Trace Element Composition of Selected Fertilizers Used in Chile: Phosphorus Fertilizers as a Source of Long-Term Soil Contamination

Anthropogenic activities like agriculture have resulted in increased concentrations of some trace elements of toxicological and environmental concern in soils. Application of fertilizers has been one of the major inputs of these contaminants to agricultural soils in developing countries. Twenty-two fertilizers, including straight nitrogen (N), phosphorus (P), potassium (K), and NK fertilizers and micronutrient sources, were analyzed by inductively coupled plasma optical emission spectrometry (ICP-OES) for arsenic (As), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo), nickel (Ni), lead (Pb), vanadium (V), and zinc (Zn). As expected, the trace element content of fertilizers was highly variable and related to the origin of the material. Phosphorus fertilizers, especially triple superphosphate, presented the highest As, Cd, Cu, Cr, Ni, V, and Zn concentrations. In some of these fertilizers, the Cr, V, and Zn contents reached values greater than 3475 mg kg^?1 of P, and the Cd content (up to 288 mg kg^?1 of P) was several times higher than the regulatory limits of different countries. Some micronutrient sources presented the highest concentrations of Mn and Pb. In the cases of N, K, and NK fertilizers, the trace element concentration was very low, sometimes below the detection limits. In some agricultural systems the input of trace elements such as As and Cd to the soil through P fertilizers application may be higher than the outputs through plant uptake and leaching; therefore the long-term use of these fertilizers may cause the trace element concentration to increase in the plow layer of agricultural soils.     

Assessment of the phytotoxicity of chromium in soils using the selective ion exchange resin extraction method

Chromium in soils is present in the form of Cr(VI) oxyanions or Cr(III) cations. The toxicity and mobility of Cr(VI) are higher than those of Cr(III), thus it is essential that the availability of Cr(VI) in soils be accurately estimated in order to assess the phytotoxicity of Cr and its resultant health hazards to animals and humans. In this study, the Cu-saturated selective ion exchange resin (DOWEX M4195) was used as an infinite sink to test the feasibility of using the resin for extracting available Cr(VI) from soil. In the experiments, the results show that the resin had a high affinity for Cr(VI) and that Cr(VI) adsorbed by resins could be desorbed by using 10% NaCl (pH 4). In addition, the adsorption and desorption of Cr(VI) were not affected by pH levels, the forms of Cr(VI) or the presence of major anions in the soil solution. The above results indicate that the Cu-saturated resin can selectively adsorb Cr(VI) from solution. In the soil extraction experiments, three Cr(VI)-spiked soils were processed using the Cu-saturated resin extraction method. The results show that amounts of soil Cr extractable by the resin had a significant negative correlation to the height of wheat seedlings grown in the Neubauer test. Comparing this to the commonly used extractant, 0.1 M HCl, the amount of soil Cr, extractable by the resin, had a higher correlation to plant height. The results suggest that the selective ion exchange resin method developed in this study is useful in evaluating the quantities of plant-available Cr(VI) in soil and can, therefore, assess the phytotoxicity of Cr in soil.     

The effects of lime and phosphorus on the function of wheat roots in acid top soils and subsoils

Two wheat varieties with differing aluminium tolerance were grown in pots of acid soil. Liming did not change significantly the amounts of chemically extractable P and K, but caused improved vegetative growth, increased inflow of P and K and reduced uptake of Al. Without lime, roots had a higher content and concentration of P than shoots; liming reversed this. Without lime the sensitive variety with a shorter root length had an Al inflow ten times that of the tolerant one: tolerance involves a mechanism for exlcuding Al. The inflow of P per unit inflow of Al (mol ratio) without lime was three times greater for the tolerant variety which therefore has more P to counteract the effects of Al. The same varieties were grown in two-layer soil columns, with a low P status and a limed topsoil and acid subsoil. Liming the subsoil improved plant growth but this was still restricted by low P availability. Addition of P to the topsoil caused good growth regardless of subsoil acidity: root growth increased in both layers and P (labelled with³²P) taken up from the topsoil was translocated to roots in the subsoil. This P inactivated root Al and allowed the roots to grow and take up more P from the acid subsoil with however a reduction in inflow. The sensitive variety was affected more by the acid subsoil and low P availability, had a similar ability to translocate P to subsoil roots but could not attain the growth rate of the tolerant wheat even with P and lime.     

Turnover of organic nitrogen in soils and its availability to crops

The root development of barley seedlings grown for one week in an aerated nutrient solution was studied in the presence of dissolved organic matter from an aqueous chestnut leaf litter extract. In particular, the different effects of low and high molecular weight fractions (small molecules: molecular weight <1000; large molecules: >10,000) of the leaf litter extract were examined. In the presence of large molecules root growth was inhibited, an irregular root tip morphology was observed, and Ca and Mg concentrations in the shoots were lower than in control plants. These phytotoxic effects were not caused by the formation of an impermeable layer of large molecules on the root surfaces that lower accessibility for nutrient cations as inferred from voltammetric experiments. A germination assay using spruce seeds, however, indicated allelochemical effects of large molecules, which exhibit a higher aromaticity than the small molecules as indicated by spectroscopic characterisation. In the growth experiments with small molecules, no influence on the root development of barley was evident, but an increase of Ca and Mg in the shoots was detected. During these growth experiments, a large amount of the small molecules, mainly simple phenols and amino acids, disappeared from the nutrient solution. The loss of small molecules was most likely the effect of mineralisation.Abbreviations DOC dissolved organic carbon - DOM dissolved organic matter - LLE leaf litter extract - MW molecular weight - HMDE hanging mercury drop electrode     

Effect of the siderophore alcaligin E on the bioavailability of Cd to Alcaligenes eutrophus CH34

Alcaligin E, the siderophore of the heavy metal-resistant A. eutrophus strain CH34, was shown to interact with Cd and consequently affect its bioavailability and toxicity. The addition of alcaligin E markedly stimulated the growth in the presence of Cd of an alcaligin E-deficient CH34 derivative. Using bioluminescence assays, this effect could be assigned to a decrease in bioavailability of Cd in the presence of alcaligin E. However, Cd-uptake studies showed no influence of alcaligin E on the cellular concentration of Cd. Furthermore, by scanning electron microscopy, the morphology of precipitated Cd crystals was shown to be altered by alcaligin E. These data suggest that alcaligin E, besides its function in iron supply to the cell, provides a protection against heavy metal toxicity. A link between the A. eutrophus CH34 siderophore system and the czc-mediated Cd-efflux system is hypothesized. Received 19 June 1997/ Accepted in revised form 14 October 1997     

Influence of dietary lead on fluoride bioavailability in the rat

A factorial experiment was conducted with weanling rats fed a purified diet to determine the influence of dietary lead (0 or 100 ppm) as either lead acetate or lead carbonate on fluoride bioavailability (2 or 10 ppm as sodium fluoride). During the 6-wk study, both forms of lead depressed weight gain, regardless of the fluoride level, despite the fact that food intake was similar for all treatment groups. Both forms of lead produced a small, but significant, reduction in femur and second molar fluoride. This effect, however, could only be demonstrated in rats fed diets containing 10 ppm fluoride, indicating a significant interaction between lead and fluoride for these indices of fluoride bioavailability. This interactive effect between fluoride and lead was also demonstrated for apparent fluoride absorption. Both forms of dietary lead significantly increased the lead concentration of plasma, femur, liver, and kidney, and both forms of lead significantly increased the urinary excretion of delta-aminolevulinic acid. The level of dietary fluoride failed to influence these measurements. We therefore conclude that, although small amounts of dietary lead reduce fluoride bioavailability, small amounts of dietary fluoride do not appear to significantly influence the utilization of dietary lead.     

The dynamics of microflora and the occurrence of phytotoxic substances in different soils with corn residues and inorganic nitrogen

In an incubation experiment the development dynamics of bacterial and fungal communities as well as the level of phytotoxicity were analysed in sand and three soils differing in mechanical structure and amended with corn residues and mineral nitrogen. Bacterial biomass was positively correlated with the degree of dispersion of the solid phase of the soil, whereas the ratio of fungal to bacterial biomass (F:B) was found to be negatively correlated. Fungi were much more tolerant to carbon or nitrogen deficit than bacteria. Introduction of the plant material alone, characterized by a broad carbon to nitrogen ratio, led to the domination of fungi in microbial communities. The level of soil phytotoxicity built up with increasing level of crop residues. Phytotoxicity was observed for the longest time period in soil with the highest silt and clay content. The narrowing of the C:N ratio at introduction of the appropriate amount of mineral nitrogen (larger in heavier soils) resulted in accelerated disappearance of phytotoxicity and at the same time favoured bacterial development. This points to a significant participation of bacteria in the degradation of phytotoxic substances in the soil.     

Development of a biological test to evaluate the bioavailability of iron in culture media

Aims:  To develop an easy-to-use and pathogen-free protocol giving reliable information on the bioavailability of iron in a medium.
Methods and Results:  In aerobic conditions, iron bioavailability is very low, and most of its forms cannot be assimilated by micro-organisms. Media with similar iron contents can differ considerably in iron bioavailability, something that is not easily achieved using conventional physicochemical methods. The assay developed in the present work is based on a pyoverdin siderophore release by fluorescent Pseudomonas in response to iron stress.
Conclusions:  The test was applied to a complex medium used for the production of diphtheria toxin (DT). A significant difference between the bioavailable iron level and the total chemical concentrations contributed by the various compounds used to make the medium could thus be detected. This can be explained by the formation of salt complexes trapping the iron, which thus cannot be used directly by the micro-organism for its metabolism.
Significance and Impact of the Study:  The assay can easily be applied to any medium designed for the production of iron-regulated compounds. This is particularly useful when dealing with processes that use pathogenic strains as was shown in the case based on DT production.     

Protein immobilization to alumina supports: I. characterization of alumina-organophosphate ligand interactions and use in the attachment of papain

The chemical adsorption of organic phosphate compounds to alumina has been used to create surface linkers for protein immobilization. A number of particulate alumina supports were screened for their physical properties and ability to bind organic phosphate compounds. Two aluminas, termed C1 and CPC, were selected based on their suitability for subsequent testing as protein immobilization supports. Papain was successfully immobilized to these supports when derivatized with phosphate compounds containing free terminal carboxyl groups. Protein binding was enhanced when support carboxyl groups were activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The level of papain immobilization was dependent upon the length of the linker used and the mass of protein exposed to the support. (c) 1992 John Wiley & Sons, Inc.     

Effect of phosphate rock,coal combustion by-product,lime, and cellulose on ryegrass in an acidic soil

Remediation of soil acidity is crucial for increasing crop production and improving environmental quality of acid infertile soils. Soil incubation and greenhouse pot experiments were carried out to examine the interactions between phosphate rock (PR), coal combustion by-product (BP), dolomitic lime (L), and cellulose (C) in an acidic soil and their effects on ryegrass (Lolium perenne L. cv Linn) growth. BP and PR application increased plant P content and dry matter yield (DMY) of shoots and roots by improving soil Ca availability and reducing Al toxicity. Application of BP at low rates (5 to 10 g BP kg^-1) with PR appeared to decrease both plant P content and DMY compared to PR application alone. The reduced DMY is due to an increased Al concentration in soil solution as a result of displacement of sorbed Al by Ca of BP. Increases in DMY were obtained by addition of lime along with PR and BP at low rates or by increasing BP application rates above 15 g kg^-1. This improved plant response was likely related to alleviation of Al toxicity by CaCO₃ contained in the BP. In addition to raising the pH to an acceptable level for plant growth, the dolomitic lime supplied needed Mg for plants, thereby maintaining a good balance between available Ca and Mg for plants in the BP- and PR-amended soils. The addition of cellulose to the BP- and PR-amended soils reduced water-soluble Al and increased DMY. Plant growth increased PR dissolution by 2.4 to 243% in a soil with low available P. Use of BP at moderate rates with PR and dolomitic lime appears to be the best combination in increasing crop yields on infertile acidic soils.     

A study of the impacts of Zn and Cu on two rhizobial species in soils of a long-term field experiment

Two agriculturally important species of rhizobia, Rhizobium leguminosarum biovar viciae (pea rhizobia) and R. leguminosarum bv. trifolii (white clover rhizobia), were enumerated in soils of a long-term field experiment to which sewage sludges contaminated predominantly with Zn or Cu, or Zn plus Cu, were added in the past. In addition to total soil Zn and Cu concentrations, soil pore water soluble Zn and free Zn²⁺, and soluble Cu concentrations are reported. Pea and white clover rhizobia were greatly reduced in soils containing ≥200 mg Zn kg^-1, and soil pore water soluble Zn and free Zn²⁺ concentrations ≥7 and ≥3 mg l^-1, respectively, in soils of pH 5.9–6. Copper also reduced rhizobial numbers, but only at high total soil concentrations (>250 mg kg^-1) and not to the same extent as Zn. Yields of field grown peas decreased significantly as total soil Zn, soil pore water soluble Zn and free Zn⁺² increased (R² = 0.79, 0.75 and 0.75, respectively; P < 0.001). A 50% reduction in seed yield occurred at a total soil Zn concentration of about 290 mg kg^-1, in soils of pH 5.9–6. The corresponding soil pore water soluble Zn and free Zn²⁺ concentrations were about 9 and 4 mg l^-1, respectively. Pea seed yields were not significantly correlated with total soil Cu (R² = 0.33) or soil pore water soluble Cu (R² = 0.39). Yield reductions were due to a combination of greatly reduced numbers of free-living rhizobia in the soil due to Zn toxicity, thus indirectly affecting N₂-fixation, and Zn phytotoxicity. These effects were exacerbated in slightly acidic soils due to increased solubility of Zn, and to some extent Cu, and an increase in the free Zn²⁺ fraction in soil pore water. The current United Kingdom, German and United States limits for Zn and Cu in soils are discussed in view of the current study. None of these limits are based on toxicity thresholds in soil pore water, which may have wider validity for different soil types and at different pH values than total soil concentrations. This revised version was published online in June 2006 with corrections to the Cover Date.     

The bioavailability of cadmium to lettuce and cabbage in soils previously treated with sewage sludges

The application of sewage sludges to soils may lead to increased soil-Cd levels. The bioavailability of Cd is determined by the interaction of a number of soil physico-chemical and plant variables, of which pH is the most important. Duplicate samples of sludge-treated soils were transferred to tubs in the field, one of each pair being limed to pH 7±0.5. Lettuce and cabbage were grown to maturity and analysed for Cd. Liming always reduced Cd uptake by the plants. Three soil extractants, 1 M NH₄NO₃, 0.05 M EDTA-(Na)₂ and 0.05 M CaCl₂ were used as indices of Cd bioavailability. CaCl₂ proved to be the most effective for both lettuce and cabbage. Multiple linear regression equations were derived to describe the uptake and accumulation of Cd by both crops. The relative influence of soil variables differed between the two species. Unlike those of a number of pot experiments conducted in glasshouses, the data from this experiment are comparable with those of crop samples taken from the field.     

Studies on the efficacy of a phytohormone producing phosphate solubilizingBacillus firmus in augmenting paddy yield in acid soils of Nagaland

Summary A super strain ofBacillus firmus (NCIM-2636) producing a phytohormone, indole-3-acetic acid, in addition to its high ability to solubilize insoluble inorganic phosphates were applied in acid soils of Nagaland, India. Rice (Oryza sativa L.) variety Jaya and IR-8 were grown in kharif season in two successive years 1980 and 1981. After proper manuring the soils received single super phosphate (S.S.P.) and Mussoorie Rock phosphate (R.P.) separately at different doses. Yield of crop in both the years increased significantly due to bacterial inoculation. Maximum grain yield was recorded in Jaya variety under S.S.P. and R.P. when treatments were at the dose of 43.75 and 17.5 kg P ha⁻¹ respectively while the same in IR-8 variety under S.S.P. and R.P. treatments were at the dose of 35 and 17.5 kg P ha⁻¹ respectively. Maximum straw yield was produced by Jaya variety when 35 and 43.75 kg P ha⁻¹ in the form of S.S.P. and R.P. respectively were applied. Highest straw yield of IR-8 variety was obtained after the application of 17.5 kg P ha⁻¹ (S.S.P. and R.P.) in combination with phosphate solubilizing bacteria. Bacterial inoculation decreased the phosphorus availability in 1 st year but increased the same in 2nd year. Phosphorus content in grains was significantly enhanced in both the trials. Maximum uptake of phosphorus by grains was noted in Jaya variety at the dose of 47.5 kg P ha⁻¹ and in IR-8 variety at the dose of 52.5 kg P ha⁻¹ under S.S.P. treatment, while 8.75 and 35 kg P ha⁻¹ in the form of R.P. yielded similar results in Jaya and IR-8 varieties respectively. Phosphorus at the dose of 35 kg ha⁻¹ was found to cause more P-uptake by straw in both S.S.P. and R.P. treatments. The various data from the experiment conclusively proved that the bacterium in combination with R.P. produced the desired effect more prominently than when bacterium applied in combination with S.S.P.     

稻麦轮作下水稻土重金属形态特征及其生物有效性

在成都平原稻麦轮作下86个土样中重金属全量和各形态含量分析的基础上,采集分析相应点位上种植的主要粮食作物小麦和水稻籽粒的重金属含量,通过描述性统计分析、相关性分析及线性回归方程的模拟,研究了土壤中重金属的形态分布特征及其生物有效性。统计分析结果表明：土壤中Cr、Cu、Pb、Zn各形态含量的分配顺序为：残渣态〉有机物结合态〉铁锰氧化物结合态〉碳酸盐结合态〉可交换态,而Cd各形态含量的分配顺序为：铁锰氧化物结合态〉残渣态〉碳酸盐结合态〉有机物结合态〉可交换态。若以活性态（非残渣态含量）而论,Cd的活性最高,Cr的活性最低。除Cr外,各元素全量与各活性形态之间的关系较为密切。土壤中全Cd含量与4种活性形态Cd含量间的相关性均达极显著水平;Cu、Zn全量分别与其铁锰氧化物和有机物结合态含量,Pb全量分别与其可交换态和铁锰氧化物含量间的相关系数达显著或极显著水平。用卫生部颁布的食品中重金属限量卫生标准评价稻麦籽粒重金属累积情况,其结果表明,小麦籽粒（旱作）中的Cd、Cr、Cu、Pb分别超标14.71%、8.70%、6.50%和17.40%,而水稻籽粒（水作）仅有Pb和Cd含量超标10.90%和8.70%。不同形态重金属对水稻和小麦籽粒中重金属累积的影响效应不同,从多元线性回归方程的回归系数来看：稻一麦籽粒中Cd含量受到土壤中各活性形态Cd含量的影响。除铁锰氧化态Cd和有机物结合态Cd含量分别对小麦和水稻籽粒中Cd的累积表现出负效应外,其余均为正效应。可交换态Cu含量对稻-麦籽粒Cu累积的效应最大,其次受碳酸盐结合态Cu含量的影响。稻一麦籽粒Pb含量的累积受土壤各活性态Pb含量影响效应的差异不甚明显。稻-麦籽粒Zn含量与土壤各活性态Zn含量间无显著线性关系。     

Response of cowpea (Vigna unguiculata) to inoculation with VA-mycorrhizal fungi and to rock phosphate fertilization in some unsterilized Nigerian soils

Summary The effects on cowpea of inoculation with vesicular-arbuscular (VA) mycorrhizal fungi and rock phosphate (RP) fertilization were studied in pots using Alagba and Araromi series soils and in the field on Alagba, Apomu and Egbeda series soils. Inoculation of the plants with VA-mycorrhizal fungi caused very rapid infection of the roots. A higher per cent mycorrhizal infection was maintained during subsequent plant growth in the field. RP application reduced the degree of infection without affecting plant growth in the field and in pot experiments. Nodulation, nitrogen fixation and utilization of RP were increased by inoculation with mycorrhizal fungi in the pot experiments but not in the field experiments. In the pot experiments, inoculated plants supplied with RP flowered earlier, and took up more phosphorus than either inoculated plants without RP or uninoculated plants. The largest response to inoculation in terms of shoot dry matter, nodule yield and nitrogen content of shoots was obtained in Alagba soil under both pot and field conditions.IITA Journal Series Paper No. 136.     

Colonisation of wheat by VA-mycorrhizal fungi was found to be higher on a farm managed in an organic manner than on a conventional neighbour

Levels of colonisation by vesicular-arbuscular (VA)-mycorrhizal fungi were compared between adjacent farms, one operated in a conventional manner and the other run according to organic farming principles. Wheat grown on the organic farm was found to have VA-mycorrhizal colonisation levels consistently 2 to 3 times higher than wheat on the conventional farm. Glasshouse and field trials indicated that the lower colonisation levels on the conventional farm were due to continual use of fertiliser containing soluble phosphorus (P). The fertiliser appeared to have an immediate negative effect on the rate of colonisation, and also appeared to have a long term negative effect through maintaining higher levels of soluble P in the soil, and by decreasing inoculum levels. Use of the relatively insoluble reactive rock phosphate fertiliser on the organic farm did not decrease levels of VA-mycorrhizas. Colonisation levels did not vary between wheat varieties, and herbicides and seed dressings were also not found to be having any significant effect on levels of colonisation.     

City refuse compost as a phosphorus source to overcome the P-fixation capacity of sesquioxide-rich soils

In sesquioxide-rich soils of tropical and subtropical areas and volcanic-ash soils with high levels of active Al(Fe), large amounts of phosphate fertilizers are needed to overcome their high P-fixation capacity (quenching strategy). A greenhouse pot experiment has been used to evaluate the effectiveness of city refuse compost (CRC) as a P-source for these variable-charge soils, compared to inorganic P. Mature CRC and K₂HPO₄ were applied at rates equivalent to 125, 250, 375, 500 and 625 kg P ha^–1 to a ferrallitic soils from Tenerife Island (Andeptic Paleudult) with a high content in active Al+Fe (4.82%) and a high P-fixation capacity (87%). Perennial ryegrass (Lolium perenne L.) was grown in pots and plants were harvested at regular intervals after seedling emergence. CRC increases plant P concentration and soil labile-P proportional to the applied rate. The best results were obtained from a compost application of 30 t ha^–1 equivalent-rate, after a residence time of at least three months. An important residual effect in the supply capacity of P in relation to the phosphate fertilizer was also observed. The relative agronomic effectiveness (RAE) in comparison to K₂HPO₄ was 66% after 6 months, considering P uptake + soil labile-P. The soil P-fixation capacity was significantly reduced from a compost application of 40 t ha^–1 equivalent-rate. Competition in adsorption between organic ligands and phosphate, in combination with net mineralization of organic P in compost, might account for the high RAE value obtained. The main conclusion is that the city refuse compost could be a suitable P-amendment for resquioxic soils due to its high RAE, and the residual effect on P-supply. ei]H. Lambers     

A feasibility test to estimate the duration of phytoextraction of heavy metals from polluted soils

The practical applicability of heavy metal (HM) phytoextraction depends heavily on its duration. Phytoextraction duration is the main cost factorfor phytoextraction, both referring to recurring economic costs during phytoextraction and to the cost of the soil having no economic value during phytoextraction. An experiment is described here, which is meant as a preliminary feasibility test before starting a phytoextraction scheme in practice, to obtain a more realistic estimate of the phytoextraction duration of a specific HM-polluted soil. In the experiment, HM-polluted soil is mixed at different ratios with unpolluted soil of comparable composition to mimic the gradual decrease of the HM content in the target HM-polluted soil during phytoextraction. After equilibrating the soil mixtures, one cropping cycle is carried out with the plant species of interest. At harvest, the adsorbed HM contents in the soil and the HM contents in the plant shoots are determined. The adsorbed HM contents in the soil are then related to the HM contents in the plant shoots by a log-log linear relationship that can then be used to estimate the phytoextraction duration of a specific HM-polluted soil. This article describes and evaluates the merits of such a feasibility experiment. Potential drawbacks regarding the accuracy of the described approach are discussed and a greenhouse-field extrapolation procedure is proposed.     

Combination of oocyte and zygote selection by brilliant cresyl blue (BCB) test enhanced prediction of developmental potential to the blastocyst in cattle

The cumulus oocyte complexes (COCs) were obtained from local abattoir. After aspiration, the COCs were allotted into four treatments to evaluation of brilliant cresyl blue (BCB) test. Control treatment (C): oocytes were cultured directly (without exposure to BCB) after recovery in in vitro production (IVP) process. Oocyte treatment (OBCB): immediately after aspiration, COCs were incubated in modified Dulbecco's phosphate-buffered saline (mDPBS) supplemented with 26 μM of BCB for 90 min and classified into two classes: oocytes with blue cytoplasm coloration (OBCB+: more competent oocytes) and oocytes without blue cytoplasm coloration (OBCB−: low competent oocytes). Directly after classification, the oocytes were maintained undisrupted in the IVP process. Zygote treatment (ZBCB): After oocyte collection, maturation and fertilization, zygotes were stained with BCB for 10 min and categorized into three ways, according to whether they were highly stained (ZBCB++: low competent zygotes), moderately stained (ZBCB+: moderate competent zygotes) and unstained (ZBCB−: more competent zygotes). Directly after classification, the zygotes were maintained undisrupted in the culture process. Oocyte and zygote treatments (OBCB/ZBCB): COCs were stained with BCB after recovery and classified into two classes (OBCB+ and OBCB−). After fertilization, the zygotes produced from OBCB+ and OBCB− oocytes were further stained with BCB for 10 min and categorized six ways (OBCB+/ZBCB++, OBCB+/ZBCB+, OBCB+/ZBCB−, OBCB−/ZBCB++, OBCB−/ZBCB+ and OBCB−/ZBCB−). Directly after classification, the zygotes were maintained undisrupted in the culture process. The selection rate produced from OBCB treatment (OBCB+; 54.3%) was greater (P < 0.05) than ZBCB treatment (ZBCB−; 44.3%). In addition, the selection rate produced from double application (combination of oocyte and zygote selection) of BCB test (OBCB+/ZBCB−: 28.8%) was less (P < 0.01) than single application of BCB test (ZBCB−: 44.3%or OBCB+: 54.3%). The percentage of blastocyst production from OBCB+ oocytes (35.7%) and ZBCB− zygotes (36.6%) were greater (P < 0.05) than that from C oocytes (25.7%), OBCB− oocytes (16.5%), ZBCB++ (13.5%) and ZBCB+ zygotes (21.3%). However, there were no significant differences (P > 0.05) in the percentages of blastocyst production between OBCB+ oocytes (35.7%) and ZBCB− zygotes (36.6%). The proportion of blastocyst production from double application of BCB test (OBCB+/ZBCB−: 48.0%) was greater (P < 0.05) than that from single application of BCB test (OBCB+: 35.7% or ZBCB−: 36.6%). In conclusion, current results confirmed that combination of oocyte and zygote selection by BCB test enhanced the efficiency of selecting for high quality embryos, compared to the single BCB test.