Reproductive toxicity of bisphenol A and cadmium in Potamopyrgus antipodarum and modulation of bisphenol A effects by different test temperature

An OECD initiative for the development of mollusc-based toxicity tests for endocrine disrupters and other chemicals has recommended three test species with respective test designs for further standardisation. Preparing a subsequent pre-validation study we performed a reproduction test with Potamopyrgus antipodarum, determining the concentration range of the selected test substances, bisphenol A (BPA) and cadmium (Cd). At 16 °C, the recommended test temperature, the number of embryos in the brood pouch was increased by BPA and decreased by Cd (NOEC: 20 μg BPA/L and 1 μg Cd/L). Coinstantaneous BPA tests at 7 °C and 25 °C demonstrated a temperature dependency of the response, resulting in lower NOECs (5 μg/L respectively). As expected, reproduction in control groups significantly varied depending on temperature. Additional observations of the brood stock showed seasonal fluctuations in reproduction under constant laboratory conditions. The recommended temperature range and test conditions have to be further investigated.     

Effects of suspended common-scale and nanoscale particles on the survival,growth and reproduction of Daphnia magna

Suspended particles are a natural component of aquatic ecosystems. This study provides a report on the survival, growth and reproduction of common-scale and nanoscale particles of Daphnia magna Straus exposed to five types of particles (i.e. KN (kaolinite), MN (montmorillonite), MNn (nanoscale MN), NP (natural particles), and NPn (nanoscale NP)). The results of the study show that the suspended particles elicited a dose-dependent toxicity in KN, MN and MNn, with the following toxicity pattern: MN > KN > MNn. On the contrary, NP and NPn did not show any harmful effects on the animals. Instead, NP and NPn, especially NPn, contributed to the survivorship of the animals. The animals were able to survive throughout the 21-day period of bioassays, and when the particle concentration reached 600 mg L⁻¹, they produced many neonates without any addition of food. This experimental results also indicated that the nanoscale particles were less toxic than the common-scale ones, both for MN and NP. Moreover, the results of the 21-day period of bioassays indicated that when the organisms were exposed to particles of different size scales, they showed different selection patterns for allocating resources. This may be due to the generation of different assimilation and digestion patterns.     

Survival and behavior of the insecticide-exposed predators Podisus nigrispinus and Supputius cincticeps (Heteroptera: Pentatomidae)

Pentatomid stinkbugs are important predators of defoliating caterpillars in agricultural and forestry systems, and knowledge of the impact of insecticides on natural enemies is important information for integrated pest management (IPM) programs. Thus, we assessed the toxicity and behavioral sublethal response of the predators Podisus nigrispinus and Supputius cincticeps exposed to deltamethrin, methamidophos, spinosad and chlorantraniliprole, insecticides commonly used to control the velvetbean caterpillar (Anticarsia gemmatalis) in soybean crops. With the exception of deltamethrin for S. cincticeps, all insecticides showed higher acute toxicity to the prey than to these natural enemies providing effective control of A. gemmatalis. The recommended field concentration of deltamethrin, methamidophos and spinosad for controlling A. gemmatalis caused 100% mortality of P. nigrispinus and S. cincticeps nymphs. Chlorantraniliprole was the less toxic and the most selective insecticide to these predators resulting in mortalities of less than 10% when exposed to 10× the recommended field concentration for a period of 72 h. Behavioral pattern changes in predators were found for all insecticides, especially methamidophos and spinosad, which exhibited irritability (i.e., avoidance after contact) to both predator species. However, insecticide repellence (i.e., avoidance without contact) was not observed in any of the insects tested. The lethal and sublethal effects of pesticides on natural enemies is of great importance for IPM, and our results indicate that substitution of pyrethroid and organophosphate insecticides at their field rates by chlorantraniliprole may be a key factor for the success of IPM programs of A. gemmatalis in soybeans.     

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.     

Influence of environmental factors on the response of a natural population of Daphnia magna (Crustacea: Cladocera) to spinosad and Bacillus thuringiensis israelensis in Mediterranean coastal wetlands

The present study was undertaken to assess the impact of a candidate mosquito larvicide, spinosad (8, 17 and 33 μg L⁻¹) on a field population of Daphnia magna under natural variations of water temperature and salinity, using Bti (0.16 and 0.50 μL L⁻¹) as the reference larvicide. Microcosms (125 L) were placed in a shallow temporary marsh where D. magna was naturally present. The peak of salinity observed during the 21-day observation period may have been partly responsible for the decrease of daphnid population density in all the microcosms. It is also probably responsible for the absence of recovery in the microcosms treated with spinosad which caused a sharp decrease of D. magna abundance within the first two days following treatment whereas Bti had no effect. These results suggest that it may be difficult for a field population of daphnids to cope simultaneously with natural (water salinity and temperature) and anthropogenic (larvicides) stressors.     

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.     

Correlations between in situ denitrification activity and nir-gene abundances in pristine and impacted prairie streams

Denitrification is a process that reduces nitrogen levels in headwaters and other streams. We compared nirS and nirK abundances with the absolute rate of denitrification, the longitudinal coefficient of denitrification (i.e., K_den, which represents optimal denitrification rates at given environmental conditions), and water quality in seven prairie streams to determine if nir-gene abundances explain denitrification activity. Previous work showed that absolute rates of denitrification correlate with nitrate levels; however, no correlation has been found for denitrification efficiency, which we hypothesise might be related to gene abundances. Water-column nitrate and soluble-reactive phosphorus levels significantly correlated with absolute rates of denitrification, but nir-gene abundances did not. However, nirS and nirK abundances significantly correlated with K_den, as well as phosphorus, although no correlation was found between K_den and nitrate. These data confirm that absolute denitrification rates are controlled by nitrate load, but intrinsic denitrification efficiency is linked to nirS and nirK gene abundances.     

Seabird feathers as monitors of the levels and persistence of heavy metal pollution after the Prestige oil spill

We measured heavy metal concentrations in yellow-legged gulls (n = 196) and European shags (n = 189) in order to assess the temporal pattern of contaminant exposure following the Prestige oil spill in November 2002. We analysed Pb, Cu, Zn, Cr, Ni and V levels in chick feathers sampled at four colonies during seven post-spill years (2003-2009), and compared results with pre-spill levels obtained from feathers of juvenile shag corpses (grown in spring/summer 2002). Following the Prestige wreck, Cu (4.3-10 μg g⁻¹) and Pb concentrations (1.0-1.4 μg g⁻¹) were, respectively, between two and five times higher than pre-spill levels (1.5-3.6 and 0.1-0.4 μg g⁻¹), but returned to previous background concentrations after three years. Our study highlights the suitability of chick feathers of seabirds for assessing the impact of oil spills on heavy metal contamination, and provides the best evidence to date on the persistence of oil pollution after the Prestige incident.     

Belowground effects of enhanced tropospheric ozone and drought in a beech/spruce forest (Fagus sylvatica L./Picea abies [L.] Karst)

The effects of experimentally elevated O₃ on soil respiration rates, standing fine-root biomass, fine-root production and δ¹³C signature of newly produced fine roots were investigated in an adult European beech/Norway spruce forest in Germany during two subsequent years with contrasting rainfall patterns. During humid 2002, soil respiration rate was enhanced under elevated O₃ under beech and spruce, and was related to O₃-stimulated fine-root production only in beech. During dry 2003, the stimulating effect of O₃ on soil respiration rate vanished under spruce, which was correlated with decreased fine-root production in spruce under drought, irrespective of the O₃ regime. δ¹³C signature of newly formed fine-roots was consistent with the differing g_s of beech and spruce, and indicated stomatal limitation by O₃ in beech and by drought in spruce. Our study showed that drought can override the stimulating O₃ effects on fine-root dynamics and soil respiration in mature beech and spruce forests.     

Arbuscular mycorrhizal fungi enhance both absorption and stabilization of Cd by Alfred stonecrop (Sedum alfredii Hance) and perennial ryegrass (Lolium perenne L.) in a Cd-contaminated acidic soil

A greenhouse pot experiment was conducted to compare the phytoextraction efficiencies of Cd by hyper-accumulating Alfred stonecrop (Sedum alfredii Hance) and fast-growing perennial ryegrass (Lolium perenne L.) from a Cd-contaminated (1.6 mg kg⁻¹) acidic soil, and their responses to the inoculations of two arbuscular mycorrhizal (AM) fungal strains, Glomus caledonium 90036 (Gc) and Glomus mosseae M47V (Gm). Ryegrass and stonecrop were harvested after growing for 9 and 27 wk, respectively. Without AM fungal inoculation, the weekly Cd extraction by stonecrop (8.0 μg pot⁻¹) was 4.3 times higher than that by ryegrass (1.5 μg pot⁻¹). Both Gc and Gm significantly increased (P < 0.05) root mycorrhizal colonization rates, soil acid phosphatase activities, and available P concentrations, and thereby plant P absorptions (except for Gm-inoculated ryegrass), shoot biomasses, and Cd absorptions (except for Gm-inoculated stonecrop), while only Gc-inoculated stonecrop significantly accelerated (P < 0.05) the phytoextraction efficiency of Cd by 78%. In addition, both Gc and Gm significantly decreased (P < 0.05) phytoavailable Cd concentrations by 21–38% via elevating soil pH. The results suggested the potential application of hyper-accumulating Alfred stonecrop associated with AM fungi (notably Gc) for both extraction and stabilization of Cd in the in situ treatment of Cd-contaminated acidic soil.     

Long-term Hg pollution-induced structural shifts of bacterial community in the terrestrial isopod (Porcellio scaber) gut

In previous studies we detected lower species richness and lower Hg sensitivity of the bacteria present in egested guts of Porcellio scaber (Crustacea, Isopoda) from chronically Hg polluted than from unpolluted environment. Basis for such results were further investigated by sequencing of 16S rRNA genes of mercury-resistant (Hg^r) isolates and clone libraries. We observed up to 385 times higher numbers of Hg^r bacteria in guts of animals from polluted than from unpolluted environment. The majority of Hg^r strains contained merA genes. Sequencing of 16S rRNA clones from egested guts of animals from Hg-polluted environments showed elevated number of bacteria from Pseudomonas, Listeria and Bacteroidetes relatives groups. In animals from pristine environment number of bacteria from Achromobacter relatives, Alcaligenes, Paracoccus, Ochrobactrum relatives, Rhizobium/Agrobacterium, Bacillus and Microbacterium groups were elevated. Such bacterial community shifts in guts of animals from Hg-polluted environment could significantly contribute to P. scaber Hg tolerance.     

Effects of elevated atmospheric CO2 and tropospheric O3 on tree branch growth and implications for hydrologic budgeting

The forest hydrologic budget may be impacted by increasing CO₂ and tropospheric O₃. Efficient means to quantify such effects are beneficial. We hypothesized that changes in the balance of canopy interception, stem flow, and through-fall in the presence of elevated CO₂ and O₃ could be discerned using image analysis of leafless branches. We compared annual stem flow to the results of a computerized analysis of all branches from the 2002, 2004, and 2006 annual growth whorls of 97 ten-year-old trees from the Aspen Free-Air CO₂ and O₃ Enrichment (Aspen FACE) experiment in Rhinelander, WI. We found significant effects of elevated CO₂ and O₃ on some branch metrics, and that the branch metrics were useful for predicting stem flow from birch, but not aspen. The results of this study should contribute to development of techniques for efficient characterization of effects on the forest hydrologic budget of increasing CO₂ and tropospheric O₃.     

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.     

Phytoextraction of heavy metal polluted soils using Sedum plumbizincicola inoculated with metal mobilizing Phyllobacterium myrsinacearum RC6b

The aim of this study was to investigate the effects of metal mobilizing plant-growth beneficial bacterium Phyllobacterium myrsinacearum RC6b on plant growth and Cd, Zn and Pb uptake by Sedum plumbizincicola under laboratory conditions. Among a collection of metal-resistant bacteria, P. myrsinacearum RC6b was specifically chosen as a most favorable metal mobilizer based on its capability of mobilizing high concentrations of Cd, Zn and Pb in soils. P. myrsinacearum RC6b exhibited a high degree of resistance to Cd (350 mg L⁻¹), Zn (1000 mg L⁻¹) and Pb (1200 mg L⁻¹). Furthermore, P. myrsinacearum RC6b showed multiple plant growth beneficial features including the production of 1-aminocyclopropane-1-carboxylic acid deaminase, indole-3-acetic acid, siderophore and solubilization of insoluble phosphate. Inoculation of P. myrsinacearum RC6b significantly increased S. plumbizincicola growth and organ metal concentrations except Pb, which concentration was lower in root and stem of inoculated plants. The results suggest that the metal mobilizing P. myrsinacearum RC6b could be used as an effective inoculant for the improvement of phytoremediation in multi-metal polluted soils.     

Effects of estrogen exposure in mussels, Mytilus edulis, at different stages of gametogenesis

Mytilus edulis were exposed to 17β-estradiol (E2) and the synthetic estrogens ethinyl estradiol (EE2) and estradiol benzoate (EB) for 10 days. Two exposures were performed to determine their effect on vitellogenin (VTG) and estrogen receptor 2 (ER2) mRNA expression at different stages of the reproductive cycle. Significant natural variation was not observed in VTG mRNA expression, though ER2 mRNA expression displayed significantly lower values during January, February and July compared with other times of the year. A significant increase in VTG and ER2 mRNA expression was observed in mussels exposed to estrogens at the early stage of gametogenesis. In contrast, mature mussels displayed no statistically significant change in the VTG or ER2 mRNA expression. The data presented suggests that the reproductive physiology of molluscs, in terms of VTG and ER2 mRNA expression, may be susceptible to damage by environmental estrogens at certain points in their gametogenesis process.     

Integration of biological responses from a suite of bioassays for the Venice Lagoon (Italy) through sediment toxicity index - Part A: Development and comparison of two methodological approaches

Marine and coastal quality assessment, based on test batteries involving a wide array of endpoints, organisms and test matrices, needs for setting up toxicity indices that integrate multiple toxicological measures for decision-making processes and that classify the continuous toxicity response into discrete categories according to the European Water Framework Directive.Two toxicity indices were developed for the lagoon environment such as the Venice Lagoon. Stepwise procedure included: the construction of a database that identified test-matrix pairs (indicators); the selection of a minimum number of ecotoxicological indicators, called toxicological core metrics (CMs-tox) on the basis of specific criteria; the development of toxicity scores for each CM-tox; the integration of the CMs-tox into two indices, the Toxicity Effect Index (TEI), based on the transformation of Toxic Unit (TU) data that were integrated as logarithmic sum, and the Weighted Average Toxicity Index (WATI), starting from toxicity classes integrated as weighted mean. Results from the indices are compared; advantages and drawbacks of both approaches are discussed.     

Effect of octylphenol on physiologic features during growth in Arabidopsis thaliana

Alkylphenol ethoxylates are widely used as detergents, emulsifiers, solubilizers, wetting agents and dispersants. Octylphenol (OP) ethoxylates, one of alkylphenol ethoxylates, represent 15–20% of the market, and their metabolic residues may be discharged to surface waters, sediments and soils as a persistent and ubiquitous pollutant. We tested the response of Arabidopsis thaliana to different concentrations of OP. OP affected the germination percentage and mean germination period. 10 d treatment with OP, especially high concentration (10 and 50 mg L⁻¹), decreased shoot and root biomass and root length of 30 d-old A. thaliana. Content of chlorophyll was decreased but that of proline was increased in leaves with OP treatment. OP caused oxidative stress in leaves; malondialdehyde content was increased, and the activities of ascorbate peroxidase, catalase and superoxide dismutase were induced. OP affects the physiologic and morphologic features of A. thaliana during growth. Because plants might be exposed to OP for a long time in the surroundings, more attention needs to be paid to the effect of OP on plants.     

A rapid bioassay for detecting saxitoxins using a Daphnia acute toxicity test

Bioassays using Daphnia pulex and Moina micrura were designed to detect cyanobacterial neurotoxins in raw water samples. Phytoplankton and cyanotoxins from seston were analyzed during 15 months in a eutrophic reservoir. Effective time to immobilize 50% of the exposed individuals (ET₅₀) was adopted as the endpoint. Paralysis of swimming movements was observed between ∼0.5-3 h of exposure to lake water containing toxic cyanobacteria, followed by an almost complete recovery of the swimming activity within 24 h after being placed in control water. The same effects were observed in bioassays with a saxitoxin-producer strain of Cylindrospermopsis raciborskii isolated from the reservoir. Regression analysis showed significant relationships between ET₅₀vs. cell density, biomass and saxitoxins content, suggesting that the paralysis of Daphnia in lake water samples was caused by saxitoxins found in C. raciborskii. Daphnia bioassay was found to be a sensitive method for detecting fast-acting neurotoxins in natural samples, with important advantages over mouse bioassays.     

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.