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Anyone in the market for a new job title or career knows firsthand the importance of a resume. After all, the first thing that is asked of you when applying for a new job is a copy of your resume. When the employer takes a look at it, this is the first impression he or she will have of you. It is the determining factor in whether or not you get called in for an interview.
There is no shame in needing some help with building your resume; no matter your skills, writing a resume doesn't come easy to everyone. If you type "top rated resume writing services" into any search engine, you'll be bombarded with hundreds of results. With so many resume builders out there, which one can you trust to get the job done?
The best resume building sites and experts will tell you that every resume should include five key components. For anyone using a service, either online or in-person, always be sure the focus of building your resume is on these five things.
Contact information (make sure it is current!)
At the top of your resume you should always include your name and contact information. The main thing to remember is to provide current information. If you provide a resume with the wrong telephone number and email, you might end up getting the job without even knowing it. Nowadays it is also smart to include social media profile information.
Summary (or objective)
Some might say that a summary is not a crucial element, but all of the best resume services will agree that it should be included. A summary – or objective – section acts as a precursor to the rest of the resume and highlights the main points you're hoping to get across.
All you need for your summary is a short paragraph (around 3 sentences) describing your skills and career goals for the future. It does not need to include your life story, but rather it allows the interviewer to get a glimpse of who you are.
Relevant skills
Including your relevant skills is the next step to any good resume, the keyword being relevant. Write about your skills that take into consideration the job you are applying for. If the position relates to customer service, include skills that demonstrate your competency in that field.
Educational background
The next component of your resume is your educational background. Include the name of the institution where you receive your highest level of education. If you have a high school degree but earned your GED, write the name of the GED organization. For college graduates, try to include the university name, your graduating year and major, and your final GPA.
Professional experience
The bulk of your resume should be in regards to your previous work experience. You don't need to go as far as writing about your high school waitressing job; just include the most recent and relevant jobs you've had. Always include the name of the company, your job title, the time you spent there, and a list of your responsibilities while working there.


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Latest News
BKR EMEA region Tax newsletter
The second edition of the BKR EMEA Tax newsletter is now available: HERE.
The intention of this publication is to keep members aware of Tax regulation and recent developments throughout the region.
This edition includes a summary on the South Africa Budget. More details on this Budget can be found in a paper prepared by the South Africa Institute of Chartered Accountants: HERE.

subset: commoncrawl


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SOMEDAY is not a Day in the Week
SOMEDAY is Not a Day in the Week
Are you a procrastinator?
Are you putting off important projects, goals, priorities? This inspiring keynote helps you identify what is important and motivates you to do it now, not someday.
Based on Sam's book that NY Times Bestselling author Geneen Roth called "joyful."


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Monthly Archives: December 2014
It's snow time
The holiday schedule is going strong now so not much has been going on the last two weeks. I got up here late last night. The fireplace pilot is still working and the water is flowing so it appears all … Continue reading
Posted in DIY old house Tagged adventures of my old house, DIY, feral cats, kittens, my old house, old house, propane, tree lighting Leave a comment


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Parasitic infection
An infection or diseases that are caused by parasites. Parasitic infection can spread in number of ways and it is mainly through the contamination of water, food or by the environment where we live. This kind of disease-causing parasites can range from single cell to multi cell such as protozoa to worms. This kind of infection is caused generally in the tropical and subtropical region. Malaria is considered as one of the deadliest diseases around the world.


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The RMWB is dealing with some shortcomings when it comes to procurement.
Wood Buffalo council learned on Tuesday that two audit reports found a lack of training and transparency with contractors was leading to inconsistent bidding processes.
"It didn't come as a big surprise that [the municipality] wasn't following our own policies and we weren't following the leading policies," said Mayor Don Scott.
MNP studied multiple projects, ranging in value from around $35,000 to $44 million, determining whether proper protocol was followed.
They found employees were treating each bid the same, despite the size, scope, and type of project. MNP says they also discovered most employees haven't received formal training.
Scott says the issue has been brought up many times by local businesses.
"Mayor and council noticed a lot of problems because we've been hearing from community members about the challenges they were having with the procurement process."
Despite multiple questions from council regarding ways to help benefit local groups, MNA says the municipality isn't allowed to favour any business over another.
"There are some trade agreements that prohibit focusing on local procurement," added Scott. "We need to start lobbying other levels of government on this issue. If there are reasons we can't start focusing more on our local businesses, that's something we need to focus on."
Meanwhile, council also passed an action plan to ensure the recommendations by MNP are followed.
"I, and I know the team at the RMWB, welcome the opportunity to learn from the findings and to move forward in a positive direction, working with all our stakeholders," added Annette Antoniak, CAO.
The municipality will have until the second quarter of 2019 to complete the plan.


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Amid the questioning of government support for remote Aboriginal communities and what PM Tony Abbott called the "lifestyle choices" of those who live there, the growing role of First Nations management of large areas of remote Australia has been overlooked. There are 1,200 small, discrete communities in Australia with various sources of income, including federal government "Working on Country" funding, as well as meagre and tightly regulated welfare payments. They fulfil a key role in populating large areas of outback Australia. Read more about Remote Indigenous communities are vital for our fragile ecosystems
More than a thousand protesters shut down traffic in Melbourne's CBD to rally against the planned closure of remote Indigenous communities in West Australia. The group initially hoped to confront Tony Abbott at the National Gallery of Victoria about his statement and support of the displacement of thousands of peoples. The crowd eventually gave up on seeing Abbott and walked from the gallery to the State Library. About half-a-dozen tram routes were disrupted and roads were closed as they made their way up Swanston Street. Read more about Protesters 'shut down Melbourne' to fight against closure of First Nation communities
The Peak body for Indigenous legal services was denied funding under the Indigenous advancement strategy as $680m in grants handed out. The NSW ALS had its two applications rejected by the IAS scheme, including one for its existing custody notification service, which provides 24-hour legal advice and welfare support for Aboriginal people arrested in NSW. Some organisations said they did not know if or how much of a grant they had been given, or if funding would continue beyond the next 12 months. Read more about First Nations legal service to shut down after losing federal funding
The Premier of Australia's wealthiest state Colin Barnett has, without notice or consultation, announced he is closing down 150 small towns in his state and has refused to meet the mayors or residents of these generally remote towns to discuss the matter. The Premier – Colin Barnett – said he had no other options after the Federal Government announced they would no longer fund essential services to the towns, instead providing a one-off payment of $90 million to the State Government to take responsibility. Read more about Plans for remote First Nations people in WA may be forced off their homelands and made homeless; Surrendering land for Mining
A group of First Nations women have established a 'Sovereign Tent Embassy' at Matargarup (Heirisson Island) WA which also hosts a "Refugee Camp" for their homeless sisters and brothers and for the displaced First Nations people from their Homelands. Elder Maureen Culbong said, "We will stand in even stronger numbers together than even the two years ago where we stood for so long. There are so many issues our peoples face that we need the Embassy as a place of sanctity for all those hurting ... " Read more about In the heart of Perth, an 'Aboriginal Sovereign Tent Embassy' and a 'Refugee Camp'


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Fixing some small bugs and add some small features - sun
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911 Memorial and Museum
You can help honor and remember those who were lost or affected by the terrible events of September 11, 2011 with a visit to the 911 Memorial and Museum
When something as tragic occurs as the events of September 11, 2011 there is a need to remember those who lost their lives directly or by helping others. It is only fitting that the grounds of the World Trade Center became a permanent memorial and museum to this tragic day.
The Memorial itself consists of 2 square memorial fountains situated in the exact footprints of the Twin Towers of the World Trade Center surrounded by a plaza landscaped with trees and, in particular, a special tree called the Survivor Tree. Each of the two fountains is surrounded by a 1/2 inch thick solid brass plate in which the names of the nearly 3000 people who lost their lives in the attacks have been not just engraved, but cut right through the thick brass so that light can pass through the letters from behind. The Survivor Tree was a callery pear tree that was rescued from the rubble, barely alive, and nursed back to health. It now stands as a symbol of hope and renewal.
Visiting the Memorial is free, however there is an admission charge to visit the museum except on Tuesday evenings when admission is free to a limited number of visitors. Please refer to the official website for more information on this before visiting the Memorial.
Visitors to the museum will enter through a pavilion that is design to resemble a partially collapsed building and then make their way down a ramp to the exhibits which are 70 feet underground. The museum will include remnants of the foundation and support columns of the World Trade Center itself along with equipment that was used in the recovery effort. It will also have a number of other artifacts related to the tragic events of that day. There are exhibits and displays to help tell the world about the many little stories that make up the larger story about this tragic event and also about the previous attack back in February of 1993.
Guided tours of the museum are available daily, but they must be pre-booked on the official website and there is an additional charge. A less expensive option is to download the free 9/11 Museum Audio Guide App for your smartphone from the AppStore or Google play.
Associated with the 9/11 Museum is the 9/11 Tribute Center located on the south side of Liberty Street just west of Greenwich Street. This is adjacent to the southeast corner of the Memorial. The 9/11 Tribute centre features a gallery and also offers guided tours of the Memorial by volunteers who where directly affected by the events of 9/11 — survivors, family members, rescue workers, recovery workers and volunteers. They share their personal insights into the horrible events and the healing process in which the Memorial serves an ongoing role. There is a modest charge for these tours which you can book below. You can get all the details from their website: 9/11 Tribute Center.
Facts For Your Visit
Official Website: 911 Memorial and MuseumFee: The 9/11 Memorial is free to visit. The 9/11 Memorial Museum has an admission fee, but is free to visit on Tuesday evenings from 5pm until closing. The free tickets are limited and are available starting at 4pm on a first-come first-serve basis.
From: $38.11 CAD...May vary slightly due to currency fluctuations
9/11 Memorial Museum Admission
The 9/11 Memorial Museum in Lower Manhattan preserves the history of the attacks on the World Trade Center, and documents the significance of those events through building remnants, personal artifacts, first-person accounts and multimedia displays. With this admission ticket, take a self-guided tour of the museum's memorial exhibition and 3-part historical exhibition, which commemorates the 2,983 people who were killed, honors those who risked their lives to save others, and reflects on the courage and compassion shown in the aftermath.
Category: Souvenir Shops
20 Vesey St
New York, NY 10007
Phone: (212) 267-0172
Jordan S. Jul-29-2018
Wow! If you are considering checking this out... definitely do it. It was worth every penny. So much history about 9/11 and all of those affected. Great... Read More
Ali G. Apr-19-2019
I was quite pleased to pick up a copy of the 9/11 Commission Report Graphic Novel when I visited this shop recently. I had read it years ago for a paper... Read More
Tiffany H. Jun-09-2019
We didn't visit the actual museum as we didn't have time, however seeing the memorial was truly touching. The place was just breath taking, it was my first... Read More
Accommodations near 911 Memorial and Museum:
|Subway Line
|Nearest Station
|Walking Time
|A, C, J, Z, 2, 3, 4, or 5
|Fulton Street
|7 minutes
|2 or 3
|Park Place
|6 Minutes
|World Trade Center
|4 minutes
|Rector Street
|6 minutes
|Cortlandt Street
|3 minutes
|Rector Street
|7 minutes
How to get to 911 Memorial and Museum by Subway
Take a A, C, J, Z, 2, 3, 4, or 5 train to Fulton Street: exit onto Fulton Street, go right on Church Street, and left on Vesey Street.
Take a 2 or 3 train to Park Place: exit and walk south on Church Street and turn right (west) onto Vesey Street.
Take an E train to World Trade Center: exit onto Church Street Walk south and turn right on Vesey Street.
Take a R train to Rector Street: exit and walk west 1 block on Rector Street and go right (north on Greenwich Street to the Memorial.
Take a R train to Cortlandt Street: walk west on Cortlandt street to the Memorial.
Take a 1 train to Rector Street: exit onto Greenwich Street and walk north.
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text: Melahnese curry was a spice.[1] This article is a stub about a food or beverage. You can help Wookieepedia by expanding it. AppearancesEdit Adventures in Wild Space: The Steal (First mentioned) Notes and referencesEdit ↑ 1.0 1.1 Adventures in Wild Space: The Steal Retrieved from "https://starwars.fandom.com/wiki/Melahnese_curry?oldid=8279158" Categories: Canon articlesFood and beverage stubsSpice variants Community content is available under CC-BY-SA unless otherwise noted.


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My Content
Membership has its privileges! Sign up or sign in to gain full access to both All About Jazz and Jazz Near You. Learn more.
The majority of big bands these days seem to be preoccupied with attempting to recreate the great sounds of a bygone era. Holland and company demonstrate a healthy respect for the tradition, but Overtime shows that is the future of the genre that interests them most.
Track Listing: The Monterey Suite (Bring It On / Free for All / A Time Remembered / Happy Jammy); Ario; Mental Images; Last Minute Man (78:44).
Personnel: Holland (leader, bass); Alex Sipiagin, Duane Eubanks, Taylor Haskins (trumpet, flugelhorn); Antonio Hart (alto and soprano sax, flute); Mark Gross (alto sax); Chris Potter (tenor sax); Gary Smulyan (baritone sax); Robin Eubanks, Josh Roseman, Jonathan Arons, trombone; Steve Nelson, vibes, marimba; Billy Kilson, drums.
Title: Overtime | Year Released: 2005 | Record Label: Dare2 Records
Start your music shopping from All About Jazz and you'll support us in the process. Learn how.


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|App Download Version
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The Star Message – Thelema Tarot
July 12, 2017
Hello lovelies, I hope you are all well today? We are continuing today to work with the Thelema Tarot. Our card for today is XVII The Star. The Star is a card of hope, inspiration and optimism. The Start Keywords: Inspiration, Luck, Optimism, Tranquility, Renewal, Fate/Hope for the future, Happiness, Spirituality and spiritual growth, Inner … More The Star Message – Thelema Tarot


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Salt Lake County home prices continued to climb in the third quarter of 2018, according to a report last week from the Salt Lake Board of Realtors. The median single-family home price in the July-through-September period increased to $355,000, up 9.2 percent compared to a median price of $325,000 in last year's third quarter.
The strong prices were led by Alpine, where the median price sits at $355,000.
The rest of this article is only available to our online subscribers. Please log in, or click here to subscribe.


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In the thick of it, I knew that this is why I'm here. I mean not necessarily for nude photography. But an extension or application of something to pull me out of my body and my mind. In the light and dark of my subconscious, I wanted to explore the realm of radical self expression, self love, vulnerability, awareness, and connection with mind-body-spirit. I wanted to be free and unchained from all of the "should's" and "shouldn't" dialogue from my past. Ironically, in stark contrast, within reality, John has me chained and tied in handcuffs with my hands above my head in blissful surrender. I'm exposed but not broken.
John shows us the pictures. I noticed my body at first and found it to not to be the main focus. It was within the faces. In Johns photography, he captures humanity. What it means to be raw and completely human and at peace.
This is Day 2 of Burning Man. This is just the beginning. I'm grateful for this experience that catapulted my understanding of self love to make room for more enriching tales to follow...
As we were dressing back up, John pulls me aside. He invites me to visit him back in Santa Clara for more individual photo-shoot sessions. I'm flattered but mostly perplexed. I had to ask, "Why? Why me?" He says something to the extent of "unconventional beauty" and how his mission is to spend the rest of his life capturing what that is for people to witness.
So a week later after Burning Man, I find myself with a strong impulse to drive to Santa Clara and connect with John. He's a wise character. And I am tenacious and relentlessly curious.
Go with the flow, explore, and find yourself along the way.
He was on board with the Q&A interview. Here is John Brennan, the man who makes people see something in themselves they never saw before. This is the man who Goes with the Flow.
Here is his life story arranged from a lovely conversation as we pick his 5 values up together.


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Want to help adoptable animals in Sacramento? Check out the latest Witherell's auction
The Sacramento SPCA and Witherell's auction house have teamed up for a benefit auction this month, with more than five dozen lots available and all proceeds going to the SSPCA.
The inaugural auction is online through July 31 at witherells.com.
The auction items were donated to the SPCA over several years, according to a news release from the SPCA and Witherell's. Some were donations to the SPCA's Adoptable Goods thrift store and some came from estate or moving donations, said Sarah Haney, the SSPCA's director of development, in the release.
When the SPCA received items that could not be sold for a fair price at the thrift store, they began setting them aside for an auction, the release said.
The lots are very diverse and include many different mediums of artwork as well as vintage items and curios, like a rare 1906 Buffalo Brewing Calendar. The calendar is one of only three known copies and is expected to sell for over $1,000, the release said.
Bidders don't need to be wealthy to benefit the SPCA; lot estimates start at just $25.
As would be expected, many of the auction lots feature animals, including paintings, photographs, and sculptures. Other lots are eclectic in medium and style, and include more than artwork, with collectibles and jewelry also up for grabs.
"The items are fascinating," said Tracy Fairchild, a member of the SSPCA board of directors who coordinated the auction effort. "They come from very different eras and sensibilities…we have everything: Sculpture, mixed media artwork. It's truly hard to describe, it's so eclectic."
Some notable items include a Grateful Dead poster, a colorful Maija Peeples painting called "Capitol Capital Corgi," and two leather footstools, one in the shape of a rhinoceros and the other a hippopotamus.
Many of the items already have dozens of bids.
For more information, to browse the lots or to register to bid, visit witherells.com.


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1990 Silverado Interior
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1990 Silverado Interior was published at April 12, 2018 at 3:18 am. It is posted at the Interior category. 1990 Silverado Interior is labelled with 1990 Silverado Interior, Interior, Silverado, 1990..
Interiorin•te•ri•or (in tēr′ē ər),USA pronunciation adj.
- being within; inside of anything;
further toward a center: the interior rooms of a house.
- of or pertaining to that which is within;
inside: an interior view.
- situated well inland from the coast or border: the interior towns of a country.
- of or pertaining to the inland.
- domestic: interior trade.
- private or hidden;
inner: interior negotiations of the council.
- pertaining to the mind or soul;
mental or spiritual: the interior life.
- the internal or inner part;
- the inside part of a building, considered as a whole from the point of view of artistic design or general effect, convenience, etc.
- a single room or apartment so considered.
- a pictorial representation of the inside of a room.
- the inland parts of a region, country, etc.: the Alaskan interior.
- the domestic affairs of a country as distinguished from its foreign affairs: the Department of the Interior.
- the inner or inward nature or character of anything.
- the largest open set contained in a given set, as the points in a circle not including the boundary.
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Pesticides are key stressors of soil microorganisms with reciprocal effects on ecosystem functioning. These effects have been mainly attributed to the parent compounds, while the impact of their transformation products (TPs) has been largely overlooked. We assessed in a meadow soil (soil A) the transformation of iprodione and its toxicity in relation to (i) the abundance of functional microbial groups, (ii) the activity of key microbial enzymes, and (iii) the diversity of bacteria, fungi, and ammonia-oxidizing microorganisms (AOM) using amplicon sequencing. 3,5-Dichloroaniline (3,5-DCA), the main iprodione TP, was identified as a key explanatory factor for the persistent reduction in enzymatic activities and potential nitrification (PN) and for the observed structural changes in the bacterial and fungal communities. The abundances of certain bacterial (Actinobacteria, Hyphomicrobiaceae, Ilumatobacter, and Solirubrobacter) and fungal (Pichiaceae) groups were negatively correlated with 3,5-DCA. A subsequent study in a fallow agricultural soil (soil B) showed limited formation of 3,5-DCA, which concurred with the lack of effects on nitrification. Direct 3,5-DCA application in soil B induced a dose-dependent reduction of PN and NO3−-N, which recovered with time. In vitro assays with terrestrial AOM verified the greater toxicity of 3,5-DCA over iprodione. "Candidatus Nitrosotalea sinensis" Nd2 was the most sensitive AOM to both compounds. Our findings build on previous evidence on the sensitivity of AOM to pesticides, reinforcing their potential utilization as indicators of the soil microbial toxicity of pesticides in pesticide environmental risk analysis and stressing the need to consider the contribution of TPs in the toxicity of pesticides on the soil microbial community.
IMPORTANCE Pesticide toxicity on soil microorganisms is an emerging issue in pesticide risk assessment, dictated by the pivotal role of soil microorganisms in ecosystem services. However, the focus has traditionally been on parent compounds, while transformation products (TPs) are largely overlooked. We tested the hypothesis that TPs can be major contributors to the soil microbial toxicity of pesticides using iprodione and its main TP, 3,5-dichloroaniline, as model compounds. We demonstrated, by measuring functional and structural endpoints, that 3,5-dichloroaniline and not iprodione was associated with adverse effects on soil microorganisms, with nitrification being mostly affected. Pioneering in vitro assays with relevant ammonia-oxidizing bacteria and archaea verified the greater toxicity of 3,5-dichloroaniline. Our findings are expected to advance environmental risk assessment, highlighting the potential of ammonia-oxidizing microorganisms as indicators of the soil microbial toxicity of pesticides and stressing the need to consider the contribution of TPs to pesticide soil microbial toxicity.
The toxicity of pesticides on nontarget organisms, including pollinators and aquatic and terrestrial organisms, is well documented (1–4). In the last 10 years, much attention has also been given to the effects of pesticides on soil microorganisms, considering their pivotal role in ecosystem functioning (5). Several studies have used advanced molecular and biochemical tools to assess the toxicity of pesticides in relation to the structure and function of the soil microbial community (6–8). The majority of them have considered the parent compound as the causal agent of the toxic effects observed, while the potential toxicity of transformation products (TPs) has attracted little attention. Recently, Karas et al. (9) demonstrated the major role of TPs of chlorpyrifos and isoproturon on the inhibition of soil microbial functions. Similarly, Papadopoulou et al. (10) showed that quinone imine, the oxidized TP of ethoxyquin (an antioxidant used in the fruit-packaging industry) was equally as toxic as, or more toxic than, the parent compound to ammonia-oxidizing microorganisms (AOM). This group has been shown to be particularly sensitive to environmental perturbations (11) including pesticide exposure (6, 9).
Iprodione is a fungicide used for the control of foliage and soilborne fungal pathogens (12). It is a potential carcinogen (13) and an endocrine-disrupting substance (14). Microbial degradation is the main process controlling its dissipation in soil and leads to the formation of 3,5-dichloroaniline (3,5-DCA) (15). 3.5-DCA is neurotoxic (16), and it is considered the most recalcitrant (17) and toxic (18) dichloroaniline isomer. Previous studies have reported significant reductions in the abundance, activity, and diversity of soil microorganisms in response to iprodione application (19–21). However, the effects observed were attributed entirely to the parent compound, while the transformation of iprodione in soil and the potential contribution of its TPs were not explored.
The main aim of this study was to assess the toxicity of iprodione on the soil microbial community and explore the role of 3,5-DCA for the potential adverse effects observed. This was achieved through a series of laboratory microcosm assays replicated in two different soils (to verify the uniformity of effects observed) where the dissipation of iprodione and the formation of 3,5-DCA were determined analytically and correlated with changes in (i) functional enzyme activity, (ii) the abundance of functional microbial groups, and (iii) the bacterial, fungal, and AOM community determined via high-throughput amplicon sequencing. Microbial taxa responsive to iprodione and 3,5-DCA were identified, and the toxicity of 3,5-DCA was further verified in soil microcosm tests with direct application of 3,5-DCA. The toxicity of iprodione and 3,5-DCA to AOM was confirmed using in vitro tests with isolated strains of terrestrial ammonia-oxidizing bacteria (AOB) and archaea (AOA).
Dissipation, transformation, and impact of iprodione on the soil microbial community: microcosm assay in the meadow soil.We initially determined in a lab microcosm study the dissipation/transformation of iprodione and its impact (applied at 1, 10, and 100 times the recommended dose [1×, 10×, and 100× dose, respectively]) on the activity, abundance, and diversity of the soil microbial community in a meadow soil. The time required for 50% dissipation (DT50) of iprodione in the different treatments was calculated with the single first-order (SFO) kinetic model, which showed the best fit to the measured data. The persistence of iprodione, as depicted by the calculated DT50 values, did not follow a dose-dependent trend and ranged from 10.2 days in the samples treated with the 10× dose to 14.0 days in the samples treated with the 1× dose (Fig. 1 and Table S1 in the supplemental material). Iprodione was hydrolyzed to 3,5-DCA, which was partially degraded in samples treated with the 1× and 10× doses but accumulated in the samples treated with the 100× dose (Fig. 1).
The application of iprodione induced significant main effects on the activity of several enzymes (Fig. S1) and potential nitrification (PN) (Fig. 2). A temporal dose-dependent trend was evident only for leucine aminopeptidase (Leu), where the 100× dose reduced activity from 3 days onward (Fig. S1g). PN activity was also significantly reduced by iprodione application, with the 10× dose transiently decreasing PN at 3 and 15 days (P < 0.05) before recovery occurred at 28 days and with the 100× dose resulting in a persistent reduction in PN from 7 days onward (Fig. 2). Pearson's correlation testing identified significant negative correlations between the soil levels of iprodione and the activity of acid phosphomonoesterase (AcP) (−0.39, P < 0.001), beta-glucosidase (Bglu) (−0.32, P < 0.01), and Leu (−0.25, P < 0.05) (Table S2). However, stronger negative correlations were evident between the soil concentrations of 3,5-DCA, formed by the hydrolysis of the applied iprodione, and the activity of phosphodiesterase (BisP) (−0.25, P < 0.05), N-acetyl-β-d-glucosaminidase (Chit) (−0.28, P < 0.05), Leu (−0.560, P < 0.001), pyrophosphatase (Piro) (−0.42, P < 0.001), and PN (−0.86, P < 0.001). Redundancy analysis (RDA) of measured enzymatic activities and PN using the ln-transformed soil concentrations of iprodione or 3,5-DCA as explanatory variables (Fig. 3) showed the following: (i) a weak but still significant effect (P = 0.008) of the concentrations of iprodione in soil, with treatment with the 100× dose clustering away from the other treatments along the iprodione concentration gradient, and (ii) a strong significant effect (P = 0.001) of the concentrations of 3,5-DCA in soil, with the samples treated with the 10× and 100× doses grouping away from samples treated with the 1× dose and the untreated samples along the 3,5-DCA concentration gradient. The application of iprodione affected the abundance of the amoA gene of AOA and the soxB gene of sulfur-oxidizing bacteria (SOB) (Fig. S2). However, the effects observed were either isolated to a single time point (i.e., at 0 days for the amoA gene of AOA where the samples treated with the 10× and 100× doses showed a significantly lower abundance than the control) or did not follow a dose-dependent pattern (i.e., the abundance of the amoA gene of AOA and of the soxB gene of SOB at 42 days was significantly lower in the samples treated with the 1× dose than in samples treated with the 10× dose).
The effect of iprodione and 3,5-DCA on the α- and β-diversities of soil bacteria, fungi, and AOM was also assessed. Good's coverage estimate for bacteria, fungi, AOB, and AOA had mean values of 82.9, 99.8, 97.1, and 99.7%, respectively, suggesting good coverage of the bacterial (dominant), fungal, and AOM operational taxonomic unit (OTU) diversity by the devoted sequencing effort. Average richness values of 7,710, 1,491, 810, and 149 OTUs were observed in all control samples for the total bacterial 16S rRNA (at 97% sequence identity, used for OTU definition), fungal internal transcribed spacer (ITS; 97% identity), and AOB amoA and AOA amoA genes (99% identity for both amoA population OTUs), respectively. No major effects of iprodione or 3,5-DCA on the α-diversity were observed (Table S3).
Hierarchical clustering analysis of the taxonomy affiliations of the soil microorganisms revealed no grouping of the samples according to the applied doses or time for any of the microbial groups studied (Fig. S3). Actinobacteria, Proteobacteria (Alpha-, Beta-, Gamma-, and Deltaproteobacteria), and bacilli dominated the bacterial community in all soil samples, while the fungal community was dominated by unclassified fungi, Ascomycetes, Basidiomycetes, and members of the family Mortierellaceae. Representative sequences of AOA and AOB, according to their abundances in each OTU, were selected and placed in previously generated phylogenetic trees (22, 23). The vast majority of AOB sequences were placed within Nitrosospira clades with the most abundant OTUs (OTU00001, OTU00002, and OTU00005, encompassing 49% of the analyzed sequences) residing in Nitrosospira sp. strains Nsp17 and Nsp2. The sister clade of the Nitrosospira multiformis/Monterey Bay C clade contained other dominant OTUs (OTU00004, OTU00006, and OTU00009) (Fig. S4). Other highly abundant OTUs resided in the Nitrosospira briensis (OTU00003), Nitrosospira sp. strain Nsp 12 (OTU00007), and Nitrosospira sp. strain Nsp 65 (OTU00008) clades. These OTUs all together accounted for 85% of the total number of analyzed sequences. AOA were dominated by OTUs whose representative sequences were mainly placed in the Nitrososphaerales γ, δ, and ε clades (Fig. S5). The most dominant OTUs, accounting for 67.7% of the total number of analyzed sequences (OTU0001, OTU0004, and OTU00005), were placed in the Nitrososphaerales ε-2.2. clade, followed by OTUs belonging to different subgroups of the Nitrososphaerales γ clade (OTU0003, OTU0006, OTU0007, OTU0008, and OTU0009), accounting for 16.1% of the total number of sequences, and OTUs of the Nitrososphaerales δ clade (OTU0002, OTU0010, and OTU0013), accounting for 12.5% of the total number of analyzed sequences.
Partial RDA (not considering the time factor) of OTUs of the different microbial groups with the measured soil concentrations of iprodione (Fig. S6a) and 3,5-DCA (Fig. S6b) as explanatory variables indicated that only the latter had a significant effect on the structure of the bacterial (P = 0.002) and fungal (P = 0.019) communities. Pearson's correlation testing showed a significant negative correlation between the soil levels of 3,5-DCA and the abundance of Actinobacteria, Hyphomicrobiaceae, Ilumatobacter, and Solirubrobacter and a positive correlation with Arthrobacter (Fig. 4). Conversely, iprodione soil concentrations were positively correlated with Arthrobacter, Marmoricola, and Xanthomonadaceae. Analysis of fungal ITS sequences revealed that 3,5-DCA concentrations were negatively correlated with Pichiaceae and positively correlated with Lasiosphaeriaceae, while iprodione showed positive correlations with Sordariomycetes. 3,5-DCA and iprodione soil levels showed negative correlations with the less dominant AOB OTUs like OTU00016 (Nsp65 group), OTU00031, and OTU00038 and positively correlated with OTU00051, all belonging to the N. briensis/N. multiformis/Monterey Bay C group. Certain AOB OTUs were affected only by 3,5-DCA (OTU00034 negatively) or iprodione (OTU00058 negatively; OTU00042 positively). For AOA, 3,5-DCA concentrations were positively correlated with dominant OTUs like OTU00003 and OTU00009 (residing in the Nitrososphaerales γ-1.2 clade), while iprodione concentrations were negatively correlated with OTU00005 (residing in the Nitrososphaerales ε-2.2 clade), one of the most dominant AOA OTUs.
Dissipation, transformation, and impact of iprodione and 3,5-DCA on the soil microbial community: microcosm assay in the agricultural soil.A replicate microcosm study in an agricultural soil (soil B) was subsequently employed to evaluate the universal nature of the effects induced by the application of iprodione to the soil microbial community. The dissipation of iprodione treated with the 1× dose was best described by the hockey stick (HS) model, whereas the SFO model provided adequate fit to the dissipation data for the 10× and 100× doses (Fig. S7). DT50 values of iprodione showed a dose-dependent trend (Table S1) and varied from 0.35 to 42.5 days in the samples treated with the 1× and 100× doses, respectively. Iprodione dissipation was accompanied by the formation of low 3,5-DCA concentrations which never exceeded 10% of the initially applied iprodione. The fungicide induced significant changes in the soil levels of NH3-N and the abundance of the amoA gene of AOB and AOA; however, no clear temporal dose-dependent patterns were observed (Fig. S7).
A follow-up microcosm study was undertaken in soil B (3,5-DCA directly applied in soil) to test the hypothesis that the lack of effects on the activity of AOM in soil B was a function of the limited formation of 3,5-DCA. The dissipation of 3,5-DCA was well described by the SFO model (Fig. 5a). The dissipation of 3,5-DCA showed a weak dose-dependent pattern, with DT50 values ranging from 4.1 to 6.6 days in the samples treated with doses of 0.6 and 60 μg g−1, respectively (Table S1). The application of doses of 6 and 60 μg g−1 induced a significant reduction (P < 0.001) in PN, which recovered to levels similar to the level of the control by day 49 only in the samples treated with a dose of 6 μg g−1 (Fig. 5b). Regardless of the treatment applied, the concentrations of NH3-N decreased with time (Fig. 5c), and this correlated with an increase in the concentrations of NO3−-N (Fig. 5d). The application of the highest dose induced a significant increase in the concentration of NH3-N and, reversibly, a significant decrease in NO3−-N concentrations, which persisted until day 49. 3,5-DCA application did not induce any clear dose-dependent temporal changes in the abundance of the amoA gene of AOB (Fig. 5e), in contrast to AOA, whose amoA gene abundance was only temporarily reduced by the application of the two higher doses of 3,5-DCA (Fig. 5f).
In vitro assessment of the toxicity of iprodione and 3,5-DCA on AOA and AOB.The superior toxicity of 3,5-DCA over iprodione on AOM was further tested in in vitro assays with selected terrestrial AOB (Nitrosospira multiformis) and AOA ("Candidatus Nitrosocosmicus franklandus" and "Candidatus Nitrosotalea sinensis"). Iprodione was not toxic to N. multiformis even at the highest concentration level tested (50 mg liter−1) (Fig. 6a). In contrast, 3,5-DCA, at concentrations higher than 0.5 mg liter−1, strongly inhibited the activity of N. multiformis, with a late recovery observed only in the cultures amended at 0.5 mg liter−1 (Fig. 6b). Iprodione inhibited the activity of "Ca. Nitrosocosmicus franklandus" at concentrations of >0.5 mg liter−1 although recovery was observed at all concentrations at the end of the incubation period (Fig. 6a). 3,5-DCA induced a significant reduction in the activity of "Ca. Nitrosocosmicus franklandus" at all concentrations; however, recovery was observed only in the cultures amended with 5 mg liter−1 or less (Fig. 6b). Iprodione induced a nonreversible inhibition of the activity of the acidophilic AOA "Ca. Nitrosotalea sinensis" at concentrations of >0.5 mg liter−1 (Fig. 6a), while 3,5-DCA halted the activity of "Ca. Nitrosotalea sinensis" at all concentration levels, and a slow recovery was observed only at 0.1 mg liter−1 (Fig. 6b). The inhibition profiles determined by measuring amoA gene abundance were concomitant with those determined via the monitoring of NO2− production (Fig. S8).
The application of iprodione in a meadow soil (soil A) significantly reduced the activity of enzymes involved in P (AcP, BisP, and Piro) and N (Chit and Leu) transformation (see Fig. S1 in the supplemental material). The activities of AcP, BisP, Piro, Chit, and Leu were strongly correlated with the soil concentrations of iprodione but mostly with the soil concentrations of its TP, 3,5-DCA (Fig. 3). In a series of studies, Zhang et al. (21, 24) observed a significant reduction in the activity of Bglu, AcP, alkaline phosphomonoesterase (AlkP), Leu, and Chit upon repeated applications of the recommended dose of iprodione (4 × 1.5 μg g−1). These effects became evident at the later stages of the incubation (from 14 days onward) and probably reflected the gradual accumulation of iprodione and/or the formation of 3,5-DCA. However, the concentration of either compound was not measured in these studies, preventing the identification of the inhibitory mechanism. The formation of 3,5-DCA was also negatively correlated with PN although this was not in agreement with the measurements of the abundances of the amoA genes of AOA and AOB, which were not altered by iprodione application. The discrepancy between PN and AOA/AOB amoA gene abundance measurements is not surprising considering that these two methods determine different attributes (potential activity versus abundance, respectively, with partial contribution to the latter of relic DNA). PN has also been identified by Crouzet et al. (25) as the most sensitive functional descriptor of pesticide effects on soil microbial activity.
The inhibitory effect of 3,5-DCA on PN (Fig. 2) led us to examine further its potential to inhibit ammonia oxidation, which often constitutes the rate-limiting step in nitrification (26). In a replicate microcosm study with a soil from a fallow agricultural field (soil B), no adverse effects of iprodione on the nitrification activity and the abundance of the amoA genes of AOA and AOB were observed (Fig. S2) and this was consistent with the limited formation of 3,5-DCA in this soil (Fig. S7). The limited formation of 3,5-DCA could be attributed either to the presence in the soil of a pool of efficient 3,5-DCA-degrading microorganisms, which actively degraded 3,5-DCA as soon as it was formed, keeping its levels low during the study, or to the operation of alternative metabolic pathways where 3,5-DCA does not constitute a major TP (27). Direct application of 3,5-DCA in soil B, at concentration levels equivalent to those formed in soil A upon application of 1, 10, and 100 times the recommended dose of iprodione, induced a significant reduction in nitrification activity, as determined by measurements of PN and NH3-N/NO3−-N soil concentrations (Fig. 5b). Together, these findings support our initial hypothesis that 3,5-DCA, and not iprodione, drives the inhibition of nitrification in soil.
The superior toxicity of 3,5-DCA over iprodione was verified by in vitro assays with selected soil AOB and AOA isolates (Fig. 6). Although their sensitivities to iprodione and 3,5-DCA varied, 3,5-DCA was consistently more inhibitory than iprodione for all strains. N. multiformis was tolerant to iprodione compared to the two AOA whose growth and activity were inhibited, either transiently ("Ca. Nitrosocosmicus franklandus") or permanently ("Ca. Nitrosotalea sinensis"), by the higher concentrations of the fungicide. In contrast 3,5-DCA showed equivalent inhibitory levels to AOB and AOA strains, with "Ca. Nitrosocosmicus franklandus" being the most tolerant, followed by N. multiformis and "Ca. Nitrosotalea sinensis," which was the most sensitive. Previous in vitro studies with classical (i.e., nonpesticide) nitrification inhibitors have also demonstrated different levels of sensitivity of AOB (N. multiformis) versus AOA (Nitrosotalea devanaterra Nd1 and Nitrososphaera viennensis) (28, 29), which is perhaps a consequence of fundamental differences between AOA and AOB physiologies. The higher tolerance of N. multiformis to iprodione might be a function of the notably high number of ABC transporters found in its genome which are known to be involved in organic solvents and multidrug efflux (30). Conversely, the consistently higher sensitivity of "Ca. Nitrosotalea sinensis" Nd2 to both compounds is reported for the first time and warrants further study. As with many other pesticides, iprodione acts at a cellular level by causing oxidative stress (31). While the exact mechanism of reactive oxygen generation by iprodione is yet unknown, it has been recently demonstrated that some AOA in culture are highly sensitive to oxidative stress, and the addition of scavenging supplements such as α-keto acids or catalase is required for growth in culture (32). Contrasting sensitivities to iprodione may therefore result from differences in physiologies of AOB and AOA with respect to oxidative stress.
In vitro assays provide a precise measure of the inherent toxicity of a compound to AOM, identify potential differences in the toxicity of a chemical to the different microbial moderators of the ammonia oxidation process (e.g., AOA versus AOB), and offer a valuable experimental platform to explore toxicity mechanisms. However, their results may deviate from soil microcosm studies due to the reduced diffusion or higher degradation of the chemicals in soil (33). To date, in vitro assays have not been used for the assessment of the soil microbial toxicity of pesticides although their inclusion in pesticide risk assessment testing as a conservative tier I step was proposed (34). In our study, 3,5-DCA inhibited the activity and growth of "Ca. Nitrosotalea sinensis" and N. multiformis at concentrations equivalent to the inhibitory concentrations of dicyandiamide (DCD) for the acidophilic AOA strain Nitrosotalea devanaterra Nd1 (28) and for N. multiformis (29). Our findings suggest that 3,5-DCA at concentration levels of 0.5 to 5 mg liter−1, expected to be found in soil pore water upon application of the recommended dose of iprodione (assuming full conversion to 3,5-DCA), could inhibit the growth and activity of AOB and AOA strains.
We further explored the potential effects of iprodione and 3,5-DCA not only on the diversity of AOB and AOA but also on the diversity of soil bacteria and fungi (Fig. S6). Multivariate analysis of the diversity matrix obtained by amplicon sequencing suggested that iprodione and 3,5-DCA induced only subtle effects on the α-diversity of bacteria, fungi, AOB, and AOA. In contrast, significant effects on the β-diversity of the bacterial and fungal communities were observed. The changes found were strongly correlated with the soil levels of 3,5-DCA and not iprodione. The abundances of Actinobacteria, especially of the genera Ilumatobacter and Solirubrobacter, and of alphaproteobacteria of the family Hyphomicrobiaceae were negatively correlated with 3,5-DCA formation (Fig. 4). Previous studies have also reported a sensitivity of these bacterial phyla to high soil concentrations of pesticides (35) and other organic pollutants (36). The positive correlation of Arthrobacter and Xanthomonadaceae with the soil levels of iprodione is in line with the assignment of all the currently known iprodione-degrading soil bacteria to the genus Arthrobacter (37, 38) and the well-documented role of members of the family Xanthomonadaceae in the degradation of pesticides (39) and antibiotics (40). Changes in fungal community structure were observed (Fig. S6), with Sordariomycetes being positively correlated with iprodione soil concentrations (Fig. 4), in agreement with Zhang et al. (20), who reported an enrichment of OTUs belonging to families of Sordariomycetes, including Cephalothecaceae, Hypocreaceae, and Cordycipitaceae. Conversely, 3,5-DCA formation favored fungi of the family Lasiosphaeriaceae and reduced the abundance of yeasts of the family Pichiaceae. The former family encompasses coprophilous fungi like Podospora and Zopfiella which are commonly found in manured soils (41), like the meadow soil studied. The Pichiaceae include yeasts with applications in agriculture (plant growth promoters or biocontrol agents) (42), food technology (alcohol fermentation), and biotechnology (43).
The AOB and AOA communities in the meadow soil were dominated by members of the genus Nitrosospira and the lineage Nitrososphaerales (clades γ, δ, and ε), respectively (Fig. S4 and S5), which are ubiquitous in the soil environment (44, 45), with Nitrososphaerales clades γ and δ representing over 65% of the currently reported soil/sediment AOA (23). Despite the inhibitory effects on soil nitrification, the β-diversity of AOB and AOA was not significantly altered by iprodione and 3,5-DCA (Fig. S6), whose soil concentrations were positively correlated with rare OTUs of the AOB community (Fig. 4). In contrast, only dominant AOA OTUs were responsive to iprodione and 3,5-DCA. These results might be an indication of the crucial functional role of the rare and dominant members of the AOB and AOA communities, respectively. Alternatively, other AOM like the comammox bacteria, not considered in the current study, might have an important functional role in the nitrification in the soil studied although their ecological role in terrestrial ecosystems is still not well defined (46).
Conclusions.To date, most of the studies that have investigated the impact of pesticides on soil microorganisms have attributed observed effects exclusively to the parent compound while the contribution of TPs was largely overlooked (47, 48). This is particularly true for iprodione, whose structural and functional effects on the soil microbial community (19, 20, 49), and particularly on AOM (50), were fully attributed to the parent compound. We provide strong evidence that 3,5-DCA, the main TP of iprodione in soil, is responsible for the significant reduction in the activity of AOM and the effects on the β-diversity of bacteria and fungi. Our findings have important practical implications for pesticide environmental risk assessment since they (i) provide strong evidence for the sensitivity of AOM to pesticides, in line with a series of previous studies (4, 6, 9–11, 25, 47), reinforcing their potential as indicators of the soil microbial toxicity of pesticides, and (ii) demonstrate that the TPs of pesticides could have a higher intrinsic soil microbial toxicity than their parent compounds; hence, assessment of their soil microbial toxicity should be an integral part of environmental risk analysis.
Soils and chemicals.The impact of iprodione and 3,5-DCA was assessed in two different top-soils (0 to 20 cm): (i) a Cambisol loam (pH 6.9; organic carbon content, 2.98%) collected from a meadow in northern Italy (Fontidella Gaverina, 45°27′55.69″N, 9°38′20.05″ E; soil A) and (ii) a Cambisol clay loam (pH 7.6; organic carbon content, 1.1%) collected from a fallow agricultural field of the Hellenic Agricultural Organization-Demeter in Larissa, Greece, with no recent history of pesticide application (39°63′27″N, 22°36′74″E, soil B). Both soils were collected from the top 20 cm according to a protocol of the International Organization for Standardization (https://www.iso.org/standard/43691.html) for collection and handling of samples. Upon collection, soils were homogenized, partially air dried, sieved (2-mm pore size), and stored at 4°C for a week before use. Iprodione and 3,5-DCA analytical standards (>97% purity) were used for analytical purposes and in vitro tests, while a commercial formulation of iprodione (Rovral 50%WP) and the analytical standard of 3,5-DCA were used in microcosm tests.
Soil microcosm experiments.Four 1-kg (each) subsamples of soils A and B were pretreated with 2.5 ml of a 0.5 M solution of (NH4)2SO4 (corresponding to 154 mg N kg−1 soil dry weight). Samples were left to equilibrate overnight and then spiked with appropriate volumes of aqueous solutions of iprodione corresponding to application of 1, 10, and 100 times the recommended dose and soil concentrations of 2, 20, and 200 μg of iprodione g−1 soil, respectively. The fourth soil sample received the same amount of water without iprodione as a control. The soil moisture content was adjusted to 40% of the water-holding capacity. Bulk soil samples were divided into 50-g subsamples which were placed in aerated plastic bags and incubated in the dark at 25°C. At regular intervals, triplicate samples per treatment were sampled and analyzed for iprodione and 3,5-DCA residues or used for determination of enzyme activity, PN, or DNA extraction.
To further explore the microbial toxicity of 3,5-DCA (in soil B), NH4+-amended soil samples were treated with 3.3 ml of methanolic solutions of 3,5-DCA at concentrations of 100, 1,000, and 10,000 μg ml−1, resulting in final soil 3,5-DCA concentrations of 0.6, 6, and 60 μg g−1 soil, respectively. These corresponded to the maximum concentrations of 3,5-DCA formed in soil by the application of 1, 10, and 100 times the recommended dose of iprodione in soil A. The fourth subsample received the same volume of methanol without 3,5-DCA as an untreated control. Samples were left for 1 h to allow evaporation of methanol and were then processed as described above. Immediately after 3,5-DCA application and at regular intervals thereafter, triplicate microcosms were sampled per treatment and analyzed for 3,5-DCA, NO3−-N, and NH4+-N concentrations or processed for DNA extraction and PN measurements.
Pesticide residue and NH3/NO3−-N analysis.Iprodione and 3,5-DCA residues were extracted from soil as described by Vanni et al. (51) with the sole modification that the final acetonitrile extract was dried under a flow of nitrogen, and the samples were resuspended in acetone containing the internal standard anthracene-d10 (m/z 188). Extracts were analyzed in an Agilent 6890 gas chromatograph-mass spectrometry (GC-MS) system equipped with an Agilent Technologies 5973 series mass selective detector. The column used was a Supelco SLB-5 MS type (30 m by 0.25-mm internal diameter by 0.25-μm film thickness). The carrier gas was high-grade helium used at a constant flow rate of 1.0 ml min−1. Injection temperature was 250°C, with an injection volume of 1 μl and a 0.5-min purge time. The GC-MS oven temperature was maintained at 70°C for 2 min and then increased at a rate of 10°C min−1 until 230°C. This temperature was maintained for 3 min and finally increased at a rate of 25°C min−1 until it reached 280°C, and it remained constant for 6 min. The detector temperatures were 150°C (MS quadrupole) and 230°C (MS source). The compounds were identified in total ion-monitoring mode, at m/z 161 for 3,5-DCA and m/z 187 for iprodione. Soil levels of NH4+-N and NO3−-N were determined as described by Kandeler and Gerber (52) and Doane and Horwáth (53), respectively.
In vitro assays.The inhibitory effects of iprodione and 3,5-DCA on soil AOB (N. multiformis) and AOA ("Ca. Nitrosotalea sinensis" strain Nd2 and "Ca. Nitrosocosmicus franklandus" strain C13) were investigated in liquid batch cultures. "Ca. Nitrosocosmicus franklandus" was isolated from a Scottish agricultural soil with pH 7.5 (54) and "Ca. Nitrosotalea sinensis" was from a Chinese acidic paddy soil (pH 4.7) (55, 56). All strains were grown aerobically in the dark without shaking. N. multiformis was grown at 28°C in Skinner and Walker's medium (57) (pH of 7.5). AOA strains were incubated at 35°C in medium supplemented with 1 mM NH4+. "Ca. Nitrosocosmicus franklandus" C13 was cultured in HEPES-buffered modified fresh water medium (pH 7.5) (54), while "Ca. Nitrosotalea sinensis" was grown in an acidic morpholineethanesulfonic acid (MES)-buffered (pH 5.2) freshwater medium (58). For each treatment, triplicate 100-ml Duran bottles containing 50 ml of growth medium were inoculated with a 1 or 2% (vol/vol) transfer of AOB and AOA culture, respectively, in exponential growth. Iprodione and 3,5-DCA were added to the cultures at four concentration levels: 0.1, 0.5, 5, and 50 mg liter−1 in 0.1% (vol/vol) dimethyl sulfoxide (DMSO) once exponential growth was observed. Triplicate cultures amended with 0.1% (vol/vol) DMSO without iprodione and 3,5-DCA served as controls. The effects of iprodione and 3,5-DCA on the growth and activity of AOM were measured regularly via determination of the amoA gene abundance and NO2− levels (59), respectively. DNA was extracted from a cell pellet obtained from 2-ml aliquots of the microbial cultures using a tissue DNA extraction kit (Macherey-Nagel, Germany).
DNA extraction from soil.DNA was extracted from ∼500 mg of soil using a FastDNA Spin kit for soil and a FastPrep instrument (MP Biomedicals, USA) and quantified using a Qubit fluorometer with a Quant-iT HS double-stranded DNA (dsDNA) assay kit (Invitrogen, USA).
qPCR analysis of functional genes.The abundances of AOB and AOA amoA genes were determined with primers amoA1F-amoA1R (60) and CrenamoA23f-CrenamoA616r (61), respectively, as described by Rousidou et al. (62), and the soxB gene using primers soxB_710f-soxB1184R as described in Tourna et al. (63). Quantitative PCR (qPCR) amplification efficiencies were between 85 and 102%, with R2 values of ≥0.993. Standard curves were obtained using serial dilutions of plasmid vectors containing amplicons of nearly full-length target genes.
Community structure analysis of bacteria, fungi, and AOM.Microbial diversity analysis for total bacteria, fungi, AOB, and AOA was performed on samples from all treatments collected at 0, 15, and 42 days via multiplex amplicon sequencing in an Illumina MiSeq sequencer generating 300-bp paired-end reads as described previously (64). Primers, primer-indexing sequences, PCR conditions, and programs are shown in Table 1 and in Table S4 in the supplemental material. Sequence screening, alignment to reference databases, and generation of OTU matrices for bacteria, AOB, and AOA were performed with mothur, version 1.36.1 (65), while the fungal ITS1 amplicon screening was performed with USEARCH, version 9 (66). Bacterial 16S rRNA gene amplicon sequences were obtained through assembly of the read pairs subsequently aligned against the Silva, version 128, bacterial database (67, 68). AOB/AOA amoA amplicons were compared with the alignments of Abell et al. (22) and Alves et al. (23), respectively, for generating OTUs. Sequences were then clustered into groups differing by 1% in identity. Chimeric amplicons were identified and removed using UCHIME, version 4.2 (69). Sequence distances were calculated for the aligned sequences, while hierarchical clustering using the average linkage algorithm (70) was performed for identifying OTUs at 0.02 sequence distances. Sequence divergences of 0.02 and 0.03 were used for bacteria and ammonia oxidizer OTU definition, respectively. Fungal ITS amplicons were clustered with the USEARCH, version 9, implemented with the UCLUST algorithm (66) using the UNITE (71), version 7.2, database ITS reference data set at a 0.03-distance OTU definition. The most abundant sequences of each OTU were selected as representatives and were classified according to the Ribosomal Database Project (RDP) with the naive Bayesian classifier module residing in mothur, version 1.36.1, for bacteria and the UNITE database for fungi with the USEARCH, version 9, native SINTAX algorithm. AOB and AOA amoA gene OTU representative sequences were placed in the previously curated phylogenies of Abell et al. (22) and Alves et al. (23), respectively. This was performed using the parsimony-based phylogeny-aware short-read alignment approach of PaPaRa, version 2.4, combined with a maximum likelihood evolutionary placement algorithm as implemented in RAxML, version 8.2.4 (72–74).
Soil enzyme activities and PN measurements.The activities of AcP (EC 220.127.116.11), AlkP (EC 18.104.22.168), BisP (EC 18.104.22.168), and Piro (EC 126.96.36.199), involved in P cycling, arylsulfatase (AryS; EC 126.96.36.199) involved in S cycling, Chit (EC 18.104.22.168) and Leu (EC 22.214.171.124), involved in N cycling, and Bglu (EC 126.96.36.199), involved in C cycling, were determined using fluorescent 4-methyl-umbelliferyl and 7-amino-4-methyl coumarin (for Leu) linked to appropriate enzyme substrates and determined in a fluorometric plate reader system as described by Karas et al. (9). PN was determined by the method of Kandeler (75). Briefly, 5-g soil samples were amended with 20 ml of 1 mM (NH4)2SO4 and 0.1 ml of 1.5 M NaClO3 and incubated under constant agitation at 20°C for 5 h, while triplicate control samples were treated in the same way and incubated at −20°C for the same period. At the end of the incubation period, NO2− was extracted from all samples with 2 M KCl. The extract (5 ml) was amended with 3 ml of 0.19 M NH4Cl and 2 ml of a colorimetric indicator prior to final determination of its adsorption at 520 nm. The PN in the soil samples was then determined with an external calibration curve prepared by measurement of the adsorption of a series of NaNO2 solutions.
Data analysis.The SFO kinetic model or the biphasic model HS was used to calculate the soil dissipation kinetics of iprodione and 3,5-DCA (76). The SFO kinetic model is based on the assumption that the change in a chemical's concentration with time (dC/dt) is directly proportional to its concentration at this time. The HS model involves two sequential first-order degradation phases with different rates (k1 and k2) and having a breakpoint between them (tb) (24). The goodness of fit was assessed using a χ2 test (<15%, for an α of 0.05), visual inspection, and the distribution of residuals.
Microbial functional measurements, in vitro assays, and α-diversity index data were analyzed by two-way analysis of variance (ANOVA) and associated post hoc tests. RDA of the PN and enzyme activities with the ln-transformed soil concentrations of iprodione and 3,5-DCA as explanatory variables was employed to identify associations between compounds and the effects observed. The OTU matrices of bacteria, fungi, AOA, and AOB were used to assess the impact of iprodione and 3,5-DCA on the α- and β-diversity values. A-diversity indices used included Good's coverage estimate (77), the Shannon index, the inverse Simpson index (78), observed richness (S), and ACE (abundance-based coverage estimator) richness estimation (79). β-Diversity analysis included hierarchical clustering using the Bray-Curtis dissimilarity and RDA on the Hellinger-transformed matrices (80–82). Correlation tests between the measured concentrations of iprodione and 3,5-DCA in soil and the OTU-assigned sequence counts identified possible effects of the two chemicals on the microbial community members. All statistical analyses were performed with R, version 3.3.2, software (83). More details on data analysis are provided in the supplemental material.
Accession number(s).The sequence data are publicly available in the National Center for Biotechnology Information (NCBI) database under BioProject accession number PRJNA472261.
This work was performed within the frame of the project SNAC, financially supported by the Cariplo Foundation (grant number 2011-1088). The Doctoral School of the Agro-Food System (Agrisystem) of the Università Cattolica del Sacro Cuore, Italy, is also acknowledged by the authors for funding. E. S. Papadopoulou was supported by the State Scholarship Foundation of Greece through the action Supporting Postdoctoral Fellows using funds from the EP Development of Human Resources, Education and Life-Long Learning and was cofunded by the European Social Fund and the Greek State. F. Papadimitriou was supported by the postgraduate program Molecular Biology-Genetics-Diagnostic Biomarkers (grant no. 3817) of the University of Thessaly, Department of Biochemistry and Biotechnology. G. W. Nicol is funded by the AXA Research Fund.
We have no conflicts of interest to declare.
- Received 22 June 2018.
- Accepted 29 August 2018.
- Accepted manuscript posted online 7 September 2018.
Supplemental material for this article may be found at https://doi.org/10.1128/AEM.01536-18.
- Copyright © 2018 American Society for Microbiology.
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Heat · 50 Cent
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Released on: 2003-01-01
Mixer, Producer: Dr. Dre
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text:
Blizzard has long decided that WoW needs to be an action-arcade game with a giant world-sized lobby with dated MMORPG mechanics, rather than an RPG with an actual world and dated MMORPG mechanics.
They just want to please really hardcore raiders while raking in the dollars from whales and addicts.
I remember very well in TBC, when I was healing on my human holy priest at the time how my mana regen was so bad at lower levels, at normal level dungeons but In time that Improved. I was able to get so much more spirit and my mana regen was so fast I'd barely run out of mana and could use more spells, more HoTs and direct heals more often. And that kind of progression that Improvement felt great. And yes, having a PRIMARY stat higher actually helped you, we should return that too, PRIMARY STATS MATTERING and not secondarywhatevertheballs stats we have now, boring as hell stats. And while at It, since Legion we're unable to look at our stats properly in the Character window, they removed our ability to do that to reduce the complexity of our classes, chars and to "Prune" everything.
Here It doesn't matter anymore, just like Ion a few days ago made a video admitting how their class design has failed these last few expansions and how such things do matter to a class, I hope they do re-consider Mana and consider removing whatever colourfuls bars other classes have, what is it now, Maelstrom for Enchancement Shaman, lightning whatever the balls for Elemental? Nobody even has mana, they know It's a boring concept because It keeps regenning so they tried to give non-mana resource bars to classes. But to this day the only classes that should have anything close to a non-mana bar are Rogues with energy, Warriors with rage and Hunters with Focus.
That's it, no more, no less. Three classes with their own special bars are fine, you can even have gauges If you like, like Final Fantasy 14 has to work into your rotation and do bigger spells, that's a cool idea.
And you really have It spot on, with today's GO GO rush mentality of not only M+ but the whole game, mana Is an annoyence people cant afford to have and Blizzard catored to those idiots who wanna just rush rush rush for free quick rewards. Unfortunately, when we look at problems the game has you realize it ties into another system blizzard implemented, and then another one and then another one... so unless they do wide sweeping changes in the next expansion nothing of significance will change and at this point they pretty much have to because games like Final Fantasy 14 with It's expansion releasing In 2-3 weeks is rivaling WoW significantly, and ESO and other games but these two are WoW's main rivals right now. And they definitely know FF14 is a big rival as It's been taking WoW subs ever since WoD has been a failure, around the time FF14's first expansion came around.
Last edited by TheVaryag; 2019-06-19 at 11:15 AM.
Banned on WoW since April 2015 because Blizzard doesn't wanna help me when my account got hacked, Is being hacked not a good reason to return my account to me, Blizzard? I'm looking to speak to representatives and members of Blizzard's CS and other departments to help me fix this situation, since CS has failed me I want to seek other official channels. Feel free to message about this
Also, matches would have an inevitable ending instead of an inorganic fake dampening.
- - - Updated - - -
I also add that Mistweaver Monks, back in MoP, had to manage mana and Chi at the same time. The more Chi you managed to use the more efficient you were manawise. Not wasting Chi = better long term performance. This was some of the funniest times I had healing in WoW.
If its done right, I think it should be a integrated to any class who uses mana and give perks for some of them. For example, disc was insanely fun to play in wrath but it relied on having the highest possible mana pool you also had to manage what you used when as a result.
Personally I always think its better in the state it is now, until I think back to the fable series. In the original fable you had mana. You used it and it regen'd slowly, you had to carry stack of mana potions to play a caster efficiently. But you could do all sorts, for example a physical barrier cost mana to cast but you could have it up indefinitely but once you were hit it drained mana. Essentially mana shield, or you could increase movement speed etc. Come fable 3, they completely ruined the system, removed mana as a resource and put the spells into gloves you had to equip. It just isn't fun to play a caster in later titles. Which thinking of that, makes me want to play mana management in wow.
It's a pointless mechanic that only frustrated players and thats why it's gone. Deal with it.
For some specs, I don't want to see it return for ret paladins or any melee DPS though personally.
Yes, mana using classes should have to manage their mana to keep dpsing. Like Monks or Rogues or hunters have to manage theirs. Giving those classes a resource than only like 2 abilities actually drain that resource is ridiculous.
If what doesn't kill you, makes you stronger. Then I should be a god by now.
Not really, having alternate resources makes classes/specs more distinct.
But i believe they should rework how resource works for DPS. DPS is too tied to Rotation and the resource is just a part of a fixed rotation for most of the time.
Tanks have to save resources for some big incs of damage, when they know they will need a selfheal that consumes resources or when they know that there will be coming a huge damage and they will need resource to activate some skill.
Healers have to control their mana, simple because its actually not enough to sustain a whole fight, so they have to know when to top everyone and burn their mana, and when they should save it for later or for some mechanic.
Now, DPS? They just sustain their damage. there's hardly any "i will burn my whole resource now and i will be useless for the rest of the fight" or "i will do less dps but i will sustain it for the whole fight". it's all based on Cooldowns and rotation. you could remove all dps resources and i wouldn't notice(at least based on the classes i have played), there is hardly any decision behind any choice of resource wasting to the point i don't even care how much my spells costs anymore.
Not like resources doesn't matter, it just doesn't give you a choice. if your rotation is "resourceBuilder > resourceSpender > resourceSpender", then you will do it everytime, i don't remember having to build up Mana/Rage/Focus for a specific part of an encounter that i will need to burn all my resources down to give more dps.
Might be wrong tho, but thats my opinion on the mana subject, mainly because i remember actually going out of mana as a Hunter back in TBC(and sadly out of arrows too sometimes... opsie!), and i remember some mages complaining about mana too. To be fair i was pretty noob back then, and didn't understand the game, so maybe i was just being a noob.
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Tfs Error Codes
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