# Role reversal in roaches/fish due to waste water treatment



## mantisdragon91 (Jun 2, 2010)

*My fiancee just forwarded me this article the abstract of which is copied below. It has some interesting/scary implications for other species(ie amphibians as well):*

Anke Lange*†, Gregory C. Paull†, Patrick B. Hamilton†, Taisen Iguchi‡, and Charles R. Tyler*† 
† University of Exeter, Biosciences, College of Life & Environmental Sciences, Exeter EX4 4PS, United Kingdom
‡ Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, and Department of Basic Biology, School of Life Science, Graduate University for Advanced Studies, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan
Environ. Sci. Technol., 2011, 45 (4), pp 1673–1679
Publication Date (Web): January 5, 2011
Copyright © 2011 American Chemical Society
*Phone: +44.1392.269298 (A.L.); +44.1392.264450 (C.R.T.); Fax: +44.1392.263700; E-mail: [email protected] (A.L.);[email protected] (C.R.T.).
Abstract
Feminized responses are widespread in wild populations of roach, Rutilus rutilus, living in UK rivers, and some of these responses have been shown to arise as a consequence of exposure to wastewater treatment works (WwTW) effluent discharges and the endocrine disrupting chemicals (EDCs) they contain. The causation of the ovotestis condition in wild roach, however, has yet to be established. Furthermore, the impact of long-term exposure to WwTW effluents on the reproductive fitness of wild fish populations is not known, and this information is crucial for population level effect assessments. We undertook a chronic exposure of roach to a treated estrogenic wastewater effluent for up to 3.5 years to assess principally for effects on subsequent reproductive fitness, as determined through parentage analysis on offspring from a competitive breeding study. In generating the fish for the breeding study we found that exposure to full strength WwTW effluent until sexual maturity resulted in sex reversal in almost all males in the population; 98% of the exposed fish were phenotypic females, containing ovaries. Furthermore, fish exposed to a 50% dilution of WwTW effluent contained ovotestis (21% of the male roach) that was absent from the control population. In competitive breeding studies, and applying DNA microsatellites to assess parentage, we show that presumptive females exposed to sexual maturity to WwTW effluent bred normally, albeit in the absence of nonexposed females, but putative sex-reversed males breeding as females contributed poorly, if at all, in a breeding population, depending on the competition. These novel findings on sex reversal add a new dimension for impact assessments of exposure to WwTW effluents on fish populations.


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## Ed (Sep 19, 2004)

This is also occuring in the US.. search intersex fish USA.. and look through the posts.. 

ScienceDirect - Marine Environmental Research : Intersexes in Mississippi River shovelnose sturgeon sampled below Saint Louis, Missouri, USA


Ed


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## earthfrog (May 18, 2008)

Many of said chemicals are components not just in pesticides but also our hygeine items, perfumes, medications. Many of these cannot be removed from the water supply effectively---take triclosan, an antibacterial agent used in soaps/sanitizers. It comes right back to you in your faucet---and to the frogs/fish/what-have-you. 
If you call your poison control hotline, they will tell you NOT to pour meds down the drain---instead, pour them into a bag of sand or kitty litter and throw them away. You may save a species.


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## Ed (Sep 19, 2004)

Actually one of the possible major causes of intersex fish are the result of human birthcontrol pills as the excess hormones enter the waste stream.. which is not set up to deal with these sorts of waste products and then they end up into the water supply.. 

Ed


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## earthfrog (May 18, 2008)

Ed said:


> Actually one of the possible major causes of intersex fish are the result of human birthcontrol pills as the excess hormones enter the waste stream.. which is not set up to deal with these sorts of waste products and then they end up into the water supply..
> 
> Ed


Not to mention the fact that the hormones stay in humans for up to 10 yrs after administration...makes you wonder what is excreted in the urine and how it affects the mind of those who take it---perhaps this is why women are characterized as illogical, moody and hormonal--the birth control makes their minds think they're pregnant perpetually, and this might affect their image in western cultures...
Ah, it seems the convenience of humans is the bane of the natural world.


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## Ed (Sep 19, 2004)

earthfrog said:


> Not to mention the fact that the hormones stay in humans for up to 10 yrs after administration...makes you wonder what is excreted in the urine and how it affects the mind of those who take it---perhaps this is why women are characterized as illogical, moody and hormonal--the birth control makes their minds think they're pregnant perpetually, and this might affect their image in western cultures...
> Ah, it seems the convenience of humans is the bane of the natural world.


I'm not buying the ten year time line.. 

Ed


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## earthfrog (May 18, 2008)

Ed said:


> I'm not buying the ten year time line..
> 
> Ed


My bad---that's the non-hormonal IUD that releases copper into the uterus. The hormone-releasing IUD lasts 5 years at a time. 
I don't know of any studies that say how long hormones linger in the body per se.


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## earthfrog (May 18, 2008)

The same thing happens to frogs---I wonder if some frogs with more extreme cases of endocrine disruption are those noted with the 'eternal tadpole syndrome' discussed here from time to time. Noting that, I try to stay away from certain plastics such as polycarbonates in general. Thankfully the food storage and baby care industries are now making bisphenol-A free products. 
Could developmental delay be in part due to polycarbonate plastic breeding trays and petri dishes? Could it be more than a feeding/nutrition issue at times?

I wish there were a way to observe the fluctuation of sex-ratios in breeding populations of the general frogging community---perhaps TWI could send out a survey of frog sexes among its breeders, and reassess that every few months for a year or two. 

Abstract from a frog study I found on ScienceDirect---if someone wants to post population effect info here, be my guest.



> During the transformation from larval tadpole to juvenile frog, there are critical periods of metamorphic development and sex differentiation that may be particularly sensitive to endocrine disruption. The aim of the present study was to identify sensitive developmental periods for estrogenic endocrine disruption in the northern leopard frog (Rana pipiens) using short, targeted exposures to the synthetic estrogen, ethinylestradiol (EE2). Post-hatch tadpoles (Gosner stage 27) were exposed over five distinct periods of metamorphosis: early (stage 27–30), mid (stage 30–36), early and mid (stage 27–36), late (stage 36–42), and the entire metamorphic period (chronic; stage 27–42). For each period, animals were sampled immediately following the EE2 exposure and at metamorphic climax (stage 42). The effects of EE2 on metamorphic development and sex differentiation were assessed through measures of length, weight, developmental stage, days to metamorphosis, sex ratios and incidence of gonadal intersex. Our results show that tadpoles exposed to EE2 during mid-metamorphosis were developmentally delayed immediately following exposure and took 2 weeks longer to reach metamorphic climax. In the unexposed groups, there was low proportion (0.15) of intersex tadpoles at stage 30 and gonads appeared to be morphologically distinct (male and female) in all individuals by stage 36. Tadpoles exposed early in development displayed a strong female-biased sex ratio compared to the controls. Moreover, these effects were also seen at metamorphic climax, approximately 2–3 months after the exposure period, demonstrating that transient early life-stage exposure to estrogen can induce effects on the reproductive organs that persist into the beginning of adult life-stages.


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## Ed (Sep 19, 2004)

earthfrog said:


> My bad---that's the non-hormonal IUD that releases copper into the uterus. The hormone-releasing IUD lasts 5 years at a time.
> I don't know of any studies that say how long hormones linger in the body per se.


The body has a pretty effective methods of controlling hormone levels using feedback, and excretion.. some of the studies show drops within hours.. and excretion in the urine and fecal route (there were studies with people using radioisotope labeled hormones). 

Ed


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## Ed (Sep 19, 2004)

earthfrog said:


> The same thing happens to frogs---I wonder if some frogs with more extreme cases of endocrine disruption are those noted with the 'eternal tadpole syndrome' discussed here from time to time. Noting that, I try to stay away from certain plastics such as polycarbonates in general. Thankfully the food storage and baby care industries are now making bisphenol-A free products.
> Could developmental delay be in part due to polycarbonate plastic breeding trays and petri dishes? Could it be more than a feeding/nutrition issue at times?
> 
> I wish there were a way to observe the fluctuation of sex-ratios in breeding populations of the general frogging community---perhaps TWI could send out a survey of frog sexes among its breeders, and reassess that every few months for a year or two.
> ...


I spoke to a exotic pathologist who was interested in it.. in what she had seen, those were usually cases where there was some abnormality of pituitary gland. (when she could actually get lucky and get it on a histopathology slide).


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## earthfrog (May 18, 2008)

Ed said:


> I spoke to a exotic pathologist who was interested in it.. in what she had seen, those were usually cases where there was some abnormality of pituitary gland. (when she could actually get lucky and get it on a histopathology slide).


I have noticed that a prenatal disruption in development or 'early programming' or development of systems can be caused by man-made drug analogs---one way this is affected in humans is the addition of synthetic oxytocin during the labor process. Some humans react by developing the inability to produce their own oxytocin--the so-called 'love hormone' that allows the brain to process feelings of empathy and care for others. The current rate of autism now is 1 in 100, with it being 1 in 70 for males.

I think it has been shown that EDCs introduced during prenatal development can alter the genetic structure of an individual, also based on biofeedback--the body getting the wrong message and constructing itself incorrectly as a result of the harmful hormones/chemicals present. 

This is from Powered by Google Docs



> In those cases in which disruption is directed
> toward programming of a function, e.g., reproductive
> health, this may interfere with early life organization, fol-
> lowed by a latent period, after which the function becomes
> ...


Now, this is for humans, but surely there could be a similar affect in anurans given the increased risk due to the 'external womb' of the egg and direct contact with synthetic estrogen in plastics...


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## Roadrunner (Mar 6, 2004)

Aren't they the reason for the low sperm counts found in Fla and the south?



Ed said:


> The body has a pretty effective methods of controlling hormone levels using feedback, and excretion.. some of the studies show drops within hours.. and excretion in the urine and fecal route (there were studies with people using radioisotope labeled hormones).
> 
> Ed


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## earthfrog (May 18, 2008)

Ed said:


> I spoke to a exotic pathologist who was interested in it.. in what she had seen, those were usually cases where there was some abnormality of pituitary gland. (when she could actually get lucky and get it on a histopathology slide).


From page 29 of previous paper:



> 2. EDC effects on sexually dimorphic brain regions and
> behavior
> 
> The regions of the hypothalamus that control repro-
> ...


From the same paper:


> B. Hypothalamic-pituitary-adrenal (HPA) effects of EDCs
> 
> As articulated by Harvey et al. (303), “The adrenal is
> arguably the neglected organ in endocrine toxicology, and
> ...


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## earthfrog (May 18, 2008)

frogfarm said:


> Aren't they the reason for the low sperm counts found in Fla and the south?


No that's due to the introduction of pythons and increased consumption of pork rinds and Miller Lite. 

I dunno, actually.


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## Ed (Sep 19, 2004)

frogfarm said:


> Aren't they the reason for the low sperm counts found in Fla and the south?


 
It has to pass through the blood stream to get to the kidneys to get to the urine... 

There are a number of endocrine disrupters ending up in the water supply 

check out the following full access papers 

http://digitalcommons.unl.edu/cgi/viewcontent.cgi?article=1064&context=usgsstaffpub

http://www.neiwpcc.org/ppcpconference/ppcp-docs/PaulStackelberg/STOTEN_Stackelberg_etal.pdf

USGS OFR-02-94 Water-Quality Data for Pharmaceuticals, Hormones, and Other Organic Wastewater Contaminants in U.S. Streams, 1999-2000


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## Ed (Sep 19, 2004)

earthfrog said:


> Now, this is for humans, but surely there could be a similar affect in anurans given the increased risk due to the 'external womb' of the egg and direct contact with synthetic estrogen in plastics...


With tadpoles the exposure would have to be during ovulation or while the egg is in the early developmental stages. In dendrobatids, there isn't a lot of exposure to outside water during that period.. (look at the use of filtered/ro or other sourced water in egg development). 

The reporting of "eternal" tadpoles is fairly rare and this needs to be compared to the number of tadpoles in the whole captive population...
*As a best guess*.. I think we are looking at spontaneous disruptions more than anything.. if we were seeing issues resulting from endocrine disruptors then the people who reported seeing one tadpole should report seeing a lot more of them.. (and they probably would given the level of interest we see when those tadpoles are reported). 


Ed


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## earthfrog (May 18, 2008)

Ed said:


> With tadpoles the exposure would have to be during ovulation or while the egg is in the early developmental stages. In dendrobatids, there isn't a lot of exposure to outside water during that period.. (look at the use of filtered/ro or other sourced water in egg development).


Yes, I was referring to eggs that were pulled and left to develop on a polycarbonate slide, which would have BPA as a synthetic estrogen. Perhaps it's a moot point, but people are also using tap water these days, and if they are it is filtered, but if it's just a high-grade carbon filter it may not remove these chemicals in sufficient quantities. 



> The reporting of "eternal" tadpoles is fairly rare and this needs to be compared to the number of tadpoles in the whole captive population...
> *As a best guess*.. I think we are looking at spontaneous disruptions more than anything.. if we were seeing issues resulting from endocrine disruptors then the people who reported seeing one tadpole should report seeing a lot more of them.. (and they probably would given the level of interest we see when those tadpoles are reported).
> 
> Ed


There are also varying degrees of damage that designate those labeled 'mentally challenged', and they manifest in different ways based on the genetic makeup of the individual---call it 'mutation susceptibility' if you like. One person may not show signs of mental delay---it may be very subtle---while another could exhibit serious symptoms---that is, not everyone responds equivalently to the same influence. This is also why some humans have allergic reactions to medications while others are not much affected at all. 

For instance, perhaps a more genetically-susceptible tadpole who is introduced to EDCs develops 'eternal tadpole syndrome', while a stronger one exposed to the same influence is able to overcome it to some degree but yet end up as the 'non-desirable vinclozolin rat' mate noted in the paper...


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## Ed (Sep 19, 2004)

earthfrog said:


> Yes, I was referring to eggs that were pulled and left to develop on a polycarbonate slide, which would have BPA as a synthetic estrogen. Perhaps it's a moot point, but people are also using tap water these days, and if they are it is filtered, but if it's just a high-grade carbon filter it may not remove these chemicals in sufficient quantities.
> 
> 
> 
> ...


When you say slide do you mean petri-dish (which are not polycarbonate, they are a stable polystyrene..) ? 

The problem with this is (as shown with zebra fish) is that you don't see just one effect.. you would see tail kinking..or spinal abnormalities or pigmentation issues along with the rare eternal tadpole... not just the eternal tadpole.. we don't hear about these sorts of morphological issues with hatching tadpoles... 

Ed


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## earthfrog (May 18, 2008)

Ed said:


> When you say slide do you mean petri-dish (which are not polycarbonate, they are a stable polystyrene..) ?


Yes, that's what I meant. Visually I can't tell the difference. I'm glad to hear that...now what about the 'critter keeper boxes' folks use...and the toolbox compartments for raising tads as well...the endocrine system is still developing then, as is the nervous system, no? I think that is still being researched since that system is so intricate and the effects on the developing systems hasn't been fully determined. 
Just did a quick look at polystyrene...what about it leaching styrene over time? Isn't that worth a look? Ever notice that plastic taste in a foam cup when you leave plain water in it for a day or so? That's what I mean...



> The problem with this is (as shown with zebra fish) is that you don't see just one effect.. you would see tail kinking..or spinal abnormalities or pigmentation issues along with the rare eternal tadpole... not just the eternal tadpole.. we don't hear about these sorts of morphological issues with hatching tadpoles...
> 
> Ed


My point was also that you may not _see_ any effects readily attributable to this one causative agent, but when mutation does occur, you can try and figure out what happened. 
What about melanistic froglets? SLS? Gills present on only one side, or bloating of fully-developed before hatching? Front legs completely absent in cases? I see abnormalites come up in the Health and Disease section from time to time...could EDCs contribute to these conditions we have not fully researched yet? Is it possible we have only attributed them to basic husbandry issues such as nutrition but not really factored these pollutants into the big equation?

Also, some things may not be possible to directly attribute to a hormone agent, such as frogs mating behavior and egg viability, since there are sometimes other factors involved. I think the endocrine disrupting chemicals have been underemphasized in general, or just not talked about at length.


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## earthfrog (May 18, 2008)

I'll have to get back to this later...but what about the use of these polystyrene dishes in heated incubators? Acidic incubation water may facilitate the leaching of styrene from polystyrene into the dishes with the developing embryos as well.
Polystyrene is synonymous with Styrofoam, and does readily leach out...so I don't know how stable it really is...relatively stable to other plastics, maybe? But, a big maybe, I think.

Also...styrene, a by-product of this polystyrene, is also an endocrine disrupting chemical (EDC). 

ScienceDirect - Biochemical and Biophysical Research Communications : Neonatal exposure to endocrine disruptors suppresses juvenile testis weight and steroidogenesis but spermatogenesis is considerably restored during puberty


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## Ed (Sep 19, 2004)

earthfrog said:


> I'll have to get back to this later...but what about the use of these polystyrene dishes in heated incubators? Acidic incubation water may facilitate the leaching of styrene from polystyrene into the dishes with the developing embryos as well.
> Polystyrene is synonymous with Styrofoam, and does readily leach out...so I don't know how stable it really is...relatively stable to other plastics, maybe? But, a big maybe, I think.
> 
> Also...styrene, a by-product of this polystyrene, is also an endocrine disrupting chemical (EDC).
> ...


Not all polystyrenes are equally stable.. high quality polystyrene plastics (like petri dishes) do not leach styrene unless you are doing something crazy with them like melting them on an open flame.. you cannot compare stryofoam with high quality polystyrene...


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## earthfrog (May 18, 2008)

Ed said:


> Not all polystyrenes are equally stable.. high quality polystyrene plastics (like petri dishes) do not leach styrene unless you are doing something crazy with them like melting them on an open flame.. you cannot compare stryofoam with high quality polystyrene...


That's good to hear. 
How long is such a petri dish good for before it begins to degrade? And would water temps of 120 degrees, such as that in a dishwasher, cause leaching in a similar way to polycarbonate?
I think we determined neoprene was good for about 10 yrs before replacement, but I didn't remember reading about the life of high-density polystyrene.

This is slightly unrelated, but has anyone designed and patented a glass, multi-compartmented tadpole-rearing station? If not---go for it and make a buck or two. 
Glass doesn't break down like plastic does over the years.


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## Ed (Sep 19, 2004)

I don't have the energy to wade through the multitude literature search on petri dish stability at different temperatures. If it is a concern.. I would suggest investing in glass petri dishes or even small culture dishes as those won't have any issues... 


Ed


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## earthfrog (May 18, 2008)

Ed said:


> I don't have the energy to wade through the multitude literature search on petri dish stability at different temperatures. If it is a concern.. I would suggest investing in glass petri dishes or even small culture dishes as those won't have any issues...
> 
> 
> Ed


I use glass jars for my tads and eggs. Later on I will do some more fishing around for longevity/stability info on polystyrene. You rest up.


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## earthfrog (May 18, 2008)

While I was looking for plastic info, I found this---I think it applies since plastic residuals end up in our water and do not readily break down, so there are implications for wildlife as well as humans:



> Plastic Drinking Receptacles and Plastics Food Containers
> 
> In 1993, at Stanford University School of Medicine, Dave Feldman, professor of medicine ran an experiment with a yeast estrogen protein that sticks to estrogen. Feldman discovered that polycarbonate bottles used for drinking water contained bisphenol-A. The polycarbonate lab flasks used to sterilize the water used in their experiments contained Bisphenol-A. Bispheol-A nicely bound to the estrogen protein found in the yeast. The same polycarbonate plastic is routinely used for the giant jugs used to ship water.
> The manufacturer knew that the bottles would leach particularly when exposed to high temperatures and caustic cleaners. The manufacturer designed a washing procedure that they thought would solve BPA leeching. However, the Stanford researchers discovered that the manufacturer could not detect Bisphenol-A samples sent from their lab. Those particular samples that were responsible for replication of estrogen responsive breast cancer cells. Why? There was a detection lower limit in the manufacturer's lab equipment. The Stanford research group discovered 2-5 parts per billion of bisphenol-A was strong enough to help the breast cancer cells to replicate and multiply.
> ...


Stanford Q&A: David Feldman on risk of bisphenol A in plastic bottles - Office of Communications & Public Affairs - Stanford University School of Medicine

This makes me wonder if the test equipment used by the manufacturers of other plastics are sensitive enough to detect leaching, esp. of styrene...still digging....


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