Spectrometry Based Metabolomic Approach for Optimization and Toxicity Evaluation of Earthworm Sub-Lethal Responses to Carbofuran

Despite recent advances in understanding mechanism of toxicity, the development of biomarkers (biochemicals that vary significantly with exposure to chemicals) for pesticides and environmental contaminants exposure is still a challenging task. Carbofuran is one of the most commonly used pesticides in agriculture and said to be most toxic carbamate pesticide. It is necessary to identify the biochemicals that can vary significantly after carbofuran exposure on earthworms which will help to assess the soil ecotoxicity. Initially, we have optimized the extraction conditions which are suitable for high-throughput gas chromatography mass spectrometry (GC-MS) based metabolomics for the tissue of earthworm, Metaphire posthuma. Upon evaluation of five different extraction solvent systems, 80% methanol was found to have good extraction efficiency based on the yields of metabolites, multivariate analysis, total number of peaks and reproducibility of metabolites. Later the toxicity evaluation was performed to characterize the tissue specific metabolomic perturbation of earthworm, Metaphire posthuma after exposure to carbofuran at three different concentration levels (0.15, 0.3 and 0.6 mg/kg of soil). Seventeen metabolites, contributing to the best classification performance of highest dose dependent carbofuran exposed earthworms from healthy controls were identified. This study suggests that GC-MS based metabolomic approach was precise and sensitive to measure the earthworm responses to carbofuran exposure in soil, and can be used as a promising tool for environmental eco-toxicological studies.     

Mortality of the earthworm Eisenia fetida (Savigny) presented to the terrestrial planarian Ártioposthia triangulata (Dendy) (Tricladida: Terricola)

Ártioposthia triangulata is a terrestrial planarian that feeds on earthworms. Up to 76% of field collected A. triangulata fed on Eisenia fetida earthworms at 10°C in the laboratory. In one experiment, the feeding rate declined from 63% of earthworms attacked to 36% and 34% in the first, second and third feeding opportunities over a week. The average number of E. fetida eaten was 1.4 per planarian per week. The gain in weight of individual A. triangulata was significantly related to the amount of earthworm tissue lost during the feeding process. Over a one week period, the amount of earthworm tissue lost was related to the total amount presented to the planarians but not to their size. Frequency of attack was, however, related to planarian size. The results are discussed in relation to an hypothetical population of earthworms in pasture. It is concluded that the results support the contention that A. triangulata could seriously deplete earthworm populations.     

Effects of earthworms on biomass production,nitrogen allocation and nitrogen transfer in wheat-clover intercropping model systems

The effects of earthworms (Lumbricidae) on plant biomass production and N allocation in model intercropping systems of winter wheat and white clover were evaluated in two pot experiments. Wheat and wheat-clover mixtures were grown in a low-organic loam soil, earthworms were added at densities comparable to field population densities and the experiments were terminated 48 and 17 d after earthworm introductions. In both experiments, earthworms significantly increased the biomass and N uptake of wheat while they had generally no effects on clover. As a result, earthworm activity increased the proportion of wheat biomass in the total plant biomass of the mixture. Nitrogen budgets of the experiment lasting 48 d indicated that additional N in the system made available by earthworm activity was primarily taken up by the wheat. Earthworms also affected intra-plant N allocation in wheat which had significantly higher shoot:root N ratios when earthworms were present. When clover was labelled with ¹⁵N in the experiment which lasted 17 d, endogeic earthworms significantly reduced the amounts of ¹⁵N excess transferred from living or decomposing clover roots to accompanying wheat plants. Earthworms assimilated small quantities of ¹⁵N tracer from decomposing clover roots but not from living clover roots. The results of these model experiments suggest that earthworms can affect the balance between intercropped cereals and legumes by altering intra- and inter-plant N allocation. This revised version was published online in June 2006 with corrections to the Cover Date.     

蚯蚓作用下污泥重金属形态变化及其与化学生物学性质变化的关系

城市污泥处理是一项世界性难题,污泥农业利用是其最简单有效的资源化利用方式之一,但污泥中较高的重金属含量限制了其实际推广应用,利用蚯蚓-超富集植物联合修复污泥重金属的方法已引起国内外研究者的关注。以新鲜城市脱水污泥为研究对象,接种赤子爱胜蚓(Eisenia fetida)进行室内培养试验,系统研究蚯蚓作用下污泥重金属形态的变化,及其与污泥氧化还原条件、化学和微生物性质变化的关系,以期为蚯蚓-超富集植物联合修复技术在污泥重金属处理中的应用提供理论依据。结果表明,试验前期蚯蚓在污泥中能正常生长和存活,前20 d总生物量增加了52%。蚯蚓可以显著促进污泥中的Cu、Zn、Cd、Ni等重金属从残渣态和铁锰态等稳定形态向交换态和水溶态等有效形态转化。还可以显著降低污泥中还原性物质的含量,减缓p H值下降速度,降低总有机碳含量,促进铵态氮向硝态氮转化,减少污泥微生物的数量并增加其种群活性。蚯蚓作用下,污泥中重金属的活化程度与还原性物质的含量呈显著负相关,而与微生物种群的活性呈显著正相关(P0.05)。综上所述,蚯蚓可以促进污泥重金属的活化,并改善污泥的肥力条件,为修复植物在污泥中的正常生长和对重金属离子的快速吸收提供有利条件。     

Carbamate Protection of AChE Against Inhibition by Agricultural Chemicals

The carbamate pyridostigmine bromide has been used as a pretreatment to protect individuals from the nerve agent soman. Previous research showed that pyridostigmine significantly protected human muscle acetylcholinesterase in vitro from soman and bovine red blood cell acetylcholinesterase from some organophosphorous pesticides. Research presented here demonstrates that pretreatment with other carbamates also protects acetylcholinesterase from inhibition by the pesticides chlorpyrifos‐oxon and diazinon‐oxon, but not from malaoxon. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:506‐509, 2012; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21456     

Pesticide Susceptibility of Two Coccinellids (Stethorus punctum picipes and Harmonia axyridis) Important in Biological Control of Mites and Aphids in Washington Hops

The susceptibility of Stethorus punctum picipes (Casey) and Harmonia axyridis Pallas larvae to pesticides used or with potential for use in Washington hops, was examined in laboratory bioassays. All pesticides tested except the miticide, hexythiazox, the insecticides, chlorpyrifos and pirimicarb, and the fungicide, mycobutanil, produced 100% mortality in S. punctum picipes at concentrations equivalent to field rates. The insecticides, pirimicarb, endosulfan, and thiamethoxam were least toxic to H. axyridis. Bifenthrin, diazinon, dimethoate, methomyl, carbaryl, malathion, phosmet, imidacloprid, and chlorpyrifos were highly toxic. The miticides, abamectin and fenpyroximate were highly toxic, milbemectin was moderately toxic but all other miticides tested were non-toxic. All fungicides had low toxicity. Selection and use of pesticides compatible with natural enemies and conservation biological control in Washington hop production is discussed.     

Human blood and environmental media screening method for pesticides and polychlorinated biphenyl compounds using liquid extraction and gas chromatography-mass spectrometry analysis

Screening assessment methods have been developed for semi- and non-volatile persistent organic pollutants (POPs) for human blood and solid environmental media. The specific methodology is developed for measuring the presence of "native" compounds, specifically, a variety of organochlorine pesticides (OCPs), organophosphate pesticides (OPPs), and for polychlorinated biphenyls (PCBs). The method is demonstrated on anonymous Red Cross blood samples as well as two potential environmental sources, tracked in soil and dog hair. This work is based on previously developed methods for semi-volatile hydrocarbon exposure from fuels usage and similarly employs liquid solvent extraction, evaporative volume reduction. and subsequent specialized gas chromatography-mass spectrometry analysis (GC-MS). Standard curves, estimates of recovery efficiency, and specific GC-MS SIM quantification methods were developed for common pesticides including diazinon. aldrin, chlorpyrifos, malathion, dieldrin, DDT, permethrin, cyhalothrin, and cypermethrin, and for seven selected PCBs. Trace levels of certain PCBs and pesticides such as permethrin, dieldrin, malathion, lindane, diazinon, and chlorpyrifos were tentatively identified in anonymous blood samples as well as in two potential environmental sources. tracked in soil and dog hair. The method provides a simple screening procedure for various media and a variety of common organic pollutants without extensive sample preparation. It is meant to complement and augment data from more specific or complex methodology, to provide initial broad spectrum guidance for designing targeted experiments, and to provide confirmatory evidence for the usual metabolic biomarker measurements made to assess human exposure.     

Local distribution of native and invasive earthworms and effects on a native salamander

North America is home to both native and invasive earthworms acting as ecosystem engineers as they build burrows that can serve as habitat for other species or otherwise alter soil structure, affecting nutrient cycling and other ecosystem processes. Here I determine where and what earthworm species commonly occur in my study area, and compare effects of native and invasive earthworms on the common woodland salamander, Plethodon cinereus, in field surveys and laboratory experiments. The native earthworm Eisenoides carolinensis was the most common earthworm, followed by two invasive species Dendrobaena octaedra and Octolasion tyrtaeum. The presence of O. tyrtaeum was associated with a narrower O-horizon (i.e., organic layer in the soil). Using structural equation modeling to explore direct and indirect pathways of these three most common earthworm species on salamanders, I found O. tyrtaeum occurrence was negatively correlated with nighttime salamander counts, a proxy for total salamander numbers, mediated by negative effects on O-horizon depth and microinvertebrate numbers. In the laboratory, O. tyrtaeum and D. octaedra consumed more leaf litter per gram of earthworm per day than the native E. carolinensis. However, salamanders consumed earthworms and used burrows of all native and invasive species of earthworms similarly. The potential for negative indirect effects of the invasive earthworm O. tyrtaeum on P. cinereus was demonstrated both in the field and laboratory, highlighting that seemingly small differences between native and invasive ecosystem engineers have the potential to significantly alter the effects of these closely related native and invasive organisms.     

Earthworms and eco-consequences: Considerations to soil biological indicators and plant function: A review

Earthworms have been well reported to have a beneficial effect on soil microbes, soil microbial biomass (SMB), fungal community, soil structure, water retention and plant growth in different terrestrial ecosystems. However, the interactions between environmental stressors and various species of earthworms and the subsequent effect on soil microbes, organic matter, soil structure and plant growth are still uncertain. The purpose of this analysis was to test 1- the impact of environmental stressors on earthworm behaviour. 2- the effect of various earthworms on soil microbes, plant growth, soil structure and the carbon cycle. We noted that less fatal temperatures are generally unknown, but higher fatal temperatures range from 25 to 48 °C. Earthworms have a role to play, depending on the nature of organic residues, in both the formation and degradation of soil aggregates. Improvements in microbial biomass and plant growth have been established according to temperature, soil toxicity, soil type, earthworms abundance, organic residues types and field conditions. We observed that although the summer temperature in the arid area was approximately (°C 48), it was found that a particular type of earthworm (Namalycastis indica) was responsible for improving soil characteristics.While a great deal of analysis has been carried out on the role of earthworms within the soil ecology, such a review identifies important knowledge gaps, particularly in the determination of the impacts of earthworm species on the soil structure, microbial biomass and plant productivity, in particular since most papers focused on European species and overlooked the role of earthworms in the arid landscape. Further research is recommended to compare the impacts of different earthworms species on soil microbes and plant growth in various soil types, earthworm abundance, field conditions, organic residues locations, inorganic fertilizers, pesticides, fertile or non-fertile soils and diverse conditions of drought and moisture.     

Determination of organophosphate and carbamate pesticides in spiked samples of tap water and fruit juices by a biosensor with photothermal detection.

The determination of organophosphate (paraoxon, chlorpyrifos, diazinon) and carbamate (carbaryl, carbofuran) pesticides in spiked drinking water and fruit juices was carried out using a photothermal biosensor. The biosensor consists of a cartridge containing immobilised enzyme acetylcholinesterase (AChE) placed in a flow-injection analysis (FIA) manifold and a photothermal detector based on thermal lens spectrometry. With this approach, 0.2 ng/ml of paraoxon can be detected in less than 15 min. Limits of detection for other organophosphate (chlorpyrifos, diazinon) and carbamate (carbaryl, carbofuran) pesticides varied, depending on their antiacetylcholinesterase (AntiAChE) toxicity, from 1 ng/ml to 4 microg/ml. The biosensor was used for the direct detection of pesticides in spiked tap water and fruit juices without any pretreatment steps. In these cases, the LOD3sigma of 1.5, 2.8 and 4 ng/ml paraoxon in tap water, orange juice and apple juice were obtained, respectively.     

Effects of transgenic Bt corn litter on the earthworm Lumbricus terrestris

A 200-day study was carried out to investigate the impact of transgenic Bacillus thuringiensis (Bt) corn on immature and adult Lumbricus terrestris in the field and in the laboratory. Another objective of this study was to develop test methods that could be used for standard testing of the impact of transgenic plants on different earthworm species in the field and in the laboratory. For this purpose two different experiments were involved, a laboratory experiment with adult L. terrestris and a field experiment with immature L. terrestris. No lethal effects of transgenic Bt corn on immature and adult earthworms were observed. Immature L. terrestris in the field had a very similar growth pattern when fed either (Bt+) or (Bt-) corn litter. No significant differences in relative weights of (Bt+) and (Bt-) corn-fed adult L. terrestris were observed during the first 160 days of the laboratory trial, but after 200 days adult L. terrestris had a significant weight loss of 18% of their initial weight when fed (Bt+) corn litter compared to a weight gain of 4% of the initial weight of (Bt-) corn-fed earthworms. Further studies are necessary to see whether or not this difference in relative weight was due to the Bt toxin or other factors discussed in the study. Degradation of Cry1Ab toxin in corn residues was significantly slower in the field than at 10 degrees C in the laboratory. Enzyme-linked immunosorbent assay results indicated that earthworms in both experiments were exposed to the Bt toxin throughout the whole experimental time.