Toxicity assessment of a common laundry detergent using the freshwater flagellate Euglena gracilis

Synthetic detergents are among the commonly used chemicals in everyday life. Detergents, reaching aquatic environments through domestic and municipal wastewater, can cause many different effects in aquatic organisms. The present study was aimed at the toxicity evaluation of a commonly used laundry detergent, Ariel, using the freshwater flagellate Euglena gracilis as a biotest organism. Different parameters of the flagellate like motility, swimming velocity, cell shape, gravitactic orientation, photosynthesis and concentration of light harvesting pigments were used as end points for the toxicity assessment. No Observed Effect Concentration (NOEC) and EC₅₀ values were calculated for the end point parameters at four different incubation times, i.e. 0, 6, 24 and 72 h. After 72 h incubation, swimming velocity of the cells was found to be the most sensitive parameter giving NOEC and EC₅₀ values of 10.8 and 34 mg L⁻¹, respectively. After 72 h exposure to the detergent, chlorophyll a and total carotenoids were significantly decreased in cultures treated with Ariel at concentrations of 50 mg L⁻¹ and above while chlorophyll b significantly decreased at concentrations above 750 mg L⁻¹. The maximum inhibitory effect on the quantum yield of photosystem II was observed after 24 h exposure and thereafter a recovery trend was observed. Motility, gravitaxis and cell shape were strongly impaired immediately upon exposure to the detergent, but with increasing exposure time these parameters showed acclimatization to the stress and thus the NOEC values obtained after 72 h were higher than those immediately after exposure.     

Evaluation of phenanthrene toxicity on earthworm (Eisenia fetida): An ecotoxicoproteomics approach

The goal of this study was to identify promising new biomarkers of phenanthrene by identifying differentially expressed proteins in Eisenia fetida after exposure to phenanthrene. Extracts of earthworm epithelium collected at days 2, 7, 14, and 28 after phenanthrene exposure were analyzed by two dimensional electrophoresis (2-DE) and quantitative image analysis. Comparing the intensity of protein spots, 36 upregulated proteins and 45 downregulated proteins were found. Some of the downregulated and upregulated proteins were verified by MALDI-TOF/TOF-MS and database searching. Downregulated proteins in response to phenanthrene exposure were involved in glycolysis, energy metabolism, chaperones, proteolysis, protein folding and electron transport. In contrast, oxidation reduction, oxygen transport, defense systems response to pollutant, protein biosynthesis and fatty acid biosynthesis were upregulated in phenanthrene-treated E. fetida. In addition, ATP synthase b subunit, lysenin-related protein 2, lombricine kinase, glyceraldehyde 3-phosphate dehydrogenase, actinbinding protein, and extracellular globin-4 seem to be potential biomarkers since these biomarker were able to low levels (2.5 mg kg⁻¹) of phenanthrene. Our study provides a functional profile of the phenanthrene-responsive proteins in earthworms. The variable levels and trends in these spots could play a potential role as novel biomarkers for monitoring the levels of phenanthrene contamination in soil ecosystems.     

Exposure to elevated ozone levels differentially affects the antioxidant capacity and the redox homeostasis of two subtropical Phaseolus vulgaris L. varieties

Ozone (O₃) has become one of the most toxic air pollutants to plants worldwide. However, investigations on O₃ impacts on crops health and productivity in South America countries are still scarce. The present study analyzed the differences on the enzymatic and non-enzymatic antioxidant system in foliar tissue of two subtropical Phaseolus vulgaris varieties exposed to high O₃ concentration. Both varieties were negatively impacted by the pollutant, but the responses between each variety were quite distinct. Results revealed that Irai has higher constitutive levels of reactive oxygen species (ROS) and ascorbate (AsA) concentration, but lower total thiol levels and catalase immunocontent. In this variety catalase protein concentration was increased after O₃ exposure, indicating a better cellular capacity to reduce hydrogen peroxide. On the opposite, Fepagro 26-exposed plants increased ROS generation and AsA concentration, but had the levels of total thiol content and catalase protein unchanged. Furthermore, O₃ treatment reduced the levels of chlorophylls a and b, and the relationship analysis between the chlorophyll ratio (a/b) and protein concentration were positively correlated indicating that photosynthetic apparatus is compromised, and thus probably is the biomass acquisition on Fepagro 26. Differently, O₃ treatment of Irai did not affect chlorophylls a and b content, and loss on the protein content was lower. Altogether, these data suggest that early accumulation of ROS on Fepagro 26 are associated with an insufficient leaf antioxidant capacity, which leads to cell structure disruption and impairs the photosynthesis. Irai seems to be more tolerant to O₃ toxic effects than Fepagro 26, and the observed differences on O₃ sensitivity between the two varieties are apparently based on constitutive differences involved in the maintenance of intracellular redox homeostasis.     

Impact of dissolved organic matter on bioavailability of chlorotoluron to wheat

Chlorotoluron (Chl) is a phenylurea herbicide and is widely used for controlling weeds. While it has brought great benefits to crop production, it has also resulted in contamination to ecosystem. In this study, we investigated accumulation of chlorotoluron (Chl) and biological responses of wheat plants as affected by dissolved organic matter (DOM). Wheat seedlings grown under 10 mg kg⁻¹ Chl for 4 d showed a low level of chlorophyll accumulation and damage to plasma membrane. The growth was inhibited by exposure of chlorotoluron. Treatment with 50 mg DOC kg⁻¹ DOM derived either from sludge (DOM-SL) or straw (DOM-ST) attenuated the chlorotoluron toxicity to plants. Both DOMs decreased activities of catalase, peroxidase and superoxide dismutase in Chl-treated seedlings. However, an increased glutathione S-transferases activity was observed under the same condition. Wheat plants treated with Chl in the presence of DOM accumulated less Chl than those treated with Chl alone. Moreover, in the presence of DOM, bioconcentration factor (BCF) decreased whereas translocation factors increased. Analyses with FT-IR spectra confirmed the regulatory role of DOMs in reducing Chl accumulation in wheat.     

Suitability of different salt marsh plants for petroleum hydrocarbons remediation

The suitability of the salt-marsh species Halimione portulacoides, Scirpus maritimus, Juncus maritimus and an association of the last two for remediation of petroleum hydrocarbons (PHC) in soil was investigated. An outdoor laboratory experiment (microcosm-scale) was carried out using contaminated soil collected in a refinery, as a complement of another study carried out in the refinery environment (mesocosm-scale). Soil samples with old contamination (mainly crude oil) and with a mixture of the old and recent (turbine oil) contamination were tested. Studies in both micro- and mesocosm-scale provided results coherent in substance. The presence of S. maritimus caused removal of old contamination which was refractory to natural attenuation (after 7 months of exposure, efficiency was 13% when only old contamination was present and 40% when the soil also contained recent contamination). H. portulacoides (only included in the microcosm-scale study) revealed also potentiality for PHC remediation, although with less efficiency than S. maritimus. Degradation of recent contamination was also faster in the presence of plants (after 7 months: 100% in the presence of S. maritimus vs. 63% in its absence). As these species are common in salt marsh areas in Atlantic coast of Europe, it is probable they will be also useful for recovering coast sediments. In contrast, J. maritimus and association did not reveal capability to remove PHC from soil, the presence of J. maritimus inhibiting the capability of S. maritimus.     

Effects of perchlorate on growth of four wetland plants and its accumulation in plant tissues

Perchlorate contamination in water is of concern because of uncertainties about toxicity and health effects, impact on ecosystems, and possible indirect exposure pathways to humans. Therefore, it is very important to investigate the ecotoxicology of perchlorate and to screen plant species for phytoremediation. Effects of perchlorate (20, 200, and 500 mg/L) on the growth of four wetland plants (Eichhornia crassipes, Acorus calamus L., Thalia dealbata, and Canna indica) as well as its accumulation in different plant tissues were investigated through water culture experiments. Twenty milligrams per liter of perchlorate had no significant effects on height, root length, aboveground part weight, root weight, and oxidizing power of roots of four plants, except A. calamus, and increasing concentrations of perchlorate showed that out of the four wetland plants, only A. calamus had a significant (p?<?0.05) dose-dependent decrease in these parameters. When treated with 500 mg/L perchlorate, these parameters and chlorophyll content in the leaf of plants showed significant decline contrasted to control groups, except the root length of E. crassipes and C. indica. The order of inhibition rates of perchlorate on root length, aboveground part weight and root weight, and oxidizing power of roots was: A. calamus > C. indica > T. dealbata > E. crassipes and on chlorophyll content in the leaf it was: A. calamus > T. dealbata > C. indica > E. crassipes. The higher the concentration of perchlorate used, the higher the amount of perchlorate accumulation in plants. Perchlorate accumulation in aboveground tissues was much higher than that in underground tissues and leaf was the main tissue for perchlorate accumulation. The order of perchlorate accumulation content and the bioconcentration factor in leaf of four plants was: E. crassipes > C. indica > T. dealbata > A. calamus. Therefore, E. crassipes might be an ideal plant with high tolerance ability and accumulation ability for constructing wetland to remediate high levels of perchlorate polluted water.     

苯污染地下水系统反硝化菌分布及其净化过程

生物降解作用是地下水系统有机污染物自然衰减过程中最重要的破坏性衰减机制。在分析中,以我国某受苯污染水源地的地下水系统为对象,研究了该地下水中反硝化菌的分布,筛选出2株土著反硝化菌,通过生物降解实验证明其具有降解苯的能力,同时结合水源地历年的水质监测数据,首次从地球化学及生物学2个方面说明了该地下水中存在反硝化菌对苯的生物净化作用,为进一步研究该地下水中苯自然衰减规律奠定基础。     

Comparative phototoxicity of nanoparticulate and bulk ZnO to a free-living nematode Caenorhabditis elegans: the importance of illumination mode and primary particle size

The present study evaluated phototoxicity of nanoparticulate ZnO and bulk-ZnO under natural sunlight (NSL) versus ambient artificial laboratory light (AALL) illumination to a free-living nematode Caenorhabditis elegans. Phototoxicity of nano-ZnO and bulk-ZnO was largely dependent on illumination method as 2-h exposure under NSL caused significantly greater mortality in C. elegans than under AALL. This phototoxicity was closely related to photocatalytic reactive oxygen species (ROS) generation by the ZnO particles as indicated by concomitant methylene blue photodegradation. Both materials caused mortality in C. elegans under AALL during 24-h exposure although neither degraded methylene blue, suggesting mechanisms of toxicity other than photocatalytic ROS generation were involved. Particle dissolution of ZnO did not appear to play an important role in the toxicity observed in this study. Nano-ZnO showed greater phototoxicity than bulk-ZnO despite their similar size of aggregates, suggesting primary particle size is more important than aggregate size in determining phototoxicity.     

Copper tolerance in the macrolichens Cladonia furcata and Cladina arbuscula subsp. mitis is constitutive rather than inducible

In this study we assessed the degree of copper (Cu) tolerance in two common lichen species (Cladonia furcata and Cladina arbuscula subsp. mitis) that grow on both uncontaminated substrata and the surface of waste heaps from abandoned old Cu-mines. Regardless of their locality, populations of these lichens contain identical strains of photobionts (Asterochloris clade A in C. arbuscula subsp. mitis and clade D in C. furcata). Therefore, it was expected that if there were differences in Cu toxicity or tolerance between populations, that the photobiont could not be a key element of Cu tolerance in these two lichen species. In laboratory experiments samples of both lichen species (from contaminated and control sites) were incubated in Cu solutions (500 μM) for 24 h. We attempted to determine whether Cu tolerance in these lichens was constitutive, or inducible form. Based on measurements of Cu accumulation, chlorophyll a integrity, chlorophyll a fluorescence, photosynthesis, respiration, measurements of the content of thiobarbituric acid reactive substances (TBARS), the content of soluble proteins, reactive oxygen species (ROS) and the amount of extracellular secondary metabolites of both lichens we found that there were no significant differences in the response of all selected populations of both lichen species to short-term exposure to these high levels of Cu. As a result, we conclude that Cu tolerance in these two lichen species is the constitutive rather than the inducible.     

Effects of sub-lethal glyphosate concentrations on growth and photosynthetic performance of non-target species Bolboschoenus maritimus

Glyphosate use has increased over the last decades for the control of invasive plant species in wetland ecosystems. Although glyphosate has been considered ‘environmentally’ safe, its repeated use could increase the toxicological risk derived from diffuse pollution of surface and groundwater on non-target vegetation. A glasshouse study was designed to determine the effect produced by the addition of different sub-lethal doses of glyphosate herbicides (5–30 mg L⁻¹) to the nutrient solution on the growth and photosynthetic apparatus of Bolboschoenus maritimus. Although B. maritimus plants were able to grow and survive after 20 d of exposure to glyphosate, the presence of this herbicide affected their growth, through a direct interaction with the root system. Particularly, at 30 mg L⁻¹ glyphosate, B. maritimus showed ca. 30% of biomass decrease. The reduction in B. maritimus growth was due to a decrease in net photosynthetic rate (A), which ranged between values ca. 11.5 and 5.5 μmol m⁻² s⁻¹ CO₂ for the control and the highest glyphosate treatment, respectively. The response of A to glyphosate could be largely accounted for by non-stomatal limitations, since stomatal conductance was similar in all glyphosate treatments. Thus, A decrease was prompted by the negative impact of herbicide on photochemical (PSII) apparatus, the reduction in the absorption of essential nutrients, the reduction of photosynthetic pigments and possibly the reduction in Rubisco carboxilation capacity. Moreover, glyphosate excess caused photoinhibitory damage. In conclusion, in this study we have shown that herbicide water pollution could be a source of indirect phytotoxicity for B. maritimus.     

Can behavioural responses of Lumbriculus variegatus (Oligochaeta) assess sediment toxicity? A case study with sediments exposed to acid mine drainage

The São Domingos mine (Portugal) is, potentially, a good site for ecotoxicological studies, due to a pH and metal gradient of acid mine drainage. In this study, the toxicity of several mine sediments was evaluated using the aquatic oligochaete Lumbriculus variegatus as a test organism. Our hypothesis was that exposure to contaminated sediments would cause behavioural early warning responses in L. variegatus. Five sites, with pH ranging from 2.5 to 6.5, and with associated metals, were investigated. The results showed poor sediment quality in most of the collected sediments and Fe, S and As were the dominant elements in the samples. High mortalities were observed, ranging from 32.6 to 100%, indicating severe contamination. The collected sediments did not support good L. variegatus growth and significantly changed its behaviour. Early warning responses consisted of decreased locomotion and decreased peristaltic movements. A behaviour inhibition will affect the ecosystem balance by limiting the organisms' ability to avoid capture, which leads to a higher risk of predation.     

Toxic and recovery effects of copper on Caenorhabditiselegans by various food-borne and water-borne pathways

Copper pollutions are typical heavy metal contaminations, and their ability to move up food chains urges comprehensive studies on their effects through various pathways. Currently, four exposure pathways were prescribed as food-borne (FB), water-borne plus clean food (WCB), water-food-borne (WFB) and water-borne (WB). Caenorhabditiselegans was chosen as the model organism, and growth statuses, feeding abilities, the amounts of four antioxidant enzymes, and corresponding recovery effects under non-toxic conditions with food and without food were investigated. Based on analysis results, copper concentrations in exposure were significantly influenced by the presence of food and its uptake by C.elegans. Both exposure and recovery effects depended on exposure concentrations and food conditions. For exposure pathways with food, feeding abilities and growth statuses were generally WFB < WCB ? FB (p < 0.05). The antioxidant activities were up-regulated in the same order. Meanwhile, the exposure pathway without food (WB) caused non-up-regulated antioxidant activities, and had the best growth statuses. For recoveries with food, growth statuses, feeding abilities and the inductions of the antioxidant enzymes were all WB ≈ WFB < WCB < FB (p < 0.05). For recoveries without food, the order of growth statuses remained WB > FB > WCB > WFB (p < 0.05), while the antioxidant activities were all inhibited in a concentration-dependent fashion. In conclusion, contaminated food was the primary exposure pathway, and various pathways caused different responses of C.elegans.     

Phosphorus availability changes chromium toxicity in the freshwater alga Chlorella vulgaris

Chromium (Cr) is one of the most serious pollutants in aquatic systems. This study examined the relationship between the toxic effects of Cr on the freshwater alga Chlorella vulgaris and phosphorus (P) availability on the algal physiology and ultrastructure. Cr inhibited C. vulgaris growth in a concentration- and time-dependent manner, and its inhibitory effect was related to the P concentration. In a low-P medium, Cr showed approximately 2.2–3.7-fold stronger toxicity than in a high-P medium. Cr was absorbed into the algal body where it disrupted the chloroplast structure and decreased the chlorophyll content. However, Cr had a weaker chlorophyll inhibitory ability and destructive power against the chloroplasts in the high-P medium than in the low-P medium due to the partial blockage of Cr absorption in high P-medium. Cr exposure also changed the metal ion and anion absorption profiles, which was also closely related to the concentration of P. Cr treatment increased the volume of the vacuole, and the larger vacuole reduced the space available for chloroplasts, as based on optical and electron microscopy results, but a higher P availability could alleviate this damage. These results suggest that high P alleviated the toxicity of Cr by decreasing Cr absorption and increasing the absorption of beneficial ions. It is, therefore, necessary to consider the phosphorus availability when the toxicity of metal compounds is evaluated.     

Generation of reactive oxygen species in cyanobacteria and green algae induced by allelochemicals of submerged macrophytes

Inhibition of phytoplankton by allelochemicals released by submerged macrophytes is reported to be one of the mechanisms that maintain a clear-water state in shallow lakes. In order to elucidate this mechanism, the ability of six polyphenols and two long-chain fatty acids to induce the generation of reactive oxygen species (ROS) in phytoplankton was studied using the ROS sensitive probe 2′,7′- dichlorodihydrofluorescein diacetate (DCFH-DA). The results showed that only (+)-catechin (CA) and pyrogallic acid (PA) could induce ROS formation in Microcystis aeruginosa and Pseudokirchneriella subcapitata. 25 mg L⁻¹ CA caused 1.2, 1.4 and 1.8 times increase of ROS levels in M. aeruginosa at 1, 2 and 4 h exposure, respectively, and, correspondingly in P. subcapitata cells, these values were 3.7, 6.2 and 7.7, respectively. PA also significantly increased the levels of intracellular ROS in P. subcapitata (P < 0.01); however, significant ROS generation in M. aeruginosa was observed at only 4 h exposure (P < 0.01). Light enhanced ROS generation in CA treated cells, but not in the cells treated with PA. CA and PA may act as redox cyclers after uptake by test organisms and produce ROS successively. These results suggest that the oxidative stress induced by the redox cycling property of allelochemicals may be one of the important causes for the inhibitory effect of some submerged macrophytes towards undesired phytoplankton in natural aquatic ecosystems.     

Isolation and characterization of lead-tolerant Ochrobactrum intermedium and its role in enhancing lead accumulation by Eucalyptus camaldulensis

In this study, the potential of rhizospheric bacteria in promoting the growth and Pb accumulation by the woody plant Eucalyptus camaldulensis under hydroponic conditions was investigated for the first time. Three Pb-tolerant bacteria were isolated from the rhizosphere of E. camaldulensis grown in Pb-contaminated soils in the Bo Ngam Pb mine, Thailand. Based on analysis of partial 16S rRNA gene sequence, the three isolates were identified as Microbacterium paraoxydans BN-2, Ochrobactrum intermedium BN-3, and Bacillus fusiformis BN-4. Among these strains, O. intermedium BN-3 showed the highest tolerance to not only Pb but also Cd and Zn. After growth in the presence of Pb, the membranes of O. intermedium BN-3 cells exhibited an increase in unsaturated fatty acid levels but a decrease in fluidity. In hydroponic studies, inoculation of O. intermedium BN-3 significantly increased the biomass and Pb accumulation by E. camaldulensis compared to the uninoculated control. The results suggested the role of the natural rhizospheric bacteria localized to the root surface of E. camaldulensis in promoting Pb accumulation and plant growth. Our results indicate that O. intermedium BN-3 and other indigenous rhizospheric bacteria have the potential to improve the efficiency of phytoremediation of Pb-contaminated sites.     

The presence of Bacillus thuringiensis (Bt) protein in earthworms Eisenia fetida has no deleterious effects on their growth and reproduction

Earthworms Eisenia fetida, bred in substances with stover of two genetically-engineered Bacillus thuringiensis (Bt) corns (5422Bt1 (Event Bt11) and 5422CBCL (MON810)) expressing Cry1Ab and their near-isogenic non-Bt corn (5422), were used to investigate the non-target effects of Bt corn on soil-dwelling organisms. Cry1Ab concentrations in substances, casts and guts of E. fetida were also investigated by Enzyme-linked immunosorbent assay (ELISA). More than 90% individuals of E. fetida survived over a period of 30 d, irrespective of whether they received Bt corn or non-Bt corn. Compared to 5422 treatments, significantly higher relative growth rate and more number of new offspring and cocoons of E. fetida were found in 5422Bt1 and 5422CBCL treatments. These results were unlikely to be directly caused by Cry1Ab released from Bt corns but rather by differences in other factors of plants such as plant components (soluble sugar, total organic carbon, total protein and available phosphorus of Bt corns were more than 5422). ELISA results indicated immunoreactive Cry1Ab was detectable in substances, and the casts, guts of E. fetida from Bt corns treatments, of which the highest levels were detected in substances under the corresponding experimental conditions. With the increase of treated time, a strong decline was observed in Cry1Ab from substances and casts of E. fetida, whereas Cry1Ab in guts of E. fetida from 5422Bt1 treatments gradually increased and that from 5422CBCL treatments increased between 14 and 30 d. Therefore, the presence of Cry1Ab in E. fetida had no deleterious effects on their growth and reproduction.     

Ecosystem engineering potential of the gastropod Terebralia palustris (Linnaeus, 1767) in mangrove wastewater wetlands - A controlled mesocosm experiment

The effect of different sewage concentrations (0, 20, 60 and 100%), vegetation (Bare, Avicennia marina or Rhizophora mucronata) and immersion periods (immersion/emersion period of 12/12 h or 3/3 days just for 100%) conditions were studied for 6 months on survival and growth rates of Terebralia palustris (Linnaeus, 1767). Gastropods' activity and ecosystem engineering preformed at bare and A. marina planted cells and 3 sewage conditions (0, 20 and 60%) were determined. Survival rates were higher than 70% in all treatments. Growth rate decreased significantly with increasing sewage concentrations (mainly at unplanted conditions) and longer immersion periods. A complete shift (from immersion to emersion periods) and a significant decrease in mobility and consequently its engineer potential, due to sewage contamination, lead to a 3-4 fold decrease in the amount of sediment disturbed. Sewage contamination, primary producers' abundance and environmental conditions may have influenced the gastropods survival, growth and its ecosystem engineering potential.