Background, aim, and scope This review deals with publications concerning the mode of action of Bt proteins and their potential synergism with extrinsic factors. The aim was to assess the impact of those factors especially regarding selectivity and efficacy of Bt toxins and to discuss possible gaps in current risk assessment of genetically engineered plants expressing Bt toxins. Main features The review shows that several extrinsic factors are able to influence the selectivity and efficacy of Bt toxins. The findings are seen as being relevant for risk assessment in Bt plants. This conclusion is derived by discussing current state of knowledge about the mode of action of Bt proteins, unexpected effects on non-target organism, and the way how modified Bt toxins are expressed in genetically engineered plants. Results Several publications have been identified that show that certain factors and synergism can impact efficacy and selectivity of Bt toxins. These extrinsic factors are various and include other Bt toxins or parts from the spore of Bacillus thuringiensis as well as certain enzymes, environmental stress, non-pathogenic microorganisms, and infectious diseases. Discussion Research on the underlying mechanism of observed synergism might help to explain some of the effects found in non-target organisms. In general, possible synergism of Bt toxins with extrinsic factors can be relevant for risk assessment of genetically engineered Bt plants since they expose a modified Bt toxin to the environment under various conditions and over a long period of time. Conclusions Risk assessment of genetically engineered plants should put into question the general assumption of a high selectivity and a linear dose-response relationship in the toxicity of Bt proteins. Both selectivity and efficacy can be influenced by synergism, which can provoke unexpected and undesired effects in non-target organisms. Perspectives It is suggested that systematic research be promoted on synergism between Bt toxins and potential extrinsic factors that could impact the spectrum of susceptible organisms. This research should become a prerequisite for risk assessment of Bt plants.

Purpose Experiments were conducted to examine the effects of mercury (Hg) on soil nitrification activities and the microbial communities of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA). Methods The soil samples spiked with different Hg concentrations were incubated for a period of 1, 2, 4, and 8 weeks in triplicate and the potential nitrification rate (PNR) of the samples was determined. The abundance of AOB and AOA was measured after an 8-week incubation by real-time polymerase chain reaction (PCR) assay of the amoA genes, while the community compositions by cloning and sequencing approaches. Results The soil PNR differed with different incubation periods. It tended to decrease with increasing soil Hg concentrations at week 1, basing on which the half-maximal effective concentration (EC50) was 1.59 mg kg⁻¹. There was no significant difference in the abundance of AOB or AOA among the treatments. The AOB community was dominated by Nitrosospira-like sequences and more than 70% of the obtained clones were affiliated with the cluster 3a.2. The percentage of cluster 3a.1 in AOB community appeared to a consistent trend of decreasing with ascending soil Hg concentrations. While all the AOA sequences in the clone libraries were grouped into cluster S (soil and sediment origin). Conclusions This study revealed that Hg could inhibit soil potential nitrification and the extent varied with incubation periods. Soil Hg pollution changed the composition of soil AOB to some extent. These findings will be helpful to recognize the effects of Hg on the activity and community composition of soil ammonia oxidizers.

Background, aim, and scope Glutaraldehyde (GA) often acts as an effective sterilant, disinfectant, and preservative in chemical products. It was found that GA had clearly acute toxicity to aquatic organisms. Furthermore, GA in natural environment could not exist as single species but as complex mixtures. To explore the toxicity interaction between GA and the other environmental pollutant, it is necessary to determine the mixture toxicities of various binary mixtures including GA. Two reference models, concentration addition (CA) and independent action (IA), are often employed to evaluate the mixture toxicity, which can be finished by comparing the concentration-response curves (CRCs) predicted by the reference models with the experimental CRC of the mixture. However, the CRC-based method cannot effectively denote the degree of the deviations from the reference models, especially at very low effect levels. Though the model deviation ratio (MDR) can be used to quantitatively evaluate the deviation of a mixture at EC50 level from the reference model, it is difficult to evaluate the deviations at the lower effect levels. Therefore, the primary aim of this study was to develop a new effect residual ratio (ERR) method to validate the deviations from the reference models at various effect levels. Materials and methods Four chemicals having possible dissimilar mode of actions with GA, acetonitrile (ACN), dodine (DOD), simetryn (SIM), and metham sodium (MET), were selected as another component in the binary mixtures including GA, which constructed four binary mixtures, GA-ACN, GA-DOD, GA-SIM, and GA-MET ones. For each binary mixture, two equipotent mixture rays where the concentration ratios of GA to another mixture component are respectively EC50 and EC5 ones were designed and their toxicities (expressed as a percent inhibition to Photobacterium phosphoreum) were determined by microplate toxicity analysis. The observed concentration-response curve (CRC) of a ray was compared with that predicted by CA or IA model to qualitatively assess the toxicity interaction of the mixture ray. To quantitatively and effectively examine the deviations at various effect levels from the reference models, a new concept, ERR at an effect, was defined, and the ERR was employed to evaluate the deviation at various effects with confidence intervals. Results For three binary mixtures, GA-ACN, GA-DOD, and GA-SIM, the CRCs predicted by IA models were almost located in the 95% confidence intervals of the experimental CRCs for both equipotent mixture rays, which indicated the independent actions between binary mixture components. However, two rays of GA-MET binary mixture displayed a little synergistic action because both CRCs predicted by CA and IA were lower than the experimental CRC. ERR showed the same results as MDR, but ERR results at low effect area were clearer than MDR ones. Discussion In CRC comparison, the deviation of CA (for GA-ACN, GA-DOD, and GA-SIM combinations) or IA (for GA-MET) model from the experimental values could be obviously observed at medium area of the CRC. However, at very low effect levels, both deviations of CA and IA and difference between CA and IA model predictions were not very apparent. Thus, it was difficult to confirm which model, CA or IA, had better predicted power at very low effect levels. MDR in many literatures often refers to a ratio at EC50 level. It was also difficult to reflect not only the deviation fact at the other ECx but also the deviation uncertainty. After we extended the definition of MDR to all ECx and examined the 95% confidence intervals based on observation, the plot of the redefined MDRs at many effect levels could better explain the deviations of CA or IA model from the observation. However, MDRs at very low effect levels did not still reflect the high uncertainty there. The ERRs defined in our paper could explicitly explain the degree of deviation from the reference models and especially reflect the high uncertainty at very low effects. It could be said that the ERR is a better indicator than MDR. Conclusions The new ERR validation method developed in our laboratory could provide us with the information about the toxicity interaction between the mixture components and quantitatively assess the accuracy of the reference models (CA or IA) at whole effect levels. The ERR method conquered the invalidation of the classical CRC comparison method on the deviation decision at low effect levels and also got the advantage over the MDR methods. Recommendations and perspectives It holds promise to become an effective method of hazard and risk assessments of chemical mixtures by well characterizing the uncertainty at very low effect levels.

Background, aim and scope Perfluorinated compounds (PFCs) are global environmental pollutants that bioaccumulate in wildlife and humans. Laboratory experiments have revealed toxic effects such as delayed development, humoral suppression, and hepatotoxicity. Although numerous human blood levels have been reported, little is known about distribution in the human body. Knowledge about PFC distribution and accumulation in the human body is crucial to understanding uptake and subsequent effects as well as to conduct risk assessments. The present study reports PFC levels in human liver and breast milk from a general population living in Catalonia, Spain. Liver and milk levels are compared to previously reported levels in blood from the same geographic area as well as to other existing reports on human liver and milk levels in other countries. Materials and methods Human liver (n = 12) and milk (n = 10) samples were collected in 2007 and 2008 in Catalonia, Spain. Liver samples were taken postmortem from six males and six females aged 27-79 years. Milk samples were from healthy primipara women (30-39 years old). Both liver and milk were analyzed by solid-phase extraction and ultra-performance liquid chromatography tandem mass spectrometry. Results Six PFCs were detected in liver, with perfluorooctanesulfonate (PFOS, 26.6 ng/g wet weight) being the chemical with the highest mean concentration. Other PFCs such as perfluorohexanesulfonate (PFHxS), perfluorooctanoic acid (PFOA), and acids with chain lengths up to C11 were also detected, with mean levels ranging between 0.50 and 1.45 ng/g wet weight. On the other hand, PFOS and PFHxS were the only PFCs detected in human milk, with mean concentrations of 0.12 and 0.04 ng/mL, respectively. Discussion While milk concentrations were similar to reported levels from other countries, liver samples contained more PFCs above quantification limits and higher PFOS concentrations compared to the only two other reports found in the literature. Differences between the results of the present study and those concerning previous investigations can be due to declining levels of some PFCs, which have been reported for the USA. The relationship between PFC concentrations in human liver, milk, and blood was assessed using blood concentrations previously determined in Catalonia. Those levels resulted in liver/serum ratios of 1.7:1, 1.4:1, and 2.1:1 for PFOS, perfluorodecanoic acid, and perfluoroundecanoic acid, respectively. Accumulation in liver is suggested for PFOS and the perfluorocarboxylic acids with carbon chain lengths C9, C10, and C11. For PFOA and PFHxS, fivefold and 14-fold higher concentrations, respectively, were seen in serum as compared to liver. The mean concentration of PFOS and PFHxS in milk was only 0.8% and 0.6% of the reported mean serum level, respectively. Conclusions The results of the present study show that several PFCs could be detected in human liver samples of subjects living in Tarragona. Concerning human milk, the mechanism by which PFCs are transferred from mother's blood to breast milk is still unclear. Considering that PFCs are strongly bound to the protein fraction in blood, the possibility of PFCs entering the milk and accumulating to levels observed in maternal plasma is limited. Recommendations and perspectives Interestingly, the potential accumulation difference for PFCs with different chain lengths might be of great importance for risk assessment. Continuing studies on the distribution of different PFCs in human tissue are therefore justified.

Background, aim, and scope Alqueva Reservoir is the biggest artificial freshwater reservoir in Europe and is an important water supply for human and agricultural consumption in the Alentejo region (Portugal). Pollution can impair environmental and human health status, and to assure water quality and ecological balance, it is crucial to frequently monitor water supplies. In this study, we used an ecotoxicological test battery to identify the potential toxicity of water from this reservoir. Materials and methods Water samples from the Alqueva aquatic system were collected bimonthly in 2006 from 11 different water points within the reservoir. Several bioassays were carried out: a 72-h growth test with Pseudokirchneriella subcapitata, a 6-day growth test with Chironomus riparius larvae, and the luminescence inhibition test with Vibrio fischeri (Microtox®). Results and discussion Algae growth was significantly inhibited in several sampling points and periods throughout the year, mainly due to the presence of pesticides. Although in some sampling points pesticide concentrations (single and sum) were still below the maximum permissible concentrations, water samples showed high toxicities to algae, especially during the summer months. In addition, several sampling points showed pesticide concentrations above the permissible level which can pose a significant risk to humans and the environment. Chironomids showed less sensitivity to the water samples, possibly due to the low concentrations of insecticides present. V. fischeri showed no sensitivity when exposed to all the water samples collected throughout the year of 2006. Conclusions Standardized laboratory bioassays can be useful tools to assess water quality from aquatic systems and can valuably complement chemical analysis evaluation. The results obtained in this study demonstrated that the most sensitive species used in this test battery was the microalgae P. subcapitata. The growth of C. riparius was less affected, which is probably due to the fact that low insecticide concentrations were measured and, furthermore, since this species lives in the sediment and not in the water column and is, therefore, usually more resistant to pollutants. Recommendations and perspectives On its own, chemical analysis is not enough to derive conclusions on the water quality and/or status, which can be valuably complemented by laboratory bioassays. Single chemical, maximum permissible values, and the sum of pesticide concentrations do not take into account possible patterns of synergism, antagonism, dose level dependencies, or even the dominance of several chemicals within a mixture. In addition, several species from different levels in trophic chains are recommended due to differences in species' sensitivities to chemical compounds that are present.

Background In the present study, we report the analytical results of pp′DDT, pp′DDE and pp′DDD determination in lake water, plankton and zooplanktivorous fish of Lake Maggiore (Northern Italy), rather recently polluted by DDT of industrial origin, in order to understand the bottom-up pollution transfer among the abiotic and biotic components of the lake ecosystem. Materials and methods Fourteen water sampling campaigns were carried out from March 2003 to January 2009 in the water column of the deepest point (Ghiffa) of Lake Maggiore. Suspended and dissolved pollutants were determined separately. Three sampling campaigns were carried out from July 2008 to January 2009 for zooplankton and pelagic fish, and DDT residues were analysed by HRGC coupled with ECD and MS. Moreover, food items were detected in fish stomachs. Results and discussion Starting from 2007, the DDT contamination along the water column became rather homogeneous, probably because no flooding or other relevant hydrological events occurred; although steady-state condition should be expected, lipid normalised concentrations of pp′DDE and pp′DDD in zooplankton exceeded the levels of the same compounds in zooplanktivorous fishes. Conclusion As this finding represents a thermodynamic paradox since bioaccumulative pollutant concentrations are expected to increase along the trophic chain, it was supposed that the abiotic and biotic lake components were not in a steady-state condition in Lake Maggiore.

BACKGROUND, AIM, AND SCOPE: This study focuses on the factors that affect trihalomethane (THMs) formation when dissolved organic matter (DOM) fractions (colloidal, hydrophobic, and transphilic fractions) in aqueous solutions were disinfected with chlorine. MATERIALS AND METHODS: DOM fractions were isolated and fractionated from filtered lake water and were characterized by elemental analysis. The investigation involved a screening Placket-Burman factorial analysis design of five factors (DOM concentration, chlorine dose, temperature, pH, and bromide concentration) and a Box-Behnken design for a detailed assessment of the three most important factor effects (DOM concentration, chlorine dose, and temperature). RESULTS: The results showed that colloidal fraction has a relatively low contribution to THM formation; transphilic fraction was responsible for about 50% of the chloroform generation, and the hydrophobic fraction was the most important to the brominated THM formation. DISCUSSION: When colloidal and hydrophobic fraction solutions were disinfected, the most significant factors were the following: higher DOM fraction concentration led to higher THM concentration, an increase of pH corresponded to higher concentration levels of chloroform and reduced bromoform, higher levels of chlorine dose and temperature produced a rise in the total THM formation, especially of the chlorinated THMs; higher bromide concentration generates higher concentrations of brominated THMs. Moreover, linear models were implemented and response surface plots were obtained for the four THM concentrations and their total sum in the disinfection solution as a function of the DOM concentration, chlorine dose, and temperature. Overall, results indicated that THM formation models were very complex due to individual factor effects and significant interactions among the factors. CONCLUSIONS: In order to reduce the concentration of THMs in drinking water, DOM concentrations must be reduced in the water prior to the disinfection. Fractionation of DOM, together with an elemental analysis of the fractions, is important issue in the revealing of the quality and quantity characteristics of DOM. Systematic study composed from DOM fraction investigation and factorial analysis of the responsible parameters in the THM formation reaction can, after an evaluation of the adjustment of the models with the reality, serves well for the evaluation of the spatial and temporal variability in the THM formation in dependence of DOM. However, taking into consideration the natural complexity of DOM, different operations and a strict control of them (like coagulation/flocculation and filtration) has to be used to quantitatively remove DOM from the raw water. RECOMMENDATIONS AND PERSPECTIVES: Assuming that this study represents a local case study, similar experiments can be easily applied and will supply with relevant information every local water treatment plant meeting problems with THM formation. The coagulation/flocculation and the filtration stages are the main mechanisms to remove DOM, particularly the colloidal DOM fraction. With the objective to minimize THMs generation, different unit operation designed to quantitatively remove DOM from water must be optimized.

Background, aim and scope The paper presents concentrations of trace elements in blood of homebred animals (cows and sheep) from Southern Serbia (Bujanovac) and the contents of natural and anthropogenic radionuclides and some heavy metals in feed. The region of Southern Serbia was exposed to contamination by depleted uranium ammunition during NATO attacks in 1999 and therefore, is of great concern to environmental pollution and human and animal health. Materials and methods Conventional instrumental and epithermal neutron activation analyses are used to measure trace elements in cow and sheep blood samples collected randomly at six locations in the region of Bujanovac (village of Borovac) in the spring of 2005. Samples of feed (grass and crops: corn, wheat and oats), collected on the same locations (households), are analysed for the contents of radionuclides on an HPGe detector (Ortec, relative efficiency 23%) by standard gamma spectrometry. The content of Hg, Pb and Cd in feed is determined by standard atomic absorption spectrometry on the VarianSpectra220/ThermoSolar GFS97 spectrometer. Results Concentrations of 29 elements (Na, Al (P), Cl, K, Sc, Cr, Mn, Ni, Fe, Co, Zn, Se, As, Br, Sr, Rb, Sb, In, I, Ba, Cs, La, Nd, Eu, Sm, Tb, Hf, Ta and Th) are determined in blood of the examined animals. In feeds, natural ⁴⁰K is found in all of the samples, cosmogenic ⁷Be and fission product ¹³⁷Cs are detected only in the grass samples, while heavy metals Hg, Cd and Pb are found in the range of 0.01-0.02, 0.84-1.15 and 0.74-7.34 mg/kg, respectively. Calculated soil-to-blood transfer factors are in a wide range of 8·10⁻⁶ to 64, as a result of varying significance of the elements in animal metabolism and feeding habits. Discussion The results of trace elements' concentrations in animal blood are in good agreement with available data for K, Ni, Zn, Se and Rb. Higher Br concentrations in animal blood are most probably caused by large biomass burning events during blood sampling. Very low concentration of Fe in cows and sheep confirms the results of previous biochemical studies on animal anaemia in the region. High concentration of As correlates with geochemical peculiarities of the Balkans and is also likely influenced by the use of pesticides in the agricultural production. For some of the elements (La, Nd, Eu, Sm, Tb, Sb, Hf, Ta, Th, In, Ba, Sr, Sc and Cs), there are few or no literature data. Therefore, some of the presented data are significant not only for the country and the region, but on a wider scale. Activities of natural radionuclides in feeds are within the average values reported for the region, while the activities of ²¹⁰Pb and ²³⁵/²³⁸U are below the limit of detection. This is in accordance with previous investigations showing no widespread contamination by depleted uranium in the area. Contents of Hg and Pb in feeds are below the nationally permissible levels, unlike the content of Cd which exceeds it, probably caused by the use of phosphate fertilisers and fossil fuel combustion in the area. Conclusions In general, the concentrations of trace elements in blood of homebred cows and sheep are in good agreement with reference materials, available literature data and the results of previous studies in the area. The exceptions are Fe, As and Br. The contents of natural and anthropogenic radionuclides in feeds are within the expected levels, and there are no signs of contamination by depleted uranium or other fission products. Apart from Cd, there are no signs of pollution by heavy metals in feeds. The highly sensitive method of instrumental neutron activation analysis provides data on the concentration of some elements in animal blood not previously reported for the region and elsewhere. Recommendations and perspectives The presented study is a part of the long term ongoing project on the health risk assessment on animals and humans in the region. The collected data is intended to provide a base for the animal and human risk assessment as well as an estimate of the general pollution status of the environment in the region. Since some of the investigated elements are classified as important trace elements for livestock, the results could also be used to balance and improve the animal diet and thus, improve the growth and reproduction rate.

Background, aim, and scope Two new high phenol-degrading strains, Micrococcus sp. and Alcaligenes faecalis JH 1013, were isolated. The two isolates could grow aerobically in mineral salts medium containing phenol as a sole carbon source at concentration of 3,000 mg L⁻¹. It was found that the binary mixed culture of the two isolates possessed good potential for phenol removal. Material and methods Phenol biodegradation using the binary mixed culture of the two isolates was studied. The optimal conditions were determined to be temperature 32°C, pH 7.0, inoculum size 10.0%, and agitation rate 150 rpm in the synthetic wastewater. In addition, the kinetics of the cell growth and phenol degradation by the binary mixed culture were also investigated using Haldane model over a wide range of initial phenol concentrations from 20 to 2,400 mg L⁻¹. Results The experimental data indicated that the binary mixed culture had pretty high phenol degradation potential, which could thoroughly degrade the phenol in the synthetic wastewater containing phenol 2,400 mg L⁻¹ within 72 h under aerobic condition. Under the optimal conditions, the phenol concentration was reduced speedily from 1,000 to below 0.28 mg L⁻¹ in the presence of the binary mixed culture, and the phenol degradation rate reached 99.97% after 16 h. It was well below the standard value 0.28 mg L⁻¹ as described by Chinese Environmental Protection Agency. It was clear that the Haldane kinetic model adequately described the dynamic behavior of phenol degradation by the binary mixed culture with kinetic constants of q max = 0.45 h⁻¹, K sq = 64.28 mg L⁻¹, and K iq = 992.79 mg L⁻¹. The phenol concentration to avoid substrate inhibition had been inferred theoretically to be 252.62 mg L⁻¹. Conclusions Phenol, as the only carbon source, could be degraded by the binary mixed culture at high initial phenol concentrations. Phenol exhibited inhibitory behavior, and the growth kinetics of the binary mixed culture could be correlated well by the simple Haldane's inhibitory model. The kinetics parameters were invariably required for the design and simulation of batch and continuous bioreactor treating phenolic wastewaters.

Background, aims, and scope Heavy metal contaminants in environment, especially in drinking water, are always of great concern due to their health impact. Due to the use of heavy metals as catalysts during plastic syntheses, particularly antimony, human exposure to metal release from plastic bottles has been a serious concern in recent years. The aim and scope of this study were to assess metal contaminations leaching out from a series of recycling plastic bottles upon treatments. Methodology In this study, leaching concentrations of 16 metal elements were determined in 21 different types of plastic bottles from five commercial brands, which were made of recycling materials ranging from no. 1 to no. 7. Several sets of experiments were conducted to study the factors that could potentially affect the metal elements leaching from plastic bottles, which include cooling with frozen water, heating with boiling water, microwave, incubating with low-pH water, outdoor sunlight irradiation, and in-car storage. Results Heating and microwave can lead to a noticeable increase of antimony leaching relative to the controls in bottle samples A to G, and some even reached to a higher level than the maximum contamination level (MCL) of the US Environmental Protection Agency (USEPA) regulations. Incubation with low-pH water, outdoor sunlight irradiation, and in-car storage had no significant effect on antimony leaching relative to controls in bottle samples A to G, and the levels of antimony leaching detected were below 6 ppb which is the MCL of USEPA regulations. Cooling had almost no effect on antimony leaching based on our results. For the other interested 15 metal elements (Al, V, Cr, Mn, Co, Ni, Cu, As, Se, Mo, Ag, Cd, Ba, Tl, Pb), no significant leaching was detected or the level was far below the MCL of USEPA regulations in all bottle samples in this study. In addition, washing procedure did contribute to the antimony leaching concentration for polyethylene terephthalate (PET) bottles. The difference of antimony leaching concentration between washing procedure involved and no washing procedure involved (AC) was larger than zero for samples A to G. This interesting result showed that higher antimony concentration was detected in experiments with no washing procedures compared with those experiments with washing procedures. Our study results indicate that partial antimony leaching from PET bottles comes from contaminations on the surface of plastic during manufacturing process, while major antimony leaching comes from conditional changes. Conclusion The results revealed that heating and microwaving enhance antimony leaching significantly in PET plastic bottles. Plastic bottle manufacturers should consider the contaminations during manufacturing process and washing bottles before first use was strongly recommended to remove those contaminants.