A psychological report is a report prepared by a psychologist. It focuses a person’s behavior, personality, and capabilities. The report can only be interpreted by a qualified psychologist or an intern. Also, psychological assessment can only be done by a professional psychologist so as to make sure that the report is error free and reliable. If the report or the evaluation is not done by either a licensed psychologist or a trainee (an intern), then it shouldn’t be used to make a decision on one’s health. On this post, we have reviewed samples of different types of psychological reports.You can also see. Psychological Assessment Report Sample. File Format • PDF Size: 43 KB Why is a Psychology Report Needed? A psychology report is needed in most places, some of these places also include school. Teachers at sometimes need to know whether their students are normal, or they have a psychological problem after seeing their behavior, having a psychology assessment carried out and brought the results to them helps them to determine whether certain students need special care or not. A psychology report is also used for legal matters and to know if someone can handle some cases in the court of law or not. Benefits of a Psychological Report A psychology report has some benefits to anyone who has undergone the psychology assessment. Some of these benefits include, they show us the current psychological state and lets us know the steps to take, whether to seek medication or not. The report gives the psychologist information on his patient, and this helps them to determine what treatment they require. Having known your psychological state, it gives one a clue on whether you should focus on personal development or treatment. When is a Psychology Report Needed? A psychology report is needed whenever one has a psychological problem, and they want it solved. Visiting a psychologist and having the assessment done, will make you eligible for the report. The report is also needed by another psychologist to know your psychological state and determine which treatment you require so as they can settle the problem. It is also -needed in the court of law to determine whether the charge is sane or insane. This helps the judge to make the charges. A psychological report is a very important document. It is used to show the current or the previous psychological state of a person. It has some benefits and among them, it helps in treatments and also in making judgment in the court of law. If you have any DMCA issues on this post, please! To create a clean and responsive WordPress theme for psychological practice personal or business websites, you need to use the flexible and modern design templates. The top location for the very best Psychologist Templates and Themes. Expertly crafted with top of the range features and on trend designs. Many people choose to work in the sphere of psychology, as they feel relief in helping others at stressful times. If you are a newbie psychologist, it may be a. I hope you do, because right now is a nice time to believe that all of your greatest and bravest ideas may be easily realized with the help of the charming psychologist WordPress templates that were made, designed and diligently featured for your future comfortable creation of a desired website. Get Social Psychology PowerPoint Template with creative backgrounds and 20 expert-quality slides from PoweredTemplate.com and create jaw-dropping PPT presentation.
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Don’t being tensed for your convenience, we’ve created an extensive list of free responsive html5 templates which have developed following site developing best practices like W3C Validation, SEO optimization, Compatible with all major browsers, precisely tailor with mobile devices, lightweight, fast loading and mind blowing design. The amazing feature of this themes, they are either the creation of expert frontend developers and established web design agencies. Let’s your head around. More Free HTML5 Website Templates Business templates list. Timer – Responsive Multi page Business Theme Agency website includes the all essential data that represents the business to the visitors precisely. But the presentation would be engaging enough when you have a clean, modern and spacious design. To serve such need Time is perfect to house all the content professionally. Time multipurpose agency template is easy to convert the programming languages for dynamic approach, tailored with agencies like travel, recruitment, government, marketing, web development, broadcasting, hiring, digital, design, job center, advertising and more. Infinity – Coming soon/under construction template Infinity is the finest example of coming soon and under construction website template. The design of this template is simple, minimal, lightweight, attractive, resourceful, glossy, catchy, and fanstic, help you to create high converting under construction website for your upcoming event, product and website. The template is equipped with striking video background, impressive countdown timer, social connection links and one page responsive layout. Minimal – Bootstrap 3 HTML5 Template You might be working for a long time but didn’t change to build your own site that attract your targeted client. But don’t miss the change now. 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The about section has a responsive timeline along with a working PHP contact form with validation. This will help you gather data on your users or to interact with them. The LESS files are included if you want to make it look better and tweak things around. Font Awesome is at work in the Services section with SCC only circle icons. Make your mark with a killer website and let Agency do all the work for you. Sedna Get your work out there with Sedna. From being a graphic designer to showing off your awesome corporate setting, this HTML5 template packs a punch in the one page that is brimmed with features to the fullest. The effects and design used are beautiful and works effortlessly for almost any kind of business. Two of the greatest features that sets it apart is the fact that it keeps user friendliness and reusability on the top of its list. It welcomes tweaking and to take it further, the Sketch file of the design is included in the package so that it can be played with. Rage – Digital Agency Free Bootstrap Multipurpose Template Designed to be a creative, sleek and beautiful expression for multipurposes, Rage is a top notch, professionally crafted template with easy on the eyes CSS Animations and typography that really pop out to make the content speak. It is built on the Bootstrap 3.3.5 framework giving it all the features that come with this particular version of Bootstrap. The code that it’s written in is extremely neat, prim and proper and you don’t ever have to worry about what browsers it might be the most adaptable to. It handles browsers effortlessly. Slab – Free Multipurpose Bootstrap Template If minimalism is your forte, Slab is your template. It uses space and fonts in the cleanest way possible so the focus on the website is exactly on what you want it on. The template contains a gigantic set of features. From being responsive to having seperate sections for videos, pricing, team and much more, it is also customization friendly so go ahead and make your website really yours and tell your story the way you want it to. For those who want to get down and get even more customized, they have access to online documentation for this and also a working contact form so that whenever your users have something to say to you, you’ll never miss a thing. Snow: Free PSD + HTML Business/Portfolio template When it comes to making a mark on the world with your work, you never want to compromise on the quality. He is robustly reliable and created with a modern and creative outlook to make sure you keep up with the rest of the world. There are two portfolio pages to choose from along with two blog pages. So go ahead and put your best foot forward to become the industry thought leader. This is a hands down awesome creation and practises minimalism to the core. Land UI Kit + Landing Page Template (HTML) Here’s a template that totally nails the combination of simplicity and performance. Rocking the Bootstrap framework and the SASS standard, you are sure to make the best use of that. The template has clean and efficient coding and it is perfect even if you’re not a coder because you can customize the way the template looks and works without having to write a single line of code. The images used are optimized so you don’t have to put extra effort to take care of that. So bring your customers to this wonderfully crafted landing page and it will take care of the rest. Attes free one page bootstrap theme Dazzle your users with your website. While that seems like a duanting task to many, Latte volunteers to help out with it’s minimalist and beautiful design. It is a one page template that is rocking the Bootstrap framework and is perfect for creatives and agencies. It makes a witty use of white space that makes readability a breeze on the website and the best part is, it is snappy and doesn’t even require you to spend hours pondering over how to make it work because it is extremely easy in the backend to modify. Oh and did we mention it’s responsive? Color HTML5 OnePage Bootstrap Template Themeinthebox seems to have come up with an amazing theme in its box- The Color Template that is a one page template that is built on the Bootstrap framework. It is specifically made out for professionals in the business industry in mainly the creative and agency sectors but the way the template looks, it can actually be used for other purposes too. The template rocks a clear and spacious look which makes the template look very clean but far from making it boring. The template also packs amazing animations that flow perfectly and are sure to impress your website visitors. Maguwohost – FREE Hosting Template This is the most different of the bunch as this template, MaguwoHost, is catered for the websites of companies that cater for other company websites. We’re talking about a hosting template here. While hosting companies do a great job at taking care of other companies, they themselves need an awesome template to set them apart from bigger or newer competitors. This is where the extremely clean, efficiently coded and extremely professional template is necessary and this is exactly what Maguwohost provides. It is a one page HTML template that enables changes to be made to the way the website looks and how the information is displayed. There is even a domain checker so that people can confirm whether a domain is available or not. The responsive, beautiful and clean looking template also lets you edit the entire look of the website from the admin panel. Mazel – Responsive Multipurpose Template (Paid) For those who wish to create successful websites with a hint of personalization, Mazel is the one for you. You can bring your A game with the 119+ different pages that you can use as you like. Crafted for almost any industry, the template really shines when it comes to showcasing a photo studio or a portfolio, even if you’re a freelancer, an agency or a blogger. Don’t forget, it also has features to pack a punch if you’re an Ecommerce business. To make it easy to understand what it is that you can expect once the template itself is done, there are 2 template demos for your use. There are over 16 variations of the Homepage so you can really go to town with the looks of the home page, and even if you’ve been using the template for a while, you can always upgrade your looks using a different variation of the home page. Not just the home page, there are also a lot of options for the other pages included in the website: around 22 one page layouts, 28 portfolio pages, 15 blog pages and much more. So there are almost more than a 100 combinations that you can make using all these features that the template provides. Also, get in touch with your customers with the working AJAX and PHP contact forms and reach to them with the newest offerings from you. Delight – Multipurpose One Page Template (Paid) The name delight is perfect for this template as it is simply delightful. It is a very clean template that uses space and minimalism perfectly and creates a wonderful combination along with all the features of Bootstrap 3 framework. The template is fully responsive so you do not have to sweat it about which screensize your viewer is displaying your website on because the template will run flawlessly on any screensize. It even has retina support. The template takes it further with viewability with the mobile first fluid grid system and even focuses heavily on making any website it is used on look beautiful with the sleek and modern effects that it rocks. Speaking of making it look great, the template also has the Magnific popup, over 2000 icons that you can pick from with the support of Font Awesome 4.4.0 and sweet CSS3 hover effects that will make you totally feel that it is a great value for its price. The documentation is up to date and you will have no difficulty figuring out how to get that paralla scrolling, the lightbox or the carousel slider work perfectly. Along with mobile first system, Delight is compatible for all major browsers along with being SEO optimized. Revelo – Multipurpose HTML Template (Paid) Revelo is an interesting experience to users and there is perhaps no heights that you cannot achieve with this template. The template packs a lot of powerful features that you can totally use to dazzle the users who visit your website. If you can dream it, this template can do it for sure. Revelo really helps to reveal your jest of business with features like the numerous header and footer options and shortcodes that can add to the stunning look of the website. There are about 6 color options that you can pick from with a promise of more coming soon. The sidebar is particularly promising for any website because it is where customers look to for more offers and there are 3 different options at play here. Headers are also particularly important for navigation and you can construct the best experience for the customers by picking what you think goes best from the 7 options that are carefully crafted for the best navigability. There is also a whooping almost giveaway of the revolution slider, the isotope filter and the form validator so you get to save as much as 63 USD with this template. There are a ton of other features included in this template such as the sticky header, the masonry layout, the coming soon pages, the mailchimp integration, the instagram feed and so much more that cannot be fit into this description so do check this template out. Versa – Multipurpose Template (Paid) Versa is well equipped with features to make any website look amazing. Be it a new website or upgrading an existing one, this template creates a fresh look for the website while keeping up to date with all the newest and greatest features in the web design industry. There are over 25 demos that you can choose from for your website whether you want a one page website or a multipage one. The templates offer options for various purposes from business startups, to youtubers to online shops or even restaurants. It can also be used for photography or fitness purposes so whatever it is that your business is, Versa has got you covered. There are about 6 sliders including master sliders and revolution fullscreen and video sliders or the static parallax if that’s your thing. The template comes with premium plugins so getting those little details perfect is easy now, and if that wasn’t enough, there are subtle and beautiful shortcodes too. This template is perfect to make almost any and every website stand out. Atropos – Responsive Website Template (Paid) Here’s a template that gets updates for its entire lifetime. If not having a bloated and clunky website is what you’re looking for, this clean and intuitive template is perfect for you and your business whether it is a business, corporate, portfolio or any other type of product. There are two skin tones or modes to choose from, one dark one light. It also ships with a Revolution slider included in the package and there are over 185 HTML files included that you can use along with the countless other features like multiple home versions, the wide and boxed layout style which makes it so pleasing to the eyes. You can have an image background or even a video background according to what fits you better. And if something is wrong, there is also a 404 page for your discretion. You can also create hype in your customers with a coming soon page or even a countdown option letting them know you’re up to something even more awesome. There are a ton of other features like the sitemap page, the services page, the support page, the ajax contact form and even a pricing table. The template also has the Google map integration to let your customers know exactly where to find you. The documentation is standard and you need not worry about understanding it. React – Business Theme (Paid) React to the changing website landscape with React. Yes, it’s a business theme from ericklvz which hones simplicity as well as customizability. Using the latest and greatest from HTML5 and CSS3, this is a very responsive template that is built with a SASS and a folder structure. It is very neat and organized for all of us clean obsessed ones. There are 3 different landing page options with 3 different kinds of features each provides so whatever you may you want, you got it. You can get a beautiful gallery with features like zoom in and zoom out so you can really put the best pictures for your products and they will look great. You can have your full business and all that it has on the website with the many pages catered to businesses like the pricings page, the comparision page, blog pages, the support page, the sign in page, the sign up page and so much more. Spacial – Responsive Website Template (Paid) Get a template that you can customize as you wish with thorough documentation. The template is fully responsive so you can view the website on any and every screensize and it will come out looking gorgeous without messing anything up in the functions department. It is perfect for businesses, creatives and agencies, ecommerce and much more. The source files are very organized so it is very easy to understand and use as well as being optimized for search engines. There are plenty of Google fonts to really make the website look modern. It is a gulp powered template with over 60 templates and has a clean and modular code. Shape – Business Template for All Niche (Paid) Shape is a refined website template for presenting your real estate, construction, finance, consulting, cleaning service or any other business made already for you. It is a well researched and constructed template that makes the best design catered right for you. There is Ajax mail that makes your form ready to use and the user experience is perfectly made for niches. The look of the template is very easy on the eyes with sleek animations. Everyone hates slow websites, make sure you do not face that problem and this template makes sure the loading of the page is blazing fast. The template has extensive and clean documentation and even then if you get into any glitch there is a Live support with real people that can give you instant feedbacks and solve your problem. There is an Awesome custom slider and is fully responsive with unlimited font icons and awesome jQuery effects. Neomax – Multipurpose Template (Paid) Neomax is a beautiful website with 15 demos that you can pick from. There is a mega menu included that will display the features on the website in an organized and clean way and it is also responsive. There are 140 HTML pages to pick from with different versions of the home, blog, shop etc. So you can pick whatever you may require. There is a product slider with thumbnails carousel. And when you pick it there is the hover effect so every time you put your mouse on it, there is a zoom in and out effect. The website can also have a parallax background ether with an image, a Youtube or Vine video background. The template also makes it very easy to get in touch with your users or let them get in touch with you with the working contact forms with a JQuery validation plugin. There is even a lightbox gallery for beautiful display of your products and even twitter feed integration so customers can get in touch with your social media. You can customize the website even further by picking the best icons from over 600 of the icons that it comes with along with different CSS animations to pick from. Halves – Responsive Versatile Template (Paid) If you need to get started with an awesome and intuitive website, halves will half your work with a gorgeous design. It has over 11 pages for different purposes starting from home to contact to a portfolio section, pricing page and much more. It makes a unique combination of typography and tables together to create a professional aura that is unparalleled to any other aspect in the industry. It rocks the Bootstrap 3.3.6 framework as well as plenty of HTML and CSS files. BigBag Store – Elegant Ecommerce Theme (Paid) Big bag store has big features packed into it. When you’re making an ecommerce website, you have to make sure that the template you have picked is perfect for it with all the features you may need for smooth operation. This powerful template has 46 page templates already with more on the horizon. It enables quick viewing of the products and also a countdown option for the new awesomeness you promise to bring them. The template is ready for any viewing on any device and has been tried and tested. The crafting of the template is done very carefully and with a lot of effort and includes awesome features like the scroll animation, the brand slider and over 700 icons to pick from. You can even guide your customers to your outlet or office with the Google map integration that you can totally customize. You do not need to sweat over cross browser compatibility either. It is built on 3.3.7 version of Bootstrap and is created with SASS. It is fully customizable with Google fonts, 4 versions of the home page and much more. Plus – Responsive E-Commerce Template (Paid) The plus point of Plus is how clean and minimal it is. Being a cross browser compatible and extremely responsive template, this template comes with over 35 HTML pages built on Bootstrap 3.3.7. There are a lot of powerful features that can enrich any browser experience and any customizing experience. You can use the mega menu along with any of the 9 color schemes. The code is validated and easy to get and the best part is, the developer promises free lifetime updates for it as well as solid support. You can use the Google fonts to create a modern and stylish look along with the tons of icons provided. There is even a coming soon page included in the template that will keep your customers on their toes with anticipation. Helena – HTML + OpenCart Store Theme (Paid) One of the most stylish ecommerce templates around, Helena is an HTML Open cart store template that is easy to integrate into any current website or work you have. It has sliders that blend perfectly in with Bootstrap as well as Navigation bars that are extremely flexible. The opencart theme comes free with it and there are different pages like the home page, the full product category, a separate page for cart and checkout. There is also a 404 page for the oh snap moments that all websites sometimes face. There is even a FAQ and blog page along with a registration page. So go right ahead and adorn your website with this template. Summary Having exploring the largest collection of Free HTML5 website templates, I know you are excited to utilized them for your next projects, right? I would like to know which HTML theme can successful catch your attention. As a quick survey to help other developers to quickly pick from, post your valuable choice in the comment section. Quick Summary For website owners who are evaluating e-commerce solutions, there are several good options for powering websites and shopping carts. Regardless of which option you chose, deciding on a design brings with it even more decisions to make. Of course, a custom design is always an option, but for those who want to keep costs down, templates are a popular choice. Premium e-commerce themes are easy to find, but they can be expensive. Free e-commerce templates are difficult to find, and quality free templates are even harder to find. We’ve done the research for you, and in this post we present 35 of the highest-quality free e-commerce templates available. There are templates specifically for WordPress,,,,,, and, as well as a number of general e-commerce templates. Please notice that these templates aren’t intended to be used 1:1, but should rather be used as a skeleton for your own shop and hence saves a lot of work, because otherwise you would have to start from scratch. For website owners who are evaluating e-commerce solutions, there are several good options for powering websites and shopping carts. Regardless of which option you chose, deciding on a design brings with it even more decisions to make. Of course, a custom design is always an option, but for those who want to keep costs down, templates are a popular choice. Premium e-commerce themes are easy to find, but they can be expensive. Free e-commerce templates are difficult to find, and quality free templates are even harder to find. You may also want to check out the following Smashing Magazine articles: • • • We’ve done the research for you, and in this post we present 35 of the highest-quality free e-commerce templates available. There are templates specifically for WordPress, Prestashop, osCommerce, Magento, Zen Cart, CubeCart, and CRE Loaded, as well as a number of general e-commerce templates. Please notice that these templates aren’t intended to be used 1:1, but should rather be used as a skeleton for your own shop and hence saves a lot of work, because otherwise you would have to start from scratch. WordPress Templates||. Zen Cart Templates| Clean Blue A collection of free CubeCart 3 Skins. More free Cubecart skins. CRE Loaded Templates| 8. General E-Commerce Templates The following templates were created for the purpose e-commerce but not built for a specific system. If you plan to use one of them, it would need to be adapted to and integrated in the e-commerce solution of your choice. 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Website Design Companies, Services and Firms Directory A directory of affordable, reliable web site design companies, services and firms offering html programming, graphic design, website templates, Free Website Templates, Free Photoshop Web Templates, Website Design Over 9000 professional website templates, flash intros and logo templates. Huge selection of affordable premium intros as well. Farmed fish escape and enter the environment with subsequent effects on wild populations. Reducing escapes requires the ability to trace individuals back to the point of escape, so that escape causes can be identified and technical standards improved. Here, we tested if stable isotope otolith fingerprint marks delivered during routine vaccination could be an accurate, feasible and cost effective marking method, suitable for industrial-scale application. We tested seven stable isotopes, 134Ba, 135Ba, 136Ba, 137Ba, 86Sr, 87Sr and 26Mg, on farmed Atlantic salmon reared in freshwater, in experimental conditions designed to reflect commercial practice. Marking was 100% successful with individual Ba isotopes at concentrations as low as 0.001 µg. G -1 fish and for Sr isotopes at 1 µg. Our results suggest that 63 unique fingerprint marks can be made at low cost using Ba (0.0002 – 0.02 $US per mark) and Sr (0.46 – 0.82 $US per mark) isotopes. Stable isotope fingerprinting during vaccination is feasible for commercial application if applied at a company level within the world’s largest salmon producing nations. Introducing a mass marking scheme would enable tracing of escapees back to point of origin, which could drive greater compliance, better farm design and improved management practices to reduce escapes. Introduction Farmed fish escapees from sea-cage aquaculture are perceived as a serious threat to wild fish populations as they can cause damaging ecological impacts. These include transfer of diseases to wild fish [], introduction and establishment of escapees as exotic species [], competition between escapees and wild stocks [,], and outbreeding depression through genetic mixing of wild and farmed populations from hybrid crosses [,]. Atlantic salmon ( Salmo salar) is the most commonly occurring farmed fish escapee from sea-cage aquaculture []. For instance, 4.6 million salmon were reported to have escaped from Norwegian fish farms from 2001–2012 () and escapes occur in all salmon farming countries. Although most farmed escaped salmon disappear, never to be observed again [–], some survive and migrate into rivers and onto the spawning grounds of native populations []. As a result of farmed salmon successfully spawning with wild salmon, genetic changes have been observed in native salmon populations in Ireland [] and Norway [], with introgression of farmed salmon estimated at 0–47% for 20 native populations spanning the entire Norwegian coastline []. Introgression of farmed salmon in native populations is of significant concern because their offspring display reduced survival in the wild compared to wild salmon [,,], and may also disrupt local adaptations []. Although fish farmers in many jurisdictions are obliged to report escapes, in some cases, escapes of farmed fish are not reported to the authorities. Under reporting is problematic, as without an understanding of why fish escape through technical investigations of escape causes, improvements to technical standards cannot be made rapidly []. Detecting escapees and determining the farm they originated from is possible through DNA-based methods [] or fatty acid profiling [], although these methods do not identify the farm in all cases. As an alternative, a permanent coded mark or tag for all farm fish applied at an industry scale would allow for a fail-safe method to trace escapees to their point of origin. Numerous methods currently exist to mark fish (e.g. Adipose fin clipping and physical tags []; otolith thermal marking []; fluorescent markers []), but all fail in one or more aspects related to the ability to deliver 100% traceability to point of origin, fish welfare considerations or cost-effectiveness at industry scale. Here, we advance a recently developed marking technique for identifying and tracing farmed Atlantic salmon escapees using stable isotope otolith fingerprint markers, delivered during vaccination [], by testing multiple combinations of seven enriched isotopes over a concentration gradient, to determine if the technique can be feasibly applied at full industrial scale where up to 500 million fish require marking each year. Marking during routine vaccination could effectively and accurately mark all farmed fish in commercial facilities with no additional manual handling or labour costs. Typically, Atlantic salmon are routinely vaccinated during the parr stage with multi-vaccines against a range of pathogens []. Otolith fingerprinting during vaccination is 100% successful using enriched stable isotopes 137Ba and 86Sr at high concentrations, and marginally successful (0 to 35%) with enriched 26Mg []. Otolith fingerprinting via larval immersion on other species suggests that the use of additional stable isotopes of Ba, Sr and Mg could produce over 100 possible otolith fingerprint combinations via vaccination [,]. Whether these combinations produce viable marks and what minimum dosages are possible for cost-effectiveness for marking during vaccination must be determined to make this marking technique financially feasible for industry-scale application. Here, we tested seven enriched stable isotopes ( 134Ba, 135Ba, 136Ba, 137Ba, 86Sr, 87Sr, and 26Mg) at 4 concentration levels (1, 0.1, 0.01 and 0.001 μg. G -1 fish) in fingerprint combinations of 1, 4 or 7, which could provide 127 unique marks. To make the experiment industry-relevant, we followed standard commercial farming practices for salmon. Moreover, we monitored the health and welfare of all marked fish until they grew to harvest size (5 kg) to determine if marked fish had similar condition and welfare to unmarked control fish and that the concentrations of stable isotopes of Ba, Sr and Mg used in this study are harmless for farmed salmon. Otolith fingerprint marking with enriched stable isotopes during vaccination Ethics statement. This study was conducted in accordance with the laws and regulations of the Norwegian Regulation on Animal Experimentation 1996. The protocol was approved by the Norwegian Animal Research Authority (Ethics permit number: 6176). Experimental location and fish. The experiment was conducted at the Institute of Marine Research, Matre Research Station, in Masfjorden, western Norway (60°N). A total of 650 Atlantic salmon (AquaGen strain) parr (mean ± SE: fork length = 19.8 ± 0.04 cm; weight = 103 ± 0.6 g) were used in the experiment. All fish were pit tagged with 11 mm Trovan ID 101 tags (BTS Scandinavia AB, Sweden) four months prior to the experiment, and reared in freshwater tanks buffered with saltwater to a salinity of 0.7 g NaCl.L -1 in standard commercial hatchery conditions. Fish in all treatments were of similar length and weight at day 1 of the experiment (one-way ANOVA; length; F 12, 649 = 1.32, p = 0.2, weight; F 12, 649 = 0.87, p = 0.6). Experimental design. We tested three combinations of the enriched stable isotopes, 134Ba, 135Ba, 136Ba, 137Ba, 86Sr, 87Sr and 26Mg (Oak Ridge National Laboratory; ) at four concentrations (1, 0.1, 0.01, or 0.001 μg of each isotope per g of parr average weight) to determine the minimum isotope concentrations required to ensure 100% mark success of isotope fingerprint tags delivered during vaccination []. Atlantic salmon parr (50 per treatment) were injected with the multi vaccine MINOVA 6 (NORVAX MINOVA 6, Global Aquatic Animal Health, Bergen, Norway) that contained either: 1) no isotope enrichment; 2) enriched 137Ba; 3) a combination of enriched 135Ba, 136Ba, 137Ba and 86Sr; or 4) a combination of enriched 134Ba, 135Ba, 136Ba, 137Ba, 86Sr, 87Sr and 26Mg (). Experimental design. Enriched stable isotopes in powder chloride form (BaCl 2, SrCl 2 & MgCl 2) used for the isotope enrichment treatments were first dissolved in Milli-Q water to make standard stock solutions of each isotope combination (i.e., 1, 4, or 7 isotope markers). The required isotope combination-by-concentration solutions were then mixed firstly by pipetting the appropriate amounts from the standard stock solutions into a 1 ml eppendorf tube and then mixing this solution with the MINOVA 6 vaccine on the day of vaccination. Final solutions were agitated for 30 seconds using a Virvel Mixer (Heidolph Instruments Gmbh & Co.KG) to ensure a stable solution. Injections (0.1 ml per fish) were given into the abdominal cavity, approximately 20 mm behind the pectoral fin on the ventral side of parr using a standard commercial vaccination gun (Socorex Swiss-167; ) fitted with a 5 mm, 27 gauge vaccination needle. On the day of vaccination, fish were anaesthetised with Benzoak VET (dose 0.2 ml L -1 of clean hatchery water), identified by their PIT tag number, then weighed, measured (fork length) and injected. After injection, fish were placed into one of five 1000 litre tanks with equal interspersion of individuals among treatments within each tank (i.e. 130 fish per tank, 10 from each treatment). The fish were reared under a 12 h light: 12 h dark photoperiod for the first six weeks post injection before being switched to 24 hours continuous light to induce smoltification. To monitor differences in growth and condition among treatments, 90 days post-injection, all fish (n = 50 per treatment) were anaesthetised, weighed, and measured (fork length). At this time, a randomly selected sub-sample of 10 fish per treatment, were euthanized by anaesthetic overdose and their otoliths were removed for isotope analysis. Sagittal otoliths (mean ± SE: maximum diameter = 3.3 ± 0.1 mm) were cleaned of any adhering tissue, air dried, and stored individually in plastic tubes. Remaining fish were transferred to a sea cage farm and grown to commercial harvest size (~ 5 kg, 570 days post-injection), then humanely culled with a quick blow to the head, measured (fork length) and weighed, to assess condition at harvest. Otolith preparation. Otoliths were prepared as per Warren-Myers et al. Sagittal otoliths were cleaned of any remaining organic tissue by immersing in a solution of ultrapure 15% H 2O 2 buffered with 0.1 M NaOH. Following immersion, otoliths were ultra-sonicated (Sonic Clean 250HT) for 5 minutes and then left for 6 hours in the cleaning solution. The cleaning solution was then aspirated off and the otoliths were transferred through three Milli-Q water rinses, each of which consisted of 5 minutes of ultra-sonification and 30 minutes resting time. Otoliths were then air dried in a laminar flow bench for at least 24 hours. Once dry, one otolith per fish was fixed, sulcus side down, onto gridded microscope slides using quick dry cyanoacrylate glue. Otolith analysis. Stable isotope analyses were done on a Varian 7700x Inductively Coupled Plasma Mass Spectrometer (ICP-MS) fitted with a HelEx (Laurin Technic and the Australian National University) laser ablation (LA) system constructed around a Compex 110 (Lambda Physik) excimer laser operating at 193 nm. 612 and 610 NIST (National Institute of Standards and Technology) glass standards doped with trace elements at known concentrations was used to calibrate the system. External precision estimates (%RSD) based on 20 analyses of a MACS3 microanalytical carbonate standard were as follows: 134Ba: 138Ba = 7.37; 135Ba: 138Ba = 0.81, 136Ba: 138Ba = 4.51, 137Ba: 138Ba = 0.72, 86Sr: 88Sr = 0.94, 87Sr: 88Sr = 1.16 and 26Mg: 24Mg = 0.60. Otoliths were run in blocks of 16 samples selected randomly from all treatments and bracketed by analyses of the standards. Samples and standards were analysed by vertically profiling in time-resolved mode, using a stationary laser with a spot size of 157 μm, an energy setting of ~ 60 mJ and a repetition rate of 10 Hz. Ablation was performed under pure He (200 ml/min) to minimise re-deposition of ablated material and the sample was then entrained into the Ar (0.95 ml/min) carrier gas flow to the ICP-MS. Using this method, we were able to quantify the concentrations of 134Ba, 135Ba, 136Ba, 137Ba, 138Ba, 86Sr, 87Sr, 88Sr, 24Mg, 26Mg and 43Ca in the outer region of salmon pre-smolt otoliths. Data were processed off-line using a specialised MS Excel template which involved a low pass filter to remove any spikes (a single acquisition value >2x the median of the adjacent acquisitions), smoothing (a running average of 3 acquisitions) and blank subtracting functions (an acquisition = a single measure of an isotope ratio while vertically profiling in time-resolved acquisition mode). A correction factor ( K = R true /R obs, where R true is the naturally occurring isotope ratio and R obs is the average isotope ratio measured in the NIST 612 and 610 standards run before and after each set of 16 samples) was applied to all sample acquisitions to correct for mass bias. The NIST 612 was used for 137Ba, 135Ba, 87Sr, 86Sr and 26Mg and NIST 610 for 134Ba and 136Ba. Isotope ratios are expressed as the enriched isotope divided by the most commonly abundant isotope for each element used so that the measure of enrichment is always expressed as an increase in the enriched isotope relative to the most common isotope. Statistical analyses were conducted on the final post-processed acquisition data values. Statistical analysis. Mark success for each treatment was evaluated using a mark detection threshold set by Warren-Myers et al. Briefly, the mark detection threshold for the isotope ratios 134Ba: 138Ba, 135Ba: 138Ba, 136Ba: 138Ba, 137Ba: 138Ba, 86Sr: 88Sr, 87Sr: 88Sr and 26Mg: 24Mg were calculated from the average isotope ratios of fish across the control treatment (i.e. Non-enrichment treatment) (). To ensure a correct classification probability of 99.94%, mark detection thresholds were set at 3.3 standard deviations above the mean observed ratio in control fish for each enriched isotope used. Because of the inherent instability in isotopic ratios measured on single-detector, ICP-based mass spectrometers, we conservatively set the criteria for detecting a successful mark in the otolith as at least three consecutive acquisitions with ratios above the detection threshold. Mark strength of 134Ba: 138Ba, 135Ba: 138Ba, 136Ba: 138Ba, 137Ba: 138Ba, 86Sr: 88Sr, 87Sr: 88Sr and 26Mg: 24Mg for each isotope enrichment concentration used (1, 0.1, 0.01, & 0.001 μg isotope g -1 fish) was analysed using a series of ANOVAs with isotope concentration and combination treated as fixed factors. The response variables used were the maximum isotope ratio observed (intensity) and the numbers of acquisitions above detection (spatial extent), in each fish otolith. The number of acquisitions above detection were ln (count + 1) transformed to improve ANOVA assumptions of equal variances. The effects of treatment on fish length (fork length), weight and condition over the experimental period were analysed with one-way ANOVAs. The response variables used were change in fish length, weight and condition over the time frame of the experiment (sampling at 90 days and harvest 570 days). Fish condition was estimated using Fulton’s condition factor ( K) calculated with the formula K = ((W/L3)×100), where W is the live body weight (g), and L is the fork length (cm) []. Mark success A six marker fingerprint combination using the enriched stable isotopes 137Ba, 136Ba, 135Ba, 134Ba, 86Sr and 87Sr was successfully created by marking during vaccination (). However, mark success was dependent on enrichment concentration and isotope combination (). 137Ba achieved 100% mark success with a minimum concentration of 0.001 μg. G -1 fish when used as a single isotope marker and 0.01 μg. G -1 fish when used in a combination of 4 or 7 isotopes. Marking with 135Ba and 136Ba was 100% successful with a minimum concentration of 0.01 μg. G -1 fish when used in combinations of 4 or 7 isotopes. Marking with 134Ba was 100% successful with a minimum concentration of 0.01 μg. G -1 fish when used in a 7 isotope combination. Sr isotopes were only successful at a concentration of 1 μg. Mark success using 86Sr was 100% successful in combinations of 4 or 7 isotopes, and 87Sr was 100% successful in the 7 isotope combination. 26Mg used in a combination of 7 isotopes produced no successful marks at any concentration level. Intensity of isotope enrichment 137 Ba, 136 Ba, 135 Ba and 134 Ba max isotope ratios. For 137Ba, an interaction between marker combination and the concentration of isotope used, showed that as concentration decreased and marker combination increased, mark strength decreased (interaction term: concentration x combination, F 6, 127 = 3.01, p = 0.009). Post hoc test for the interaction term highlighted that 137Ba used as a singular marker produced higher maximum ratios than combinations of 4 or 7 isotopes, depending on the enrichment concentration used (, Tukey HSD: 1 μg, 1 marker > 4 markers = 7 markers; 0.1 μg, 1 marker > 7 markers; p. Intensity of mark using multiple combinations of isotopes. There was no interaction between combination and concentration for 136Ba or 135Ba (F 3, 87 = 0.2 & 1.2 respectively, p > 0.3 for both). Mark strength for 136Ba and 135Ba used in combinations of 4 or 7 isotopes decreased as concentration decreased (, F 3, 87 = 341, p 0.1 μg > 0.01 μg > 0.001 μg = 0 μg, p 0.1 μg > 0.01 μg > 0.001 μg = 0 μg, p 0.1 μg = 0.01 μg = 0.001 μg = 0 μg, p 0.1 μg = 0.01 μg = 0.001 μg = 0 μg, p. Spatial extent of isotope enrichment Analysis of the total number of acquisitions observed above the detection threshold were only analysed for the Ba and Sr enrichment treatments. Mg enrichment did not produce enough acquisitions above the detection threshold to warrant further analyses. No control fish had 3 consecutive acquisitions above the detection threshold for any Ba or Sr isotope, so the analysis was restricted to the isotope enriched vaccine treatments. 137 Ba, 136 Ba, 135 Ba and 134 Ba acquisitions above detection. There were no interactions between concentration and combination for 137Ba (F 6, 117 = 0.85, p = 0.5), 136Ba or 135Ba (F 3, 87 = 0.04 & 0.29 respectively, p > 0.5 for both). Separately, isotope concentration and combination did influenced the total number of acquisitions above the detection threshold for 137Ba (F 3, 117 = 77.9, p 0.1 μg > 0.01 μg > 0.001 μg, p 4 markers = 7 markers, p 0.1 μg > 0.01 μg > 0.001 μg, p. Spatial extent of mark using multiple combinations of isotopes. 86 Sr and 87 Sr acquisitions above detection. There was no interaction between combination and concentration for 86Sr (F 3, 77 = 0.2, p = 0.8).The number of acquisitions above detection for 86Sr were 10 times higher in the high concentration (1μg. G -1 fish) compared to the 3 lower concentrations (, F 3, 77 = 86.6, p 0.1 μg = 0.01 μg = 0.001 μg, p 0.1 μg > 0.01 μg = 0.001 μg, p. Mortality and growth There was no effect of treatment on mortality, with only 3 mortalities out of a total of 650 injected fish during the first 90 days before sea-transfer. Prior to sea transfer, overall, experimental fish increased in weight by 53.9 ± 0.6 g (mean ± SE) and fork-length by 3.9 ± 0.02 cm. No differences were detected among treatments for fish growth (weight: F 12, 646 = 1.18, p = 0.3; fork length: F 12, 646 = 1.27, p = 0.2) or fish condition (Fulton’s condition factor ( k): F 12, 646 = 1.02, p = 0.4). However, average condition of fish across all treatments was approximately 10% lower at 90 days post injection compared to day 1 (Fulton’s condition factor ( k): Day 1 = 1.30 ± 0.002; Day 90 = 1.16 ± 0.003). Fish harvested at 570 days post injection (5.21 ± 0.06 kg, fork-length 72.2 ± 0.3 cm, condition factor ( k) 1.36 ± 0.007) showed no difference in length, weight or condition among treatments (, weight: F 12, 408 = 1.11, p = 0.3; fork length: F 12, 408 = 0.84, p = 0.6; Fulton’s condition factor ( k): F 12, 408 = 0.82, p = 0.6); p = 0.4, 0.6, 0.6 respectively). Mortality per treatment during the sea cage stage averaged 7.4 ± 1%, with no difference among treatments (χ 2 12 = 7.2, p > 0.1). Mark success and strength We have successfully produced a six marker stable isotope fingerprint and effectively determined the minimum optimal concentrations required for the 63 possible combinations of four Ba isotopes and two Sr isotopes for fingerprint marking Atlantic salmon otoliths during vaccination (.). Creating a single Ba isotope fingerprint mark using 137Ba with 100% mark success is achievable at a concentration of 0.001 μg. However, as the number of isotopes used in a fingerprint combination increases to four ( 137Ba, 136Ba, 135Ba & 86Sr) or seven ( 137Ba, 136Ba, 135Ba, 134Ba, 87Sr, 86Sr & 26Mg) the required concentration of each Ba isotope needed to ensure 100% mark success increases to 0.01 μg. Sr isotopes used in multiple fingerprint marks required higher concentrations in comparison to Ba isotopes (1 μg vs 0.01 μg. G -1 fish, respectively) to guarantee 100% mark success, and for Sr there was no difference between fingerprint combinations of four or seven. These results demonstrate that the minimum concentration of isotope required to mark an individual fish during vaccination is 2 (for Sr) to 2000 (for 137Ba) times lower than the initial concentrations of 2 μg. G -1 fish trial by Warren-Myers et al. [], depending on the isotope used and the isotope combination. This suggests that stable isotope marking during vaccination with Ba isotopes, in particular, has the potential to be economically feasible at an industry scale where costs per fish must be as low as possible. Mark strength, measured using maximum isotope ratios (intensity) and number of acquisitions above detection (spatial extent) declined as isotope concentration was reduced. However, ratios did not decrease by an order of magnitude as one would predict, and for the Ba isotopes the intensity of marks was higher for single mark compared to 4 or 7 multiple marks at the highest concentration (1 μg. This would suggest there is a possible facilitation, competition, or dilution effect influencing the degree of marker incorporation in multiple marker fingerprints above a threshold concentration. Facilitation of Ba uptake when Sr is present is known to occur in some fish species when the Sr:Ca ambient concentration in brackish or sea water is greater than 20 μmol. However, if facilitation was occurring this should have increased the intensity of Ba marks when Sr was present in multiple mark fingerprints, not decreased mark intensity. Reduced mark intensity when using multi-isotopes markers compared to a single marker due to competition has not been reported before, but maybe a plausible explanation if 1 μg. G -1 fish (highest concentration tested) is a threshold at which competition among isotopes of the same element occurs. Dilution is most likely the cause for reduced mark intensity when multiple isotopes from the one element (e.g. 137Ba, 136Ba, 135Ba, 134Ba) are used together. A dilution effect could result from the residual amount of 138Ba impurities in the enrich isotopes used ( 138Ba impurities in: 137Ba = 17.4%; 136Ba = 2.4%; 135Ba = 3.6%; 134Ba = 5.3%, Oak Ridge National Laboratory; ). A dilution effect of added residual 138Ba would also explain why mark success for 137Ba, which was 100% when used as a single isotope marker at the lowest concentration (0.001 μg. G -1 fish), dropped to 80% when used in combination with 2 other Ba isotope markers ( 135Ba, 136Ba) and to 70% when used with 3 other Ba isotope markers ( 135Ba, 136Ba, 134Ba). Hence, an increase in marker concentration from 0.001 to 0.01 μg. G -1 fish for multiple Ba marks is required to ensure mark intensity and spatial extent is strong enough that 100% unique marks are created. Mark success with 26Mg was unsuccessful even at the highest concentration of 1 μg. Poor mark success with Mg isotopes has been reported for marking via vaccination [], or larval immersion [] and may be due to self-regulation of Mg in fish [] or a slow exchange rate of Mg ions into the endolymph fluid that surrounds the otolith []. Alternatively, natural levels of Mg in water and food may be too high for the introduction of an enriched Mg spike to significantly shift the natural Mg isotope ratios at the concentrations Mg has been tested. Greater concentrations than used in this study, even if successful, would make Mg too costly and hence unsuitable for marking farmed salmon. Fish condition and survival Stable isotope marking with Ba, Sr and Mg did not affect growth, condition or survival, among treatments over the 570 days between injection date and harvest date. This is consistent with other stable isotope marking studies that used transgenerational and larval immersion techniques, and which similarly detected no negative effects on survival and growth due to marking [,,,]. Although average condition of all fish in the experiment dropped initially (~10%) over the first 90 days, the photoperiod regime used in the trial typically induces a decrease in condition factor similar to that normally seen during the parr–smolt transformation of Atlantic salmon and other salmonid species [,]. In addition, growth rates often reduce in the short-term in vaccinated Atlantic salmon [,], which is associated with loss of appetite post vaccination []. At harvest, fish condition was slightly higher compared to condition at injection date and no differences were found among treatments suggesting there are no long-term detrimental effects of stable isotopes on Atlantic salmon when delivered during vaccination. Application of otolith fingerprinting with enriched stable isotopes during vaccination We have demonstrated that isotope marking delivered during vaccination can effectively mark farmed salmon and enable detection of the mark with 99.94% accuracy. Moreover, the concentrations required are sufficiently low that cost-effectiveness is high compared to all other common salmonoid mass marking techniques (). The amount of isotope required to mark a fish delivered during vaccination is between 0.01 and 0.001 μg. G -1 fish for Ba isotopes and 1 μg. G -1 fish weight for Sr isotopes. Typically, Atlantic salmon parr average 40 g at vaccination time, meaning the total amount of enriched isotope required for marking ranges between 0.04 and 1.6 μg of Ba isotope per fish depending on the fingerprint combination used () and 40 μg per fish for a single Sr isotope. The optimal isotope concentration delivered during vaccination used in the present study is lower for Ba, but higher for Sr when compared with a larval immersion study on Murray cod ( Maccullochella peelii) [], which used the equivalent of 2 to 3 μg of Ba, and 5 to 8 μg of Sr per individual. Hence, for marking during vaccination at the concentrations we have demonstrated, isotopes of Ba are the most suitable and cost effective given current prices (isotope source = Oak Ridge National Laboratory; ), which range from $US 0.0002 to $US 0.02 per fish depending on the combination of Ba isotopes used. Concentrations at which Sr isotopes are effective render them less economically viable for delivery during vaccination (from $US 0.48 to $US 1.72 per fish depending on the combination of Sr isotope). Sr isotopes may be more cost effective for marking using other techniques, such as immersion with osmotic induction (e.g. De Braux et al. Ba isotope otolith fingerprinting. In addition to marking, there is an additional analysis cost of identifying marked fish. The cost per sample to analyse based on this study this was between $US 15 to 20 per fish. Hence, analytical costs for monitoring for compliance of correct application of marks at an individual fish farm, or an assessment of a group of fish thought to have come from an escape event could be done for as little as $US 300 to 400 (N = 20 fish) due to the high accuracy (99.94%) of the enriched stable isotope marking method. Industry-scale marking with isotopes of Ba For a mass marking technique to work at an industry scale and to be successful in driving compliance, escaped marked fish need to be traceable to a point that assigns accountability for an escape event. Stable isotope fingerprint marking could ensure accountability if each company within major producer nations was assigned its own unique marker combination. Mass marking would also allow for an accurate assessment of the level of integration between escapees and wild fish. For example, the Scottish salmon industry produces 180 000 tonnes of salmon a year from 13 main companies, hence, using the 13 cheapest of a possible 15 Ba marker combinations () would enable each company in Scotland to have its own unique salmon identification mark at a median cost of $US 0.012 per fish. Canadian salmon farming produces 100 000 tonnes of salmon per year from 6 main companies, meaning only the 6 cheapest of a possible 15 Ba marker combinations are required to mark at the company level for the Canadian salmon industry at a median cost of $US 0.0008 per fish. The Faroe Islands produces 61 000 tonnes of salmon per year from just 3 companies, requiring just 3 unique Ba markers at a median cost of $US 0.0003 per fish. Currently the biggest producer of salmon worldwide is Norway, with an estimated annual production of 1.28 million tonnes per year from 83 companies. 15 Ba markers are insufficient to assign a unique mark at the company level. More markers would need to be tested, for example 132Ba and 131Ba to ensure enough unique combinations. However, recent legalisation in Norway now allows for greater industry amalgamation; individual stakeholders may now obtain up to a 40% share of Norway’s total production (increased from 25%). Hence, if the total number of companies is reduced in the future to less than 60 through the amalgamation of smaller industry partners, marking with Ba and Sr stable isotopes during vaccination would become viable for the Norwegian salmon industry. An alternate solution that could produce hundreds of marks using only the most cost-effective barium isotopes would be to combine marking during vaccination at the parr stage (this study) with marking via immersion during the larval stage [] to produce multiple fingerprint marks in different parts of the otolith. Although confirmation that this approach doesn’t cause cross-contamination of marks is required, it would allow for a possible 255 unique fingerprints. We have shown that stable isotope fingerprint marking during vaccination using isotopes of Ba is an economically viable method for uniquely identifying fish to the company level for the major salmon production regions worldwide. Importantly, the marks are permanent, unique, relatively easy to detect, and can be incorporated into standard salmon hatchery production with no additional production or welfare issues for fish grown to full commercial production size. |
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April 2018
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