Trimetal-based nanomaterials induced toxicity to plants: Does it differ from the toxicity of mixed and single-element nanoparticles?

Advanced materials comprising multiple metal alloys have made their way into the market. Trimetal-based nanomaterials (TNMs) are an example of advanced materials which have gained significant traction and are now employed in a wide array of products. It is essential to raise the question if the toxicity of advanced nanomaterials like TNMs differs from the joint effects as manifested by exposure to the single component nanoparticles (NPs). To answer this question, a trimetal-based nanomaterial: bismuth cobalt zinc oxide (BiCoZnO) was tested. This TNM had a mass ratio of 90 % ZnO NPs, 7 % Bi2O3 NPs and 3 % Co3O4 NPs. Nanoparticle-exposed lettuce seedlings (Lactuca sativa L.) showed decreases in relative root elongation (RRE) and biomass production after 21 days of exposure. The 50 % of maximal effective concentration (EC50) value of the TNMs for biomass production was 1.2 mg L-1 when the exposure period was 240 h. This is of the same magnitude as the EC50 values found for ZnO NPs (EC50 = 1.5 mg L-1) and for the mixture of components NPs (MCNPs) which jointly form the TNMs (EC50 = 3.7 mg L-1) after 10 d of exposure. The inhibition of plant root elongation by the TNMs was partially (65 %) attributed to the release of Zn ions, with the actual concentration of released Zn ions being lower in TNMs compared to the actual concentration of Zn ions in case of ZnO NPs. It is therefore to be concluded that the concentration of Zn ions cannot be used as a direct measure to compare the toxicity between traditional and advanced Zn-related nanomaterials. The EC50 values could be assessed within a factor of two; which is helpful when developing advanced alloy nanomaterials and assessing prospective the effects of trimetal-based nanomaterials.

Impact of PpSpi1, a glycosylphosphatidylinositol-anchored cell wall glycoprotein, on cell wall defects of N-glycosylation-engineered Pichia pastoris.

IMPORTANCE: Engineering of biological pathways in various microorganisms is a promising direction for biotechnology. Since the existing microbial cells have evolved over a long period of time, any artificial engineering may cause some unexpected and harmful effects on them. Systematically studying and evaluating these engineered strains are very important and necessary. In order to produce therapeutic proteins with human-like N-glycan structures, much progress has been achieved toward the humanization of N-glycosylation pathways in yeasts. The properties of a P. pastoris strain with humanized N-glycosylation machinery were carefully evaluated in this study. Our work has identified a key glycoprotein (PpSpi1) responsible for the poor growth and morphological defects of this glycoengineered strain. Overexpression of PpSpi1 could significantly rescue the growth defect of the glycoengineered P. pastoris and facilitate its future industrial applications.

Production of potassium-enriched biochar from Canna indica: Transformation and release of potassium.

Potassium (K) is one of the essential macronutrients for plant growth, while most agricultural soils are suffering from K deficiency worldwide. Therefore, it is a promising strategy to prepare K-enriched biochar from biomass waste. In this study, various K-enriched biochars were prepared from Canna indica at 300-700 °C by pyrolysis, co-pyrolysis with bentonite, and pelletizing-co-pyrolysis. The chemical speciation and release behaviors of K were investigated. The derived biochars showed high yields, pH values, and mineral contents, which were affected by the pyrolysis temperatures and techniques. The derived biochars contained a significant amount of K (161.3-235.7 mg/g), which was much higher than the biochars derived from agricultural residues and wood. Water-soluble K was the dominant K species in biochars with a proportion of 92.7-96.0%, and co-pyrolysis and pelletizing promoted the transformation of K to the exchangeable K and K silicates. In comparison with the C. indica derived biochars (83.3-98.0%), the bentonite-modified biochar showed a lower cumulative release proportion of K (72.5% and 72.6%) in a 28-day release test, meeting the Chinese National Standard for slow-release fertilizers. In addition, the pseudo-first order, pseudo-second order, and Elovich models well described the K release data of the powdery biochars, and the pseudo-second order model was the best fit for the biochar pellets. The modeling results indicated that the K release rate decreased after the addition of bentonite and pelletizing. These results indicated that the biochars derived from C. indica could be used as potential slow-release K fertilizers for agricultural application.

Effects of combined nitrification inhibitors on soil nitrification, maize yield and nitrogen use efficiency in three agricultural soils.

Application of nitrification inhibitors (NIs) with nitrogen (N) fertilizer is one of the most efficient ways to improve nitrogen use efficiency (NUE). To fully understand the efficiency of NIs with N fertilizer on soil nitrification, yield and NUE of maize (Zea mays L.), an outdoor pot experiment with different NIs in three soils with different pH was conducted. Five treatments were established: no fertilizer (Control); ammonium sulfate (AS); ammonium sulfate + 3, 4-dimethyl-pyrazolate phosphate (DMPP) (AD); ammonium sulfate + nitrogen protectant (N-GD) (AN); ammonium sulfate + 3, 4-dimethyl-pyrazolate phosphate + nitrogen protectant (ADN). The results showed that NIs treatments (AD, AN and ADN) significantly reduced soil nitrification in the brown and red soil, especially in AD and ADN, which decreased apparent nitrification rate by 28% - 44% (P < 0.05). All NIs treatments significantly increased yield and NUE of maize in three soils, especially ADN in the cinnamon soil and AD in the red soil were more efficiency, which significantly increased maize yield and apparent nitrogen recovery by 5.07 and 6.81 times, 4.39 and 8.16 times, respectively. No significant difference on maize yield was found in the brown soil, but AN significantly increased apparent nitrogen recovery by 70%. Given that the effect of NIs on both soil nitrification and NUE of maize, DMPP+N-GD was more efficient in the cinnamon soil, while N-GD and DMPP was the most efficiency in the brown and red soil, respectively. In addition, soil pH and soil organic matter play important role in the efficiency of NIs.

Similarity assessment of metallic nanoparticles within a risk assessment framework: A case study on metallic nanoparticles and lettuce.

Similarity assessment is one of the means of optimally using scarcely available experimental data on the fate and hazards of nanoforms (NFs) for regulatory purposes. For a set of NFs that are shown to be similar it is allowed in a regulatory context to apply the information available on any of the NFs within the group to the whole set of NFs. Obviously, a proper justification for such a similarity assessment is to be provided. Within the context of exemplifying such a justification, a case study was performed aimed at assessing the similarity of a set of spherical metallic NFs that different with regard to chemical composition (three metals) and particle size (three different sizes). The endpoints of assessment were root elongation and biomass increase of lettuce (Lactuca sativa L.) seedlings and exposure assessment was performed in order to express the actual exposure concentration in terms of time-weighted average particle concentrations. The results of the study show that for the specific endpoints assessed, chemical composition is driving NF toxicity and this is mostly due to impacts on the fate of the NFs. On the other hand, particle size of Cu NFs had a negligible impact on the dose-response relationships for the specific endpoints assessed. It is thus concluded that hazard data available on spherical Cu NF tested in our case can be used to inform on the hazards of any spherical Cu NF within the size range of 25-100 nm, but only applies for the certain endpoints. Also, toxicity data for the Cu2+-ion are suited for such a similarity assessment.

Polyethyleneimine-reinforced Sn/Cu foam dendritic self-supporting catalytic cathode for CO2 reduction to HCOOH.

In this work, a novel catalytic cathode of polyethyleneimine (PEI)-Sn/Cu foam with dendritic structure was prepared by electrodeposition and impregnation. It was used in the electrocatalytic reduction of CO2 to HCOOH, and its performance in this process was evaluated. At -0.97 V vs. RHE, the faradaic efficiency and current density reached 92.3% and 57.1 mA cm-2, respectively, in a 0.5 M KHCO3 electrolyte. The HCOOH production rate reached 890.4 µmol h-1 cm-2, which exceeds those for most reported Sn catalysts. Density functional theory calculations showed that use of Sn/Cu foam is more conducive to HCOOH formation than use of Cu or Sn alone, and *OCHO is the main intermediate in HCOOH formation. The results of OH- adsorption experiments confirmed that the introduction of PEI enhanced the catalytic capacity of the Sn/Cu foam, stabilized CO2·- intermediates, and promoted HCOOH generation. These results will provide an attractive strategy for developing efficient catalysts with excellent activities and stabilities for CO2 electroreduction.

In Situ Ti6Al4V/TiB Composites Prepared by Hydrogen-Assisted Sintering of Blends Containing TiH2 and Ball-Milled Ti+TiB2 Powders.

In the present study, 98.6-99.5% dense in situ reinforced Ti6Al4V/TiB composites were manufactured with a newly developed approach based on hydrogen-assisted blended elemental powder metallurgy (BEPM). The approach includes the activation milling of titanium powder produced with hydrogenation-dehydrogenation (HDH-Ti powder) with finer TiB2 additives, following blending with TiH2 and master alloy (MA) powders, and final press-and-sinter operations. Scanning electron microscope (SEM) observations prove the formation of microstructures with improved density and homogeneous distribution of TiB reinforcements in a sintered Ti6Al4V matrix. Hardness and compressive tests validated the high mechanical characteristics of produced composites. The effect of preliminary milling time over 2-6 h and the ratio of hydrogenated and non-hydrogenated titanium powders used (TiH2 vs. HDH Ti) on microstructure and mechanical properties were studied to further optimize the processing parameters. Test results indicate the above approach can be regarded as a promising route for the cost-effective manufacturing of Ti6Al4V/TiB composite with reduced porosity, tailored microstructure uniformity, acceptable impurity level and, hence, mechanical characteristics sufficient for practice applications.

Combining electrokinetic treatment with modified zero-valent iron nanoparticles for rapid and thorough dechlorination of trichloroethene.

In situ injection of nanoscale zero-valent iron (nZVI) slurry is a promising method to treat chlorinated solvents represented by trichloroethylene (TCE) in groundwater. In this study, the effects of sulfidation and emulsification treatment on the performance of nZVI reductive dechlorination of TCE under enhancement by an external electric field were evaluated. The hydrophobic oil film on the surface of sulfidized and emulsified zero-valent iron (S-EZVI) can sequestrate more than one-fifth of the unreacted TCE in the early stage of the experiment (at 5 min). The FeS layer formed on the surface of S-EZVI can facilitate the electron-transfer process and reduce the degree of corrosion of Fe0 with water by 94.0%. Electric-field-enhanced S-EZVI technology can remove more than 93.1% of TCE in the pH range 6.0-9.0, and the performances in overly acid and overly alkali environments both improved. Under the optimal conditions, the TCE removal rate and reaction constant of the applied electric field group reached 96.7% and 1.6 × 10-2 L g-1 min-1, respectively, which were much higher than those of the group without an electric field (53.2% and 3.3 × 10-3 L g-1 min-1) owing to rapid concurrent hydrogenolysis of dichloroethenes and vinyl chloride, or another transformation pathway, such as direct oxidation by the anode. Thereby, this method avoids accumulation of chlorinated intermediates, especially toxic vinyl chloride. This work shows that combination technology has many characteristics that are favorable for field application, and it is expected to provide a new reference and have application value for development of in situ efficient and thorough treatment of TCE-contaminated groundwater.

The complete mitochondrial genome sequence of Schizothorax dulongensis (Cypriniformes, Cyprinidae) from Irrawaddy River in China.

Schizothorax dulongensis Huang (1985) is an endemic species in China, which is mainly distributed in the Irrawaddy River and its tributaries. In this study, the complete mitochondrial genome was sequenced. Its length is 16,579 bp, containing 22 tRNAs, two rRNAs, and 13 PCGs. The phylogenetic results showed that S. dulongensis and these species from Brahmaputra River clustered a clade, and supported the Brahmaputra River and the Irrawaddy River have been reorganized of drainage.

The influence of elevated CO2 on bacterial community structure and its co-occurrence network in soils polluted with Cr2O3 nanoparticles.

Elevated CO2 (eCO2) and nanoparticles release are considered among the most noteworthy global concerns as they may impose negative effects on human health and ecosystem functioning. A mechanistic understanding of their combined impacts on soil microbiota is essential due to the profound eCO2 effect on soil biogeochemical processes. In this study, the impacts of Cr2O3 nanoparticles (nano-Cr2O3) on the activity, structure and co-occurrence networks of bacterial communities under ambient and eCO2 were compared between a clay loam and a sandy loam soil. We showed that eCO2 substantially mitigated nano-Cr2O3 toxicity, with microbial biomass, enzyme activity and bacterial alpha-diversity in clay loam soil were much higher than those in sandy loam soil. Nano-Cr2O3 addition caused an increase in alpha-diversity except for clay loam soil samples under eCO2. 16S rRNA gene profiling data found eCO2 remarkably reduced community divergences induced by nano-Cr2O3 more efficiently in clay loam soil (P < 0.05). Network analyses revealed more complex co-occurrence network architectures in clay loam soil than in sandy loam soil, however, nano-Cr2O3 decreased but eCO2 increased modularity and network complexity. Rising CO2 favoured the growth of oligotrophic (Acidobacteriaceae, Bryobacteraceae) rather than the copiotrophic bacteria (Sphingomonadaceae, Caulobacteraceae, Bacteroidaceae), which may contribute to community recovery and increase available carbon utilization efficiency. Our results suggested that the degree to which eCO2 mitigates nano-Cr2O3 toxicity is soil dependent, which could be related to the variation in clay and organic matter content, resilience of the resistant bacterial taxa, and microbial network complexity in distinct soils.

Bioavailability and cytotoxicity of Cerium- (IV), Copper- (II), and Zinc oxide nanoparticles to human intestinal and liver cells through food.

Anthropogenic nanoparticles (NPs) are emitted to the environment and may be present in vegetables for human consumption. However, the toxicity of NPs exposure through food lack systematical investigations. In order to propose a systematical study, lettuce grown in a Cerium- (IV), Copper- (II) and Zinc oxide NP contaminated environment were digested. This digestate was used to culture human intestine cells (i.e. epithelial colorectal adenocarcinoma cells, Caco-2). The basolateral juice produced by the intestinal cells was then used to culture normal human liver (HL-7702) cells. Bioavailability and biotoxicity of the NPs in the vitro models were assessed. NPs were found to be taken up from the environment by vegetables, and may thus be transferred to humans through oral exposure. Bioavailability and the effect of their concentration in the digestate medium differed in regards to NP materials. The levels of NPs found in the digestate were detrimental to intestine cells, while the liver cells exposed to lower concentrations of NP in the bodily fluid showed no statically significant change in cell necrosis. A closer assessment of the detrimental effect of the studied NPs to Caco-2 cells revealed that the damage was mainly related to the solubility of the NPs. This may partly be due to that the more soluble NP material (ZnO > CuO > CeO2) render higher metal ion release and thus higher bioavailability. This appeared to cause more cell death, and even lead to local intestinal inflammation. Although no liver cells died, there was an increase of ROS level, causing ROS-related DNA damage prior to cell necrosis. The findings in this study enhances our understanding of the relative detrimental effect of different types of NPs, and the mechanisms causing their biotoxicity in human cells through food.

Enhancement of phytoextraction of Pb by compounded activation agent derived from fruit residue.

Chelate-assisted phytoextraction is an attractive strategy to remove toxic metals from soil. However, there is lack of an effective and sustainable chelating agent. In this study, 11 kinds of fruit residue were extracted and selected to combine with N, N-bis (carboxymethyl) glutamic acid (GLDA) (0.7%) and tea saponin (4%) for the compounded activation agent (CAA), and its enhancement on Pb phytoextraction by Sedum alfredii was further evaluated by pot experiment. Among 11 fruit residue extracts, lemon residue showed the highest ability (34.7%) to extract Pb from soil. Through combining with GLDA (0.7%) and tea saponin (4%) at the optimal volume ratio of 15:2.5:2.5, the CAA removed Pb most effectively (57.1%) from soil and increased the solubility of three Pb mineral (PbS, PbCO3 and PbSO4) by 8.7-56.4 times. In pot experiment, the addition of high dosage (15 mL) CAA increased the biomass of S. alfredii by 52% and doubled the Pb accumulation. In addition, CAA-assisted phytoextraction also increased both water-soluble and acid-soluble Pb in soil, while reduced the proportion of the immobile Pb (oxidizable and residual). Generally, the compounded activation agent derived from lemon residue could be considered as-a promising enhancer for Pb phytoextraction.

Role of Vertical Transmission of Shoot Endophytes in Root-Associated Microbiome Assembly and Heavy Metal Hyperaccumulation in Sedum alfredii.

The transmission mode of shoot-associated endophytes in hyperaccumulators and their roles in root microbiome assembly and heavy metal accumulation remain unclear. Using 16S rRNA gene profiling, we investigated the vertical transmission of shoot-associated endophytes in relation to growth and Cd/Zn accumulation of Sedum alfredii ( Crassulaceae). Endophytes were transmitted from shoot cuttings to the rhizocompartment of new plants in both sterilized (γ-irradiated) and native soils. Vertical transmission was far more efficient in the sterile soil, and the transmitted endophytes have become a dominant component of the newly established root-associated microbiome. Based on 16S rRNA genes, the vertically transmitted taxa were identified as the families of Streptomycetaceae, Nocardioidaceae, Pseudonocardiaceae, and Rhizobiaceae. Abundances of Streptomycetaceae, Nocardioidaceae, and Pseudonocardiaceae were strongly correlated with increased shoot biomass and total Cd/Zn accumulation. Inoculation of S. alfredii with the synthetic bacterial community sharing the same phylogenetic relatedness with the vertically transmitted endophytes resulted in significant improvements in plant biomass, root morphology, and Cd/Zn accumulation. Our results demonstrate that successful vertical transmission of endophytes from shoots of S. alfredii to its rhizocompartments is possible, particularly in soils with attenuated microbiomes. Furthermore, the endophyte-derived microbiome plays an important role in metal hyperaccumulation.

Successive phytoextraction alters ammonia oxidation and associated microbial communities in heavy metal contaminated agricultural soils.

Phytoextraction is an attractive strategy for remediation of soils contaminated by heavy metal (HM), yet the effects of this practice on biochemical processes involved in soil nutrient cycling remain unknown. Here we investigated the impact of successive phytoextraction with a Cd/Zn co-hyperaccumulator Sedum alfredii (Crassulaceae) on potential nitrification rates (PNRs), abundance and composition of nitrifying communities and functional genes associated with nitrification using archaeal and bacterial 16S rRNA gene profiling and quantitative real-time PCR. The PNRs in rhizosphere were significantly (P < 0.05) lower than in the unplanted soils, and decreased markedly with planting time. The decrease of PNR was more paralleled by changes in numbers of copy and transcript of archaeal amoA gene than the bacterial counterpart. Phylogenetic analysis revealed that phytoextraction induced shifts in community structure of soil group 1.1b lineage-dominated ammonia-oxidizing archaea (AOA), Nitrosospira cluster 3-like ammonia-oxidizing bacteria (AOB) and Nitrospira-like nitrite-oxidizing bacteria (NOB). A strong positive correlation was observed between amoA gene transcript numbers and PNRs, whereas root exudates showed negative effect on PNR. This effect was further corroborated by incubation test with the concentrated root exudates of S. alfredii. Partial least squares path model demonstrated that PNR was predominantly controlled by number of AOA amoA gene transcripts which were strongly influenced by root exudation and HM level in soil. Our result reveals that successive phytoextraction of agricultural soil contaminated by HMs using S. alfredii could inhibit ammonia oxidation and thereby reduce nitrogen loss.

Effects of elevated CO2 and endophytic bacterium on photosynthetic characteristics and cadmium accumulation in Sedum alfredii.

Elevated CO2 and use of endophytic microorganisms have been considered as efficient and novel ways to improve phytoextraction efficiency. However, the interactive effects of elevated CO2 and endophytes on hyperaccumulator is poorly understood. In this study, a hydroponics experiment was conducted to investigate the combined effect of elevated CO2 (eCO2) and inoculation with endophyte SaMR12 (ES) on the photosynthetic characteristics and cadmium (Cd) accumulation in hyperaccumulator Sedum alfredii. The results showed that eCO2 × ES interaction promoted the growth of S. alfredii, shoot and root biomass net increment were increased by 264.7 and 392.3%, respectively, as compared with plants grown in ambient CO2 (aCO2). The interaction of eCO2 and ES significantly (P < 0.05) increased chlorophyll content (53.2%), Pn (111.6%), Pnmax (59.8%), AQY (65.1%), and Lsp (28.8%), but reduced Gs, Tr, Rd, and Lcp. Increased photosynthetic efficiency was associated with higher activities of rubisco, Ca2+-ATPase, and Mg2+-ATPase, and linked with over-expression of two photosystem related genes (SaPsbS and SaLhcb2). PS II activities were significantly (P < 0.05) enhanced with Fv/Fm and Φ(II) increased by 12.3 and 13.0%, respectively, compared with plants grown in aCO2. In addition, the net uptake of Cd in the shoot and root tissue of S. alfredii grown in eCO2 × ES treatment was increased by 260.7 and 434.9%, respectively, due to increased expression of SaHMA2 and SaCAX2 Cd transporter genes. Our results suggest that eCO2 × ES can promote the growth of S. alfredii due to increased photosynthetic efficiency, and improve Cd accumulation and showed considerable potential of improving the phytoextraction ability of Cd by S. alfredii.

Effects of Cr2O3 nanoparticles on the chlorophyll fluorescence and chloroplast ultrastructure of soybean (Glycine max).

Chromic oxide nanoparticles (Cr2O3 NPs) are widely used in commercial factories and can cause serious environmental problems. However, the mechanism behind Cr2O3 NP-induced phytotoxicity remains unknown. In this study, the effects of Cr2O3 NPs on the growth, chlorophyll fluorescence, SEM-EDS analysis, and chloroplast ultrastructure of soybean (Glycine max) were investigated to evaluate its phytotoxicity. The growth of soybean treated with various Cr2O3 NP suspensions (0.01, 0.05, 0.1, and 0.5 g L-1) was significantly inhibited. Specially, shoot and root biomass decreased by 9.9 and 46.3%, respectively. Besides, the maximum quantum yield of PSII (Fv/Fm) as well as the photochemical quenching (qP) decreased by 8-22 and 30-37%, respectively, indicating that the photosynthetic system was damaged when treated with Cr2O3 NPs. Moreover, the inhibition was confirmed by the reduction of Rubisco and MDH enzyme activity (by 54.5-86.4 and 26.7-96.5%, respectively). Overall, results indicated that the damage was caused by the destruction of chloroplast thylakoid structure, which subsequently reduced the photosynthetic rate. Our research suggests that Cr2O3 NPs can be transported and cause irreversible damage to soybean plants by inhibiting the activity of electron acceptors (NADP+) and destroying ultrastructure of chloroplasts, providing insights into plant toxicity issues. Graphical abstract ᅟ.

Nitrogen Fertilizer and Straw Applications Affect Uptake of 13C,15N-Glycine by Soil Microorganisms in Wheat Growth Stages.

This study investigated the influence of nitrogen (N) fertilizer and straw on intact amino acid N uptake by soil microorganisms and the relationship between amino acid turnover and soil properties during the wheat growing season. A wheat pot experiment was carried out with three treatments: control (CK), N fertilizer (NF) and N fertilizer plus rice straw (NS). We used stable isotope compound-specific analysis to determine the uptake of 13C,15N-glycine by soil microorganisms. In the NF treatment, microbial 13C,15N-glycine uptake was lower compared with CK, suggesting that inorganic N was the preferred N source for soil microorganisms. However, The application of straw with N fertilizer (in NS treatment) increased microbial 13C,15N-glycine uptake even with the same amount of N fertilizer application. In this treatment, enzyme activities, soil microbial biomass C and microbial biomass N increased simultaneously because more C was available. Soil mineral N and plant N contents all decreased substantially. The increased uptake of intact 13C,15N-glycine in the NS treatment can be attributed to direct assimilation by soil microorganisms to satisfy the demand for N when inorganic N was consumed.

[Mass spectrometry assay for denitrifying enzyme activity measurement].

Nitrification inhibitors exert inhibition function in soil nitrification process (NH4(+)-N to NO3(-)-N) and are widely applied in order to improve N fertilizer use efficiency. Before the new nitrification inhibitor is used, its effects on denitrification process must be investigated and denitrifying enzyme activity (DEA) is an effective indicator to show this process. In the present paper, a mass spectrometery (MS) method was taken to measure the denitrifying enzyme activity in the new nitrification inhibitor 3, 4-dimethylpyrazole phosphate (DMPP) incubation system. The results showed that the method measured the concentration of N2O in the incubation system accurately and the concentration measured by MS had marked correlation with that measured by gas chromatogram (GC) (MS(N2O) = -0.45 + 1.03GC(N2O, R2 = 0.995). At the same time, enrichment of 15 N2O and 15 N2 was measured to discriminate the nitrate reductase and N2O reductase. Differed with traditional method, the enzymatic inhibitor-acetylene was not appended. The results showed that DMPP had no influence on the denitrifying enzyme activity and indicated that the denitrification process was not influenced by DMPP.