# What is everyones stand on chytrid and testing??



## kristy55303 (Apr 27, 2008)

ok...i am polling to see what is everyones stand on chytrid and properly testing. For me, i believe it may be atleast important to test non cb PDF's for sure( i.e. imports /wc/farm raised), and maybe to run a few tests throughout our collection to see if we do in fact have chytrid among ours. what are your thoughts, arguments for, and against? kristy


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## dwdragon (Aug 14, 2008)

I don't currently have frogs but I voted I think it's beneficial to test and will be testing any frogs I get both fecal and chytrid while in quarentine.

From what I have read there is just not enough information as to if a frog can have chytrid and not die from it or have symptoms but if this is possible then it could infect others that cannot withstand it.


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## kyle1745 (Feb 15, 2004)

I removed the public showing of who posted what. You may get a better sample that way.


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## kristy55303 (Apr 27, 2008)

cool kyel my first poll whatever you think will get better attention to this thread is great with me

yes i think we should chytrid test. i wonder if a company would allow dendroboard 2 test our darts anonymously so we could test our frogs at a discounted price if we had enough interest and were going to test more than they would say on average receive? just an idea. and maybe have the results emailed to each individual? could be a bad idea.....but thinking from a prospective of knowing individually if we in fact do have chytrid in our collections. 

we fecal. why not test for the most devastating disease out there? if caught early enough there are treatments now i have read. just my thoughts. kind of on a conservative level i guess kristy


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## Dancing frogs (Feb 20, 2004)

I voted 1,2, and 4.

I would also like to see/have available, a test for soils or wood.


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## afterdark (Jan 16, 2007)

I voted the same as Brian. While I think it's important to test - my discovery was that the cost was a bit prohibitive.


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## Roadrunner (Mar 6, 2004)

Man, testing for chytrid is cheaper than fecals!
A test kit, which can hold 8 swabs from different animals is only $25 + 10 for the supplies. Cheaper if you buy the alcohol and swabs and packaging yourself. This means you can swab 8 frogs from 8 tanks and get them pooled into 1 test to see if anything has it. Then you can test individually or treat them all. That`s less than $5/frog or tank tested.


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## kristy55303 (Apr 27, 2008)

frogfarm said:


> Man, testing for chytrid is cheaper than fecals!
> A test kit, which can hold 8 swabs from different animals is only $25 + 10 for the supplies. Cheaper if you buy the alcohol and swabs and packaging yourself. This means you can swab 8 frogs from 8 tanks and get them pooled into 1 test to see if anything has it. Then you can test individually or treat them all. That`s less than $5/frog or tank tested.


absolutely correct. i was a little confused on pooled samples. and individual testing myself. but if you just want to know it is in your collection, you can pool and get the cheaper end of the deal, if you want to know which frogs individually are infected than i think it would be more. but hey, atleast "knowing" it is in your collection and treating is better than not imo. kristy


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## kristy55303 (Apr 27, 2008)

kyle was kind enough to give me this link. at the bottom are testing sites for those that do not know where to get the tests done. as i was informed, you need to test the frog itself as it is more concentrated but i had the same opinion on whether or not or rather why we cant just swab our tanks because chtrid i thought would be in the tanks themselves. whether soil can or can not be tested i dont know and maybe contacting these companies at the bottom of this link kyle gave me which is excellent will get you an answer Chytrid Fungus
kristy


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## Dane (Aug 19, 2004)

I have a question; what is everyones stance on the failure rate of the tests? Pisces lists it at 3 out of 15. Has anyone had a false positive/negative? I think that false + is more common due to the amplification of present Bd DNA (possibly due to cross contamination). I've been sending in 2 samples for each group of frogs that I want to test to decrease the possibility of a false result. Does anyone think that there is merit in that?


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## kristy55303 (Apr 27, 2008)

Dane said:


> I have a question; what is everyones stance on the failure rate of the tests? Pisces lists it at 3 out of 15. Has anyone had a false positive/negative? I think that false + is more common due to the amplification of present Bd DNA (possibly due to cross contamination). I've been sending in 2 samples for each group of frogs that I want to test to decrease the possibility of a false result. Does anyone think that there is merit in that?


good thinking dane. 3 out of 15 failure rate is high. i think there is merit in sending two samples in. makes perfect sense to decrease the failure rate. just my opinion. kristy


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## kristy55303 (Apr 27, 2008)

now thinking about it .....wouldnt three tests like asn protocols for fecaling be more reliable? say you got one neg. and one pos. from the same frogs. that third one i would think might be more beneficial then? kristy


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## sports_doc (Nov 15, 2004)

frogfarm said:


> Man, testing for chytrid is cheaper than fecals!
> A test kit, which can hold 8 swabs from different animals is only $25 + 10 for the supplies. Cheaper if you buy the alcohol and swabs and packaging yourself. This means you can swab 8 frogs from 8 tanks and get them pooled into 1 test to see if anything has it. Then you can test individually or treat them all. That`s less than $5/frog or tank tested.


Well..... actually.... You will have to buy a kit of 10, which is 100$ with shipping. True, that is only 10$ each for the 'pooled' sample kit.

Then USPS Express overnight, small box. 25$.

37$ to test the pooled sample. More if you send individual samples that they 'pool' themselves.

20% surcharge if you dont use a supplied kit tube, and 250$ if you 'homemade' kit spills in their machine [so buy the true kit in other words]

That all said...I'm in favor of testing.

Shawn


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## afterdark (Jan 16, 2007)

Bingo! Thanks Shawn - my costs would actually be higher since I would have to have everything shipped to Canada from the US.
I would love to test my frogs to be sure they are not infected, but at the moment I can't justify the cost. I am planning to bring in 5-8 more frogs in the next month or so from Understory, so with more $$ invested in my collection I may be more inclined to shell out for the testing.

Does anyone know if there are (or will be) any labs in Canada offering the test?

Cheers,


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## kristy55303 (Apr 27, 2008)

i am looking to test definately. in fact i received a shipment i am very concerned with today and when you do get larger collections, the potential for it being an issue and risk increases. these darts i received will be quarantined and even serviced last in i wish a whole different facilty! grrr. in saying that they are not even going into my quarantine room, i will have to place them somewhere in a whole separate room. great post sports doc. i am so in favor of it. I do know a specific vet that tests it and also have called some companies and you are right, we should really do it the right way, avoid error, and buy the kits, do as instructions say to avoid these possibilities of cross contamination etc. as for canada i dont know but i can look into it. i do have more of a list now. i'll just need to check into it. kristy


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## kristy55303 (Apr 27, 2008)

afterdark said:


> Bingo! Thanks Shawn - my costs would actually be higher since I would have to have everything shipped to Canada from the US.
> I would love to test my frogs to be sure they are not infected, but at the moment I can't justify the cost. I am planning to bring in 5-8 more frogs in the next month or so from Understory, so with more $$ invested in my collection I may be more inclined to shell out for the testing.
> 
> Does anyone know if there are (or will be) any labs in Canada offering the test?
> ...



my advice to anyone bringing in more frogs is this, and by no means getting down on you, we have a responsibility to our collection, whether it be darts or what not. if we can not IMO justify the cost of testing our darts and taking for responsibility for them, then we shouldn't be buying them in the first place and save that money to test the ones we do have. in sayting this, i will look into the canada PCR testing for ya as soon as i can. kristy


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## afterdark (Jan 16, 2007)

Thanks very much for looking into it Kristy! I think there would be a number of people in Canada interested.


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## nish07 (Mar 16, 2008)

If you do two tests (better at different times) your odds go from 20% false negative to 4% false negative chance. If you pool swabs of a collection housed together, you multiply .2 by number of frogs to get your chance of a false negative within the sample (minus 'possible' error caused by testing pooled samples which are probably negligible within the same type of method <swab or skin patch, etc...>). 

This is assuming environmental effects don't change the amount of chytrid zoospores on the frog's skin. If they do and it's significant with testing then it 'might' be good to drop the temps down a tad so spores can reproduce before you sample. (I don't know the time it takes for this to happen).

-Nish




Dane said:


> I have a question; what is everyones stance on the failure rate of the tests? Pisces lists it at 3 out of 15. Has anyone had a false positive/negative? I think that false + is more common due to the amplification of present Bd DNA (possibly due to cross contamination). I've been sending in 2 samples for each group of frogs that I want to test to decrease the possibility of a false result. Does anyone think that there is merit in that?


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## kristy55303 (Apr 27, 2008)

yep, i'll look into it afteredark, cant make any guarntees but i'll try. nish great post and good example of the odds and environmental factors involved. kristy


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## Roadrunner (Mar 6, 2004)

sports_doc said:


> Well..... actually.... You will have to buy a kit of 10, which is 100$ with shipping. True, that is only 10$ each for the 'pooled' sample kit.
> 
> Then USPS Express overnight, small box. 25$.
> 
> ...


Unless you bought individual kits from me when I was selling them at the shows for $10ea. which means Marcus since he`s the only one who bought any. And I have 20 or so left. So, much more expensive for kits if you think the people at IAD and other frog shows would care to get their animals tested and buy some to sell to recoop the cost and end up w/ dusty test kits.
If they are $37ea from Pisces then prices went up from last summer when they were $25ea. 
This must be a new thing to have to send them overnite also since mine was 2 day or just priority, can`t remember which.
Of course if they have to pool them it`s more, I mentioned blanket testing in groups of 8, which is what the test kits are set up for, 8 swabs in one vial. You wouldn`t have them pool them unless you expected it in some animals and wanted to have individual tests done to find the exact culprit, if present. I thought we were talking a cheap, is it there, test.


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## nish07 (Mar 16, 2008)

I don't think they need to be overnight expressed. Even if the chytrid dies, the dna remains. That's all they need.


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## nish07 (Mar 16, 2008)

You can purchase all of the "recommended" supplies to pool samples from Fisher and the cost will end up being around 75 bucks for enough supplies to do at least 25 pooled tests and have left over alcohol and swabs. If you have access to pure grain alcohol (everclear), you can make a 70% v/v solution with that and it'll be acceptable (and drastically reduce the price).

You can purchase separate 25 tube blocks of centrifuge tubes directly from fisher as well so no need to order a box/case.

Swabs are cheap.

Just double ziploc bag the tubes and if they break open in shipping (somehow), they'll notify you and won't charge you the clean up charge.

-Nish


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## TDK (Oct 6, 2007)

So, are there visible indications of chytrid and if so someone please post photos.


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## nish07 (Mar 16, 2008)

Search out my post "Leucs with Chytrid" in health and disease thread. Realize that the leuc in the pic probably would have died if I hadn't been very careful with it and babied it until I did the lamisil treatments. You can see the results of the lamisil treatments in the photos. The first pic (sick) was indicative of the health of the frog for a full 4-6 weeks before the treatment. The pic after the treatment (healthy) was 7-10 days after treatment. Other frogs in the group had it and looked fine but wouldn't eat.

The thread says it all, I think.

-Nish


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## kristy55303 (Apr 27, 2008)

nish showed a blatant very good symptom of it, but as i have heard chytrid can go unnoticed and die all of a sudden w/o any noticeable symptoms in dart frogs. they can have it for a couple of weeks and die up to years and it just depends on the immune system and also envoronmental factors i believe. kristy


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## kristy55303 (Apr 27, 2008)

is it ok for me or against the rules to post where we can get chytrid testing done and maybe investigate the costs for everyone etc. and post the telephone number of facilty and/or vet involved that does do PCR testing for chytrid publicly?


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## TDK (Oct 6, 2007)

There's a couple or more videos on youtube--the last golden forg 1 and 2 also some testing video.


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## kyle1745 (Feb 15, 2004)

Yes it is allowed and it was posted in this thread:
http://www.dendroboard.com/forum/ge...someone-elaborate-signs-symptoms-chytrid.html


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## kristy55303 (Apr 27, 2008)

what about DVM's that do it?


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## nish07 (Mar 16, 2008)

I haven't seen another group that's currently doing pcr chytrid testing publicly but I hear they exist. Pisces molecular is the only one I know of. You need a lab that has a pcr machine (which is very expensive in terms of someone just picking one up for their office). All of the reagents would be very expensive as well unless purchased in large quantities. Until I hear otherwise, Pisces is where I'm going and I trust their ability.

Unfortunately, because of the time of year they're a bit backlogged. If you find someplace else, feel free to post it as it could be cheaper with less of a wait time but I get the feeling that it'd be an "I know a group of people who can and happen to do it but not for everyone" type thing.

-Nish

P.S. I've worked with PCR before and I could probably do it myself if I had access to a machine but the reagents (primers too) would end up costing me -far- more than sending it to Pisces.


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## kristy55303 (Apr 27, 2008)

reason i asked is i happen to know a dvm that does have the ability to do it. wonder if the time it takes to get test results back then would be sooner? kisty


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## nish07 (Mar 16, 2008)

It's up to the individual and their workload. I would imagine sooner.

-Nish


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## kristy55303 (Apr 27, 2008)

that was pretty much my thought. kisty


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## Ed (Sep 19, 2004)

kristy55303 said:


> nish showed a blatant very good symptom of it, but as i have heard chytrid can go unnoticed and die all of a sudden w/o any noticeable symptoms in dart frogs. they can have it for a couple of weeks and die up to years and it just depends on the immune system and also envoronmental factors i believe. kristy




Not counting species that are known carriers (like bullfrogs) a frog can be infected with chytrid and not die as long as the temperatures stay above about 75 F.. or the frog can bask to increase its body temperature above 75 F... this can allow frogs to remain infected and infectious for years.. 

This is where some of the concerns come into play.. for example if you have chytrid in your collection and are discarding water and materials from the enclosure into your backyard then you are inoculating the local enviroment with it.


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## kristy55303 (Apr 27, 2008)

Ed said:


> Not counting species that are known carriers (like bullfrogs) a frog can be infected with chytrid and not die as long as the temperatures stay above about 75 F.. or the frog can bask to increase its body temperature above 75 F... this can allow frogs to remain infected and infectious for years..
> 
> This is where some of the concerns come into play.. for example if you have chytrid in your collection and are discarding water and materials from the enclosure into your backyard then you are inoculating the local enviroment with it.


absolutely true. i wish we could get some more symptoms that we should be aware of if it does show. obviously nish had one that showed. but what is the tally for all of the symptoms in case they do show even though many circumstances they may not. and we may be contributing, like you said to spreading chytrid into our local environment without even knowing it. i wish pices was open today or i could pool some samples. anybody know what rush testing costs are as well with pices? say i wanted the results next day? afterdark(member) found a place in canada and I encouraged him to also post where we could test if we live in canada. i know one dvm that also tests. i will post the doc with permission but not sure if that is against rules. kristy


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## nish07 (Mar 16, 2008)

In the absence of a local incinerator, I burned all my stuff (tubs etc...) in a garbage can. I could have gotten in trouble for it but after asking a local medical waste disposal company what to do, the guy just said burn it in a small fire. The closest operational incinerator was outside of the state and I imagine it would have cost me a fortune to have it properly disposed of. I bleached all of the solution used for treating the frogs before dumping it out.

-Nish


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## rozdaboff (Feb 27, 2005)

nish07 said:


> You can purchase all of the "recommended" supplies to pool samples from Fisher and the cost will end up being around 75 bucks for enough supplies to do at least 25 pooled tests and have left over alcohol and swabs. If you have access to pure grain alcohol (everclear), you can make a 70% v/v solution with that and it'll be acceptable (and drastically reduce the price).
> 
> You can purchase separate 25 tube blocks of centrifuge tubes directly from fisher as well so no need to order a box/case.
> 
> ...


I wanted to mention that you should make sure to contact the company/facility performing the test to find out their preferred testing supplies. IIRC - Pisces likes to have the samples stored in 70% ethanol, but I know of other facilities performing the testing that would prefer to receive the samples dry.



> I don't think they need to be overnight expressed. Even if the chytrid dies, the dna remains. That's all they need.


Again - get the recommendations from the person doing your testing, but the DNA should be stable for a long time at room temperature. One study identified that chytrid DNA was stable for 18 months when stored at room temperature or less.

Additionally - be careful when pooling samples. It was found that for one PCR assay, pooling more than 5 samples decreased the detection sensitivity of the assay.

The data that I am mentioning came from a very nice paper by Hyatt _et al._ (2007) that examined the different types of chytrid testing and variables of testing. For anyone interested - you can find the article open access by searching Google Scholar for "Diagnostic assays and sampling protocols for the detection of Batrachochytrium dendrobatidis".

>>> Edit - let's see if this link works: http://www.int-res.com/articles/feature/d073p175.pdf

One thing I have read that I don't believe is mentioned in the Hyatt paper is the types of swabs that have shown to be the best for sampling. Using polyester-tipped swabs with plastic handles is associated with better chytrid detection. So if you can, avoid cotton-tipped, wooden handle applicators.

Oz


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## nish07 (Mar 16, 2008)

You might want to contact Pisces on the swab choice. They recommend wooden swabs with cotton tips and while they might not mind, they seem to be pretty strict about what to use insofar as alcohol and centrifuge tube type.

-Nish



rozdaboff said:


> I wanted to mention that you should make sure to contact the company/facility performing the test to find out their preferred testing supplies. IIRC - Pisces likes to have the samples stored in 70% ethanol, but I know of other facilities performing the testing that would prefer to receive the samples dry.
> 
> 
> 
> ...


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## rozdaboff (Feb 27, 2005)

nish07 said:


> You might want to contact Pisces on the swab choice. They recommend wooden swabs with cotton tips and while they might not mind, they seem to be pretty strict about what to use insofar as alcohol and centrifuge tube type.
> 
> -Nish


Of course - any one who is using Pisces should send the samples as they want them; that was one of the points I was making in my last post - make sure your samples are in line with what they want. There isn't a blanket method for sample collection and submission.

But - I was pointing out that even small things such as the type of swab being used can have effects on the assay result. The testing of wild populations for the presence of chytrid is a hot-button issue in the literature - so there are constantly advances being made.


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## kristy55303 (Apr 27, 2008)

oz whats your stand on testing enclosures? wouldnt the chytrid be on say the walls of a viv or enclosure ? as grabbing say a thumbnail may be proven very difficult to test...and as i have proposed to twi, OFP, testing my waters in my pond for chytrid...possible right? or no? or just that swabbing is only method on darts and most reliable and enclosure testing cant be done? forgot if i asked this question before i started typing this. excuse me if i have and repeated myself again. kristy


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## rozdaboff (Feb 27, 2005)

I don't feel it would be reliable enough. 

Any test has its limits of detection. By testing enclosure walls, there is a chance that you may pickup the fungus or a zoospore - but there is also a very good chance (most likely a much greater chance) - that you won't. This type of testing will result in a lot of false negatives (a negative test result when there is a chytrid infection). When you swab the skin of the frog - you are directly collecting a sample the point of infection and replication (the skin). So - if infected, the chance of collecting chytrid/zoospore are much higher. This will result in fewer false negatives.

To detect chytrid in water - you are facing a similar situation. How much dilution is occurring? What is the ratio of chytrid to the volume of water? If there is ALOT of fungus or zoospore in the enclosure, then you might be able to detect it in a small water sample. But again, you are still facing an issue of dilution. 

As skin scraping is a simple and non-invasive sampling technique, given sampling of captive amphibians, it is a much better option than tank or water sampling.


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## kristy55303 (Apr 27, 2008)

got it thanks oz. imbedde now in my memory kristy


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## afterdark (Jan 16, 2007)

Hi all - I found a lab in Canada that does PCR testing. I'm waiting to hear back from them if they have a test for chytrid set up. In the meantime, here is the lab's info if anyone is interested:
Animal Health Centre - Charges for Laboratory Services

Cheers,


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## Ed (Sep 19, 2004)

rozdaboff said:


> I
> 
> To detect chytrid in water - you are facing a similar situation. How much dilution is occurring? What is the ratio of chytrid to the volume of water? If there is ALOT of fungus or zoospore in the enclosure, then you might be able to detect it in a small water sample. But again, you are still facing an issue of dilution.


When the pcr test originally came out, there was a lot of hope that you could simply wash down a frog and test the water unfortunately this did not work out as the available zoospores were too diluted and the frogs need to be swabbed to get any level of accuracy.


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## Ed (Sep 19, 2004)

nish07 said:


> In the absence of a local incinerator, I burned all my stuff (tubs etc...) in a garbage can. I could have gotten in trouble for it but after asking a local medical waste disposal company what to do, the guy just said burn it in a small fire. The closest operational incinerator was outside of the state and I imagine it would have cost me a fortune to have it properly disposed of. I bleached all of the solution used for treating the frogs before dumping it out.
> 
> -Nish


This level of scorched earth policy is probably not warranted.. At this time through drying of items that cannot be disinfected by bleach (wood, cork, substrate) kills chytrid (although there is speculation about a possible resting spore but to my knowledge, there has not been one reported in the literature to date). 

Also disposal into an approved landfill should be safe as the landfills should collect and process leachates so they can deal with toxic residues (like heavy metals etc). 

Ed


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