Optimization of assay conditions to quantify ECOD activity in vivo in individual Daphnia magna. Assay performance evaluation with model CYP 450 inducers/inhibitors.

Screening the activity of the cytochrome P450 (CYP450) mixed function oxidase system in aquatic invertebrates received seldom applications in ecotoxicology due to low baseline enzymatic activities characteristic for these organisms. In this study, an existing in vivo spectrofluorometric assay method based on quantifying the cytochrome P450 mediated conversion of 7-ethocycoumarin (EtC) used as substrate to the product 7-hydroxycoumarin (HCm) called: ethoxycoumarin-O-deethylase (ECOD) activity, initially applicable on pooled samples of Daphnia magna, was optimized for use on individual organisms. Optimal assay conditions have been established for as small as 3- and 6 days old individuals, and the limits of spectrofluorometric detection of HCm excreted by daphnids in the incubation media were defined. The modified assay was tested by screening the modulation of ECOD activity in daphnids following 24 h exposure to ß-naphthoflavone (ß-NF, reference CYP450 inducer) and to prochloraz (PCZ), a potent CYP450 inhibitor. Maximal ECOD activity levels in daphnids were recorded following 2 hours of incubation to 200 nM EtC. The limit of spectrofluorometric detection of HCm in the incubation media was 6.25 nM, achieved by more than 80% of three days old daphnids and all six days old individuals. Exposure of daphnids to ß-NF demonstrated a bell-shaped ECOD activity induction potential, while PCZ elicited partial (60%) inhibition of ECOD activity. This optimized in vivo ECOD activity assay may serve as a cost-effective tool to study the responsiveness of Phase-I metabolism in D. magna to toxic pressure and its applicability to other aquatic invertebrates is also worth for consideration.

Interactive transgenerational effects of parental co-exposure to prochloraz and chlorpyrifos: Disruption in multiple biological processes and induction of genotoxicity.

The application of different types of pesticides can result in the coexistence of multiple pesticide residues in our food and the environment. This can have detrimental effects on the health of offspring across generations when parents are exposed to these pesticides. Therefore, it is imperative to understand the long-term effects that can be inherited by future generations when assessing the risks associated with pesticides. To study the genotoxic effects of commonly used pesticides, prochloraz (PRO) and chlorpyrifos (CHL), and assess whether their combined exposures have a different toxic effect, we modeled the transgenerational effects of parental (F0-generation) and/or offspring (F1-generation) exposures on zebrafish embryos in the F1-generation. Following the exposures, we proceeded to assess the impacts of these exposures on a range of biological processes in F1-generation zebrafish. Our results revealed that exposure to PRO and CHL altered multiple biological processes, such as inflammation, apoptosis, oxidative stress, and thyroid hormone synthesis, and detoxification system, providing molecular targets for subsequent studies on toxicity mechanisms. Notably, our study also found that the biological processes of F1-generation zebrafish embryos were altered even though they were not exposed to any pesticide when F0-generation zebrafish were exposed to PRO or CHL, suggesting potential genotoxicity. In conclusion, we provided in-vivo evidence that parental exposure to PRO and/or CHL can induce genotoxicity in the offspring. Moreover, we observed that the toxic effects resulting from the combined exposure were interactive, suggesting a potential synergistic impact on the offspring.

Baseline sensitivity and toxicity mechanisms of prochloraz to Alternaria alternata strains associated with maize leaf blight in Heilongjiang province in China.

Alternaria species are fungal pathogens that can infect maize, causing leaf blight disease and significant economic losses. This study aimed to determine the baseline sensitivity to prochloraz of A. alternata isolates obtained from diseased maize leaves collected from Heilongjiang province by assessing the half-maximal effective concentration (EC50) values. The EC50 values of prochloraz ranged from 0.0550 µg/mL to 2.3258 µg/mL, with an average of 0.9995 ± 0.5192 µg/mL. At EC50 (1.2495 µg/mL) and 2EC50 (2.4990 µg/mL), prochloraz increased the number of mycelial offshoots, disrupted the cell membrane integrity of conidia and mycelia, and resulted in a reduced ergosterol content in the mycelia. Prochloraz significantly affected the mycelial cell membrane permeability and increased the malondialdehyde (MDA) content and superoxide dismutase (SOD) activity. No cross-resistance was detected between prochloraz and other fungicides. These data demonstrate that prochloraz is a promising fungicide for managing maize leaf blight caused by A. alternata and provide novel insights into understanding the mechanism of prochloraz toxicity against A. alternata isolates.

Decamethylcyclopentasiloxane affects estradiol production in female rats but not H295R cells.

Sex hormones, such as androgens and estrogens, are predominantly produced in the gonads (ovaries and testes) and adrenal cortex. Endocrine-disrupting chemicals (EDCs) are substances that mimic, block, or interfere with hormones in the endocrine systems of humans and organisms. EDCs mainly act via nuclear receptors and steroidogenesis-related enzymes. In the OECD conceptual framework for testing and assessment of EDCs, several well-known assays are used to identify the potential disruption of nuclear receptors both in vivo and in vitro, whereas the H295R steroidogenesis assay is the only assay that detects the disruption of steroidogenesis. Forskolin and prochloraz are often used as positive controls in the H295R steroidogenesis assay. Decamethylcyclopentasiloxane (D5) was suspected one of EDCs, but the effects of D5 on steroidogenesis remain unclear. To establish a short-term in vivo screening method that detects the disruption of steroidogenesis, rats in the present study were fed a diet containing forskolin, prochloraz, or D5 for 14 days. Forskolin increased plasma levels of 17ß-estradiol (E2) and testosterone as well as the mRNA level of Cyp19 in both the adrenal glands and ovaries. Prochloraz induced the loss of cyclicity in the sexual cycle and decreased plasma levels of E2 and testosterone. D5 increased E2 levels and shortened the estrous cycle in a dose-dependent manner; however, potential endocrine disruption was not detected in the H295R steroidogenesis assay. These results demonstrate the importance of comprehensively assessing the endocrine-disrupting effects of chemicals on steroidogenesis in vivo.

Biochemical and Molecular Characterization of Prochloraz Resistance in Lasiodiplodia theobromae Field Isolates.

Stem-end rot (SER), caused by Lasiodiplodia theobromae, is one of the most critical diseases of mango in China. The demethylation inhibitor fungicide prochloraz has been widely used in China to control mango diseases. Isolates (n = 139) of L. theobromae were collected in 2019 from six mango-producing regions in Hainan Province, China. The fungicide sensitivity of L. theobromae isolates to prochloraz revealed that the EC50 (50% effective concentration) values ranged from 0.0006 to 16.4131 µg/ml. In total, 21 of the 139 isolates were categorized as resistant to prochloraz. The resistant isolates sprayed with prochloraz could not be effectively controlled in detached fruit. The mycelial growth, conidia germination, and ability to grow at temperatures ranging from 12 to 35°C of resistant isolates decreased, suggesting fitness penalties. The experiment showed that, after treatment with prochloraz at 10 µg/ml, the content of ergosterol in the mycelia of the sensitive isolate decreased by 80.23%, whereas the resistant strain decreased by only 57.52%. The damage to membranes in the sensitive isolates was more serious than for resistant isolates. The target gene CYP51 and the ATP-binding cassette (ABC) subfamily ABCG gene were cloned but no mutation was found. When treated with prochloraz, the expression of CYP51 and ABCG in resistant isolates was significantly higher than in the sensitive isolates. Thus, induced expression of its target gene combined with the induction of expression drug efflux transporters appeared to mediate the prochloraz resistance of L. theobromae.

Occurrence and Chemical Control Strategy of Wheat Brown Foot Rot Caused by Microdochium majus.

Wheat brown foot rot (WBFR), caused by a variety of phytopathogenic fungi, is an important soilborne and seedborne disease of wheat. WBFR causes wheat lodging and seedling dieback, which seriously affect the yield and quality of wheat. In this study, 64 isolates of WBFR were isolated from different wheat fields in Yancheng city, Jiangsu Province, China. The internal transcribed spacer, elongation factor 1α, and RNA polymerase II subunit were amplified and the sequencing results of the fragments were analyzed with BLAST in NCBI. Through morphological and molecular identification, all of the isolates were identified as Microdochium majus. Verification by Koch's postulates confirmed that M. majus was the pathogen causing WBFR. The antifungal activities of fludioxonil and prochloraz against 64 isolates of M. majus were determined based on mycelial growth inhibition method. The results showed that fludioxonil and prochloraz had good antifungal activity against M. majus. The mean 50% effective concentration values of fludioxonil and prochloraz against M. majus were 0.2956 ± 0.1285 µg/ml and 0.0422 ± 0.0157 µg/ml, respectively. Control efficacy for seed-coating treatments conducted in a greenhouse indicated that M. majus severely damaged the normal growth of wheat, while seed coating with fludioxonil or prochloraz significantly reduced the disease incidence and improved the seedling survival rates. At fludioxonil doses of 7.5 g per 100 kg and prochloraz doses of 15 g per 100 kg, the incidence was reduced by 22.26 and 25.33%, seedling survival rates increased by 25.37 and 22.66%, and control efficacy reached 70.02 and 72.30%, respectively. These findings provide vital information for the accurate diagnosis and effective management of WBFR.

Dissipation and Dietary Risk Assessment of Prochloraz in Strawberries under Greenhouse Conditions.

Prochloraz and its metabolites in strawberries have not been determined until now. Meanwhile, few reports in the literature have concerned the dissipation behavior and risk assessment of prochloraz and its metabolites in strawberries under greenhouse conditions in Beijing. A method for the determination of prochloraz and its metabolites in strawberries was developed using QuEChERS in combination with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Prochloraz and its metabolites recovered from strawberries were present in concentrations of 73.06% to 116.01%, their RSDs ranged from 1.12% to 9.17%, and their limits of detection ranged from 0.1 to 1 µg kg-1. Then, a study was conducted on the dissipation of prochloraz in strawberries under greenhouse conditions. The dissipation of prochloraz in strawberries followed the first-order kinetic equation, and its half-life was 8.06 days. The health risk associated with prochloraz in strawberries was evaluated using the target hazard quotient (THQ) method and EFSA PRIMo model. The results showed that the THQ values, %ARfD values, and %ADI values were less than 1. These results indicate that no health concerns of prochloraz are associated with the consumption of the studied strawberries. The government can use the results of this study to support the establishment of a maximum residue level for prochloraz in strawberries.

[Sensitivity baseline establishment and resistance risk assessment of Botrytis cinerea from Panax ginseng to prochloraz].

This study aimed to establish the baseline sensitivity of Botrytis cinerea from Panax ginseng to prochloraz, and ensure the fitness of prochloraz-resistant mutants and the cross-resistance of B. cinerea to prochloraz and commonly used fungicides for the prevention and control of gray mold including boscalid, pyraclostrobin, iprodione, and pyrimethanil. The sensitivity of B. cinerea from P. ginseng to fungicides was determined by the mycelial growth rate method. The prochloraz-resistant mutants were screened out through fungicide domestication and ultraviolet(UV) induction. The fitness of resistant mutants was determined through the stability of subculture, mycelial growth rate, and pathogenicity test. The cross-resistance between prochloraz and the four fungicides was determined by Person correlation analysis. The results showed that all B. cinerea strains tested were sensitive to prochloraz, and the EC_(50) value ranged from 0.004 8 to 0.062 9 µg·mL~(-1), with an average of 0.022 µg·mL~(-1). The sensitivity frequency distribution diagram showed that 89 B. cinerea strains were located within the main peak with a continuous single peak curve, and the average EC_(50) value of 0.018 µg·mL~(-1) was taken as the baseline sensitivity of B. cinerea to prochloraz. The fungicide domestication and UV induction obtained 6 resistant mutants, among which 2 strains were unstable and the other 2 strains showed decreased resistance after multiple generations of culture. Furthermore, the mycelial growth rate and spore yield of all resistant mutants were lower than those of their parents, and the pathogenicity of most mutants was lower than that of their parents. In addition, prochloraz had no obvious cross-resistance with boscalid, pyraclostrobin, iprodione, and pyrimethanil. In conclusion, prochloraz has great potential for controlling gray mold in P. ginseng, and the resistance risk of B. cinerea to prochloraz is low.

Monitoring and characterization of prochloraz resistance in Fusarium fujikuroi in China.

Rice bakanae disease, caused by Fusarium fujikuroi, is a destructive seed-borne disease throughout the world. Prochloraz, a DMI (C-14α-demethylase inhibitor) fungicide, has been registered in China for >20 years. Prochloraz resistance in F. fujikuroi was severe in China with resistance frequencies of 34.56%, 45.33%, and 48.45% from 2019 to 2021. The fitness of prochloraz-resistant population was lower than that of sensitive population, with an average CFI of 2.86 × 106 and 4.56 × 106, respectively. No cross-resistance was detected between prochloraz and tebuconazole or hexaconazole, and the prochloraz-resistant isolates were still sensitive to fludioxonil, phenamacril, and pydiflumetofen. S312T mutation in Ffcyp51b or overexpression of Ffcyp51a and Ffcyp51b was detected in the highly resistant isolates. AS-PCR primers were designed to detect the prochloraz-resistant isolates with S312T mutation in the field. Resistant isolates carrying S312T mutation were the dominant group in prochloraz-resistant population with frequencies of 43.26%, 23.59%, and 71.20% from 2019 to 2021, which indicated that more attention should be paid to this genotype when monitoring and managing the prochloraz resistance in F. fujikuroi.

Characterization of Prochloraz Resistance in Fusarium fujikuroi from Heilongjiang Province in China.

Prochloraz is widely used to control rice bakanae disease caused by Fusarium fujikuroi. The current study was aimed at monitoring the development of F. fujikuroi resistance to prochloraz in the Heilongjiang Province and analyzing the fitness of F. fujikuroi strains with different resistance levels. The results indicated that most of the 89 F. fujikuroi strains collected from the Heilongjiang Province were resistant to prochloraz, with resistance frequency reaching 92.1%. To assess the field resistance risk of prochloraz, 21 F. fujikuroi strains with different resistance levels were selected to investigate their biological characteristics and assess their fitness. Mycelial growth, sporulation, and germination rates were significantly different among the tested strains. However, when grouped into two subpopulations, no significant difference was tested between prochloraz-resistant and prochloraz-sensitive strains. Pathogenicity assays revealed that the disease severity index of prochloraz-resistant strains was higher than that of prochloraz-sensitive strains. Cross-resistance assays showed no cross-resistance between prochloraz and five other fungicides, namely phenamacril, ipconazole, tebuconazole, carbendazim, and fluopyram. Ffcyp51A gene overexpression was observed in the prochloraz-resistant F. fujikuroi strains after exposure to prochloraz. Collectively, these results indicated that F. fujikuroi resistance against prochloraz was severe. Furthermore, prochloraz-resistant strains were highly fit and could potentially become a dominant population in rice fields, consequently resulting in yield loss.

Synergistic Activity of Ketoconazole and Miconazole with Prochloraz in Inducing Oxidative Stress, GSH Depletion, Mitochondrial Dysfunction, and Apoptosis in Mouse Sertoli TM4 Cells.

Triazole and imidazole fungicides represent an emerging class of pollutants with endocrine-disrupting properties. Concerning mammalian reproduction, a possible causative role of antifungal compounds in inducing toxicity has been reported, although currently, there is little evidence about potential cooperative toxic effects. Toxicant-induced oxidative stress (OS) may be an important mechanism potentially involved in male reproductive dysfunction. Thus, to clarify the molecular mechanism underlying the effects of azoles on male reproduction, the individual and combined potential of fluconazole (FCZ), prochloraz (PCZ), miconazole (MCZ), and ketoconazole (KCZ) in triggering in vitro toxicity, redox status alterations, and OS in mouse TM4 Sertoli cells (SCs) was investigated. In the present study, we demonstrate that KCZ and MCZ, alone or in synergistic combination with PCZ, strongly impair SC functions, and this event is, at least in part, ascribed to OS. In particular, azoles-induced cytotoxicity is associated with growth inhibitory effects, G0/G1 cell cycle arrest, mitochondrial dysfunction, reactive oxygen species (ROS) generation, imbalance of the superoxide dismutase (SOD) specific activity, glutathione (GSH) depletion, and apoptosis. N-acetylcysteine (NAC) inhibits ROS accumulation and rescues SCs from azole-induced apoptosis. PCZ alone exhibits only cytostatic and pro-oxidant properties, while FCZ, either individually or in combination, shows no cytotoxic effects up to 320 µM.

The FgCYP51B Y123H Mutation Confers Reduced Sensitivity to Prochloraz and Is Important for Conidiation and Ascospore Development in Fusarium graminearum.

Fusarium graminearum is one of the most important causal agents of Fusarium head blight disease and is controlled mainly by chemicals such as demethylation inhibitor (DMI) fungicides. FgCYP51B is one of the DMI targets in F. graminearum, and Tyrosine123 (Y123) is an important amino acid in F. graminearum CYP51B, located in one of predicted substrate binding pockets based on the binding mode between DMIs and CYP51B. Previous studies suggest that resistance to DMI fungicides is attributed primarily to point mutations in the CYP51 gene and that the Y123H mutation in F. verticillioides CYP51 confers prochloraz resistance in the laboratory. To investigate the function of FgCYP51B Y123 residue in the growth and development, pathogenicity, and DMI resistance, we generated and analyzed the FgCYP51B Y123H mutant. Results revealed that the Y123H mutation led to reduced conidial sporulation and affected ascospore development; moreover, the mutation conferred reduced sensitivity to prochloraz. Quantitative PCR and molecular docking were performed to investigate the resistance mechanism. Results indicated that Y123H mutation changed the target gene expression and decreased the binding affinity of FgCYP51 to prochloraz. These results will attract more attention to the potential DMI-resistant mutation of F. graminearum and increase our understanding of the DMI resistance mechanism.

Transcriptome analysis of fungicide-responsive gene expression profiles in two Penicillium italicum strains with different response to the sterol demethylation inhibitor (DMI) fungicide prochloraz.

BACKGROUND: Penicillium italicum (blue mold) is one of citrus pathogens causing undesirable citrus fruit decay even at strictly-controlled low temperatures (< 10 °C) during shipping and storage. P. italicum isolates with considerably high resistance to sterol demethylation inhibitor (DMI) fungicides have emerged; however, mechanism(s) underlying such DMI-resistance remains unclear. In contrast to available elucidation on anti-DMI mechanism for P. digitatum (green mold), how P. italicum DMI-resistance develops has not yet been clarified. RESULTS: The present study prepared RNA-sequencing (RNA-seq) libraries for two P. italicum strains (highly resistant (Pi-R) versus highly sensitive (Pi-S) to DMI fungicides), with and without prochloraz treatment, to identify prochloraz-responsive genes facilitating DMI-resistance. After 6 h prochloraz-treatment, comparative transcriptome profiling showed more differentially expressed genes (DEGs) in Pi-R than Pi-S. Functional enrichments identified 15 DEGs in the prochloraz-induced Pi-R transcriptome, simultaneously up-regulated in P. italicum resistance. These included ATP-binding cassette (ABC) transporter-encoding genes, major facilitator superfamily (MFS) transporter-encoding genes, ergosterol (ERG) anabolism component genes ERG2, ERG6 and EGR11 (CYP51A), mitogen-activated protein kinase (MAPK) signaling-inducer genes Mkk1 and Hog1, and Ca2+/calmodulin-dependent kinase (CaMK) signaling-inducer genes CaMK1 and CaMK2. Fragments Per Kilobase per Million mapped reads (FPKM) analysis of Pi-R transcrtiptome showed that prochloraz induced mRNA increase of additional 4 unigenes, including the other two ERG11 isoforms CYP51B and CYP51C and the remaining kinase-encoding genes (i.e., Bck1 and Slt2) required for Slt2-MAPK signaling. The expression patterns of all the 19 prochloraz-responsive genes, obtained in our RNA-seq data sets, have been validated by quantitative real-time PCR (qRT-PCR). These lines of evidence in together draw a general portrait of anti-DMI mechanisms for P. italicum species. Intriguingly, some strategies adopted by the present Pi-R were not observed in the previously documented prochloraz-resistant P. digitatum transcrtiptomes. These included simultaneous induction of all major EGR11 isoforms (CYP51A/B/C), over-expression of ERG2 and ERG6 to modulate ergosterol anabolism, and concurrent mobilization of Slt2-MAPK and CaMK signaling processes to overcome fungicide-induced stresses. CONCLUSIONS: The present findings provided transcriptomic evidence on P. italicum DMI-resistance mechanisms and revealed some diversity in anti-DMI strategies between P. italicum and P. digitatum species, contributing to our knowledge on P. italicum DMI-resistance mechanisms.

Comparing response of buff-tailed bumblebees and red mason bees to application of a thiacloprid-prochloraz mixture under semi-field conditions.

Recent studies have reported interspecific differences in how bee species respond to various stressors. Evaluating the exposure and responses of different bee species to plant protection products is considered an essential part of their risk assessment. This study was conducted to assess the impacts of thiacloprid-prochloraz mixture on buff-tailed bumblebees (Bombus terrestris) and red mason bees (Osmia bicornis) in a worst-case scenario under semi-field conditions. Bumblebee colonies or solitary bee trap nests were confined in tunnels with flowering oilseed rape. The recommended maximum application rates of 72 g thiacloprid/ha and 675 g prochloraz/ha were applied as a tank mixture during bee flight in full flowering oilseed rape. Several parameters such as flight and foraging activity, population parameters, and exposure level were investigated. Our results show adverse effects of the combination of thiacloprid and prochloraz on the reproductive performance of red mason bees. The number of cocoons produced by O. bicornis was significantly reduced in the treatment compared to the control group. Regarding bumblebees, we found no effects of the thiacloprid-prochloraz mixture on any observed parameters of colony development. The maximum detected concentrations of both active substances three days after application were higher in O. bicornis pollen mass compared to B. terrestris stored pollen. We conclude that this worst-case scenario of thiacloprid-prochloraz exposure poses a high risk to solitary bees and thus the use of such mixture should be restricted.

A beta-cyclodextrin-functionalized magnetic metal organic framework for efficient extraction and determination of prochloraz and triazole fungicides in vegetables samples.

A ß-cyclodextrin-functionalized magnetic zinc-metal organic framework (M-MOF/ß-CD) was synthesized via a facile one-pot reaction. M-MOF/ß-CD was used as a magnetic porous absorbent for the extraction and determination of prochloraz and three triazole fungicides in vegetable samples. M-MOF/ß-CD was prepared by creating MOF layers on the surface of a Fe3O4-graphene oxide (GO) nanocomposite and bonding them with ß-CD molecules. Characterization suggested that a 3D porous structure was formed, with M-MOF/ß-CD exhibiting high superparamagnetism and a large surface area. As a new strategy, integrating MOFs with Fe3O4-GO could improve their water-resistance and mechanical strength by providing a rigid nanosupport interface. Combining M-MOF and ß-CD resulted in excellent selective adsorption capacities for prochloraz and three triazole fungicides. The static adsorption process was evaluated and the results were in good agreement with the Freundlich model. Subsequently, M-MOF/ß-CD was applied to extracting prochloraz and triazole fungicides from tomato and lettuce vegetables, followed by HPLC-MS/MS determination. The limits of detection for the above fungicides were found to be 0.25-1.0 µg/L at a signal-to-noise ratio of 3, with spiked recoveries of 74.13%-119.83%, indicating that M-MOF/ß-CD was promising for application to the extraction and determination of fungicides in complex matrices.

Residual behavior and risk assessment of prochloraz in bayberries and bayberry wine for the Chinese population.

The bayberry is an important economic fruit as well as a minor crop in China, and few pesticide products are registered for bayberry. Prochloraz is a widely used fungicide with a high detection rate on bayberry. This study evaluated the potential dietary risk of prochloraz for different populations in China based on field trial data and market surveillance. The results indicate that one-time applications at dosages of 1000 and 1500 mg/kg with a recommended preharvest interval of 20 days do not pose a chronic or acute dietary risk. However, applying the above dosages twice will cause a potential short-term dietary risk. Risk assessment results conducted on surveillance samples indicated acceptable long-term risks for the general population, with a hazard quotient < 0.82. Furthermore, simulated washing and wine production processes were performed to mimic household practices to investigate residue transfer and distribution. We found that rinsing with tap water for 1 min was an effective way to remove residue, and the processing factors of prochloraz for both bayberry and wine were < 1, indicating that wine production could reduce residue levels. Prochloraz had a strong capacity to transfer to wine due to its high log Kow value, with transfer percentages up to 43%. This study supports the recommendation on good agricultural practices for prochloraz application and provides a guide for safe consumption.

Comparing effect levels of regulatory studies with endpoints derived in targeted anti-androgenic studies: example prochloraz.

Prochloraz is an imidazole fungicide, and its regulatory toxicological data package has been primarily generated in the 1990s. More recently, studies have been published demonstrating an interaction with hormone receptors/steroidogenesis and effects with an endocrine mode of action. In the present study, prochloraz has been investigated in a comprehensive in vivo study including relevant elements of current regulatory reproduction toxicity studies and additional mechanistic parameters. Prochloraz was administered per gavage in oil from GD 6 to PND 83 to pregnant and lactating Wistar rats and their respective offspring, at doses of 0.01 mg/kg bw/day (acceptable daily intake of prochloraz), 5 mg/kg bw/day [expected no-observed-effect-level (NOEL)] and 30 mg/kg bw/day. At 30 mg/kg bw/day maternal and offspring effects (decreased viability, lower number of live offspring) were seen including a delayed entry into male puberty (+1 day) accompanied by lower male offspring body weights, increased anogenital distance/index in females and transiently retained nipples in males at PND 12 (not seen at PND 20). The only finding at the "expected NOEL" was increased incidences of transiently retained nipples in males which are not considered adverse. No effects were seen in the low-dose group. There was no evidence for a non-monotonic dose-response curve or effects at low levels.

Residue Dynamics and Risk Assessment of Prochloraz and Its Metabolite 2,4,6-Trichlorophenol in Apple.

The residue dynamics and risk assessment of prochloraz and its metabolite 2,4,6-trichlorophenol (2,4,6-TCP) in apple under different treatment concentrations were investigated using a GC-ECD method. The derivatization percent of prochloraz to 2,4,6-TCP was stable and complete. The recoveries of prochloraz and 2,4,6-TCP were 82.9%-114.4%, and the coefficients of variation (CV) were 0.7%-8.6% for the whole fruit, apple pulp, and apple peel samples. Under the application of 2 °C 2.0 g/L, 2 °C 1.0 g/L, 20 °C 2.0 g/L, and 20 °C 1.0 g/L treatment, the half-life for the degradation of prochloraz was 57.8-86.6 d in the whole fruit and apple peel, and the prochloraz concentration in the apple pulp increased gradually until a peak (0.72 mg·kg-1) was reached. The concentration of 2,4,6-TCP was below 0.1 mg·kg-1 in four treatment conditions and not detected (

A novel major facilitator superfamily transporter in Penicillium digitatum (PdMFS2) is required for prochloraz resistance, conidiation and full virulence.

OBJECTIVES: To clone a novel major facilitator superfamily (MFS, a large protein family with diverse physiological functions in all kingdoms) transporter gene, Pdmfs2, and characterize its function in Penicillium digitatum. RESULTS: A novel MFS transporter gene, Pdmfs2, was isolated from P. digitatum. The full-length DNA of Pdmfs2 had a 1590 bp ORF encoding a full-size MFS transporter with 529 amino acids. In a prochloraz-resistant strain (PdHS-F6), Pdmfs2 transcript level was up-regulated compared with the prochloraz-sensitive strain (PdHS-E3) and could be induced by 7 µg prochloraz/ml. The deletion of Pdmfs2 (ΔPdmfs2) in PdHS-F6 led to increased susceptibility to prochloraz and lower EC50 value (the concentration of prochloraz producing 50 % growth inhibition) compared with the PdHS-F6 or complementation strain (COPdmfs2). The ΔPdmfs2 strain was defective in conidia yield and virulence towards citrus fruits, while the complementation of Pdmfs2 could restore the phenotypic features to a large extent. CONCLUSIONS: Pdmfs2 is the second MFS transporter gene in P. digitatum and is required for prochloraz resistance, conidiation and full virulence.

Is UV radiation changing the toxicity of compounds to zebrafish embryos?

At ecosystems level, environmental parameters such as temperature, pH, dissolved oxygen concentration and intensity of UV radiation (UVR) have an important role on the efficiency of organisms' physiological and behavioral performances and consequently on the capacity of response to contaminants. Insignificant alterations of these parameters may compromise this response. In addition, these parameters can additionally alter chemical compounds by inducing their degradation, producing thereafter other metabolites. Understanding the combined effects of chemicals and environmental parameters is absolutely necessary for an adequate prediction of risk in aquatic environments. According to this scenario, this work aims at studying the combined toxicity of UVR and three xenobiotics: the biocide triclosan (TCS), the metal chromium (as potassium dichromate, PD) and the fungicide prochloraz (PCZ). To achieve this goal zebrafish (Danio rerio) embryos (3h post fertilization (hpf)) were exposed to several concentrations of each chemical combined with different UV intensities; mortality and eggs were recorded every 24h for the all test duration (96 h). Results showed different response patterns depending on the toxicant, stress levels and duration of exposure. The combination of UVR and TCS indicated a dose ratio deviation where synergism was observed when UVR was the dominant stressor (day 2). The combination of UVR and PD presented a dose level dependency at day 3 indicating antagonism at low stress levels, changing with time where at day 4, a dose ratio deviation showed statistically that synergism occurred at higher PD concentrations. Finally, UVR combined with PCZ indicated a dose ratio at day 3 and dose level deviation at day 4 of exposure, suggesting a synergistic response when PCZ is the dominant stressor in the combination. The obtained results in this study highlighted the importance of taking into account the possible interaction of stressors and time of exposure to better predict environmental risk.