Low Concentrations Of Atrazine Research Articles

Polyaniline and polystyrene sulfonic acid modified on conductive biochar nanoparticle with strong signal enhancement for trace atrazine detection

A label and reagent free and cost effective immunosensing system was newly developed for detection of trace leveled atrazine in water. This was constructed by synthesis of polyaniline (PA) and polystyrene sulfonic acid (PSS) modified biochar (BC) nanoparticle composite (PA-PSS-BC) and coating them on screen printed electrode as conductive and electrochemically active layer, and covalently immobilizing anti-atrazine antibody on electrode for specific target binding. The low cost biochar from sugarcane was highly conductive and favorable for attaching PA and PSS due to large surface area and sp2 carbon structure. Crediting to dopant PSS, PA-PSS-BC achieved significant enhancement of redox current signal even in phosphate buffered saline of neutral pH compared to that of without PSS. And redox current of PA was extremely stable with negligible signal change even after twenty cycles repeated reactions owning to protection by BC. These essentially ensured sensing platform being sensitively performed in neutral solution with exemption of adding external redox reagent. As a result, fairly low concentrations of atrazine could be detected within 5 min, with limit of detection being 0.407 ng/L in a dynamic detection range varying from 1 to 100 ng/L. Moreover, the immunosensor exhibited reasonable signal recoveries for detecting trace leveled atrazine in real water samples, high selectivity, and good stability with dry and cold storage.

Does atrazine induce changes in predator recognition, growth, morphology, and metamorphic traits of larval skipper frogs (Euphlyctis cyanophlyctis)?

Atrazine, an info disruptor, interferes with the olfaction of aquatic organisms by impairing the chemosensory system. Consequently, it affects behavior, physiology, and growth increases mortality and infections, and suppresses the immune system of aquatic animals. In this study, we wanted to determine the sensitivity of larval Euphlyctis cyanophlyctis to different concentrations of atrazine by assessing their antipredator behavior, growth, morphology, and metamorphic traits. The results indicate that exposure to atrazine did not affect the survival of tadpoles. However, it caused retarded growth at higher concentrations. Interestingly, the antipredator behavior of tadpoles toward conspecific alarm cues decreased in a dose-dependent manner with an increase in the concentration of atrazine. Tadpoles exposed to low concentrations of atrazine had deeper, wider bodies and tails while those exposed to higher concentrations had shallower and narrower bodies with shallower tail muscles. However, at low and moderate concentrations atrazine did not affect size at metamorphosis, it extended the larval duration at higher concentrations.

Atrazine modifies markers of melanocyte maturation and apoptosis in primary skin cultures

Atrazine (ATZ) is part of a group of herbicides called triazines. ATZ is widely used in agricultural areas of Mexico, commonly used for the selective control of weeds in corn and sorghum crops. The exposure to ATZ can have serious human health effects since its use was associated with the development of cutaneous melanoma in an epidemiological study. The aim of this study was to evaluate the expression of maturation and apoptotic markers in primary skin cultures exposed to ATZ. The primary skin cultures were exposed to 0.1, and 10 µM ATZ with or without ultraviolet (UV) radiation and the expression of maturation and apoptotic markers were evaluated by RT-qPCR. We observed a significant increase in all the melanocyte maturation markers in cells exposed to ATZ with or without UV, with SOX-9 and FAK (melanoblast markers) being the highest. Also, the expression of BCL-2 (anti-apoptotic marker) was the most increased gene in cells exposed to ATZ with or without UV. Low concentrations of ATZ and UV radiation induce genetic changes associated with the development of immature melanocytes and activate mechanisms associated with the inhibition of apoptosis characteristics of malignant cell transformation, which will allow proposing new therapeutic targets and generating new restrictions or care in farmers exposed to pesticides such as the ATZ.

Short-term lethal and sublethal atrazine effects on Litoditis marina: towards a nematode model for marine toxicity assessment?

Organochlorine pesticides are still widely used in many regions of the world, despite their toxic properties to many organisms, including humans, and their high environmental persistence in waters and sediments. In order to properly assess contaminant effects across trophic levels in aquatic communities, the responses of different model organisms should be investigated. Particularly for marine benthic environments, relatively few model organisms for the assessment under controlled laboratory conditions of both lethal and sublethal effects are available. Here we investigate the use of a cryptic species of the free-living nematode Litoditis marina as a model organism for short-term tests of the impacts of contaminants, using the case of the herbicide atrazine. Its ease of culturing, short generation time and high fecundity render L. marina PmIII a good candidate to assess contaminant effects for both a rapid assessment of impacts and more detailed investigations of the mechanisms behind those effects. The LC50 of atrazine to adult L. marina PmIII was 3.3 ± 0.5 mg/L after a 5-day exposure, suggesting a comparatively higher sensitivity of this species compared to other aquatic invertebrate model species, mainly crustaceans. This LC50 was less than half the value after a 4-day exposure and was 22-fold lower than the 48 h LC50, demonstrating the importance of performing lethality tests for as long an exposure time as possible. Sublethal effects were observed at much lower atrazine concentrations and encompassed, from 0.8 mg/L onwards, impairment of life-history traits (reduced fecundity and extended egg deposition and development times) and population parameters (abundances of juveniles and adults, adult sex ratio). These sublethal effects were broadly consistent among different life-history traits and population parameters, but fecundity and the maximum abundances of juveniles, adults and total nematodes were the most sensitive response variables. We therefore suggest that an adequate assessment of sublethal contaminant effects on L. marina requires a combination of different life-history traits and population-level parameters. We conclude that L. marina PmIII is a suitable model species for ecotoxicological research encompassing both lethal and sublethal effects, and can as such contribute to environmental risk assessment in marine and estuarine systems.

Preconcentration and chromatographic detection of atrazine in real water sample using aqueous two‐phase system based on tetrahydrofuran and glycerol

AbstractEnvironmental contamination by atrazine leads to serious environmental impact. The low concentrations of atrazine in aqueous media render its identification and quantification difficult. In this context, aqueous two‐phase systems based on tetrahydrofuran (THF) + glycerol were tested as simultaneous recovery and preconcentration platforms of atrazine from real water samples. The phase diagrams were determined, and it was reported that the phase separation ability largely depends on the number of hydroxyl groups present in each polyol. Posteriorly, the atrazine was partitioned to the THF‐rich phase and presented recovery values between 70.9 (KATZ = 2.9) and 96.5% (KATZ = 15.2). The computational predictive method (COSMO‐RS) was used, aiming to confirm the atrazine partitioning for the THF‐rich phase. In addition, a 250‐fold preconcentration factor was achieved using a mixture point of 3.27 wt% of THF + 93.98 wt% of glycerol + 2.75 wt% of real water sample. This preconcentration factor and subsequent use of UFLC‐TOF permitted the identification and quantification of atrazine in real samples of water collected in the hydrographic basin from the Japaratuba River, in the state of Sergipe, Brazil. The protocol presented values of 95‒2400 ng L−1 of atrazine with a limit of detection of 50 ng L−1.

Trifluralin and Atrazine Sensitivity to Selected Cereal and Legume Crops

Soil-applied herbicides can persist in sufficient concentrations to affect the growth of crops in rotations. The sensitivity of wheat, barley, oat, lucerne and lentil to trifluralin and atrazine residues were investigated with three glasshouse experiments in 2018 and 2019. Each bioassay crop species was tested against different concentrations of trifluralin and atrazine in sandy soil using a full factorial design. Shoot and root parameters of the tested crop species were fitted in logistic equations against herbicide concentrations to calculate effective doses for 50% growth inhibition (ED50). Results revealed that both shoot and root parameters of all the test crop species were significantly affected by trifluralin and atrazine. Trifluralin delayed crop emergence at the lower concentrations examined, while higher concentrations prevented emergence entirely. Low concentrations of atrazine did not affect emergence but significantly reduced plant height, soil–plant analyses development (SPAD) index, shoot dry weight, root length, root dry weight and number of nodules of all the crop species. At high concentration, atrazine resulted in plant death. Legumes were found to be more sensitive than cereals when exposed to both trifluralin and atrazine treatments, with lucerne being the most sensitive to both herbicides, ED50 ranging from 0.01 to 0.07 mg/kg soil for trifluralin; and from 0.004 to 0.01 mg/kg for atrazine. Barley was the most tolerant species observed in terms of the two herbicides tested. Lucerne can be used to develop a simple but reliable bioassay technique to estimate herbicide residues in the soil so that a sound crop rotation strategy can be implemented.

Configuration of molecular imprinted polymer for electrochemical atrazine detection

This paper addresses the specific ability of molecular imprinted polymers (MIPs) to bind the template. MIPs for atrazine with various monomer:template and crosslinker:template ratios were prepared using bulk polymerization process. In order to obtain conditions closer to real wastewater test cases, the degree of the imprinting factor by the selection of solvent (methanol and water) was studied and compared. The polymer tested in methanol did not demonstrate good recognition of atrazine. In addition, the imprinting factors increased significantly when the imprinted polymers were tested in water solvent by 45%. Based on Scatchard analysis, the MIP was dependent upon low atrazine concentration (up to 40 mg/L) as the cavities formed in the MIP proved the cavities effects. An electrochemical analysis was done in order to prove that the method can be used as a quantitative analysis of atrazine concentration detection in water solution.

Oxidative stress response induced in an atrazine phytoremediating plant: Physiological responses of Pennisetum glaucum to high atrazine concentrations

ABSTRACTThis research presented here, for the first time, elucidates the responses of several antioxidants in Pennisetum leaves exposed to varying concentrations of atrazine (0–200 mgkg−1). Pennisetum has been reported to be resistant to atrazine; however, its physiological response to high concentrations (≥50 mgkg−1) of atrazine is not well documented. The contents of reduced (AsA) and oxidized (DHA) ascorbate increased significantly with increase in atrazine concentration and exposure time; but the increase was more evident under higher (50 and 100 mgkg−1) atrazine concentrations. Increase in atrazine concentration to 200 mgkg−1 significantly decreased AsA, but increased DHA content, throughout the experiment. Seedlings treated with 200 mgkg−1 atrazine showed significantly lowest reduced glutathione (GSH) content, while oxidized glutathione (GSSG) was not significantly affected, after 68 d. Seedlings treated with 100 mgkg−1 atrazine showed increased glutathione-S-transferase (GST) activity after 48 d and 68 d, while treatment with 200 mgkg−1 atrazine significantly increased glutathione reductase (GR) after 58 d. This result suggests that Pennisetum may tolerate lower atrazine concentrations. However, higher concentrations (≥50 mg kg−1), which could have longer residency period in the soil, could induce more physiological damage to the plant.

Typical agricultural diffuse herbicide sorption with agricultural waste-derived biochars amended soil of high organic matter content

Biochar application has been identified as the effective soil amendment and the materials to control the diffuse herbicide pollution. The atrazine was selected as the typical diffuse herbicide pollutant as the dominant proportion in applications. The biochar treated from four types of crops biomass were added to soil with high organic matter content. The basic sorption characteristics of biocahrs from corn cob (CC), corn stalk (CS), soybean straw (SS), rice straw (RS) and corn stalk paralyzed with 5% of ammonium dihydrogen phosphate (ACS) were analyzed, along with the comparison of the sorption difference of the raw soil and soil amended with biochars at four levels of ratio (0.5%, 1.0%, 3.0% and 5.0%). It was found that the linear distribution isotherm of raw soil was much effective due to the high organic matter background concentration. The addition of five types of biochars under two kinds of initial atrazine concentration (1 mg/L and 20 mg/L) demonstrated the sorption variances. Results showed the soil amended with RS and CS biochar had the biggest removal rate in four regular biochars and the removal rate of the ACS was the biggest. The sorption coefficient and the normalized sorption coefficient from Freundlich modeling presented the isothermal sorption characteristics of atrazine with soil of high organic matter content. The normalized sorption coefficient increased with the equilibrium concentration decreased in the biochar amended soil, which indicated the sorption performance will be better due to the low atrazine concentration in practice. Results showed that biochar amendment is the effective way to prevent leakage of diffuse herbicide loss.

Toxicity of atrazine, glyphosate, and quinclorac in bullfrog tadpoles exposed to concentrations below legal limits.

This work sought to ascertain survival and possible changes in levels of glycogen, triglycerides, total lipids, cholesterol, protein, and lipid peroxidation in gills, liver, and muscle of bullfrog tadpoles (Lithobates catesbeianus) exposed to low concentrations of atrazine (2.5μgL(-1)), glyphosate (18μgL(-1)), and quinclorac (0.025μgL(-1)) at laboratorial conditions. Tadpoles showed a reduction of glycogen and triglyceride in all organs and an increase in lipid peroxidation (LPO) compared with control animals. Total lipid in gills and muscle increased in exposure to atrazine, and gills alone in exposure to glyphosate, but decreased in gills, liver, and muscle after quinclorac. Cholesterol increased in gills and liver after atrazine, in gills and muscle after glyphosate, and decreased in liver after quinclorac. Total protein in gills decreased after exposure to all herbicides, increased in muscle after atrazine, and in liver and muscle after quinclorac. These findings show that at concentrations of these herbicides tested can lead to an increase in energy expenditure to maintain homeostasis and survival of these animals despite the increase in lipid peroxidation levels in all organs analyzed. Responses observed can be one of the factors responsible for the decline in the number of amphibians around the world.

Bioremediation strategies for removal of residual atrazine in the boreal groundwater zone

Strategies for bioremediation of atrazine, a pesticide commonly polluting groundwater in low concentrations, were studied in two boreal nonagricultural soils. Atrazine was not mineralized in soil without bioremediation treatments. In biostimulation treatment with molasses, up to 52% of atrazine was mineralized at 10 °C, even though the degradation gene copy numbers did not increase. Incubations with radioactively labeled atrazine followed by microautoradiographic analysis revealed that bioremediation strategies increased the relative proportion of active degraders from 0.3 up to 1.9% of the total bacterial count. These results indicate that atrazine degradation might not solely be facilitated by atzA/trzN-atzB genes. In combined biostimulation treatment using citrate or molasses and augmentation with Pseudomonas citronellolis ADP or Arthrobacter aurescens strain TC1, up to 76% of atrazine was mineralized at 30 °C, and the atrazine degradation gene numbers increased up to 10(7) copies g(-1) soil. Clone libraries from passive samplers in groundwater monitoring wells revealed the presence of phylogenetic groups formerly shown to include atrazine degraders, and the presence of atrazine degradation genes atzA and atzB. These results show that the mineralization of low concentrations of atrazine in the groundwater zone at low temperatures is possible by bioremediation treatments.

Quantitative Fractal Evaluation of Herbicide Effects on the Water-Absorbing Capacity of Superabsorbent Polymers

The water absorption capacity of superabsorbent polymers (SAPs) is important for agricultural drought resistance. However, herbicides may leach into the soil and affect water absorption by damaging the SAP three-dimensional membrane structures. We used 100-mesh sieves, electron microscopy, and fractal theory to study swelling and water absorption in SAPs in the presence of three common herbicides (atrazine, alachlor, and tribenuron-methyl) at concentrations of 0.5, 1.0, and 2.0 mg/L. In the sieve experiments it was found that 2.0 mg/L atrazine reduces the capacity by 9.64–23.3% at different swelling points; no significant diminution was observed for the other herbicides or for lower atrazine concentrations. We found that the hydrogel membrane pore distributions have fractal characteristics in both deionized water and atrazine solution. The 2.0 mg/L atrazine destroyed the water-retaining polymer membrane pores and reduced the water-absorbing mass by modifying its three-dimensional membrane structure. A linear correlation was observed between the fractal analysis and the water-absorbing mass. Multifractal analysis characterized the membrane pore distribution by using the range of singularity indexes Δα(relative distinguishing range of 16.54–23.44%), which is superior to single-fractal analysis that uses the fractal dimensionD(relative distinguishing range of 2.5–4.0%).

Environmentally-relevant concentrations of atrazine induce non-monotonic acceleration of developmental rate and increased size at metamorphosis in Rhinella arenarum tadpoles

Despite of the various studies reporting on the subject, anticipating the impacts of the widely-used herbicide atrazine on anuran tadpoles metamorphosis remains complex as increases or decreases of larval period duration are almost as frequently reported as an absence of effect. The aim of the present study was to examine the effects of environmentally-relevant concentrations of atrazine (0.1, 1, 10, 100, and 1000μg/L) on the timings of metamorphosis and body size at metamorphosis in the common South American toad, Rhinella arenarum (Anura: bufonidae). None of the atrazine concentrations tested significantly altered survival. Low atrazine concentrations in the range of 1–100μg/L were found to accelerate developmental rate in a non-monotonic U-shaped concentration–response relationship. This observed acceleration of the metamorphic process occurred entirely between stages 25 and 39; treated tadpoles proceeding through metamorphosis as control animals beyond this point. Together with proceeding through metamorphosis at a faster rate, tadpoles exposed to atrazine concentrations in the range of 1–100μg/L furthermore transformed into significantly larger metamorphs than controls, the concentration–response curve taking the form of an inverted U in this case. The no observed effect concentration (NOEC) was 0.1μg atrazine/L for both size at metamorphosis and timings of metamorphosis. Tadpoles exposed to 100μg/L 17β-estradiol presented the exact same alterations of developmental rate and body size as those treated with 1, 10 and 100μg/L of atrazine. Elements of the experimental design that facilitated the detection of alterations of metamorphosis at low concentrations of atrazine are discussed, together with the ecological significance of those findings.

DNA damage and effects on glutathione‐S‐transferase activity induced by atrazine exposure in zebrafish (Danio rerio)

This study was undertaken to investigate the protective effect of atrazine (2-chloro-4-(ethylamino)-6-(isopropylamino)-S-triazine) on the activity of glutathione-S-transferase (GST) and DNA damage in males and females of adult zebrafish (Danio rerio). Zebrafish were exposed to control and three treatments (0.01, 0.1, and 1 mg/L) of atrazine for 5, 10, 15, 20, and 25 days. The results indicated that, for males, the GST activity at lower atrazine concentrations (0.01 and 0.1 mg/L) was markedly higher than that of the controls throughout the duration of the experiment while there was a significant inhibition of the GST activity at 1 mg/L atrazine at days 5 and 20. For females, a significant increase was detected at 0.1 mg/L on the days 5 and 15 and at 0.01 mg/L on day 20. The DNA damage in zebrafish was evaluated using the comet assay; the olive tail moments obtained for hepatopancreas were enhanced after treatment with different concentrations of atrazine on days 5, 10, 15, 20, and 25. The DNA damage increased with increasing atrazine concentrations, indicating that genotoxicity of atrazine and significant differences was found compared to the controls. In conclusion, these findings provide further evidence of the effects of atrazine on aquatic ecosystems.

Evidence for taxonomic and functional drift of an atrazine-degrading culture in response to high atrazine input

We evaluated the effects of variations in atrazine input on the evolution of a bacterial culture adapted to a low atrazine concentration. This initial culture (M3-K) was subjected to weekly subculturing in the presence of a high concentration of atrazine as the only N source (100 mg l(-1)). After four subculturing, M3-K evolved to a new bacterial culture (M3) which exhibited a significant increase in the extent of atrazine mineralization in comparison with the initial culture. Molecular analyses of M3-K and M3 cultures by cloning, restriction analysis, and sequencing of the 16S rRNA genes revealed significant differences in culture structure and composition. M3-K culture comprised mainly Actinobacteria (40%), β-Proteobacteria (26%), and Bacteroidetes (16%). After exposure to a high atrazine concentration, the dominance of Actinobacteria decreased (14%), Bacteroidetes increased (27%), and β-Proteobacteria were replaced by γ-Proteobacteria (32%). Quantitative PCR revealed that the abundance of atzB and atzC genes relative to total bacteria decreased by a factor of 3-4 following the increase in atrazine concentration, while the relative abundance of trzD increased significantly (≈400 times). Presented study shows that variations in atrazine input drive both functional and compositional shifts in the atrazine-degrading bacterial culture.

Adsorption and removal at low atrazine concentration in an MBR pilot plant

Atrazine is a persistent organic pollutant and it has been widely used in agriculture and forestry in the world for more than fifty years. Atrazine shows ecotoxicity effects in aquatic ecosystems even at very low level concentrations with endocrine disruptor activity. Few studies were carried out on atrazine removal performances in drinking and waste-water by biological treatments, especially in membrane bio-reactors (MBRs). MBR technology might be more efficient than the conventional one in the removal of micro-pollutants. The fate of atrazine in wastewater treatment plants and its influence on the biomass activity was evaluated in this study. The experimental work was divided in three different phases: inhibition studies on different types of biomass (by means of microcalorimetry); adsorption studies on different sludges (conventional activated sludge (CAS) - and MBR) calculating adsorption isotherms and, finally, atrazine removal in an MBR pilot plant (simulating a treatment of atrazine and nitrate contaminated groundwater). The absence of significant inhibition was observed; higher atrazine adsorption on MBR sludge was detected for lower atrazine concentration (<50 µg L(-1)); the removal efficiency in the MBR pilot plant was lower than 25% but higher than the theoretical one (based on adsorption isotherms).

Low concentrations of atrazine, glyphosate, 2, 4-dichlorophenoxyacetic acid, and triadimefon exposures have diverse effects on Xenopus laevis organ morphogenesis

Abstract Many chemicals are released into the environment, and chemical contamination has been suggested as a contributing factor to amphibian declines. To add to a growing body of knowledge about the impact of individual chemicals on non-target organisms, we examined the specificity of deformities induced by exposure to four pesticides (atrazine, 2,4-dichloropheoxyacetic acid (2,4-D), triadimefon, and glyphosate) in the model amphibian species, Xenopus laevis. We focused on the period of organ morphogenesis, as it is frequently found to be particularly sensitive to chemical exposure yet also commonly overlooked. We found similar levels of intestine malformations and edemas, as well as disruption of skeletal muscle, in atrazine and triadimefon exposed tadpoles. The effects of 2,4-D were only apparent at the highest concentrations we examined; glyphosate did not induce dramatic malformations at the concentrations tested. While researchers have shown that it is important to understand how chemical mixtures affect non-target organisms, our results suggest that it is first crucial to determine how these chemicals act independently in order to be able to identify consequences of individual pesticide exposure.

Label-free impedimetric immunosensor for sensitive detection of atrazine

Impedance spectroscopy transduction combined with the immunosensor technology has been used for the determination of atrazine, a herbicide The sensor electrode was based on the immobilization of anti-atrazine antibody by affinity binding onto a polypyrrole film N-substituted by nitrilotriacetic acid NTA electrogenerated on a gold electrode. The poly NTA film was previously modified by the coordination of Cu2 ions by the chelating NTA centers. The anti-atrazine antibody Fab fragment K47 modified with histidine-tag was then anchored by affinity interactions between the histidine-tag and the coordinated Cu2. Cyclic voltammetry experiments confirm that the antibody immobilization and the resulting immunosensor were applied to the impedimetric detection of atrazine without reagent and labelling step. The immunoreaction of atrazine on the attached anti-atrazine antibody directly triggers an increase in the charge transfer resistance proportional to the atrazine concentration, allowing the detection of extremely low atrazine concentration, namely 10 pg mL −1.

Environmental levels of atrazine decrease spatial aggregation in the freshwater mussel, Elliptio complanata

The commonly used herbicide atrazine (ATR), may cause estrogen-like alterations and, like other xenoestrogens, may alter behavior. Here effects on animal aggregation were tested in freshwater mussels exposed short-term to ecologically relevant ATR concentrations between 1.5 and 150 μg/L. The aggregation index (AI), the fraction of mussels in the most densely populated tank section, was evaluated and showed higher AI at later times in both 6- and 72-h trials ( p<0.001). After 72 h, there was also less aggregation in estradiol- and mid-ATR-treated animals, 26% and 24% less than control, respectively ( p<0.04). These data suggest that freshwater mussels tend to aggregate, that 72-h exposure to ecologically relevant ATR concentrations decreases aggregation, and that estradiol exposure has a similar effect. Given that bivalve aggregation may be related to reproduction, the results suggest that low concentrations of ATR may have ecological consequences on animal populations in this species.

Research Article: Differential acute sensitivity of wild Rana sylvatica and laboratory Xenopus laevis tadpoles to the herbicide atrazine

Abstract Atrazine is one of the most used herbicides in the United States and has previously been implicated in local amphibian declines. The purpose of this study was to examine the influence of atrazine on 2 frog species—the amphibian model species Xenopus laevis and the wood frog Rana sylvatica. Tadpoles just before hindlimb emergence were exposed to either 1 part per billion (ppb) or 25 ppb atrazine in their water. Behavior was observed daily, and at the end of 15 days exposure the tadpoles were weighed, developmentally staged, and measured for head width and tail width. Differences were found among all of these for the X. laevis (p < 0.002), with the difference occurring only for the low dose animals (and not the high dose animals) for weight, stage, and tail width. Head width was significantly different for the X. laevis exposed to both the high and low concentrations of atrazine. No morphological differences were seen in R. sylvatica, but increased mortality and behavioral differences were seen in ...