Influence of soil structure on contaminant leaching from injected slurry.

Animal manure application to agricultural land provides beneficial organic matter and nutrients but can spread harmful contaminants to the environment. Contamination of fresh produce, surface water and shallow groundwater with the manure-borne pollutants can be a critical concern. Leaching and persistence of nitrogen, microorganisms (bacteriophage, E. coli, and Enterococcus) and a group of steroid hormone (estrogens) were investigated after injection of swine slurry into either intact (structured) or disturbed (homogeneous repacked) soil. The slurry was injected into hexaplicate soil columns at a rate of 50 t ha-1 and followed with four irrigation events: 3.5-h period at 10 mm h-1 after 1, 2, 3, and 4 weeks. The disturbed columns delayed the leaching of a conservative tracer and microorganisms in the first irrigation event compared to the intact columns due to the effect of disturbed macropore flow paths. The slurry constituents that ended up in or near the macropore flow paths of the intact soil were presumably washed out relatively quickly in the first event. For the last three events the intact soil leached fewer microorganisms than the disturbed soil due to the bypassing effect of water through the macropore flow path in the intact soil. Estrogen leached from the intact soil in the first event only, but for the disturbed soil it was detected in the leachates of last two events also. Leaching from the later events was attributed to higher colloid transport from the disturbed soils. In contrast, NO3-N leaching from the intact soil was higher for all events except the first event, probably due to a lower nitrification rate in the disturbed soil. A week after the last irrigation event, the redistribution of all slurry constituents except NO3-N in most of the sections of the soil column was higher for the disturbed soil. Total recovery of E. coli was significantly higher from the disturbed soil and total leaching of mineral nitrogen was significantly lower from the disturbed soil. Results demonstrate how manure-borne constituents injected into undisturbed soil columns respond more as expected in the field, in terms of leaching and persistence, than do the same constituents injected into typically constructed columns of disturbed soil.

Aneuploidy is a key causal factor of delays in blastulation: author response to 'A cautionary note against aneuploidy risk assessment using time-lapse imaging'.

In a previous paper, we had reported use of time-lapse monitoring to develop an aneuploidy risk classification model after identifying significant periblastulation delays in aneuploid embryos compared with euploid embryos. The model was validated subsequently in a second paper by retrospective assessment of transferred blastocysts that had also undergone time-lapse monitoring in which clinical pregnancy or live birth outcomes were established. A significant difference was seen for both outcome measures between embryos classified as low and medium risk by the model. Here we respond to the commentary entitled 'A cautionary note against embryo aneuploidy risk assessment using time-lapse imaging', which presented a case for our conclusions being unsound on the basis that maternal age, rather than aneuploidy, might be the cause of the developmental delays observed. We demonstrate that this is not the case and strengthen the argument that ploidy is a key factor influencing morphokinetics of blastulation. We also describe why the arguments made in the commentary based on comparisons between static standard observations and timings of the preimplantation embryo compared with those obtained from dynamic or time-lapse methodologies are inexact.

Effect of red light on the development and quality of mammalian embryos.

PURPOSE: To assess irradiance and total energy dose from different microscopes during the in-vitro embryonic developmental cycle in mouse and pig and to evaluate its effect on embryonic development and quality in pig. METHOD: Spectral scalar irradiance (380-1050 nm) was measured by a fiber-optic microsensor in the focal plane of a dissection microscope, an inverted microscope and a time-lapse incubation system. Furthermore, the effect of three different red light levels was tested in the time-lapse system on mouse zygotes for 5 days, and on porcine zona-intact and zona-free parthenogenetically activated (PA) embryos for 6 days. RESULTS: The time-lapse system used red light centered at 625 nm and with a lower irradiance level as compared to the white light irradiance levels on the dissection and inverted microscopes, which included more energetic radiation <550 nm. Even after 1000 times higher total energy dose of red light exposure in the time-lapse system, no significant difference was found neither in blastocyst development of mouse zygotes nor in blastocyst rates and total cell number of blastocysts of porcine PA embryos. CONCLUSIONS: Our results indicate that red light (625 nm, 0.34 W/m(2)) used in the time-lapse incubation system does not decrease the development and quality of blastocysts in both mouse zygotes and porcine PA embryos (both zona-intact and zona-free).

Persistence and leaching potential of microorganisms and mineral N in animal manure applied to intact soil columns.

Pathogens may reach agricultural soils through application of animal manure and thereby pose a risk of contaminating crops as well as surface and groundwater. Treatment and handling of manure for improved nutrient and odor management may also influence the amount and fate of manure-borne pathogens in the soil. A study was conducted to investigate the leaching potentials of a phage (Salmonella enterica serovar Typhimurium bacteriophage 28B) and two bacteria, Escherichia coli and Enterococcus species, in a liquid fraction of raw pig slurry obtained by solid-liquid separation of this slurry and in this liquid fraction after ozonation, when applied to intact soil columns by subsurface injection. We also compared leaching potentials of surface-applied and subsurface-injected raw slurry. The columns were exposed to irrigation events (3.5-h period at 10 mm h(-1)) after 1, 2, 3, and 4 weeks of incubation with collection of leachate. By the end of incubation, the distribution and survival of microorganisms in the soil of each treatment and in nonirrigated columns with injected raw slurry or liquid fraction were determined. E. coli in the leachates was quantified by both plate counts and quantitative PCR (qPCR) to assess the proportions of culturable and nonculturable (viable and nonviable) cells. Solid-liquid separation of slurry increased the redistribution in soil of contaminants in the liquid fraction compared to raw slurry, and the percent recovery of E. coli and Enterococcus species was higher for the liquid fraction than for raw slurry after the four leaching events. The liquid fraction also resulted in more leaching of all contaminants except Enterococcus species than did raw slurry. Ozonation reduced E. coli leaching only. Injection enhanced the leaching potential of the microorganisms investigated compared to surface application, probably because of a better survival with subsurface injection and a shorter leaching path.

Sorption of 17ß-estradiol to pig slurry separates and soil in the soil-slurry environment.

Contamination of freshwater by estrogens from manure applied to agricultural land is of grave concern because of the potentially harmful effects on aquatic life and human health. Recent developments in liquid manure (slurry) management include partial removal of particulate slurry dry matter (PSDM) by separation technologies, which may also remove parts of the estrogens and enhance infiltration of the slurry on field application and hence the interaction between estrogens and the soil matrix. This study investigated how 17ß-estradiol (E2), a natural estrogen commonly found in pig manure, sorbs to agricultural soils, to different size fractions of pig slurry separates, and to soils amended with each size fraction to simulate conditions in the soil-slurry environment. A crude fiber fraction (SS1) was prepared by sieving (<500 µm) the solids removed by an on-farm separation process. Three other size fractions (SS2 > SS3 > SS4) were prepared from the liquid fraction of the separated slurry by sedimentation and centrifugation. Sorption experiments were conducted in 0.01 mol L(-1) CaCl(2) and in natural pig urine matrix. Sorption in 0.01 mol L(-1) CaCl(2) was higher than that in pig urine for all solids used. Sorption of E2 to soil increased with its organic carbon content for both liquid phases. The solid-liquid partition coefficients of slurry separates were 10 to 30 times higher than those of soils, but the organoic carbon normalized partition coefficient values, reflecting sorption per unit organic carbon, were lower for slurry separates. Mixing slurry separates with soil increased the sorption of E2 to the solid phase significantly in the order: SS1 < SS3 < SS2 for both liquid phases. In contrast, SS4 reduced the sorption of E2 to the solid phase by increasing the sorption to suspended or dissolved organic matter. The study suggested that potentially 50 to 75% of E2 in slurry can be removed from the liquid fraction of slurry by physical separation.

Transport and distribution of Salmonella enterica serovar Typhimurium in loamy and sandy soil monoliths with applied liquid manure.

A leaching experiment, where liquid manure spiked with Salmonella enterica serovar Typhimurium (Tet(+)) DSM554 was applied to soil surfaces, was conducted on intact soil monoliths (60 cm in diameter and 100 cm long). A total of 6.5 x 10(10) CFU was applied to each column. We found that Salmonella serovar Typhimurium could be transported to a 1-m depth in loamy soil at concentrations reaching 1.3 x 10(5) CFU/ml of leachate. The test strain was found in concentrations ranging from 300 to 1.3(5) cells/ml in loamy soil throughout the 27 days of the experiment, while concentrations below 20 cells/ml were sporadically detected in the leachates from sandy monoliths. Real-time PCR targeting invA DNA showed a clear correspondence between the total and culturable numbers of cells in the leachate, indicating that most cells leached were viable. On day 28, distribution of Salmonella serovar Typhimurium at five depths in the four monoliths was determined. The highest recovery rate, ranging from 1.5% to 3.8% of the total applied inoculum, was found in the top 0.2 m.

Transport and fate of estrogenic hormones in slurry-treated soil monoliths.

The naturally occurring hormones, such as 17-beta-estradiol, 17-alpha-estradiol, and estrone, present in livestock manure may have detrimental environmental effects if released into surface waters. In areas where manure application is intensive, estrogens have been found in surface waters in concentrations known to affect the endocrine system of fish and amphibians. How the estrogens reach the surface waters is unclear. To investigate whether leaching through the soil profile plays a significant role, we conducted leaching experiments on intact soil cores. Lysimeter soil monoliths (60 cm in diameter and 100 cm long) were excavated from two sites in Denmark (one loamy and one sandy soil). The soil monoliths were treated with pig slurry containing estrogenic hormones and amended with an estrogen tracer (17-alpha-ethinylestradiol) and a conservative tracer (bromide). 17-alpha-ethinylestradiol is a synthetic analog of 17-beta-estradiol with sorption characteristics and molecular structure similar to those of the naturally occurring estrogens in slurry. The monoliths were exposed to a short-term irrigation event (12 h) followed by a long-term semi-field experiment (16 wk), during which leaching of natural estrogens and tracers was followed. Estrogens from slurry were transported to a depth of 1 m in loamy soil and sandy soil. The estrogen concentrations in the leachate were at a level known to affect the endocrine system of aquatic organisms.

Leaching of cyanogenic glucosides and cyanide from white clover green manure.

Use of crops for green manure as a substitute for chemical fertilizers and pesticides is an important approach towards more sustainable agricultural practices. Green manure from white clover is rich in nitrogen but white clover also produces the cyanogenic glucosides (CGs) linamarin and lotaustralin; CGs release toxic hydrogen cyanide (HCN) upon hydrolysis which may be utilized for pest control. We demonstrate that applying CGs in the form of a liquid extract of white clover to large columns of intact agricultural soils can result in leaching of toxic cyanide species to a depth of at least 1m. Although degradation of the CGs during leaching proceeded with half lives in the interval 1.5-35 h depending on soil characteristics, a fraction of the applied CGs (0.9-3.2%) was recovered in the leachate as either CGs or toxic cyanide species. Detoxification of the HCN formed was rapid in soil and leachate from both sandy and loamy soil. However, 30% of the leachate samples exceeded the EU threshold value of 50 micrgl(-1) total cyanide for drinking water and 85% exceeded the US threshold of 5 micrgl(-1) for cyanide chronic ecotoxicity in fresh water. This study demonstrates that even easily degradable natural products present in crop plants as defense compounds pose a threat to the quality of groundwater and surface waters. This aspect needs consideration in assessment of the risk associated with use of crops as green manure to replace chemical fertilizers and pesticides as well as in genetic engineering approaches to design crops with improved pest resistance.

Redistribution and persistence of microorganisms and steroid hormones after soil-injection of swine slurry.

The redistribution and fate of contaminants in pig slurry after direct injection were investigated at two field sites, Silstrup (sandy clay loam) and Estrup (sandy loam), in Denmark. Intact soil samples were collected for up to seven weeks after slurry injection and concentrations of Salmonella Typhimurium Bacteriophage 28B (phage 28B), Escherichia coli, steroid hormones and other slurry components (water, volatile solids, chloride and mineral N) determined in and around the injection slit. The two experiments at Silstrup and Estrup differed with respect to slurry solid content (6.3 vs. 0.8%), as well as soil clay content (27 vs. 15%) and differed considerably with respect to the initial redistribution of slurry-borne contaminants in soil. The transport of microorganisms from the slurry injection slit to the surrounding soil was much lower than that of mineral N and chloride due to attachment and entrapment. The redistribution of E. coli was more affected by site-specific conditions compared to phage 28B, possibly due to the larger cell size of E. coli. The overall recovery of phage 28B was 0.8-4%, and of E. coli 0.0-1.3% in different samples, by the end of the study. Nine different steroid hormones were detected in the slurry slit, and a slow redistribution to the surrounding soil was observed. Overall recovery of estrogens was 0.0 to 6.6% in different samples. The study showed that the combination of soil and slurry properties determined the initial spreading of contaminants, and hence the potential for subsequent leaching.