<%BANNER%>

Organochlorine Pesticide Reproductive Effects in Fathead Minnows (Pimephales promelas): Comparison of Embryo and Materna...

xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID E20101112_AAAAAY INGEST_TIME 2010-11-12T08:10:00Z PACKAGE UFE0014367_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES
FILE SIZE 925 DFID F20101112_AAAMKF ORIGIN DEPOSITOR PATH huge_d_Page_55.txt GLOBAL false PRESERVATION BIT MESSAGE_DIGEST ALGORITHM MD5
b9d98129de2eb2b05e1f16a515a363be
SHA-1
b59cf82a229d9123235c51331488b09223a56cb1
96213 F20101112_AAAMUA huge_d_Page_33.jp2
b5dfbbf29511c573dbdb819ea5d51023
7a945f0581f3c580c961feaa46e21bcd294b0284
52348 F20101112_AAAMWY huge_d_Page_38.jp2
307dd831349dec90b5e1ad6696559848
85a130c4e2ae213ce4fc9785779adf88a34cfaed
510105 F20101112_AAAMPD huge_d_Page_55.jp2
b2363cce439c8c1d934a268220597818
96e54bfcdec9c78c264168d4dba35be1aa8508fc
1053954 F20101112_AAAMKG huge_d_Page_66.tif
11b36129828913b51b7d7038598f94fc
6c9f39aac77910d48d1b9511d1e772f08ec6319e
6716 F20101112_AAAMUB huge_d_Page_63thm.jpg
d3784833e0c5200ab2588832bad2f296
0a10ed5795c1895fc660cf62a6d511513e47a5b7
71775 F20101112_AAAMWZ huge_d_Page_39.jp2
6949b7ba1d0083b8bb1db7aa345cff4f
f55a5657905fd2bc70b860a576fd983048938402
107372 F20101112_AAAMPE huge_d_Page_35.jp2
4bd01469d16148b939483636534ac6e6
f33c5c31ca4fecfd3a39439e8fec0023902b482d
49734 F20101112_AAAMKH huge_d_Page_25.pro
6ed5a8a7f2ee83f1939194da903513d6
9975dedc22464173f10b6d1e61f3207a82071497
2014 F20101112_AAAMUC huge_d_Page_50.txt
1f787be2ff8cd55d5158a92e5d579fcb
b78a96cd7af879ac618ad6a2a4a98fcdd4352460
53657 F20101112_AAAMPF huge_d_Page_51.jpg
1485a7322a4e5500f6c4623258619410
0d057a70788161ef6e3f70546a6ef367ae6f2e43
12455 F20101112_AAANBA huge_d_Page_66.QC.jpg
d0f854b056d3b0d86a3c5c0ce45f7e0d
9bffb3e644ae66af705ba8d18173356f929f7166
2000 F20101112_AAAMZA huge_d_Page_25.txt
f4ec6431b8c1d16f646534f106aa8e11
6b7573becab74768889128d88b9cce3c8a38bb84
23817 F20101112_AAAMKI huge_d_Page_44.QC.jpg
9e01cf2247bf36afde71e65616cd9801
30f5a802af67c64bb3a5d4ecadbbee3970792c04
6534 F20101112_AAAMUD huge_d_Page_16thm.jpg
9a815b4c6509f072d38b55e10d13a769
362708748d827f435d9f51ed01e5faa7cc8e8def
920 F20101112_AAAMPG huge_d_Page_30.txt
bf0ad46fdfd85a426425c823bb052658
5870ec92afd8841ebc33838be580b26d996573c9
77520 F20101112_AAANBB UFE0014367_00001.mets FULL
33344a3a61f3d7aaa3df5b47ba063b47
77d438e44326b0a6620637f5828faf3ab145cc1c
2035 F20101112_AAAMZB huge_d_Page_26.txt
8079b14a863b192d6f37af067ce1496e
05f774b7f6f12cb83e436f90be314524800e0691
73790 F20101112_AAAMKJ huge_d_Page_19.jpg
8ce5c5abe9211e9dcc6a6755677e21bb
854819bccab16ec3ef07f0bd2baf6d7edc85ae0b
72613 F20101112_AAAMUE huge_d_Page_45.jpg
597ace7c25cb3425ea8bf30b353a5538
49941032eeddde234b95e5b029a10ce1df0b6eef
11455 F20101112_AAAMPH huge_d_Page_06.QC.jpg
13c723df4b58aee6c43118235dad443d
fe2a0f6324c93326e642f5b28e9b87765529c1d1
1981 F20101112_AAAMZC huge_d_Page_29.txt
7fff4313aeccae2f32c4805d4aa7f473
2d84496be95e76cdec6ac8905af8395a8f7dbd54
101427 F20101112_AAAMKK huge_d_Page_60.jp2
75ca6b1348d4bf3fe532393bce56f82a
0979c75be4c0df8235de6d96f3d0e5b5751778ed
6533 F20101112_AAAMUF huge_d_Page_43thm.jpg
c441f222a87f09968a1e9dc16a4ed576
57d1996e16918329bb2f1c1dee152fccfd6a17d5
1939 F20101112_AAAMZD huge_d_Page_32.txt
23ad2dbbaca6ba4b23cf67b01e3da203
3f4f8cc5da1d9f387296b58ebd5802152a4683c1
69835 F20101112_AAAMUG huge_d_Page_12.jpg
6e73a2228e29a28d4af2070ed14df1dd
965e41748da533e2c341f61343464e61f4964d03
2050 F20101112_AAAMPI huge_d_Page_19.txt
6e993a3044a88c598733237cb482703b
74541d8161f3a358e35c664ecc70b8149244475b
2008 F20101112_AAAMKL huge_d_Page_44.txt
cd2781cd42235d8e99f037e91718d4f9
99d382e341ae70bc560dd74de2fccc6ac25ac177
1421 F20101112_AAAMZE huge_d_Page_39.txt
e01b3775f51e936051ec671fa01c126c
d66682862f848c5b19bc60d93a914056cf2d584f
45762 F20101112_AAAMUH huge_d_Page_40.pro
cca890aac40057c2b0dc29183577bedc
27b9469872862e102b21b8d0f7a700037ab9beb6
F20101112_AAAMPJ huge_d_Page_38.tif
3524de39b8f01a82698d70e858e3a6f3
077930d097ee85bcb1c27469f1af6d44724a343b
F20101112_AAAMKM huge_d_Page_03.tif
6e1e9ef86da69af41b42cd0c3e951bfd
c8219ccaeb381d88f5e4e5154159fccf36e9aae5
2027 F20101112_AAAMZF huge_d_Page_46.txt
13bc186bc98b0d0aa691db0d62f1ef8c
93bc606f936ca020c9d72447d52a96d6ba8ad343
2541 F20101112_AAAMUI huge_d_Page_01thm.jpg
f1b186d3eb97357385d9f94d51a9b53f
96a8338f2526c7f4f33358661f07c894aa5a8107
F20101112_AAAMPK huge_d_Page_12.tif
be25aead6eef231d11304fd2f3c921f3
d0cda42d5b12346e54e68825caef445895b80f0a
22953 F20101112_AAAMKN huge_d_Page_36.QC.jpg
174639c85f546672f521b80abf787434
ed5c893b11fe5bf946b5fc9cb47423575eae8a94
1991 F20101112_AAAMZG huge_d_Page_48.txt
0a244e34cceeb65d3cd4e5a2e8ea1acf
3461c604a710fb5b49667cde24ff9d1cbe987fae
F20101112_AAAMUJ huge_d_Page_34.tif
28954bf24aceda0dc45d2a3201dbd10c
22d20314b6754f1b512a12a3d2fe60df0460f2cb
108085 F20101112_AAAMPL huge_d_Page_25.jp2
6c02fa0af4cd9e8851cc3831896d62d8
36f0b63c79aeb3d35bb3f9eaa2824e71c9e729e2
19475 F20101112_AAAMKO huge_d_Page_09.QC.jpg
680e7c5cf97a8022ac8e86bccaa1cb6a
d1113c6a39ecee8afe5999658e2a78c9d454f2b5
1082 F20101112_AAAMZH huge_d_Page_52.txt
73527c7dd630a0026486db731139dc7c
a266693c574680ede68ba211a6cb3b687cb62d0f
94436 F20101112_AAAMUK huge_d_Page_63.jpg
53a85ee4663cdf592dab2807fc4b56ea
3b9cacff4f64f014e75ad34fd09e11e1c448768f
53091 F20101112_AAAMPM huge_d_Page_21.pro
3dcf716652886306837739e5ccc26644
5ce614f5245b78726db3bec1d0899db66416a536
107470 F20101112_AAAMKP huge_d_Page_18.jp2
f289d672b9c62c8f215b4ccf58281f29
a51edb9b6f523cb3fe72f9442a044db034f1fbe8
1470 F20101112_AAAMZI huge_d_Page_56.txt
c64a1d7ecba8a4fe6e143c7ef3d50b13
093e0e40b9e06e228dc0a89aa75ff458550aa8c4
F20101112_AAAMUL huge_d_Page_51.tif
60acdede27436fe85eef44483b4811e8
fcf3e978b615611c9b66d2b9d5a2d01ca2ed312c
22252 F20101112_AAAMPN huge_d_Page_12.QC.jpg
bade47609b9e9981022e7d71c4b4ae81
92a909abd2409d7cf13179ce9488a659fdae765c
26670 F20101112_AAAMKQ huge_d_Page_63.QC.jpg
b59851503d3dd358f118f058530c1e54
b538bd39d1f2cf8650a180b4045d3cb1d5dc89fd
2744 F20101112_AAAMZJ huge_d_Page_62.txt
4ed0e3e689bf219bdc55da2dc2c58d84
a18a0af162ac50a685194e0fa411f7dbe7c41681
2033 F20101112_AAAMUM huge_d_Page_42.txt
ae21035f0c3c58810ded216f67d10b52
1f8ef9ed034d200263ff4e1dbef79175b38959aa
12999 F20101112_AAAMPO huge_d_Page_05.jpg
157c83c129f6adf9c68ceb8f63735a86
f6f60007e7a27d59cffd54b718f90d5ba1d0e2a7
7082 F20101112_AAAMKR huge_d_Page_62thm.jpg
3fd1f3113f7a7e63a71888fc1c003a8f
1e5186eed5a345721c2aae20a8d40b1e9c6f228e
2187 F20101112_AAAMZK huge_d_Page_65.txt
fb2179062827c196cabfd875ceb683c7
ed95b317bc96d1eac0acf64e040c843db4bd9494
20074 F20101112_AAAMPP huge_d_Page_08.QC.jpg
1b5026f253805d7ca64a72a1c10f9012
c4854a506e139f282528afc45f916526599fd58a
50071 F20101112_AAAMKS huge_d_Page_26.pro
9c97b5afcd961afd6a668748e3f6d625
0624e90fc0fc68253d5940004b0899cc3bf91bd4
383618 F20101112_AAAMZL huge_d.pdf
d00609bd282b367970ddfe2fe8266df3
269a7cdaa67f14e4e4ff551e2ed8a98dd19bef12
1762 F20101112_AAAMUN huge_d_Page_09.txt
028529263c64fb835411017616c37e66
0cc87eeb04c62630576760351530101814cb5877
67475 F20101112_AAAMPQ huge_d_Page_07.pro
e018ad8f4917054fae468175c3afc134
d57e847eabfeb19cfdebb9e2eb5b0cb29562dadc
89529 F20101112_AAAMKT huge_d_Page_31.jp2
3a0f234d042a344ba082cc866bbfa719
396ce90bbadb481f229db19cc4462ed0166d3772
3970 F20101112_AAAMZM huge_d_Page_03thm.jpg
63b38b218cd182d876157a706bbb2ea2
0e50fb683422460b1b91dce4220a6b901b67c7be
1885 F20101112_AAAMUO huge_d_Page_34.txt
bac836d8ec2514588f9f71878951eb2e
d8b9d38be024657c1e050a60712ae4cd9044f467
1051964 F20101112_AAAMPR huge_d_Page_04.jp2
5764b84e92b8715d08e2d67fc18891b7
20de17be075f078dfc0241dc631c4653a66b23b6
65789 F20101112_AAAMKU huge_d_Page_56.jpg
852d7bb641792b8ae557c4812cf07fa7
4df026c10c7eee763b4bbb237513d1fe53c874aa
3871 F20101112_AAAMZN huge_d_Page_05.QC.jpg
3faa2d0c04cf793eecdc209ab6e63c76
f73aeac289433d8af9e581646dd197a98e9d669c
5035 F20101112_AAAMUP huge_d_Page_51thm.jpg
6b49256c6e1ff537116da7ec87f3424e
ad3ae5ffd8ff41ded86367a1f017224bbdf7f588
6522 F20101112_AAAMPS huge_d_Page_20thm.jpg
ddc02c64fe2b9f3da91f00720de0ea6c
31d7bf926067b0ebad610a24cbe8a03a1847b918
73778 F20101112_AAAMKV huge_d_Page_49.jpg
ef6670be03ea6f2c1cde314770025121
0b69a67ded480337fe057fa762c0cc9bf071e513
3270 F20101112_AAAMZO huge_d_Page_06thm.jpg
0df95638d446876eac0e823582c11d5d
6f544947e30c1664e5aa7ca6e22147a6e773fd7d
113529 F20101112_AAAMUQ huge_d_Page_46.jp2
b1b9080e2de85b6021ac5713c8ddce93
4e517ff721e80d2c91009d948a0271902d37c0e8
4275 F20101112_AAAMPT huge_d_Page_04thm.jpg
c1070730b9b0334d254febe8b3809279
2abf023f5b768a46a3f8e0a3f3fa52c00521f7a7
50185 F20101112_AAAMKW huge_d_Page_41.pro
8a99d015dee66765569164f1381f6da6
04c5f6dee95f1220bd8c316eb6ea9583d8571793
27489 F20101112_AAAMZP huge_d_Page_07.QC.jpg
84bb07c2fbd0ecde3a4d51f3b98d04ad
1bded2e92426409c065b725831764fd1de969611
20594 F20101112_AAAMUR huge_d_Page_58.QC.jpg
e2b19fb626ba16532f2dbb2045c5d242
35df45db4e85ae88a8798053cb416b9511efde4e
52587 F20101112_AAAMPU huge_d_Page_30.jp2
3ab7a806b1ef785cc387d004f77ebc60
4d5bb6d40fd98e5983adb50c67c3585637d0c95e
62522 F20101112_AAAMKX huge_d_Page_09.jpg
acd5e797093222ab52d918557262c05c
e6a3b6542475a026c2a47634b5361731d003f45e
6458 F20101112_AAAMZQ huge_d_Page_07thm.jpg
42e6b77dcfbfe08d71f895e324c34492
6a1eddaca2543cace2b61d81ab6e51d714391463
6062 F20101112_AAAMUS huge_d_Page_56thm.jpg
da46543e60c54bdbdbc0b93b7f457c65
260b731df11950735abad6ce05bffd949eed8045
60489 F20101112_AAAMPV huge_d_Page_23.jpg
e6772e659a876cc3afd1532c4404839c
fb83ce9675e0aaa8e02de9a135d5598ff8537156
72807 F20101112_AAAMKY huge_d_Page_48.jpg
70c25c664771fba0d037679535cc6ebf
eda4ebeebedd19b8ef5d626298c5cb454f92da9f
6196 F20101112_AAAMZR huge_d_Page_12thm.jpg
e22fc9f2e3056405459189c63c15af56
479f5e5a9b5fdcd207699115026f3734d0264569
50428 F20101112_AAAMUT huge_d_Page_47.pro
67000be474d365b6a775a8d75ee6646f
b4c4c8eee0d4d90fd27c7445fee8a035036edecc
6692 F20101112_AAAMPW huge_d_Page_21thm.jpg
cc811d93b78e7cdfd8b16163f039c21c
a7cf0f025450d74d632aff9f70ad20e3beb891b0
77167 F20101112_AAAMKZ huge_d_Page_08.jpg
2e60df5c939ad17127c7f7df36d2c76f
47dceb44164b91c364b2776e4cd6138e8538d3e1
6465 F20101112_AAAMPX huge_d_Page_48thm.jpg
21615ee25b06e6831757b20f3942fce2
ef94f583cd01701da9d7e6c6ef1c0d8e83852634
717768 F20101112_AAAMUU huge_d_Page_56.jp2
79b319543578ef99ed27532e398b8e3f
96a5f125d1726a8896e87f1cd942c347e45fab2a
22830 F20101112_AAAMZS huge_d_Page_13.QC.jpg
eeafd3a406efd846112a92e0e67b22c2
cad1e44c8386c5d0df3cd51e7010ae07324b6111
F20101112_AAAMNA huge_d_Page_33.tif
94a73cb41a9695078623982d791e73f9
a8c7e1f7f99c6e91461a71788c458b451aa74731
1392 F20101112_AAAMPY huge_d_Page_51.txt
b2865d592ff0a8d8c8768681f78b0cf0
4dc10988bbfd86d1f444bdfdb110193a38e5fb0c
48535 F20101112_AAAMUV huge_d_Page_60.pro
0e2d163b7ffb65d2ce32fb582ba23a9f
221bd468736d840c90a62354dea29cb86bad6a17
6235 F20101112_AAAMZT huge_d_Page_13thm.jpg
2ddaaa67a58bb847007e0e2d7be87199
8e322aaa0281266620475803a1d062f3055fff86
91883 F20101112_AAAMNB huge_d_Page_62.jpg
f294e8b8ffbe5a1b993972d4e850024d
a7bfb3834431423be15680d13697efe4edc23bdd
5337 F20101112_AAAMPZ huge_d_Page_54thm.jpg
bb6ca1265c19951aac582c5a498fb4f3
06712b27f60f042340948fe8278fc90f88cb4f10
48838 F20101112_AAAMUW huge_d_Page_36.pro
7a5bd5d4dc9f12085521cf90ecdab9f9
5f9b67043b7a1f7d974cba3021e8472ef656f684
6502 F20101112_AAAMZU huge_d_Page_14thm.jpg
44147e849bf94f51fbb01dc54913720d
bbff6ab54b7a865724a33688b6108971fc71042a
F20101112_AAAMNC huge_d_Page_29.tif
88f6af343bf06a1202cde28905619e1f
49224703e73cb8ee8bb4077c17c7b0918845370e
50685 F20101112_AAAMUX huge_d_Page_48.pro
e56fca303b411e8879a5ee16e0b1cc03
5a086c9113c295d90ee403a701968ffd6ee0ccd6
6577 F20101112_AAAMZV huge_d_Page_15thm.jpg
8a42f61c73bd86b6c8f60e394b6b24f6
b640ecfe7ed3b16f0f1246eeb0dd1dfa770df349
24120 F20101112_AAAMND huge_d_Page_54.pro
5a66c2dc6a375b0c33c25139c11c7506
6bb7d5dbd9aad117bb12a8bd15c8c3f5c7392af7
1873 F20101112_AAAMSA huge_d_Page_13.txt
816913a5a949affd27320552ebc49574
d689bfbfb4ffcaa16b14c04abed1bb02964a9c3c
21843 F20101112_AAAMUY huge_d_Page_30.pro
d24f794f43be73383d98049535e79974
9e1b1ca78413acc2c054f6457141baa3303c42b7
6558 F20101112_AAAMZW huge_d_Page_17thm.jpg
1282a07eb07e415d56d870a6ba8dc87e
b3223e9ce5e045dcff866165e1ebc6480800d5c9
12635 F20101112_AAAMNE huge_d_Page_57.QC.jpg
25bce89e54d0c026110dc884d01b0618
0eb0dac2e70593d663af02dff59ec81f1ab259e8
918 F20101112_AAAMSB huge_d_Page_57.txt
0e7bade45a39a827c21dfd03ac2ddede
525f6d29df9e203849b578e7ea966bba90691c0d
1990 F20101112_AAAMUZ huge_d_Page_60.txt
cbe681c7f44270452a8e5ed3bde59dc7
941a88adb54f2ec233ea34572e849a0866426680
23572 F20101112_AAAMZX huge_d_Page_22.QC.jpg
ad8dab309005edea5debb46325a986f9
486b3d06836ff9438bc0298171d15c5e1f7157b2
69528 F20101112_AAAMNF huge_d_Page_14.jpg
ad19b1603a2f1484994ab8c72bcace85
2d5c298549268268dbd2d1a721a75fb1cf649e0e
13423 F20101112_AAAMSC huge_d_Page_03.QC.jpg
e9f97c30339ee3f2bcb54d11a18d0203
3ff141ba720b36ff56d5f68a73155ed6b7e699ac
19742 F20101112_AAAMZY huge_d_Page_23.QC.jpg
1eefa1fcb4bfaeea29f8f5c23e2a90e7
7a0761f72ffba4691e2b865e901bfe8f12ee318a
113059 F20101112_AAAMXA huge_d_Page_42.jp2
163e6d59ecb9e60c55ee44c646264f39
19bab5662a8b9ba27ce9c51926e79c9339d7f531
109188 F20101112_AAAMSD huge_d_Page_26.jp2
c963ec5dba419786e82cc5deb41b6796
1ec06cc080058f46cb6d461767ace42c2865c0bf
5668 F20101112_AAAMZZ huge_d_Page_24thm.jpg
b052a1bbda763b910e28048c4f6bd09a
d4ce30430e4799b2b650061195b779a290120545
6641 F20101112_AAAMNG huge_d_Page_49thm.jpg
fddba537fb68d7cdb9c60f2d9e17753c
211cdae9a9252568581ed753fa4825ebbe82037a
111894 F20101112_AAAMXB huge_d_Page_47.jp2
7f32a2062b7a0b18fef11b2cf560ab38
70f4ae4ed1f064bccad4ffc434331edb97894cd8
24163 F20101112_AAAMSE huge_d_Page_42.QC.jpg
dea1db0065a73aebbd2a117509bc96a6
487f63101f738e762b999f4572fd0d5ab7708b04
112757 F20101112_AAAMXC huge_d_Page_49.jp2
77883fe37159214dc1c9e0cafd4b1054
104591f0a8af32820800d4830eea2f6b1ecff34b
17113 F20101112_AAAMSF huge_d_Page_54.QC.jpg
06509a1b05ce588017690b1125721ae3
27fd4ee33483f742e6db0376d47f03b79dde2469
6420 F20101112_AAAMNH huge_d_Page_35thm.jpg
f978d7c4c33394c70956b4f702ca24df
5941806d0866ed51e5c98edcf73eb4708b968efa
640289 F20101112_AAAMXD huge_d_Page_54.jp2
ffa594d3062097a022d9d266436c07b8
ebd3dbb25c7d0377e4918372168142a71a30be1c
6549 F20101112_AAAMSG huge_d_Page_18thm.jpg
1f0930f8742a34e49fa150978c313069
db3ca8b38884b51bdf1decff07e081b775fb1178
75331 F20101112_AAAMNI huge_d_Page_21.jpg
455a66c430f389d7fa19715711360d2e
976d1ab63895ac24338a21572a796419be6600f6
143081 F20101112_AAAMXE huge_d_Page_61.jp2
14852c249ada94d67ea5c767c9af2cb4
91f228b1027cbfb04dec963ec76cb17c49d098e9
136962 F20101112_AAAMSH huge_d_Page_63.jp2
73efaa4ff60cf518aa5f8b94258096ba
5db6e0f362898f0e65abe2a45ff6aec04aced1d2
F20101112_AAAMNJ huge_d_Page_17.tif
d9e6e26336c7387395b034b452cf9006
7c359292ac7fe0b4f30ddb6aa411c8a690041d4c
50034 F20101112_AAAMXF huge_d_Page_66.jp2
91e11523b62c473d055b81cceeac8728
a575e42e6e1e97337af92b8b70a24b9a87d582bb
F20101112_AAAMSI huge_d_Page_42.tif
9056f8fc0ff85374c3f00841ae2a8326
36b5fb579feb0a35eebb40ca35b90280546e4602
23840 F20101112_AAAMNK huge_d_Page_19.QC.jpg
8637bd73c622e2e90ab219297b72549e
124b8e0d1fd4d2fefa9d70d60a9193f551281bdc
F20101112_AAAMXG huge_d_Page_02.tif
74eba989384f75971745c0ac4dda96d4
5eca9b54e422f5c57dd7976a399528ddb9b662ef
5590 F20101112_AAAMSJ huge_d_Page_53thm.jpg
e3c18ccb200afa47bc2d244b864cc869
f7c6e6ab15cdd36f06a6793c46db0878d67ea8fe
F20101112_AAAMNL huge_d_Page_18.tif
9c0eaef6bc2410e5fa81376e2e45ed7e
cb95661627e83e1b37d0793a728f1ff6a332ac38
25271604 F20101112_AAAMXH huge_d_Page_04.tif
92f07a1c52c5ac6991aaf00b9f681300
f5160814427f7b31b3c5ec8a52c16c4a1e891080
5909 F20101112_AAAMSK huge_d_Page_33thm.jpg
1445f0b21909128e369bff12c6423951
14d66c774a69ebb8af6326bd032595b7e9e678b5
114120 F20101112_AAAMNM huge_d_Page_65.jp2
0840b721eeb6c9dbdd7d025d0574f18d
0d857f63949edfac66850536c65a39042b724521
F20101112_AAAMXI huge_d_Page_11.tif
66ca097a2c7cf490ed5e67cc2ea818be
88a0ca1ac9e1faa84c818509ccbea7d7a2379e5f
19536 F20101112_AAAMNN huge_d_Page_31.QC.jpg
4743c93ab85a86baf5009be588a96e73
71609e6dbf34506a987cf898c3f5ad2e159b2108
F20101112_AAAMXJ huge_d_Page_13.tif
51ceaaae2b390f2d5fc1314820454a36
661b361f6db9796a49a2ed77494c378f11b9092f
F20101112_AAAMSL huge_d_Page_14.tif
93e864f5285118dbdb82c7e5dde36c1d
bd1a8bfe5aef69c3c7978af07043f94c0ef472a0
41721 F20101112_AAAMNO huge_d_Page_38.jpg
ca9c1b065e04ff3393db466d10afa3da
63ebf841b569a112526829c8b20b28206ef3e84b
F20101112_AAAMXK huge_d_Page_15.tif
7461f1974e28c20bd2a5430efb51fc80
2b958e27d317b5a825ce6405a6fd3a33e9324cab
71963 F20101112_AAAMSM huge_d_Page_22.jpg
7eaa1eae059d9d793d0ac61fe7cf3b12
7876fa75c85d0212db41eee16310e26ad95bf65a
6603 F20101112_AAAMNP huge_d_Page_46thm.jpg
e13db711881b4c29025a879e4f700774
fd86a246de777d690a610c32b0e2ce2286b9ec95
F20101112_AAAMXL huge_d_Page_16.tif
7eb202732a12d8b5e8f01eff628036f7
f18292a65c18a4a50088fefcbd5386a0876ed4c0
42024 F20101112_AAAMSN huge_d_Page_03.jpg
af99ab4c7bb37b5b81c4c28a063c3630
9248e36ec016afdf1cb9a19ba7f58f5314114b47
74370 F20101112_AAAMNQ huge_d_Page_20.jpg
a1387976ad845fbc67d253e6842ba686
7ed605f1193eaf1cda76d844f71db7f4a53a2ded
F20101112_AAAMXM huge_d_Page_20.tif
49235982dc6e1ee5f134a4b53696e17c
be796a8a05d5e3bad95aaaf9d46a37c0c36fd611
1074 F20101112_AAAMSO huge_d_Page_03.txt
caaf548c42cafeb60230170d8acc6145
cefbaa0fcc8732c285412070c7bb937490535963
6275 F20101112_AAAMNR huge_d_Page_36thm.jpg
80ba9b5b208cff0ff8a503e525d6dc49
c3d2fc68c8269b8c9e7ac0398626fdb2cd82e217
F20101112_AAAMXN huge_d_Page_23.tif
d7574220fd3bf5c5ae5a36ad2c3be06c
126f1135895b33d146c372fe718ddcbd78cf5d5c
64277 F20101112_AAAMSP huge_d_Page_64.pro
b628ade457969de457dd2ca6036a3e7f
385310bb79ae5583926c2397f627406788ef6c9d
1016 F20101112_AAAMNS huge_d_Page_10.txt
dbb93093c0bebc71445a5e17272acb66
058fc8e707c870a7af327f47a2013d47b48686e3
F20101112_AAAMXO huge_d_Page_25.tif
211256ba5a050ec648ca4538c0bb18f2
57122444a9f627adbe1edb55d6a403e754d5fe3e
F20101112_AAAMSQ huge_d_Page_33.txt
eb963ad58847a49e837700aac56edea5
36fe64c9d4ad2044ae6e4d27d38bad569e418dae
113 F20101112_AAAMNT huge_d_Page_02.txt
0acff65ca471cc5807d6ca0e0965a059
d1d0fdd0f871d50d6f717f8c6f0c1ef0bc374884
F20101112_AAAMXP huge_d_Page_28.tif
c3c2df7ab6b34e42a8ceae012cda2437
5a9dc76c9850a1d5af66ac02c57c723f5c33ad56
2017 F20101112_AAAMSR huge_d_Page_45.txt
7cbb9086212f557b33d6212f781d53b8
d50f5f02a3fc27de39d475967840f0a9c9711241
F20101112_AAAMNU huge_d_Page_27.tif
19eef3ca123a2c46a3bd7dac5e56a409
1e8721a0d58b04d596b0e8ee60ae231de0e676e9
22391 F20101112_AAAMSS huge_d_Page_65.QC.jpg
ca3dd95c821806de4857798eebf9802d
24f8c69bde0aea8e35310aa3207894e534b5d161
F20101112_AAAMNV huge_d_Page_56.tif
178b72e2dae4163f360e87a6bfa95226
a4a6a7389cf220dd30232a65b67a71ff8ff665bd
F20101112_AAAMST huge_d_Page_58.tif
8f9e577ec8c455f79f0c8b51b632cb1c
eed0d011d2731c8e70d4fc4c880697ff64334d25
F20101112_AAAMNW huge_d_Page_07.tif
b26403076746de4892fc47dff2fa1824
1e12d298473afa5c96849679224b32aee8daa90a
F20101112_AAAMXQ huge_d_Page_36.tif
d6d90a8aab15495ec727f457b00ed942
3df1dafdcbce3e22cfbdc709572a411b40124976
F20101112_AAAMSU huge_d_Page_08.txt
32923174a900dfe106c63e3bec454966
7f0cfe0e5eb6c547a151cb1537385178e436a350
22833 F20101112_AAAMNX huge_d_Page_14.QC.jpg
18926ca1fcd58406522913baaca0d87d
a50a37f9a0a0edb33633075f26afd1a0a9be5260
F20101112_AAAMXR huge_d_Page_37.tif
4e4d9eb0d22794179b768a0c4733ce13
0889228509413021426d52035744ccc0c3d0e33c
24313 F20101112_AAAMSV huge_d_Page_20.QC.jpg
df59b353c8eb141514d538f4e70e2272
5d3ec3152a4518ba0e3a42f1c3cd65702864b4f4
6636 F20101112_AAAMLA huge_d_Page_29thm.jpg
11b4a4325034a25f4843390b0c8fe549
abc73044289d74dffc2c6f8955548b0b3bf01f44
13744 F20101112_AAAMNY huge_d_Page_10.QC.jpg
b2e2305d6a38a000730b638aa747225c
d2c0950cd9fc8a89cfac44f7bbfd0e3182205cf7
F20101112_AAAMXS huge_d_Page_39.tif
902de54349f8292ce795144865c5af50
4f0c267aeb1a0f200529db3593552d285347cd5d
6434 F20101112_AAAMSW huge_d_Page_44thm.jpg
634842fb83c7e04a74c1ff11c42de612
981d73c217b1dc92ef159d0f7a6ca7f378983627
F20101112_AAAMLB huge_d_Page_43.tif
8acb3015bff16aa04c0d8fd8fe01d7bb
dba1ac80a20482cd6ae82a5b5098bb5e7659adb7
66112 F20101112_AAAMNZ huge_d_Page_40.jpg
be533e37b98b6a06a4a9d133e2d3866e
860be2a6e04ef700b01e37ff6db27bed46095c91
F20101112_AAAMXT huge_d_Page_45.tif
335ad70256acaca33a9d9c1264deaf8a
187d4684cca132f92577628e4433c09d921559b2
39482 F20101112_AAAMSX huge_d_Page_09.pro
9a4d19e0b307b46bb92a42fd59f7bfe2
3defc840472fb4aebed11f357af27c878fc56800
22722 F20101112_AAAMLC huge_d_Page_34.QC.jpg
a48137dc5210e8b34344fc8bf8692b5c
9eaebfce20499b098074ea5597b73e57070621d6
F20101112_AAAMXU huge_d_Page_48.tif
1f138449c121ce457ac5185ef0415ff6
4b86d3a2fe9060e7d7e869ebc6f6bc10122afc74
5601 F20101112_AAAMQA huge_d_Page_55thm.jpg
0292986f3a8b169baf2bc1328547ac41
57af20e00c2a9d6b7c5efc3bdef04ea667f2b271
1982 F20101112_AAAMSY huge_d_Page_15.txt
07a8589be48bc8e782c3b80c25c1bc7f
ec1acb0c554de7410d41702e77da4af8414754a5
74536 F20101112_AAAMLD huge_d_Page_46.jpg
efc09308148f85c5657589d2e25750b4
7405ebbc2f9bcf8336ff2b303ddb8266a687cbfd
F20101112_AAAMXV huge_d_Page_53.tif
84c598e5d52a383ecb1cebd52c256d1b
2f29720c1c0dd7ca4bf9e38a9f8bee674b96e8f5
60986 F20101112_AAAMQB huge_d_Page_53.jpg
efca2bf0a572ba572da3b4550cc07dde
ef18e3b4bbffa93e00ac3e547a63a2e3b6c7c3a1
23652 F20101112_AAAMSZ huge_d_Page_17.QC.jpg
2ff7eedcdbdd5f5797f0642401bd3edf
77f3a6a96cd239995d792cb1437159c728b82c2b
F20101112_AAAMXW huge_d_Page_54.tif
53cff216493a202e3c9ca10111abbd22
1e8e50ffb19ce5490c07ca39d3f418e683a1e761
34924 F20101112_AAAMQC huge_d_Page_51.pro
471ed0748df810fee4211e4a250a9592
eeef616b00b2560d47e7db48f68e7914776b837d
26632 F20101112_AAAMLE huge_d_Page_01.jpg
c2edddbb989cab3668220ccc99253f77
6ad9c3941f41561a8d334b09fa541c1f985d00c9
F20101112_AAAMXX huge_d_Page_57.tif
5e0505fac06ad8df430ac6e9f4f3f171
29bdb6100becd58cd492ee2f34e102235ff30d63
F20101112_AAAMQD huge_d_Page_46.tif
c610c6660eadfcc77a224ae23cef61dc
2e84a59371df54520901d8929c127a0e2a0a00ff
32032 F20101112_AAAMLF huge_d_Page_39.pro
f952303444f5e2efdd853422583babf2
bef39b4d7cb240bb6e94063f2a260145c8856f7d
F20101112_AAAMVA huge_d_Page_55.tif
606503ccf3d00c5c7fe67aedb9260441
32ece6df19f44ec2d6bb044c24fef8ab3d98a7aa
F20101112_AAAMXY huge_d_Page_60.tif
892d59f58b19f53e26c5a50bdbd556a3
5d29167a102f96d157f52602f5d4ab1841fb5ebc
850 F20101112_AAAMLG huge_d_Page_37.txt
c340e6a0b23e5af5a1f713d7b1bdd76a
7aca9922d021cf49d43b13d34f81271e080d8ea2
2045 F20101112_AAAMVB huge_d_Page_22.txt
a8e186713cc5cdfc42883fdf05cf909a
7319e7b88396a1faa9473f268709516c18f2938c
F20101112_AAAMXZ huge_d_Page_61.tif
423f09e255e6f402b5b8c0cd09a1c0b9
ea290fbacec4b9144a021c0a10c8e5bd41942bd3
1389 F20101112_AAAMQE huge_d_Page_02thm.jpg
e32ccc391bd360f1b8506198ccb8bd5e
c7e892d43fa43c96eaa9004abc336e7d7e1cf305
54816 F20101112_AAAMLH huge_d_Page_54.jpg
3abc541993d60d391433663b93f82192
407b50c3b9b72e10d6c38e91ad63abde695885ac
5379 F20101112_AAAMVC huge_d_Page_09thm.jpg
093c6fd6c965a912f84b47553ae9f44e
6012bb7473ead2ef6e6d957a349cf63e58d5701c
110364 F20101112_AAAMQF huge_d_Page_28.jp2
ff14b03bdbdf36609552086c2acd75ae
764abf74bbf61dd524c84ef267115c4dc46b7e31
72489 F20101112_AAAMLI huge_d_Page_29.jpg
114ad766e5db3ef047e16ba74096b8c7
e4e5be3e52b471d538fef8e31cccf1804ffbf09d
6686 F20101112_AAAMVD huge_d_Page_19thm.jpg
07099c6a96d69ae0b85d37f64c6928b8
491a1d2988be958b759fb680858b2e2e662e887d
24357 F20101112_AAAMQG huge_d_Page_21.QC.jpg
d225ebfed036863af9314b6263011474
508106b08818ecdd7babe740823eaab24b8c34a6
66980 F20101112_AAAMLJ huge_d_Page_34.jpg
80db4b403057e66b9eb57cc2c165cc64
ba136f2dfb6bae66402c9b42fe2bee79d98d5173
60568 F20101112_AAAMVE huge_d_Page_10.jp2
c4b978e4d272e93c5cc6e516bc1c87be
ac4e24701a3bb68f92615f9362ab399036787a42
1221 F20101112_AAAMQH huge_d_Page_02.pro
bcc089a0a963f391aa4532205befb607
527605ac0cfaffb667046b7b3e661a212fc68b5c
2866 F20101112_AAAMLK huge_d_Page_61.txt
26278bae95605bf7b9c2f54bfa9f5188
a72d867d61848d97dc70e4465abebae438e099a0
53473 F20101112_AAAMVF huge_d_Page_65.pro
f59e44149e3a25749ddf89d1b254c4f4
2b821af504a7a9e3786009ed3d8ef9c02be8300e
F20101112_AAAMQI huge_d_Page_35.tif
136d56427880d10e1632f5e95296e81e
85bc436093670a13e0088337a3eaff325af20fe1
6570 F20101112_AAAMLL huge_d_Page_26thm.jpg
18d55a9ddded43525d42bc5f02f0345e
67e9c2997763fb205ae377d9752401fbebabbc51
1646 F20101112_AAAMVG huge_d_Page_23.txt
d7777d1a3af2634a8b80d6c21f464cfb
2a1703f76cb5ec3b24914615cdd3f1926bc19678
1051866 F20101112_AAAMLM huge_d_Page_08.jp2
d1c2a824e8f239bd7e212aa33e289dc3
6f0c0c18967708e4b4b50ced15b815558400a9c3
5515 F20101112_AAAMVH huge_d_Page_23thm.jpg
839d136e1b0e4c512faa02f52bcd7532
9207000c6ca73b20afb61e9b9e67f7fa34e615cd
989372 F20101112_AAAMQJ huge_d_Page_06.jp2
3e8dff2eb3c018edee188e3711c52ae7
84da5c3931dee63a58c0cfe7b32db7b501de39af
753671 F20101112_AAAMLN huge_d_Page_53.jp2
ccba915416fff5bf5ce85f4a4e9a2005
8005fa61c336a1dfe24b273acfadddfeb7dcebb7
4970 F20101112_AAAMVI huge_d_Page_08thm.jpg
c8be35676329553dd50220428047dc59
f0a5db55825a2892c520dce7f61070225b427986
27440 F20101112_AAAMQK huge_d_Page_01.jp2
9a86314be8bb0613659e00ff04fcb117
e7a5ddb1ff9c50b23f158463d10b84ccb9b06df2
6573 F20101112_AAAMLO huge_d_Page_42thm.jpg
b09f98a2d700a3f3e6e5a05a9d6bfd76
957f0b29a9bbf187806181226d52db1808b6b766
25377 F20101112_AAAMVJ huge_d_Page_10.pro
9a6f8d0e807703ff857b8012e6aa6bc1
136069a1dea11d21a070ee8b39c82faad7b194ca
51244 F20101112_AAAMQL huge_d_Page_45.pro
e836349452e2a0944034aa39582181b3
abfafd0a286852a9cdfceefc7c4105995ff9e7e3
26830 F20101112_AAAMLP huge_d_Page_64.QC.jpg
a4c6019bcdf59ba773979b96c61558b6
2cc3b3c211251169e94b7e5114aa9b61d4759831
51281 F20101112_AAAMVK huge_d_Page_22.pro
2e284ea73ea4b7444074e432fbeaf4d5
fe92577a5ebe5534c603c40a241ff353262415d3
F20101112_AAAMQM huge_d_Page_06.tif
03b53b52fd718d5fdd42e1971600b3ad
180c6373e81861e9dd4bf4d6d96a329addff53e9
24170 F20101112_AAAMLQ huge_d_Page_46.QC.jpg
01f29960898d35182e51a826ddb09714
0246cdb58f63b5bbe856aa48c9302348dbfe6ebc
20434 F20101112_AAAMVL huge_d_Page_56.QC.jpg
d6365d1644f861298f74f52ccd70a72a
40edec6deb6a223b4422007d57c18db81fa1044a
6151 F20101112_AAAMQN huge_d_Page_40thm.jpg
0d3aab5b713ce0db2ea757fb3b9f502b
c704f9daecd465a953c40329065ddba194355443
F20101112_AAAMLR huge_d_Page_64.tif
40ce2b1e5adbb3c8fa97e33cb3d72b09
8a27d0bdda131aff6f81ca26b237cb9c3d191d18
110880 F20101112_AAAMVM huge_d_Page_45.jp2
42e46afcb58ae26c5f0226a1152f1018
4ec6c55b37215dff09fca1d51d87cd0132063b38
110036 F20101112_AAAMQO huge_d_Page_48.jp2
eb4b7f61d37ad3ea40b021e89360543b
bcefd0b21cb36949cd7894311aca1ab0e17242d1
1702 F20101112_AAAMLS huge_d_Page_31.txt
3ea806b35f666571bc9c792a17babfd0
b82c06c55716fc83f064f104430182d05cef00cb
62112 F20101112_AAAMVN huge_d_Page_04.jpg
f4a10b64747bd725609ea758e3b082b7
7fa9dc886eb6ae82d57a2e7701883909e151a701
31810 F20101112_AAAMQP huge_d_Page_53.pro
90c8f4e7240ff540c630455940d6989b
f03a7a1025eb6dc0ce051d0d56d4df3d09887267
F20101112_AAAMLT huge_d_Page_44.tif
c05778066364dffed3414870a3936cf7
b88eb1b35fd46e87b6eefd9656fd33cd584a6c56
53410 F20101112_AAAMQQ huge_d_Page_04.pro
3c14cb619343a4263f2d1a3fd66ebc73
03d013fa4a17ef403b9010b51718862c6b612bfe
17875 F20101112_AAAMLU huge_d_Page_51.QC.jpg
de8f28ac926942e2b10f41e3a9b2dd91
9340ad72e47fc5b91ddef16d1ba2ef2c9dc45adc
20816 F20101112_AAAMVO huge_d_Page_66.pro
0a2ab3ddf080afb9c529c7c649f9cfa1
aaa673cbc36d8c4170eddf1815ca15b76fc168b5
F20101112_AAAMQR huge_d_Page_24.tif
04e464fe5db111c49c3c6f49574e4d90
2e706ccd150d52c8539143e950fd7bc896d00230
60304 F20101112_AAAMLV huge_d_Page_31.jpg
86595012fbb89c5e95e95b2babf53d11
8f8e9cfe7ccea14d1fc42e10c46affada4774c8d
23448 F20101112_AAAMVP huge_d_Page_18.QC.jpg
b425e8f05b3edb569b04e3eb1b8d406c
91792372fce103ef4e1a494606f04e66e788a647
21215 F20101112_AAAMQS huge_d_Page_37.pro
b6d9c8259b6ba79e9fd68760fe406adf
4c480513419ed961c9f96580bf54aea5904afcf5
68393 F20101112_AAAMLW huge_d_Page_27.jpg
5295aa20a5bdf9ef9f6c2345d7a9b546
4dd6341898c879763d8188956f7f6abbb989d4e6
133608 F20101112_AAAMVQ huge_d_Page_64.jp2
06aa15bc5443129b4627a4e0b831b4d8
bcb667c2a688546d58ebbb53e2d17c9dcf73b8c0
73180 F20101112_AAAMQT huge_d_Page_28.jpg
a9be6d67ed41a818d9f27dcb551e3d58
ddaa653dee903f2b5198d103efbad5deff987191
2076 F20101112_AAAMLX huge_d_Page_21.txt
9e84592973418f4b9b0422b9d510674c
bf0ab9b5833af40d604f4dd06829b0280ed972e2
11687 F20101112_AAAMVR huge_d_Page_37.QC.jpg
022c9d326b8dabc0eb1ec84aedcaf933
83f9914340e8c81bc38004a53cd2aceb29717e28
23733 F20101112_AAAMQU huge_d_Page_50.QC.jpg
48387d04de6206c61b972737313a7366
c6e9548a09ab9afbc76cd05f6a5de1737218b4d0
1459 F20101112_AAAMLY huge_d_Page_53.txt
153f592b25a014952243d5e176fa1e9e
cb79ca3ea580f8b8101e983ef08e17dc938b65fa
100063 F20101112_AAAMVS UFE0014367_00001.xml
e28f2de34b7ba9df9b9283f2f9de4783
2f1c6b876951a341cdb0a2ebce5f833cd3ced508
51718 F20101112_AAAMQV huge_d_Page_49.pro
52da80df62c9afa4141b727b2cf6ab27
25d3e87dc74bb5306228adee91c6ae9d9b0df7bc
2595 F20101112_AAAMLZ huge_d_Page_64.txt
d5de4e91a2a0b82da1872561aa41dcaa
dc86bbd03cbf16cc4edaac291364237f246e45c9
78834 F20101112_AAAMQW huge_d_Page_51.jp2
bc2028aff21b020143c2ed9433ef95a0
e6e11ef3f9b76cd32af4056e2abfdf5cf4e0322c
F20101112_AAAMQX huge_d_Page_40.tif
5d1b947882628daca54f04da93caca41
28956559b6fdb4cd101778a763be962691236a5e
10423 F20101112_AAAMVV huge_d_Page_02.jpg
3cda0ad250ac7d029d0db4a3dbcb248e
b4af049363acfc9859b0bbd7b90c7bae5f32a78a
F20101112_AAAMOA huge_d_Page_08.tif
0102fa5fba77e6a352d73867020c3748
d14143e35c9542a364553880cdd26f5df7324b7d
F20101112_AAAMQY huge_d_Page_31.tif
f3c45d7fb3eaea8403e8cb0fc918a121
22a1b60a53c0aade9ae9bb78b7baa58b4d13757b
62565 F20101112_AAAMVW huge_d_Page_11.jpg
3e75860f374769e7ce9b4a0e69c468ac
495aade6be5173b1bbc764a26f5351bb53ee176f
51806 F20101112_AAAMOB huge_d_Page_59.jp2
c89f18da35f36999c4e7fb1f3fd99d5f
af59919d68b4de6f60c8908378359d2d51c7e06d
F20101112_AAAMQZ huge_d_Page_47.txt
c43851768aa0a754376ba4374aa378fe
e0292bf3de15395e1a8e94a3a46261cd4331fcc9
23921 F20101112_AAAMJD huge_d_Page_45.QC.jpg
16b1b9ef7e7e943a608eb4f90815be1f
debe94942c3268b915fdcf04a2d1299fb6aa41ec
68474 F20101112_AAAMVX huge_d_Page_13.jpg
6ac4a1231b30bf0c19febbda897ede01
1eccd8552bc84d12f27fcf69091cc161beec4425
369 F20101112_AAAMOC huge_d_Page_05.txt
5533755f3b684de81a7f3e3da624bf68
c9d433ba7ccb44d928e401776caa0aaf16eba2bd
394657 F20101112_AAAMJE huge_d_Page_57.jp2
68a174229d28bd12af9f471db8c81980
9186ff14838c1f1cc00bf295512b61454c04b23e
71221 F20101112_AAAMVY huge_d_Page_25.jpg
e4bf2e031d5ed542d098a132d450a645
b730ea16d4d99cc71f42f65e3e9ff69e7cb37ce6
877 F20101112_AAAMOD huge_d_Page_66.txt
b4a6cf3d5aa478f4988647dadc984f1d
79245014e1da709270a5f0ef0628ff1098861f86
5636 F20101112_AAAMJF huge_d_Page_11thm.jpg
6ffefe83fb1ae765ae89e1e6d79a8127
19ff3efb595a1d9b3a75b73cd6a13b2851994e43
3681 F20101112_AAAMTA huge_d_Page_66thm.jpg
f0b03d53ac92abe349ef9459305813c5
8086ce99d3df621d047a0a16e04e14595dee04ea
36191 F20101112_AAAMVZ huge_d_Page_30.jpg
9f212d8eae542ad29b00c4607b29bc65
fc9d86ab8ebaa596d606c8bb67c65b3d2555bae1
5207 F20101112_AAAMOE huge_d_Page_05.pro
a28c280ca41ab765747fc52306dd9ce6
0b8a71ad24bfc4601b956aa6ddc18fa2d3be096e
996 F20101112_AAAMJG huge_d_Page_06.txt
fdd8eb3de515b525a0706086b8ee05bc
4ba8142dc1c7620371ee74a751657647f7b84fc3
72478 F20101112_AAAMTB huge_d_Page_18.jpg
e0a5677626abc06501f1592b0d0185ca
b35135ccbccf6a5d1b284ad596fc735602b540e2
F20101112_AAAMOF huge_d_Page_41.tif
fd2c8a2dac6d8e536b84b4e006773f06
cba6dd2c75c612777408f54dacc3f9ef87b3edfc
F20101112_AAAMJH huge_d_Page_49.tif
527c4b1069f2b7fc273b61a22b6cd69e
485741ac58d2984e45916dc7a84d9d6d3d071d5e
46434 F20101112_AAAMTC huge_d_Page_08.pro
cc635d38fae90859b4f581365132d261
215378acaa81b5fc6eaddeda10bbab28fd20f48e
23942 F20101112_AAANAA huge_d_Page_26.QC.jpg
5da561fe72ef95e45013f13e3d17421c
3b6a7e66fa2cf623119bb598dc966fcd3c9910e3
23082 F20101112_AAAMOG huge_d_Page_06.pro
27f7a90370926f26b8437b8e58be3c41
e7cdf219c47a0a7700ce959989ea00b9e7bec611
F20101112_AAAMYA huge_d_Page_62.tif
8994ee2419cefc28ea123d06555862b8
7ebda5aeeff9fa79413b748faa9c4418ba6b8d8b
112081 F20101112_AAAMJI huge_d_Page_22.jp2
074577b36f07894755e107cefcbdd156
a42d5e14e72b4b0374b3ee7569685232dec5d380
105131 F20101112_AAAMTD huge_d_Page_13.jp2
3be247a9cfe7737cd6d240d1ac27c54f
fdd42dd9292c3f6222f6965f3a0dc09adf728433
22279 F20101112_AAANAB huge_d_Page_27.QC.jpg
7658f085b535abf8675037bb4226ba69
1cd9e5a3e002d9e3689031e7fcfe6eebd4c12ab3
F20101112_AAAMYB huge_d_Page_63.tif
b0c8f41eb89342a7ea67cc2b4e0df29e
e3c4c2dc8be7325e0fd2a62a944b8b0fc2b4dce8
20390 F20101112_AAAMJJ huge_d_Page_24.QC.jpg
7bb39785176b081a175c68a8cdf55af8
15aa32943834b3fb153b3c644db58ad2d2c240ea
50031 F20101112_AAAMTE huge_d_Page_17.pro
23974e79ed5ba3101ea0bfb640ef333f
cc208e1bb0162b97062433de86f0a6025f84e7c3
6368 F20101112_AAANAC huge_d_Page_27thm.jpg
822a79af68d55164e29025b146ae8f2f
3d937748708886a80ee622477bc748fe4b367785
F20101112_AAAMOH huge_d_Page_30.tif
a40ab7afa740e66fb03bf1c63af9910d
c6c0d3cf9f2ae2c984fdd6f0a14ef34a666c5208
F20101112_AAAMYC huge_d_Page_65.tif
1b096917154cc1a92a1ef07c587c5d71
696ce047d6ef3f614d45f11deded00631af7c04d
22243 F20101112_AAAMJK huge_d_Page_59.pro
22d6015a8de6778744a11599529f121c
d2158b34812deb1a997dd4b52a09e1eaab9296c8
27393 F20101112_AAAMTF huge_d_Page_61.QC.jpg
d5506b7ae68e08c40de54ed51af17e22
d38afaefc6af9a822c7d03de3376588fffd07b7a
6630 F20101112_AAANAD huge_d_Page_28thm.jpg
b974479e42459919a45f402bf6ea013a
6e01ea390e8d832564220ceff825921b2093719a
5808 F20101112_AAAMOI huge_d_Page_02.jp2
6f19aac38fd0cdca5f4f9b055a92f095
3d30650fedd321514cd75437dd9fbec687127d34
25588 F20101112_AAAMYD huge_d_Page_03.pro
a2b0e93c88ad87d53bdf41c574c86923
8056923a751d71bdb52accb9671b8871fac86058
1880 F20101112_AAAMJL huge_d_Page_40.txt
cb19b2b0a1a1619c864b081e8b80c7b7
169d6d2c824d4445fa0258305bd81317f0a01c80
1996 F20101112_AAAMTG huge_d_Page_28.txt
065850bd32e953c41239b087c43a3d80
68c9ec2311435b2b808e0bf8453821ac7e7941fe
3814 F20101112_AAANAE huge_d_Page_30thm.jpg
f224ea8573ed83b64c467a3870e2e8af
ad3122ab8920419dcf87f1ffcbd28cdc002c3fa2
111944 F20101112_AAAMOJ huge_d_Page_50.jp2
17d26e1cb2acb5d2086f16aaeef88cd9
fd206bfdbbd23e2014d6928cff1ddceb53d5cc2f
42218 F20101112_AAAMYE huge_d_Page_11.pro
720598676c40acac7cb481958c04920a
98c54d0031dbd7147203ae654338973818dafb5d
62343 F20101112_AAAMJM huge_d_Page_24.jpg
b2a54c91d6bf0aad546407f029bc2a65
a6e605a54adcb68f4a05f3597e77a8ccabec072b
74039 F20101112_AAAMTH huge_d_Page_16.jpg
3e11c5904f107bca4f0824ea9361b90a
85e3563986bee3c9c2efe32a2003f61398bc02e3
5494 F20101112_AAANAF huge_d_Page_31thm.jpg
f3387c20ad5e30159e9e25d90f48a74b
391b554f40889a01f22c1f173c2920595e4abdb4
F20101112_AAAMOK huge_d_Page_26.tif
213a1e403602313b0380976277de7574
244da1c8f87d6204f6dc50f672745616f8173e42
50427 F20101112_AAAMYF huge_d_Page_15.pro
0a13c2c377b7cdff5816da7ff71d3000
e2b22c04df84e7b1014dfb5ceb5a04525d6f52df
51113 F20101112_AAAMJN huge_d_Page_44.pro
51626d50968c206599f9fbd95bf7c141
9c0d6ebcee4ea38685086712ee49efbf69165d64
51338 F20101112_AAAMTI huge_d_Page_43.pro
ccb8074dcedaabb323c6fdab37f42643
b2f821542795307b65fe317f29367159f2a61a86
23004 F20101112_AAANAG huge_d_Page_32.QC.jpg
8bd1f00a3cea615bd3f365d9d4f0a396
751502791160d53157f68bd83ef9546e2af7ccb3
6537 F20101112_AAAMOL huge_d_Page_50thm.jpg
924ae5d055724234bb7c97f77926b5f9
85190d33a10aba8a262f4f0324c9968ceb9b5871
49970 F20101112_AAAMYG huge_d_Page_18.pro
2c7a89e97e3102fd7b951c03e0febf2d
970949c386339d7b98066d04134faee3dd627be1
73025 F20101112_AAAMJO huge_d_Page_44.jpg
8c79b2680723a9b426e8a4f37e7ca00a
d966093790c5015991bb28f72c83cad03a1b6242
9381 F20101112_AAAMTJ huge_d_Page_01.pro
cfc7efbef76b5513b3d47662fb1f9978
7d6b0071b9862a1c99b5c8bf0a5ce6983a638d1f
23584 F20101112_AAANAH huge_d_Page_35.QC.jpg
0700169906b05cef11fbb99fcde978c3
aed9cc0aaac3b2c7c9a330ed91a643e4e0ad1569
48528 F20101112_AAAMOM huge_d_Page_12.pro
1dcee1f95a657fd030b0372d897e2cf3
6f8230dc168cdce80a4eb4282b1d86852a10c601
40941 F20101112_AAAMYH huge_d_Page_24.pro
fe39abe67c2b9698fdcd940bdac5bbd3
fdc99586d08cb83e1f07273f62e7e0b2cecbf1fb
112414 F20101112_AAAMJP huge_d_Page_43.jp2
4dd742017bf1f132e4e5e6a2a574fb78
8e205b26248a478a4351aaa5d33e8a0e411c5869
41489 F20101112_AAAMTK huge_d_Page_10.jpg
fa690f7cffccd38e317e27d92ad1ca01
bd96bd4426e4d2fd66c3f2d754df16b507093034
3586 F20101112_AAANAI huge_d_Page_37thm.jpg
d7cdde3c4104ae67590a4bab759647d2
3129127553fa781b7f80937a00c513b25a9f6825
19827 F20101112_AAAMON huge_d_Page_11.QC.jpg
922aee8995f8f7b0154d8f86efc6546e
eb4e0b7d6a153b91595170d3ebbf0a9f27b5cde1
47085 F20101112_AAAMYI huge_d_Page_27.pro
c96cace960e5251a903453509c01c955
45b4ab8f8bdeab32078d4f1fbbac4ababc288397
1748 F20101112_AAAMJQ huge_d_Page_24.txt
81d94bac18d96420b9ecd74e63bd33d9
349a27fe9d4c4a14b5f1cf7123955b4b771c528e
100890 F20101112_AAAMTL huge_d_Page_40.jp2
9d3a078fcc7399b04607f729529129fd
981d2b5a2fa6a0c9663102f03abf0e0e751fa3f9
14599 F20101112_AAANAJ huge_d_Page_38.QC.jpg
b14480dfccf825132270d2c9a3c3bde3
2747e131faa6ad41ef0623328c038272d29adc8c
3341 F20101112_AAAMOO huge_d_Page_02.QC.jpg
d327dbb4a5a9ce25cac7c1ea54f56677
152f7eedbb1af21449329a57f19aa22d3f6b5f66
F20101112_AAAMYJ huge_d_Page_28.pro
a482bdf20bf6b4331fc30377a6eccb5f
a4b2d11553971318aa468cd6328e6a15fb9bcdca
2038 F20101112_AAAMJR huge_d_Page_49.txt
b8b21cef3b6e9f74546de737c0f7991b
a1c1ff37d0e5bdac1c85155acd88054aaaf60de9
4668 F20101112_AAANAK huge_d_Page_38thm.jpg
2eaa9b797b1cce0fb1771fc04f7be5a4
9d1afa01490788976a7891a2da62a8af88c46c27
20812 F20101112_AAAMOP huge_d_Page_33.QC.jpg
4d71909160b43b5ed2bd2bc0191af676
8ce569de16dbae7e4940819c1885d38ac3ffb16b
49219 F20101112_AAAMYK huge_d_Page_32.pro
830dd243296eb9f23b230090e9e70c66
6fb80bb44dceb85c7d1f53de5c45a349b387190b
110727 F20101112_AAAMJS huge_d_Page_41.jp2
91724a58c475620e70b3f7fe917e9b25
56b1a0e003e8834c91cdc5555d1c0f5a6bc85d9b
2075 F20101112_AAAMTM huge_d_Page_16.txt
881f71b4ebc6d2b1b22d8d1238727a74
b35a0a7b734d9dc7dca6dfcf5e04f19fbd8adb60
21340 F20101112_AAANAL huge_d_Page_40.QC.jpg
a5a71e3440dd9d98315f022509ed8acb
1fe53fdac7d77f999a5a22fb80dc2f84b80efb23
2005 F20101112_AAAMOQ huge_d_Page_35.txt
7f4af393c6167d722b372b64b2f7464f
8f60ed131e65534a19f6e155c0ebc3b1af48496e
44296 F20101112_AAAMYL huge_d_Page_33.pro
fc192939b206278c934a70ec17b2ef99
c228079d9aca004fe0b487e43d896b4808d0e3be
894 F20101112_AAAMJT huge_d_Page_59.txt
103ffcd9faabdc5374514cca55113401
6dac927239c24b2e1cf2a20482e52076e7dc6857
F20101112_AAAMTN huge_d_Page_09.tif
d65007e76da832c3a13cc2d7faa5c1b1
f865fe02fb624f4dd533ff46a7ad1effa13b7e08
23757 F20101112_AAANAM huge_d_Page_43.QC.jpg
142b71c9f368923824d693b34ff5198f
d8e898f51297a6b62a93da7a9a60b399f3177719
103097 F20101112_AAAMOR huge_d_Page_27.jp2
84cc0a00d03ffe5876c4fa1ac43380a5
d9d3dacb5f817c56b90aba0509648a42cf433501
46777 F20101112_AAAMYM huge_d_Page_34.pro
001eb55bb28ab24e96448df5105c3b99
9c5db61992b4131290d496fc7eb0e426a01b4c07
72227 F20101112_AAAMJU huge_d_Page_26.jpg
9c63a774ddca4a50a07111a2bf2bfada
a41396bbaa1cf54331ba30bafd6cc883a37e1fc8
110186 F20101112_AAAMTO huge_d_Page_15.jp2
1fc328331e7b8af691c29063598bba0f
a5ef357edd6dc508bb4c3bb567f1623661bae66a
6561 F20101112_AAANAN huge_d_Page_45thm.jpg
4e509e53bf376c4bd4eec9a3a75af88e
14b75df1928105afe5c36cf5ad1a581678a6d5ea
6519 F20101112_AAAMOS huge_d_Page_41thm.jpg
9abddb38275f9ca22dbb6a9b58ec5e91
447f605f82f0ace41f06754577375573efc0a393
20053 F20101112_AAAMYN huge_d_Page_38.pro
2a67c26a1f36b7bfad65be0424e9c493
548f51e99e91017f84a5f3fac967fb36aadd64ab
16102 F20101112_AAAMJV huge_d_Page_55.QC.jpg
b32bba3566d4639cfe97fe9e13b6afd2
985ede8e494b5c5b27f38aa26caf282f01dc89b9
1558 F20101112_AAAMTP huge_d_Page_05thm.jpg
38cd6a67628c29a79428799bf044d1b9
d0a4b602a02ba5247318952b4aee0ce741879ecb
23665 F20101112_AAANAO huge_d_Page_48.QC.jpg
500067385604261653dbc64b9b947de3
d35ec8e7f8a804ee175361300e2130b80d1a2abe
36630 F20101112_AAAMOT huge_d_Page_66.jpg
f98b5370d1d8c795476d29f12a8ad9dd
f22da851be5709acaaf48314b14e9f05fb5cb544
23470 F20101112_AAAMYO huge_d_Page_52.pro
94d11c8ffffcdabffbd9a963b87c878e
cc4c220fc80544312ac5adcb64a7eceb86db8773
2049 F20101112_AAAMJW huge_d_Page_20.txt
b258d2e2868ab82206b895343dc5c236
02994e4361c3c6f1302e59f80399bf09827d509e
1913 F20101112_AAAMTQ huge_d_Page_12.txt
6bf653d460ace920caeecfdb4fefb89e
b547a70089b6cb66b3cb8d8cc094182e332a3203
23883 F20101112_AAANAP huge_d_Page_49.QC.jpg
d67b5a8b1519fd07a7d0ae5d83fb3f73
a60c845d8e51fa5b32ac006720c067a677f53902
16913 F20101112_AAAMYP huge_d_Page_55.pro
a417d3479e5764cfdbedfdbefec0f835
3b119b4aa768d758b9651954777dd41a424d2aa4
48627 F20101112_AAAMOU huge_d_Page_14.pro
87ded4e7eecdb2aa781534850adcd41a
50b715d02fc6cfbf57b5fb9305248902afa5ffbd
96303 F20101112_AAAMJX huge_d_Page_61.jpg
2cf380f2d325d278490f8e8870f3b994
eebe8e71bad269279def5244993b516127e0c386
11813 F20101112_AAAMTR huge_d_Page_30.QC.jpg
8d535f437c2057582a249ec66b585b9c
fe5900d931d4784db3bcbd69a15b94e458e1a11f
16687 F20101112_AAANAQ huge_d_Page_52.QC.jpg
af598dbc32f62fcb2f7af32bbbc5acb0
35f3ad9a737a587ff1b4a476e163be85a721b47e
23091 F20101112_AAAMYQ huge_d_Page_56.pro
9c1a6d6f83fc218b263d2b1555cb13e1
644cd77fe622e329945f355d09ce9087a348844b
2329 F20101112_AAAMOV huge_d_Page_04.txt
4ad06466475a8c9c5a83b5a5574eca90
2e21ed166036edc6f59632c986daab47f2193dbb
52085 F20101112_AAAMJY huge_d_Page_20.pro
c0ddf6c5f1f38a41174d80ff8d453312
6d3bc04cbd83a54936668c3388f686527b892414
942 F20101112_AAAMTS huge_d_Page_38.txt
ed36f9bbbe9031a6086c7caeb2bbc579
8ecc8be4a7eec0e099b49f5753af8189054a023a
17950 F20101112_AAANAR huge_d_Page_53.QC.jpg
120f366ceea695ff0744c3c42d5b0d50
74ee9af62dcb2391fd1179772e5ce5c76aaa5b9c
7193 F20101112_AAAMOW huge_d_Page_61thm.jpg
f13b9e7866fed7c188d93b9cf94a7436
9a6afb15ea5c99f8dcc072d8c537fa4bff03feec
51396 F20101112_AAAMJZ huge_d_Page_19.pro
fbea701d589854d82e4dabc161eba896
845a3a4c6aa951a1a202f1ce58d8062ae3597fd9
1775 F20101112_AAAMTT huge_d_Page_11.txt
168a6ee460c7485f7b85b74710bd2112
230596746b6a2d16c3cde32c814e121d5b4b3774
3765 F20101112_AAANAS huge_d_Page_57thm.jpg
0b8cc8889f8e0dfafbddf09f45f43784
bbab5cc18a5887217d7805306667e4e9c5456d16
16042 F20101112_AAAMYR huge_d_Page_57.pro
b1303d857deb1ad315d2d9f76d90e3a9
5cdf7c6138a1b5854f76f19adf6a60381d790270
71805 F20101112_AAAMOX huge_d_Page_35.jpg
3032d28d2e30dec452e3237fa3f9a8f0
05378caaff3404e568443d6c3fa64dc6e89527ca
3940 F20101112_AAAMTU huge_d_Page_10thm.jpg
4f45c2872fb0f09c4f04a82d6b07f6f7
cacfdfdb5daf3bef82b33e351022b7215af884db
5765 F20101112_AAANAT huge_d_Page_58thm.jpg
ea8c73f2a82e59418112d589d680471d
ff78303b1266b6064e124733e45371152c6497b7
70794 F20101112_AAAMYS huge_d_Page_61.pro
1b4fdbc833023b70b6df821e5403770d
b4452296d0c28a53d798a7a2178220137128f162
52358 F20101112_AAAMOY huge_d_Page_52.jpg
69a915ef50c5ad48cfd38dba6b0d8e5f
c58b20a71a2c9cd205f559d67a8ca853370a3f7e
F20101112_AAAMTV huge_d_Page_32.tif
949d6e14803e7bcbea6f37b0b384fe69
5b8a0ddb057207fd0c6be36577ab0f2e70c3f5c9
F20101112_AAAMMA huge_d_Page_22.tif
4b12280b769aead90999fbabbf6e4bef
bb877e9eb29f851e7f64d893a0713cff1972cc51
11894 F20101112_AAANAU huge_d_Page_59.QC.jpg
142c4040746ab9c7726c869130ed5393
f112c25ea84ae680f96d6d8db019906253045a64
67992 F20101112_AAAMYT huge_d_Page_62.pro
00db77d0ceec7f1e6d24f70a20f1cef5
f18bd045ad039d3c8a4bd979722d6c5ab7515ef9
51629 F20101112_AAAMOZ huge_d_Page_46.pro
7967039068826800a5409145dd3a4466
f50e6ab43e22cd2aa50218e7e916f5a1a367755b
43095 F20101112_AAAMTW huge_d_Page_58.pro
401437b1c7981d57d4f58716c25c1aa9
d107c0e1bc04cdd9209fd02aab1e668d981d926d
23892 F20101112_AAAMMB huge_d_Page_47.QC.jpg
fb3d1a64c9b8005d080019f914cbf3a9
d00bf610547b1fb3f6024783d8b58eba0947d7e0
3561 F20101112_AAANAV huge_d_Page_59thm.jpg
db90e44bf144f04ec50ff170417a8933
2d0cb7f9dceb76b8db3deeae83c8bc0babaa4e24
67333 F20101112_AAAMYU huge_d_Page_63.pro
f760a0216814767bf1c1cafb22ab76fc
adacec7dafb4d269c37a99b191af6abd9c75ab75
6509 F20101112_AAAMTX huge_d_Page_22thm.jpg
5cbf46a98bf0bc1487db0a37b3adedef
ba3648073e5c2fc21840e2419c4761c79977cad1
71773 F20101112_AAAMMC huge_d_Page_17.jpg
579a5b6e7bdf1e2190e01374643ee8f5
17b39efa4aeafbd898cfe68bd3e4f103891c9206
20379 F20101112_AAANAW huge_d_Page_60.QC.jpg
9bab7cd3e60f95b6d349c04f9d504983
1e0cd372a4eaf943954bd20e7bcf7e52469d1a05
511 F20101112_AAAMYV huge_d_Page_01.txt
44994db2694267663940b0263ef961d7
50d1386f11366e3f537f01cf4b2d2abd76e42834
641738 F20101112_AAAMRA huge_d_Page_52.jp2
4c92d383837d7e14c9812ae16b07088a
4c4e3fa911fc1905f2c1151a5130f4e3f7d069cd
40345 F20101112_AAAMTY huge_d_Page_31.pro
358b83107312e92ec63ab5df98d35c0d
a917cf19fe9085d21a613d657ebad8a7f3187b58
1862 F20101112_AAAMMD huge_d_Page_27.txt
3bc37663b5e4dd23743b53815c1c4907
37e6a87f00ac7d7d5efef87c53559c338ce57001
26419 F20101112_AAANAX huge_d_Page_62.QC.jpg
f04daea216e743a6593bbe4b9d1449f2
4c35636323976dd34e93542caa5686d7480f4c81
2834 F20101112_AAAMYW huge_d_Page_07.txt
4ebe0cc656ee1bdce8a8fe2e709b80dc
80de68fdd33cc70a120a08cfd3862d89c009ad46
59145 F20101112_AAAMRB huge_d_Page_03.jp2
89c715e6e84067b977b75db13687d0f4
fbebf04e5aadfa5b8a70f4956048ee468ad37fab
F20101112_AAAMTZ huge_d_Page_50.tif
0b0fde345e7a9f5b27cd67495d5f6e85
29a08050ed7cd4ff12ad9e26dec5ce8c400253d0
153550 F20101112_AAAMME huge_d_Page_05.jp2
90560240dc5975798c065df85ffdee00
2431b683295d8ad2d1e160e9bf7382d2305e0370
1956 F20101112_AAAMYX huge_d_Page_14.txt
bfb4359774dabec7f3079c304cd972d7
dcf718c9857866bceb4c82e22d3ed3ca873c7149
112133 F20101112_AAAMRC huge_d_Page_19.jp2
556dcc90a1d7e33b64403562a7b33464
a5cd8afd0ab5e887aae6189d8699fea761d94355
7048 F20101112_AAANAY huge_d_Page_64thm.jpg
56377b00661dd340c4b5a179ad8d4fd5
9a3f220cfbe201abdddc7984ed2d842187314bac
F20101112_AAAMYY huge_d_Page_17.txt
073e696ddaa5e603984537f8907ff7d0
efac1aabda094f5042e05a648814ef8996bb9279
7973 F20101112_AAAMRD huge_d_Page_01.QC.jpg
c1bfc04e1313fe59ba763107c29adf6b
40e251426bd958cd17c934d3359a3c3a01516370
23866 F20101112_AAAMMF huge_d_Page_29.QC.jpg
02f818bb3dc1fd1576589fd6ae2f7107
392b2d89a6ad5e24890e29ec0390c9df282db2eb
69983 F20101112_AAAMWA huge_d_Page_32.jpg
71bb35f3bfdb24635100d665e361864c
9d02a6bd4233ab249105792b3b642dc6ccdbd395
6016 F20101112_AAANAZ huge_d_Page_65thm.jpg
e5264197630c1f790fc54a04408566b8
06caf0e6703dfad7d5bc7019e07d1a3c67cc949a
1972 F20101112_AAAMYZ huge_d_Page_18.txt
7cb86e4ae8bb2761b585e73a856b4b77
1661dfeaedee184bd1113092f32fb69b3bf83fd0
1976 F20101112_AAAMRE huge_d_Page_41.txt
ad02f1b466b5e9f80573ab2c83ad92c1
d0d9fcfe2a948340db57b8237e3adc5d0d0be30b
51818 F20101112_AAAMMG huge_d_Page_42.pro
4857f63c79c9143cbea970a73bd8094d
0c87e7abe728b24fe7d3c1e3178be6198c73283e
64146 F20101112_AAAMWB huge_d_Page_33.jpg
4dc37cd9ff7fafc9cff7bcf7ff1a7c02
02792b6c7e6f7e1d8868e3f7d65829a3a6f5d6ba
89714 F20101112_AAAMRF huge_d_Page_23.jp2
808c1b459bc9ef71c6fd0a3ef76204bb
5d71b847e72fa1c2810d01b3cc3efb6a5fdfe4c9
5500 F20101112_AAAMMH huge_d_Page_52thm.jpg
f8ce64bb23c3de079bc611a41539d7a7
4f70c8d9255d06374817dfa4e63da7c61ec4a99e
70402 F20101112_AAAMWC huge_d_Page_36.jpg
c96d4440b8306dc2acffb5ecb0979dbf
7de57ada03ecf5e2cf44bc85a189853012dbb734
2734 F20101112_AAAMRG huge_d_Page_63.txt
98f9912dc5933139f01ccb9db0ee2e1d
af898d92ad0a60486a28789f003194604b40ac8d
F20101112_AAAMMI huge_d_Page_59.tif
fe563ed1780998dd9ce2b8f263ad378b
8b8de342a7f633af3d256856817f4dd032a23e88
36006 F20101112_AAAMWD huge_d_Page_37.jpg
7ca68e19c4ab4e2a6764b251089ecf65
9c9977476efb668cb4dbb96457d7843141148004
24104 F20101112_AAAMRH huge_d_Page_28.QC.jpg
373c5f5c504ec6499be781902c757b81
bbfa993b7e1f9102f12658924399f254d067b326
90241 F20101112_AAAMMJ huge_d_Page_09.jp2
0406e5d20b90d1cba721fb07aa2102aa
4dda1e8434c56af79467b8fec3bb9a4b50df0d45
72500 F20101112_AAAMWE huge_d_Page_41.jpg
1f40a431281c5d2f13882c985cf76a84
49226f145f4cc5c3b37c417672dadf4ec29196b5
74119 F20101112_AAAMRI huge_d_Page_42.jpg
89cdf07d3e32f8f2efd1eedbb68a860f
092b13cf13c24c663450b91081a6e16062ff9295
37133 F20101112_AAAMMK huge_d_Page_59.jpg
35344bfb0dc8688b93b156276f781112
ef939bdc5c32b0b9b7136afb6e54487ec70369c5
72260 F20101112_AAAMWF huge_d_Page_43.jpg
e638e8da36a3ca02422fbeaabcafbf87
1524e7a7ad07d85935a02a1b84152f7fbf3a341e
F20101112_AAAMRJ huge_d_Page_47.tif
51311ed316b4cf3d9c784b97b341eba5
8aa039bb16e31ee6725dccf0bfc5768ead76a9b5
92719 F20101112_AAAMML huge_d_Page_24.jp2
033bfcdb994749a2ea7f1b880ba49a7a
1200dd03cd570857f01291acd69bdee9c5af3d3b
73417 F20101112_AAAMWG huge_d_Page_50.jpg
a8c905691d0c6a4714af3abd0180c68d
51bd665ad3d808055349684001f64826ad9f1784
4806 F20101112_AAAMMM huge_d_Page_39thm.jpg
fe85e025eea7b3abfe6d5d19545cbfb4
fbcb3d5281f925edbae8e5205918231781e6b0bc
46226 F20101112_AAAMWH huge_d_Page_55.jpg
3a6f0e4cda970c47c281e7eb6b7949f7
c3b6e7343de299df3edc594d25aa0aa61e10f062
23642 F20101112_AAAMRK huge_d_Page_41.QC.jpg
9389158586dce5b28092cfec26282693
78d1e069ec86d6f44192129dc3d77845feed9e99
6485 F20101112_AAAMMN huge_d_Page_47thm.jpg
40dd48b726d858631dd1ea3f39887b9c
f89ec13663c189ced6ca1959b4904127619dd942
39200 F20101112_AAAMWI huge_d_Page_57.jpg
dbb5dfc7d06c06ddcacfd3867d411794
6ffa74762b9ec946343cfb62fd7a8c67e31ef5eb
39207 F20101112_AAAMRL huge_d_Page_06.jpg
1ba276e3cf112cfb9b97c79bc9d1149e
75789828f70a8e9302a687d42e9ae71d4612b261
49229 F20101112_AAAMMO huge_d_Page_29.pro
59335cf4fe4cc758cf478e5d5798ebb7
0538cc6dc211ab15638e11a9f7d49f5fc2a80e50
62812 F20101112_AAAMWJ huge_d_Page_58.jpg
cae9ab1c1b551a37815f9b7d747c3359
50a1dcb53f41fd8440027db85dd43217d3bc125a
F20101112_AAAMRM huge_d_Page_52.tif
34f72e1316482c713ec8469be1001b08
fe37a65213c742b254185ea8465a4f81575540f2
108171 F20101112_AAAMMP huge_d_Page_07.jpg
5d2c3eb5222196293feb1c2e981fb7db
7930d5b35d0739af7a233bc2c538fe7d161be9f7
71130 F20101112_AAAMWK huge_d_Page_60.jpg
64a3558a50147964fd1b5d4b918f159d
14c1f221b5f4523a66b4868b45929f2a24cd2f49
23784 F20101112_AAAMRN huge_d_Page_25.QC.jpg
738a64b96101d55635791141a6cfbb89
d86432cde5fb1dc408c6c0e55d65cbe3848af6f7
24074 F20101112_AAAMMQ huge_d_Page_16.QC.jpg
acc7d6b6855e0e0bf7a1723d33b443c7
f079f5872338271a8557555566463025303f8a1e
96225 F20101112_AAAMWL huge_d_Page_64.jpg
7c9a1e63577edfaff140dd2fe7f182e9
55d9b247802b2ced3c089ae1c2fe6f2b823f54ed
93267 F20101112_AAAMRO huge_d_Page_11.jp2
4133f95f9f30e15238757012f90584ec
f68fe484861c36835c88826408084509f612faf6
111907 F20101112_AAAMMR huge_d_Page_16.jp2
f737208f76c8ec820f7d40eb13907774
fada59dd528e54f28b42f5fab1b7f70c426d48fb
79395 F20101112_AAAMWM huge_d_Page_65.jpg
af0628cac39ebdb186e6b9934511d288
67079bae5960ad928fd572e67734bbbcd942bca7
5468 F20101112_AAAMRP huge_d_Page_60thm.jpg
bb3d5ddac2704695a1f98fae442d4b39
b04086d37066756e5c279b3d67b00f7ba183bc63
54345 F20101112_AAAMMS huge_d_Page_39.jpg
2a3341d6f6e1626aa48896a8f0270561
642a5cdc74a38159a2236e0f81049980bc53e141
1051976 F20101112_AAAMWN huge_d_Page_07.jp2
7f93f79fa67fe9dc15be97f701c19d2d
6751a9f2d562329bc550cc8466f129b642108569
72804 F20101112_AAAMRQ huge_d_Page_15.jpg
0dcb40491d8605df9c225f289a5ce63c
25b14b4c0d93aa0a76209f29b1c34b796ad7ba8f
46728 F20101112_AAAMMT huge_d_Page_13.pro
8548d195ad2ad00ee2e81d98f850100d
ca621731346af759aa8f5e1e0bdbd282c9f1a263
108675 F20101112_AAAMWO huge_d_Page_12.jp2
199519e23426e51c4a1ee57974bda155
dd5cf67a1c735209772b25359fd28bf65e290204
40069 F20101112_AAAMRR huge_d_Page_23.pro
7efdcb46ab1863542fc3ad6e16b54360
02f2b9d4000ec8ae19e827a1aca94879f788bcc6
2021 F20101112_AAAMMU huge_d_Page_43.txt
11124effd6be07c10637d904f97d0c4f
f4297e26f89de263a77df248f0fff9adc0f3da92
1119 F20101112_AAAMRS huge_d_Page_54.txt
6a9c9bd1a37702c1832db880d8efcf1d
da62db51abf6ec195bb3bc4657012f156d5baf06
109441 F20101112_AAAMMV huge_d_Page_44.jp2
d3ab2e4deba184a9caceee73ad2b35f2
a8c7741d8af11fa2984cb6f5cfe25f6ea9f9a21c
106944 F20101112_AAAMWP huge_d_Page_14.jp2
7b060d1312a9c69c0373ff484a8001dc
abbc339bfbd0629b1ed3566ca5bbb535bf752021
6383 F20101112_AAAMRT huge_d_Page_34thm.jpg
c955a58e0b91fc2cfa5f420a5c1f3c19
a771619037d674ef7781cd60f1255ba6f6df4d8b
6714 F20101112_AAAMMW huge_d_Page_25thm.jpg
2e01e842b17345bf4beccb1df8223607
f202a1a4e037c0284c5c982ac69270ca04313f67
109068 F20101112_AAAMWQ huge_d_Page_17.jp2
8580951972f241613b329476a839d1c6
ebe9e03513046d597ead3917a93ea28e88b6d45a
73159 F20101112_AAAMRU huge_d_Page_47.jpg
649f3b2f6c7207f062a22c0f579dc29f
d9111a35a74327463daf8ed44d97a52270905a44
50636 F20101112_AAAMMX huge_d_Page_35.pro
e39f390a7d60bdfa14df04cc13bccf78
7e935ff07bf7ac02a8cb464105d2176d1d0945d6
113338 F20101112_AAAMWR huge_d_Page_20.jp2
5612602ebcd45f03e4462db684d62d5c
9a1747f71216e5f3b72af0deafb1b1509f3d2bbc
F20101112_AAAMRV huge_d_Page_10.tif
c9741538bc4ba82c65a30c72e9f64db3
7f47b897520341930a4f06ba0385acfdc07f2725
51414 F20101112_AAAMKA huge_d_Page_50.pro
dd0f5db50c7e0539b6a5f87fa18b0fdc
430ce57e97a675abf13250ef61d6fe424886fcab
F20101112_AAAMMY huge_d_Page_21.tif
5dc5d9c54a007b6f63f9e7d006f90805
ad672ba388251fe537037bc42a19c7bff5ee1fbf
114308 F20101112_AAAMWS huge_d_Page_21.jp2
b2c7bec1942fa7fb789feb20aa28a724
95a64af02f001248646a87ca2b617581a7aca0e9
6379 F20101112_AAAMRW huge_d_Page_32thm.jpg
39d83a130e0b97652576dc339046ef1c
ab373cb7a783a4134b4e6fdc0899be99de9219cd
17125 F20101112_AAAMKB huge_d_Page_39.QC.jpg
8e6126ccbbae10f761d0d52146ad9842
6efc5dffcddb1c2d5a8770084a4ceae4346ac6bd
52932 F20101112_AAAMMZ huge_d_Page_16.pro
a6aa194c7266617996046905b406af06
2d0654b7d26da526e3e1e674543f19ceb5060ce4
109733 F20101112_AAAMWT huge_d_Page_29.jp2
30d5f07faf9fa86c598e4fbc29cea2c6
463fdf569e5f391ad1037f596aded529f82faca3
1781 F20101112_AAAMRX huge_d_Page_58.txt
6cabd1c59f48c41a1367e9d53f9e4722
12b45c025de8ed769efb2439acf85c6e50369d63
1931 F20101112_AAAMKC huge_d_Page_36.txt
59c2b6237d724328c214bf7bb288b472
7ab450213a7e0f7ad12f04951d6d966d7d00a602
106708 F20101112_AAAMWU huge_d_Page_32.jp2
97d535bad60e7609a806606b5458bb83
5b9d27c037862c87a401c206edc5e1b4916c71db
101855 F20101112_AAAMWV huge_d_Page_34.jp2
2b585364707c5f307fc627d0ff6693cc
303edaf89e1478ee630b11fa0fbcacf4de10ec5b
95968 F20101112_AAAMPA huge_d_Page_58.jp2
b9c37ca77196e7c5f9036443c2fbfabc
84cdea59fcad45d2210e8fdb60245e401f954df0
136949 F20101112_AAAMRY huge_d_Page_62.jp2
bd356a21a7aab393bb41b5fbcdd8efe3
e0b8bef863bf1ed415fb912d2c99ead3e274f1f4
F20101112_AAAMKD huge_d_Page_05.tif
e265c751990b76b69dd263b28d4c9d99
bd0c79d27b1cd57ac54b772933d72058f373a5d6
106443 F20101112_AAAMWW huge_d_Page_36.jp2
9ff295dad283a6cb2838d1fce1201dae
90a40b50fbab9ac243f01f8e80859b7f7e8d1a3c
23692 F20101112_AAAMPB huge_d_Page_15.QC.jpg
ddf4a05f560fdffe168d361c7035eebf
9f6c063ade441ad04b1625c8ba346356613d9ecf
F20101112_AAAMRZ huge_d_Page_01.tif
5a8115b9d901ebeb90fc734bc74092ec
381a97772833caa10a8144043b423cc63cf63490
F20101112_AAAMKE huge_d_Page_19.tif
90f04f635c1e23fc0377eec21cbbbe59
d5bd18702d65b1aa16ff827f4a01fedfa09b682b
49102 F20101112_AAAMWX huge_d_Page_37.jp2
f395fd767f7aee9b2382ace5def4dd7f
4ff6118624f42a03f9090b60438d9c40c8b139e6
16028 F20101112_AAAMPC huge_d_Page_04.QC.jpg
83ee9f3641f38b7c56b8cc981cb87f85
a78c88bfe0e55c80cf10e6febef4cc8a86b1b809



PAGE 1

ORGANOCHLORINE PEST ICIDE REPRODUCTIVE EFFECTS IN FATHEAD MINNOWS ( Pimephales promelas): COMPARISON OF EMBRYO AND MATERNAL EXPOSURE By DANE HOLLAND HUGE A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

PAGE 2

Copyright 2006 by Dane Holland Huge

PAGE 3

ACKNOWLEDGMENTS I extend my gratitude to Dr. Timothy S. Gross for providing the resources and guidance required for me to succeed as a graduate researcher and graduate student. Special thanks go to all the staff at the United States Geological Survey-Florida Integrated Science Center, Ecotoxicology Lab (Gainesville, Florida). Special thanks go to Dr. Richard H. Rauschenberger, Dr. Maria S. Sepulveda, Carla Weiser, Janet Scarborough, Travis Smith, and Jon Wiebe. I would also like to thank Dr. David Barber for his invaluable assistance in my thesis revision. My research was supported from a grant to Dr. Timothy S. Gross from the National Institutes of Environmental Health Sciences. I would also like to thank my friends, family, and especially my wife Tina who supported me throughout my career as a graduate student. I would also like to thank my grandfather (Edwin J. Kent), who exposed and explained to me the irreplaceable value of our aquatic ecosystems. iii

PAGE 4

TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................iii LIST OF TABLES .............................................................................................................vi LIST OF FIGURES ..........................................................................................................vii ABSTRACT .......................................................................................................................ix CHAPTER 1 INTRODUCTION........................................................................................................1 Background...................................................................................................................1 Selection of Animal Model...........................................................................................3 Fathead Minnow Reproductive Cycle..........................................................................4 OCP Characteristics......................................................................................................7 OCP Exposure Responses in Fish.................................................................................9 Significance of this Study...........................................................................................12 2 MATERIALS AND METHODS...............................................................................14 Experimental Animals................................................................................................14 Chemicals and Dosing................................................................................................14 Pilot Study..................................................................................................................15 Nanoinjection Experiment..........................................................................................16 Maternal Transfer Experiment....................................................................................16 Analysis of Fathead Minnow Tissues and Eggs for OCPs.........................................18 Gonad Histology.........................................................................................................19 Statistical Analysis......................................................................................................20 3 RESULTS...................................................................................................................21 Maternal Exposure......................................................................................................21 Nano-Injection Experiment........................................................................................24 4 DISCUSSION.............................................................................................................30 5 CONCLUSION...........................................................................................................48 iv

PAGE 5

LIST OF REFERENCES...................................................................................................50 BIOGRAPHICAL SKETCH.............................................................................................56 v

PAGE 6

LIST OF TABLES Table p age 1 Day 30 GC-MS results of flake feed OCP concentrations (ng/g). N.D. is defined as not detected.............................................................................................28 2 Day 30 GC-MS results of female whole body burden OCP concentrations (ng/g). N.D. is defined as not detected....................................................................28 3 Day 30 GC-MS results of Egg OCP co ncentrations (ng/g). N.D. is defined as not detected..........................................................................................................28 4 Day 30 GC-MS results of OCP con centrations in eggs (ng/g) taken during Days 1-4 (early clutches), 13-16 (mid clut ches) and 27-30 (late clutches). N.D. is defined as not detected.............................................................................................29 5 Day 30 GC-MS results of OCP con centrations in male and female gonads (ng/g). N.D. is defined as not detected....................................................................29 vi

PAGE 7

LIST OF FIGURES Figure p age 1 The percent of spawning pairs among treatment groups. Control, low, middle, and high treatment groups had 10 potential spawning fathead minnow pairs. Mean standard error results are shown. Significant differences between control middle and high treatment groups using One-way ANOVA (P<.05). Asterisks indicate differences in relation to controls...............................................42 2 The mean number of eggs laid per spawn among treatment groups. Control, low, middle, and high treatment groups had 10 potential spawning fathead minnow pairs. Mean standard error results are shown. Significant differences between low and control treatment groups, low and middle treatment groups using One-Way ANOVA (P<.05). Asterisks indicate differences in relation to controls.....................................................................................................................42 3 The mean number of eggs hatc hed among treatment groups. Control, low, middle, and high treatment groups had 10 potential spawning fathead minnow pairs. Mean standard error results are shown. Significant differences between low and control treatment groups, low and high treatment groups, and low and middle treatment groups using One-Way ANOVA (P<.05). Asterisks indicate differences in relation to controls.............................................................................43 4 The percent of clutch viability among treatment groups. Control, low, middle, and high treatment groups had 10 potential spawning fathead minnow pairs. Mean standard error results are shown. Clutch viability = No. of eggs yielding a live hatchling / Fecundity x 100. Significant differences between low and middle treatment groups, low and high treatment groups, control and middle treatment groups, control and high treatment groups, and middle and low treatment groups using One-Way ANOVA (P<.05). Asterisks indicate differences in relation to controls.............................................................................43 5 The percent of la rvae survived to Day 7 among treatment groups. Control, low, middle, and high treatment groups had 10 potential spawning fathead minnow pairs. Mean standard error results are shown. Significant differences between control and high treatment groups and control and middle treatment groups using One-Way ANOVA (P<.05). Asterisks indicate differences in relation to controls....................................................................................................44 6 The percent of la rvae survived to Day 14 among treatment groups. Control, low, middle, and high treatment groups had 10 potential spawning fathead vii

PAGE 8

minnow pairs. Mean standard error results are shown. Significant difference between control and middle treatment groups using One-Way ANOVA (P<.05). Asterisks indicate differences in relation to controls...............................................44 7 Mean GSI among male treatment groups. Control, low, middle, and high treatment groups had 10 potential spawning fathead minnow pairs. Mean standard error results are shown. No significant differences using One-Way ANOVA (P<.05)......................................................................................................45 8 Mean GSI among female treatment groups. Control, low, middle, and high treatment groups had 10 potential spawning fathead minnow pairs. Mean standard error results are shown. No significant differences using One-Way ANOVA (P<.05)......................................................................................................45 9 Percent of Females Spawning vs. Nu mber of Spawns. Control, low, middle, and high treatment groups had 10 potential spawning fathead minnow pairs. Mean standard error results are shown. Note: Decreased number of spawning females for treated versus controls and altered patterns across treatments..............46 10 Mean Number of Eggs Laid vs. Number of Times Spawned. Control, low, middle, and high treatment groups had 10 potential spawning fathead minnow pairs. Mean standard error results are shown. Note: Lower number of eggs produced for treated versus controls and altered patterns across treatments...........46 11 Percent of Clutch Viability vs. Number of Time s Spawned. Control, low, middle, and high treatment groups had 10 potential spawning fathead minnow pairs. Mean standard error results are shown. Note: Lower clutch viability produced for treated versus controls and altered patterns across treatments...........47 viii

PAGE 9

Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science ORGANOCHLORINE PESTICIDE REPRODUCTIVE EFFECTS IN FATHEAD MINNOWS (Pimephales promelas): COMPARISON OF EMBRYO AND MATERNAL EXPOSURE By Dane Holland Huge May 2006 Chair: Timothy S. Gross Major Department: Veterinary Medicine Previous studies from this laboratory have documented decreased reproductive efficiency and altered hormone concentrations in wild populations of American alligators (Alligator mississippiensis) and largemouth bass (Micropterus salmoides) from sites contaminated with organochlorine pesticides (OCPs). These bioaccumulate significantly in fish. Our purpose was to determine whether adverse effects to reproductive fitness are caused by OCP-induced effects on maternal reproductive physiology. We investigated maternal transfer of the OCPs dichlorodiphenyldichloroethylene (DDE), dieldrin, chlordane, and toxaphene from adult fathead minnows (Pimephales promelas) to egg and larvae. Adult female fathead minnows were dosed at rates of high, middle, and low. The high dose was a complex mixture comparable to concentrations found in blue tilapia (Oreochromis aurea) from a historically contaminated lake in central Florida. The middle dose was 50% of the high dose, and the low dose was 25% of the high dose. We recorded the frequency of spawns, the number of eggs laid per ix

PAGE 10

spawn, the number of eggs hatched, and the number of larvae surviving to Day 14. At the end of the dosing period, we made histology slides of male and female gonads to determine stage of sexual maturation. We calculated GSI on male and female gonads. We used GC-MS analysis to analyze concentration levels of OCPs in fathead minnow eggs, male and female gonads, and whole body burdens. Statistical analysis was completed by using a One-way ANOVA to compare treatment groups. Significant differences among control and treatment groups were detected when examining egg counts, the number of eggs hatched, clutch viability percentage, survivorship percentage to Day 7, and survivorship percentage to Day 14. No significant differences were detected between treatment groups when examining GSI. In conclusion, laboratory populations of fathead minnows maternally exposed to OCPs spawn less frequently, have a decreased hatch rate, and have decreased survivorship. x

PAGE 11

CHAPTER 1 INTRODUCTION Background The group of chemicals known as organochlorine pesticides (OCPs) consists of structurally diverse compounds used to control pests that have potential to damage agricultural crops, and to serve as vectors for human diseases. Because of the chemical stability, lipophilicity, and low water solubility of OCPs, these compounds and their metabolites persist in the environment. The OCPs can bioaccumulate in vertebrate species, causing negative effects in developmental and reproductive potential. OCPs alter enzyme activities (such as Ca2 + -ATPase and phosphokinase), and alter electrophysical properties (such as K + Na + ion exchange of nerve cell membranes), thus affecting neural transmission. Because of their environmental persistence and their ability to biomagnify in food webs, the U.S. Environmental Protection Agency (USEPA) began to restrict (or in some cases, ban) the use of OCPs on agricultural lands between 1978 and 1983. Growing concern has arisen that many environmental pollutants have the capability to interfere with normal function of human and animal endocrine systems. This type of pollutant is known as an endocrine disruptor. The endocrine system is composed of numerous types of tissues and can briefly be described as any tissues or cells that release chemical messengers or hormones that signal or trigger a physiological response from a target tissue (Thomas and Thomas, 2001). The endocrine system is integrated with many other biological functions and is widely dispersed throughout entire organisms. Because 1

PAGE 12

2 of this, a wide range of toxicological responses may develop after exposure to endocrine disrupting compounds (EDCs) (Newman and Unger, 2003). Responses may include sexual developments, sexual differentiation, and success rates of reproduction. Reproductive processes in fish and other species of wildlife are largely controlled by complex hormonal pathways, thus giving endocrine disruptors the potential to target reproductive organs. The USEPA developed a tiered testing paradigm, and paralleled assays to identify potential EDCs such as OCPs (USEPA, 1998). Some OCPs alter endocrine pathways controlled by thyroid hormones, estrogens, and androgens. Recommendations for the initial (Tier 1) screening assays include 3 assays using male or female rats at different life stages, using the amphibian (Xenopus laevis) as metamorphosis test, and a short-term reproduction test with the fathead minnow (Pimephales promelas) (USEPA, 1994). In general, Tier 1 screening assays include exposing fathead minnows to the chemical of concern for up to 21 days. Post exposure, the survival, behavior, fecundity, and secondary sexual characteristics are assessed. Fertility and early development of the F1 generation may also be evaluated. At the end of the test, plasma concentrations of sex steroids (-estradiol, testosterone, 11-ketotestosterone [11-KT]) and vitellogenin (Vtg) are measured. Gonadal status is also assessed using a gonadosomatic index (GSI) and histopathology. Although a general reduction in use of OCPs has been observed, several field studies suggest that OCPs adversely affect endocrine function in fish. Thus indicating aquatic and semi-aquatic organisms are continuously being exposed to levels of toxicants capable of altering reproductive parameters. Marburger et al. (2002) tested OCP levels in

PAGE 13

3 soil from the Emeralda Marsh Conservation Area (on the north-east shore of Lake Griffin, Florida, United States), a historically contaminated lake. The lake contained concentrations of p,p-DDE, dieldrin, and toxaphene over 3,000, 500, and 40,000 ng/g, respectively. The same study showed concentrations of OCPs in largemouth bass (Micropterus salmoides) ovaries and fat reached levels of 4,000 and 17,000 ng/g respectively, for total DDT derivatives, over 100 and 700 ng/g for dieldrin, and over 4,000 and 20,000 ng/g for toxaphene. Selection of Animal Model Numerous reasons exist for selecting the fathead minnow as a model species for EDC screening (Ankley, 2000). The fathead minnow is a member of the Cyprinidae family. Cyprinidae represent the largest fish family in the world. Over 2,000 species of Cyprinids or true minnows make up 25% of all freshwater fish and 9% of all fish species (Nelson, 1984). The Cyprinids are distributed in the fresh waters of North America, Europe, Asia, Africa, and Australia. They exist in virtually every freshwater habitat including swamps, sloughs, springs, ponds, lakes, large rivers, and tiny creeks. Although considered a freshwater species, some Cyprinids have been known to frequent tidal fresh and brackish water (Jenkins and Burkhead, 1994). North America contains approximately 295 described species of Cyprinids, 9 being exotic (Miller, 1996; Robins et al., 1991). Although most minnows are commonly considered small, the Cyprinidae family contains the smallest American minnow, the Mexican shiner (Notropis saladonis), reaching 150 mm total length (TL), as well as the endangered Colorado squawfish (Ptychocheilus lucius) which is known to reach a TL of 1800 mm and weigh as much as 45 kg (Smith, 1945).

PAGE 14

4 Specifically, the fathead minnow represents an excellent model for the recognition of contaminant accumulation for numerous reasons. Adult fathead minnows are omnivores. Different populations have shown that one ate primarily insects, another only algae, another entirely detritus (Cahn 1927; Coyle, 1930 in Scott and Crossman, 1973; Starrett 1950) and yet another consumed microcrustaceans, insects, algae, and detritus (Pearse, 1918). Because of the fathead minnows range in diet, they possess a moderately coiled gut which is an intermediate between the long and coiled gut of detritivores and herbivores and the S-shaped gut found primarily in carnivores. Their wide variety in diet insures they have the potential to eat, digest, and possibly bioaccumulate environmental contaminants located in various levels of the food web. Approximately 11,080 developmental and survival tests in support of regulatory programs in North America and Europe (USEPA 1982, 1989, 1991, and 1994) have been established, giving an extensive background in sub-lethal and lethal effects on fathead minnows caused by agents and metabolites in numerous classes of environmental toxicants. Fathead Minnow Reproductive Cycle Because the fathead minnow is widely distributed across the United States and North America, specific times of maturation and spawning temperatures are difficult to pinpoint. The fathead minnow is native to the central portion of North America (Scott and Crossman, 1973). However, numerous populations exist in California, Arizona, Texas, and into the New England states; all of which possess different maturation times and spawning temperatures. Generally, spawning occurs in water temperatures of 15 to 32 C (Markus, 1934). Because the population distribution of the fathead minnow ranges from southern Texas to

PAGE 15

5 northern Minnesota, actual months of the year that the fathead minnow spawns will vary tremendously. When photoperiod and temperature reach acceptable limits, males develop dark vertical bands, turn a rusty brown, and develop white breeding tubercles that are prominent on the tip of the snout and top of the head. Once a dominant male is selected, he establishes a territory which he defends against other subordinate males in the local vicinity. He will then clean the nesting site which may be the underside of a flat rock or submerged vegetation, stones, logs or other acceptable substrates. Spawning typically takes place between dawn and 10:00am. After engaging in courtship, the female will deposit eggs onto the prepared surface of the selected substrate. Gale and Gerard (1982) showed in a study that female fathead minnows laid between 9 and 1,136 eggs per session (clutch). They also showed that between May 22 and August 22 five pairs of fish produced 16 to 26 clutches of eggs. Post fertilization, the male will assume responsibility of nest guarding. Again, depending on water temperature, the eggs will hatch into larvae in approximately six days and remain in the nesting area for several more days until their yolk material is absorbed. Along with environmental cues such as photoperiod and temperature, teleost fish reproductive cycles are also regulated by endogenous hormonal cues (Gross et al., 2002). A combination of these factors stimulate the hypothalamus of the fish to release gonadotropin-releasing hormone (GnRH), norepinephrine (NE), as well as other neuropeptides to stimulate the pituitary gland which releases the primary teleost gonadotropins GTH-I and GTH-II (Van Der Kraak et al., 1998). These two gonadotropins are similar to the mammalian lutenizing hormone (LH) and follicle stimulating hormone (FSH) (Redding and Patino, 1993). GTH-I is the gonadotropin

PAGE 16

6 associated with stimulation of events leading to vitellogenesis and spermiogenesis as well as early gonadal development. GTH-II is involved in the stimulation of events that will eventually lead to the final oocyte maturation and ovulation in females, as well as spermiation in males. The primary sex steroids involved in regulation of gametogenesis in the majority of male and female teleost fish are 11-KT and 17-Estradiol. An increase in plasma concentrations of these hormones has been shown to be associated with the onset of seasonal reproductivity (Gross et al., 2002). In females, an increase in estrogen (E 2 ) levels within the blood stimulates the liver to produce vitellogenin, a phosphoglycolipoprotein that serves as a precursor to yolk production in oviparous vertebrates (Wahli et al., 1981). Vitellogenin is then released into circulation where it travels to the gonad and is used as a nutrient source for developing oocytes. Within the promoter region of the vitellogenin gene lies an estrogen responsive element which is transcribed in response to an estrogen receptor (ER) complex (Wahli et al., 1981). The surface of the oocyte contains vitellogenin receptors, which once these receptors are occupied, the oocyte is cleaved into smaller yolk proteins. The yolk proteins are embodied into yolk granules which will in turn constitute majority of the mature oocytes. The yolk granules are stored and will serve as the nutrient source for developing embryos (Wahli et al., 1981). Once oocytes have reached their properly developed size, vitellogenesis stops, and the oocytes complete maturation. During this maturation phase, follicles increase in size due to hydration, as well as collect additional vital proteins. At the time of germinal vesicle breakdown, protein uptake ceases. Due to hydration, the follicle volume continues to increase. It is at this time the cellular envelope that surrounds an egg in preparation for ovulation known as the chorion, begins to

PAGE 17

7 develop. The time of ovulation is species specific and will take place when follicles reach a specific size (Wallace and Selman, 1981). Although male and female fish contain the vitellogenin gene, concentrations of E 2 normally only found in females are needed to produce measurable levels of vitellogenin (Wallace, 1970; Ryffel, 1978; Hori et al., 1979). Presence of vitellogenin in males can then be used as an indicator of estrogenic compound exposure (Pelissero et al., 1993; Sumpter and Jobling, 1995; Shelby et al., 1996; Okoumassoun et al., 2002). OCP Characteristics OCPs are synthetic compounds that contain certain amounts of chlorine substituted for hydrogen on a hydrocarbon backbone, also known as chlorinated hydrocarbons. However, also included in this group are a few compounds such as dieldrin and methoxychlor that have oxygen incorporated in their structure. Most OCPs do not resemble naturally occurring organic compounds in their structure, hence, they degrade slowly within their environment (Newman and Unger, 2003). Because OCPs have high molecular weights and are non-polar, they have low water solubilities and are soluble in lipids that are found within living organisms. The lipophilicity of OCPs causes bioaccumulation and possibly biomagnification. One of the first widely used OCPs was dichloro-diphenyl-trichloroethane (DDT). DDT became a popular pesticide for numerous reasons. DDT has the capability to control or kill a wide range of insect pests, it is persistent in the environment, and it has low mammalian toxicity. DDT was used during World War II to control fleas, lice, flies, and mosquitoes which are vectors for malaria and typhus transfer to servicemen and civilian populations. Based on the success of DDT during World War II, DDT became the pesticide of choice for agricultural and commercial use. Gildersleeve et al. (1985)

PAGE 18

8 and Bryan et al. (1989) were able to demonstrate through laboratory studies that in ovo treatment with DDT to Japanese quail resulted in female progeny that lay eggs without shells on a regular basis, as well as, have reproductive tract malformations. These, along with numerous other studies on exposure of DDT to wildlife led to the banning of DDT by the Environmental Protection Agency (EPA) in the United States at the end of 1972. DDT undergoes degradation in the environment to the metabolites DDD and DDE. Both of these metabolites have a half life on the order of years. Most samples taken in the environment today contain predominately the DDE and DDD isomers. Another class of insecticides commonly found in the environment that act similar to DDT are the Cyclodienes. Included in the Cyclodiene class are toxaphene, dieldrin, and chlordane. One complex mixture of polychlorobornanes and camphenes is known as toxaphene. The chlorination of technical camphene or -pinase can consist of over 300 congeners (Di Giulio and Tillitt, 1997). Between 1972-1975, toxaphene was widely used in the United States on cotton crops. Toxaphene was banned in the United States in 1982, however, each structurally different compound in the mixture will have a specific set of chemical properties, and thus predicting its environmental fate proves difficult. Dieldrin is a common name of an insecticide that was routinely used in the 1950s to the early 1970s. It was used in agriculture for soil and seed treatment and in public health to control disease vectors such as mosquitoes and tsetse flies. It was also used as a sheep dip and has been used in the treatment of wood and woolen products. Most uses of dieldrin were banned in 1975 and currently it is not produced or imported by the United States. Studies have shown that oral uptake, as well as, exposure through fish gills can

PAGE 19

9 adversely affect fish nitrogen content in the liver and reduce swimming speed at which fish can maintain themselves (Mayer, 1971). Chlordane is another insecticide within the Cyclodiene class that was used in the United States between 1948 and 1988. Chlordane is not a single chemical, but is a mixture of many related chemicals of which approximately 10 are major components including trans-chlordane, cis-chlordane, heptachlor, and trans-nonachlor. Prior to 1978, chlordane was used as a pesticide on agricultural crops. From 1983-1988, chlordane was only approved to be used as a pesticide to control termites in homes. Because of concerns over cancer risk and adverse affects in wildlife, the EPA cancelled the use of chlordane on food crops and phased out other above-ground uses. OCP Exposure Responses in Fish Numerous studies have linked disturbances in reproductive function to exposure of different classes of toxic chemicals (Jobling et al., 1998). However, when looking specifically at OCPs, much research has been concentrated on endocrine disruption (Rauschenberger et al., 2004; Ree and Payne, 1997; Russell et al. 1999; Gleason and Nacci, 2001). It is thought that one area of reproductive success that is affected by OCP exposure is circulating sex steroids. Studies have shown that abnormalities exist in concentration levels of circulating sex steroids in fish exposed to OCPs. Johnson (2005) found that Florida largemouth bass (Micropterus salmoides floridanus), exposed to p,p-DDE and dieldrin for a 120-day period, led to depressed concentrations of plasma E 2 for female largemouth bass, increases in 11-KT in female largemouth bass and a lack of consistent increases and/or depressions of male largemouth bass E 2 and 11-KT concentrations. Female largemouth bass in another study (Muller, 2003) also showed higher circulating E 2 concentrations when exposed to 2.5 mg of DDE compared to

PAGE 20

10 control groups. Male largemouth bass when exposed to 5.0 mg of DDE also consistently showed higher circulating levels of E 2 Not only have laboratory studies shown altered sex steroid hormones, but also fish captured from wild populations. Largemouth bass collected from the Emeralda Marsh Conservation Area (EMCA) located on the north-east shore of Lake Griffin, Florida, USA, demonstrated depressed levels of sex steroid hormones 17-estradiol and 11-KT concentrations that are possibly a result of a disruption in the hypothalamus-pituitary-gonad axis which is responsible for stimulating synthesis and secretion of sex steroid hormones (Gross et al., 2003). Another example of altered circulating sex steroids was demonstrated by (Kelce et al., 1995). They demonstrated that p,p-DDE acts as a potent anti-androgen in rats by binding to the androgen receptor (AR), preventing testosterone synthesis, and causing demasculinization. This phenomenon has also been demonstrated in several fish species including white sturgeon (Acipenser transmontanus) (Foster et al., 2001) and goldfish (Carassius auratus) (testes only), (Wells and Van Der Kraak, 2000). Numerous mechanisms of action exist for EDCs and may interrupt multiple pathways along the hypothalamic-pituitary-target-tissue axis. These interruptions may disturb the transport, binding, release, biotransformation, elimination or normal synthesis of natural hormones. EDCs may alter the hypothalamic-pituitary axis which could have a cascading affect on the endocrine system downstream of the hypothalamus. EDCs may interfere with neurotransmitters that control GnRH secretion resulting in decreased levels of GnRH production, as well as a reduction in gonad size, and may be responsible for the alterations in the concentrations of circulating sex steroids (Jansen et al., 1993). Specific endocrine tissues synthesize hormones that are secreted into the bloodstream where they

PAGE 21

11 are bound to proteins and transported to target tissues where they interact with receptors, bring about responses, or may be metabolized or degraded. EDCs may block or enhance the function of these hormones by interfering with one or several of these steps. EDCs may cause synthesis failure of certain hormones or limit the uptake of critical precursors to produce hormones. Additionally, EDCs may alter the rate at which hormones are metabolized as is the case with the super family of enzymes, CYP 450 which are critical in the synthesis and metabolism of steroid hormones. EDCs may also induce hormone-like effects due to the alternating rates of degradation. EDCs may interfere with hormones binding to transport proteins thus preventing delivery to target tissues (Darnerud et al., 1996). Some EDCs have the ability to mimic estrogens or androgens. These EDCs may bind to globulin proteins, thus displacing and possibly increasing the elimination of endogenous circulating sex steroids (Rosner, 1990). EDCs may also have the potential to bind to hormone receptors and either activate, (agonize), (Flouriot et al., 1995) or inhibit (antagonize) (Danzo, 1997) receptor function. Particular research interest has been focused on EDCs and their ability to bind to estrogen receptors (ER). Most ER are located in the nucleus of target cells. ER-DNA complexes interact with chromosomal proteins and transcription factors in order to induce or inhibit transcriptions of specific genes, thus enabling endocrine specific responses. It is possible for EDCs to enhance or block the function of a hormone or endocrine target tissue by interfering with one or several critical steps in the transcription process (Hoffman et al., 2003). Unlike ER that have a specific E 2 response element, EDCs that have androgenic activities may exert broader effects than those attributed to a simple androgen mimic. Endocrine-disrupting effects may also occur due to direct or indirect toxicities on specific target

PAGE 22

12 tissues. Lipophilic EDCs such as OCPs will accumulate primarily in fatty tissues (Raushenberger, 2004) such as gonads and the liver, potentially interfering with the mobilization and synthesis of lipids, thus inhibiting specific endocrine related functions such as vitellogenesis. Significance of this Study Numerous oviparous vertebrates become subjects for studies because they are sensitive to adverse effects of chemical contaminants released into the environment, highly visible by the public, or obtain interest by organizations because they are viewed as limited natural resources. However, a species that attracts attention by the public or is federally listed may not be considered appropriate for laboratory studies. It is crucial when assessing risks to wildlife populations that not only must one understand environmental exposures, but also dose-response relationships using measured endpoints. Endpoints may be measured as lethal (mortality) or as sub-lethal effects such as adverse changes in reproduction or development. In many instances use of a surrogate species may be appropriate to assess possible risks involved in exposure to chemical contaminants. Biological endpoints can be used to help express potential for chemicals to cause negative ecological effects. Surrogate species are often used to in laboratory tests to complete acute or chronic toxic effects for specific chemicals (Zeeman and Gifford, 1997). Most surrogate species are selected because they are easily cared for and cultured under laboratory settings and provide an inexpensive and reliable assessment of acute and chronic toxicity of potential chemical contaminants. Identifying potential ecological and physiological characteristics related to the susceptibility to effects of contaminants, along with their exposure routes is important in managing wildlife populations for the optimal use by humans (Rauschenberger, 2004).

PAGE 23

13 The ability to develop the fathead minnow into a biological model would provide wildlife managers a standard by which to measure ecological impacts brought about by anthropogenic chemicals dispersed throughout aquatic environments. The objectives of this study were: 1) to determine if OCPs are maternally transferred from the adult female fathead minnow to her offspring; 2) to determine if maternal OCP exposure adversely affects hatch rate and larval mortality; and 3) to determine if oral exposure of OCPs cause masculinization in female fathead minnow gonads and/or feminization in the gonads of male fathead minnows. Previous studies have shown that OCPs may act as endocrine system modulators affecting reproductive success in numerous animal species (Gallager et al., 2001; Gross et al., 1994; Mills et al., 2001; Muller et al., 2004). We hypothesized that oral exposure to fathead minnows by a mixture of p,p-DDE, dieldrin, toxaphene, and chlordane at increasing concentrations would bioaccumulate in muscle tissue and in the gonads of both male and female fathead minnows due in part to the highly lipophilic nature of these compounds, and thus be maternally transferred to fathead minnow eggs. Exposure to the OCP mixture would also demonstrate an enzyme inducing and/or estrogenic properties that would directly or indirectly increase larval mortality, adversely affect clutch size and frequency of spawns per fathead minnow pair, and would cause feminization or masculinization of fathead minnow gonads.

PAGE 24

CHAPTER 2 MATERIALS AND METHODS Experimental Animals Male and female fathead minnows of 5 to 7 months of age were obtained from Aquatic Bio Systems Inc. in March 2005. The fish were transported to the USGS-FISC laboratory where they were maintained in 10-gallon aquariums with a flow-through water system supplied by on-sight well water and aeration. Upon arrival, all fish looked healthy, disease free, and weighed between 2.20 and 4.13 grams (g). Fish were fed a diet of Ziegler Prime Tropical 45-9 Flake Feed ad libitum once per day. The fish were allowed two weeks to acclimate to their new environment prior to dosing. Water quality parameters measured included temperature, pH, and dissolved oxygen. Water quality parameters remained within acceptable ranges for the duration of the experiment. Temperature ranges were from 21.30 to 22.30 C. Dissolved oxygen ranges were from 5.30mg/L to 6.35mg/L. pH levels were from 7.90 to 8.70. Chemicals and Dosing The organochlorine pesticide 1,2,3,4,10,10-hexachloro-6,7-epoxy-1,4,4a,5,6,7,8,8a-octahydro-1,4,5,8-dimethanonaphthalene (dieldrin, Lot#77H3578) was obtained from Sigma (St. Louis, MO). The organochlorine pesticide 2,2-bis(4-chlorophenyl)-1,1-dichloroethylene (p,p-DDE, Lot#0902KU) was obtained from Aldrich Chemical Co. (Milwaukee, WI). The organochlorines chlordane (Lot#303-16B) and toxaphene (Lot#302-125B) were purchased from Chem Service (West Chester, PA). The chemicals were added to 1690.0 ml of Yelkin oil provided by Ziegler Bros., Inc. at the 14

PAGE 25

15 following concentrations: dieldrin 5.0 mg, p,p-DDE 12.0 mg, chlordane 10.0 mg, toxaphene 95.0mg. The contaminated Yelkin oil was then mixed with 25.0 pounds (lbs.) of Prime Tropical Flake Feed by Ziegler Bros., Inc. Pilot Study Prior to the conduction of the experiment, a pilot study was undertaken to establish body burdens and possible lethal doses of the experimental fish, as well as contaminant concentration amounts present in the treated and control feeds. The control and treated feed was analyzed by the Center for Environmental and Human Toxicology, University of Florida, Gainesville, Florida. Screen assays were performed to establish limits of detection, as well as limits of quantification (Table 1). When determining the amount of contaminants to add to the flake feed, an attempt was made to achieve target parts per million (ppm) values comparable to what is seen in blue tilapia (Oreochromis aureus) stocked at the north shore restoration area in Lake Apopka, Florida, a historically hypereutrophic and contaminated lake (Pollman et al., 1988). A biomagnification factor of 15 was used based on previous studies performed at the USGS-FISC, Gainesville, Florida, laboratory. Post feed analysis, the control and treated feed was mixed to establish a high, middle, and low dose. One hundred twenty grams of control feed was added to a 64.0 ounce (oz.) clean plastic container. One hundred twenty grams of the high dose feed was also added to a 64.0 oz. clean plastic container. The medium dose was established by mixing 60.0 g of control feed with 60.0 g of treated feed in a 64oz. clean plastic container. The low dose was established by mixing 90.0 g of control feed with 30.0 g of treated feed in a 64.0 oz. clean plastic container. The middle and low doses were then shook to ensure thorough mixing and stored in a refrigerator at 5 C.

PAGE 26

16 Five female fish of each treatment group and the control were placed in separate 10-gallon aquariums. Fish were then fed approximately 2.5% of their body weight once per day for 30 consecutive days. At the end of 30 days, all fish were separately wrapped in foil, frozen, and delivered to the Center for Environmental and Human Toxicology, University of Florida, for chemical analysis. During the analysis, 2 fish from each treatment group were individually assayed, and the remaining 3 were analyzed via a composite assay with the exception of the low dose treatment group and control group where each sustained one mortality. In this case, 2 individual fish from the low dose treatment group and control group were analyzed and the remaining 2 fish from each group were analyzed via a composite assay. The results of the individual and composite assays are located in Table 2. Nanoinjection Experiment A nanoinjection experiment was performed that involved four replicate trials. Each trial contained five treatments. Eggs were injected at a low, middle, and high dose rate at 5, 10, and 20 ppm of p,p-DDE at a quantity of 83.5-116.9 micrograms. A control that was not injected, as well as a vehicle control that was injected with the same quantity of triolein was used. Eggs were injected with a Kanetec MB-B manipulator and pre-made needles comprised of aluminosilicate 0.68-mm capillary tubes. Eggs were held in place for injection by agar placed on microscope slides acting as a substrate. Once the eggs were injected, they were placed in a flow-through incubator for hatching. The number of hatched larvae, as well as the number of surviving larvae were monitored to Day 11. Maternal Transfer Experiment Ten female fathead minnows per treatment group and control were randomly selected and placed in separate 10-gallon aquariums supplied by flow-through, on-sight

PAGE 27

17 well water and aeration. Water quality parameters were measured, and remained within acceptable levels throughout the experiment. Photoperiod was gradually increased from an 8 light/16 dark hour day to a 12 light/12 dark hour day. Temperature was gradually increased from 22 C ( 1 C) to 25 C ( 1 C). The fish were dosed with the same feed mixture as in the pilot study. Based upon the amount of food consumed by the fish during the pilot study, a reduction was made in the amount of food provided to the fish to approximately 2% of the fishs body weight. The fish were then dosed for a consecutive 30 days. At the end of the dosing period, male fish were separated from each other, and randomly placed in 10-gallon aquariums. Female fish were then randomly paired with the male fish. Each treatment and control groups contained 10 pairs of male and female fish. Water quality was monitored throughout the experiment and aquariums were siphoned as needed. Control fish were fed .18 .02 g per pair of Ziegler Prime Tropical 45-9 Flake Feed once per day; while the 3 treatment groups remained on their diet of the previously mixed treated feed at .18 .02 g per pair once per day. Spawning substrates consisted of PVC pipe with a 4-inch diameter cut in half to 4 inches long. The substrates were inspected for the presence of eggs daily at approximately 10:30am. Substrates that contained eggs were removed, and the eggs counted under a bench magnifying glass. The substrates with eggs were then placed into a 64 oz. plastic container with 1000 micron mesh attached to the sides in order to allow water flow-through. The container was then placed into a flow-through water bath at 22.2 C with a constant drip consisting of aerated well water. A 2-inch airstone

PAGE 28

18 supplied heavy aeration into the container to prevent fungus from attaching to the eggs. Egg substrates were replaced to aquaria. Larvae began hatching 7 to 11 days after spawning. Hatched larvae were transferred to a clean 64 oz. container and individually counted. This process was repeated on Days 7 and 14 after hatch. A composite assay cont aining 0.2 g to 0.4 g of eggs with all four treatment groups was performed to confirm that contaminants were being maternally transferred. This assay was performed by the Center for Environmental and Human Toxicology, University of Florida, Gainesville, Florida. The trial was repeated dosing 40 female fathead minnows 7-9 months of age for 30 consecutive days. At the end of the 30-day dosing period, female fathead minnows were paired with male fathead minnows 7 to 9 months of age into individual 10-gallon aquariums with the same flow-through water system. Each pair of fish was fed 0.4 g of control and treated feed once daily for an additional 30 consecutive days. A spawning substrate constructed of a 4-inch long, 4-inch diameter PVC pipe cut in half was placed in the aquarium. Spawning substrates were checked daily for the presence of eggs. Any eggs present were individually counted and recorded. A composite sample containing 0.2 g to 0.4 g of the control and 3 treatment group eggs were collected at the beginning, middle, and end of the 30-day trial. The Center for Environmental and Human Toxicology, University of Florida, Gainesville, Florida, performed an OCP screen on the eggs to affirm the presence of contaminants. Analysis of Fathead Minnow Tissues and Eggs for OCPs Analysis was conducted by the Center for Environmental and Human Toxicology, University of Florida. Briefly, the OCPs in a weighed, homogenized portion of sample 2.0 g were extracted into ethyl acetate. Sample clean-up included use of C18 and NH2

PAGE 29

19 solid-phase extraction cartridges prior to analysis by GC-MS. Each analyte was quantified against a standard curve having at least five points and a correlation coefficient > 0.995. For the flake feed analysis, the Est LOD-LOQ range was 0.3-7.5 (ng/g) for chlordane, 0.1-7.5 (ng/g) for DDE (metabolites), 0.8-1.5 (ng/g) for dieldrin, and 63-1500 (ng/g) for toxaphene. Fathead minnow body burdens had an Est LOD-LOQ range of 0.3-1.5 (ng/g) for chlordane, 0.1-1.5 (ng/g) for DDE (metabolites), 0.5-1.5 (ng/g) for dieldrin, and 41-1500 (ng/g) for toxaphene. The fathead minnow gonads had an Est LOD-LOQ range of 1.2-7.5 (ng/g) for chlordane, 0.1-7.5 (ng/g) for DDE (metabolites), 0.4-1.5 (ng/g) for dieldrin, and 42-1500 (ng/g) for toxaphene. Fathead minnow eggs had an Est LOD-LOQ range of 0.2-1.5 (ng/g) for chlordane, 0.1-1.5 (ng/g) for DDE (metabolites), 0.8-1.5 (ng/g) for dieldrin, and (ng/g) 42-1500 for toxaphene. Gonad Histology Tissue staining with hematoxylin and eosin (H&E), sectioning and slide mounting was performed by Histology Tech Services (Gainesville, Florida). Ovaries were observed under a light microscope at 40X and stage of sexual maturation was assigned (Seplveda, 2000). Briefly, stage 1 ovaries are undeveloped with mostly primary phase follicles. Stage 2 ovaries are pre-vitellogenic with primary and secondary phase follicles, but have no vitellogenic follicles. Stage 3 ovaries are early vitellogenic with some vitelline granules in follicles of varying size and no fully developed eggs. Stage 4 ovaries are late vitellogenic with a majority of follicles containing numerous vitelline granules and fully developed eggs are present (Muller, 2003). Stage 1 testes are non-spermatogenic, with an extremely thin germinal epithelium and no sperm present. Stage 2 testes show low spermatogenic activity, with a thin

PAGE 30

20 epithelium that contains scattered proliferation and maturation of spermatozoa. Stage 3 testes show moderate spermatogenic activity, thick germinal epithelium and diffuse to moderate proliferation and maturation of sperm. Stage 4 testes show thick germinal epithelium, high proliferation and maturation of sperm. Statistical Analysis All statistical analyses were performed using Statistical Analysis system (SAS) Enterprise Reporter software version 2.6. The experimental design required statistical analysis comparing each treatment group by a One-Way ANOVA. ANOVAs were performed and significance was set at alpha = 0.05. Results are represented as means standard error. A Tukeys Multiple Comparison test (P < 0.05) was used to compare treatment groups and the stage of the gonad in both male and female fathead minnows.

PAGE 31

CHAPTER 3 RESULTS Maternal Exposure Maternal exposure to contaminant mixture led to a decrease in the percent of spawning pairs in all three treatment groups when compared to the control. The percent of females spawning per treatment group resulted in the following: Control group 80%, Low treatment group 60%, Middle treatment group 40%, and High treatment group 50%. Significant differences were observed between the control and the middle treatment group and the control and the high treatment group when using a One-way ANOVA (P<.05). The total number of potential spawning pairs for the control and treatment groups were 10 pairs over a 30-day period. Exposure to the contaminant mixture led to an increase in the mean number of eggs laid per spawn in the low treatment group 350 vs. 220, and in the high treatment group 270 vs. 220, but a decrease in the number of eggs laid per spawn in the middle 190 vs 220. The mean and standard error of eggs laid for each treatment group is represented in Figure 2. The mean number of eggs laid for the control group standard error was 220.04 32.36. The mean number of eggs laid for the low treatment group was 351.67 41.16. The mean number of eggs laid for the middle treatment group was 188.73 34.41. The mean number of eggs laid for the high treatment group was 271.68 29.41. The controls laid the most total number of eggs at 5501, the low treatment group laid the second highest amount of eggs at 5275, the high treatment group laid the third most 21

PAGE 32

22 number of eggs at 4890, and the middle treatment group laid the fewest number of total eggs at 2831. Exposure to the contaminant mixture led to an increase in the mean number of eggs hatched in the low treatment group, but a decrease in the mean number of eggs hatched in the middle and high treatment groups when compared to the control. This is the same phenomenon witnessed in the mean number of eggs laid. The mean and standard error number of hatched eggs for each treatment group is represented in Figure 3. The mean number of hatched eggs for the control group and standard error was 144.64 25.77. The mean number of eggs hatched for the low treatment group was 265.60 31.98. The mean number of eggs hatched for the middle treatment group was 69.67 21.66. The mean number of eggs hatched for the high treatment group was 82.95 28.06. Significant differences were detected among the low vs. mid treatment groups, low vs. control treatment groups, and low vs. high treatment groups. Exposure to the contaminant mixture also led to an increase in clutch viability for the low treatment group and a decrease in clutch viability for the middle and high treatment groups when compared to the control. This is the same phenomenon in witnessed in the mean number of eggs laid and hatched. The mean and standard error of clutch viability percentage is represented in Figure 3. The mean number of clutch viability percentage for the control group was 64.98 6.19. The mean number of clutch viability percentage for the low treatment group was 76.39 3.72. The mean number of clutch viability percentage for the middle treatment group was 30.64 9.71. The mean number of clutch viability percentage for the high treatment group was 28.57 8.35. Significant differences were detected among the low vs. middle treatment groups, low vs.

PAGE 33

23 high treatment groups, control vs. middle treatment groups, and control vs. high treatment groups. Exposure to the contaminant mixture led to a decrease in the percent of larvae surviving to Day 7 in all three treatment groups when compared to the control. The mean and standard error of surviving larvae to Day 7 percentage is represented in Figure 4. The mean and standard error of surviving larvae to Day 7 percentage in the control treatment group was 43.59 6.98. The mean number of surviving larvae to Day 7 percentage in the low treatment group was 31.35 5.98. The mean number of surviving larvae to Day 7 percentage in the middle treatment group was 8.35 4.91. The mean number of surviving larvae to Day 7 percentage in the high treatment group was 15.71 7.09. Significant differences were detected in the control vs. middle treatment groups and the control vs. high treatment groups. Exposure to the contaminant mixture led to a decrease in the percent of larvae surviving to Day 14 in all three treatment groups when compared to the control. The mean and standard error of surviving larvae to Day 14 percentage is represented in Figure 5. The mean and standard error of surviving larvae to Day 14 percentage in the control treatment group was 32.52 8.14. The mean number of surviving larvae to Day 14 percentage in the low treatment group was 39.67 9.62. The mean number of surviving larvae to Day 14 percentage in the middle treatment group was 7.60 4.57. The mean number of surviving larvae to Day 14 percentage in the high treatment group was 7.06 4.03. Significant differences were detected between the control vs. middle treatment groups.

PAGE 34

24 With the exception of the high treatment group in regards to female GSI, the mean GSI for the low and middle remained approximately the same when compared to the control, while the high treatment group increased. The mean and standard error for GSI in the female control groups was 10.44 5.57. The mean and standard error for GSI in the female low treatment group was 9.44 1.79. The mean and standard error for GSI in the female middle treatment group was 9.50 1.99. The mean and standard for GSI in the female high treatment group was 14.12 1.63. Mean GSI for male and female fathead minnows showed no effect. With the exception of the middle treatment group in regards to male GSI, the mean GSI for the low and high groups remained approximately the same when compared to the control, while the middle treatment group decreased. The mean and standard error for GSI in the male control treatment group was 1.36 0.79. The mean and standard error for GSI in the male low treatment group was 1.33 0.15. The mean and standard error for GSI in the male middle treatment group was 0.66 0.13. The mean and standard error for GSI in the male high treatment group was 1.36 0.23. No significant differences were observed among treatment groups or sex in regards to GSI. Nanoinjection Experiment Nanoinjection trials indicated that fathead minnow eggs injected with p,p-DDE were adversely affected by the chemical. Trials 1, 2, and 3 showed eggs injected with 20, 10, and 5 ppm of p,p-DDE had lower hatch and survivorship when compared to control eggs. Trial 4 showed hatch and survivorship to be higher in the p,p-DDE injected eggs than the controls. Overall, hatch and survivorship rates during this trial were lower than previous trials.

PAGE 35

25 The percent of hatched eggs for the control group was 81 with a standard error of 10. The triolein group showed a percent of hatched eggs at 67 7. The treatment group at 5 ppm of p,p-DDE had a percent of hatched eggs at 49 17, the treatment group at 10 ppm had a percent of hatched eggs at 56 18, and the treatment group at 20 ppm had a percent of hatched eggs at 54 2. The percent of hatched eggs when comparing treatment groups to the controls showed no significant differences. The percent of fry surviving to pre-swim-up stage for the control group was 68 with a standard error of 11. The triolein group showed the percent of survived fry to pre-swim up at 83 8. The treatment group at 5 ppm of p,p-DDE had a percent of survived fry to pre-swim up stage at 60 9, the treatment group at 10 ppm had a percent of survived fry to pre-swim up stage at 56 18, and the treatment group at 20 ppm had a percent of survived fry to pre-swim up stage at 54 2. The percent of fry surviving to post-swim-up stage for the control group was 93 with a standard error of 4. The triolein group showed the percent of survived fry to post-swim up at 93 4. The treatment group at 5 ppm of p,p-DDE had a percent of survived fry to post-swim up stage at 90 6, the treatment group at 10 ppm had a percent of survived fry to post-swim up stage at 88 6, and the treatment group at 20 ppm had a percent of survived fry to post-swim up stage at 97 3. The percent of survivorship to Day 30 for the control group was 51 with a standard error of 11. The triolein group showed the percent of survivorship to Day 30 at 51 6. The treatment group at 5 ppm of p,p-DDE had a percent of survivorship at 26 7, the treatment group at 10 ppm had a percent of survivorship to Day 30 at 28 10, and the treatment group at 20 ppm had a percent of survivorship at 31 9. Although I observed a

PAGE 36

26 lower percentage of hatched larvae in fathead minnow eggs injected with p,p-DDE, there was not a decrease in survivorship. Use of a Tukeys Multiple Comparison Test showed that there were no significant differences in male or female fathead minnow gonads when comparing the stage of the ovary and/or testis to the treatment group when the p value was declared at equal or lower than 0.05. Only 1 sample collection was performed for both male and female fathead minnows at the end of the 30-day exposure. Control flake feed did exhibit some levels of OCP contaminants however, this is expected due to the fact that the flake feed is comprised of wild-caught fish and shellfish that naturally contain some levels of contaminants. The levels of OCPs in the contaminated feed were within the selected range while attempting to achieve ppm values comparable to blue tilapia (Oreochromis aureus) located at the north shore of Lake Apopka, Florida, USA, with a biomagnification factor of 15 (Table 1). Again, the controls of body burdens exhibited minimal levels of OCP contaminants, while the treatment groups displayed a dose-response relationship in all contaminants in the mixture (Table 2). When examining the levels of OCPs maternally transferred to the eggs in treatment groups, it was demonstrated that individual contaminants either did not transfer from the female to the egg or did not show a dose-response pattern. In some cases, the amount of contaminant transferred to the control group was indeed higher than those transferred to the treatment group (Table 3). Because these patterns were unexpected, a second round of maternal transfer from dosed female fathead minnows to eggs was performed to confirm our findings (Table 4). In early clutches (Days 1-5), again, the controls showed a higher amount of contaminant was

PAGE 37

27 transferred in the controls than in the treatment groups. In the middle clutches (Days 13-17), there were no dose-response patterns observed with some contaminants being higher in the controls than the treatment groups, some contaminants being higher in the medium treatment groups, and some contaminants being the mid-range of the high treatment groups. The late clutches (Days 25-30), di splayed no obvious dose-response relationship throughout all contaminants. While the chlordane did show a dose-response in the control and treatment groups, p,p-DDE, dieldrin, and toxaphene did not. Gonad OCP concentrations displayed a dose-response relationship across all contaminants with the only exception being the low treatment group of chlordane and the medium treatment group of chlordane (Table 5).

PAGE 38

28 Table 1. Day 30 GC-MS results of flake feed OCP concentrations (ng/g). N.D. is defined as not detected. Control Treated Chlordanes 5.57 105.74 DDE (metabolites) 44.61 528.67 Dieldrin N.D. 200.0 Toxaphene 3850.37 6447.13 Table 2. Day 30 GC-MS results of female whole body burden OCP concentrations (ng/g). N.D. is defined as not detected. Control Low Middle High Chlordane 3.54 8.5 21.07 40.07 DDE (metabolites) 7.07 93.44 102.42 206.57 Dieldrin N.D. 6.8 15.06 33.92 Toxaphene N.D. N.D. N.D. N.D. Table 3. Day 30 GC-MS results of Egg OCP concentrations (ng/g). N.D. is defined as not detected. Control Low Middle High Chlordane N.D. N.D. N.D. 2.79 DDE (metabolites) 298.11 117.19 79.3 103.26 Dieldrin 3.77 N.D. N.D. 2.79 Toxaphene N.D. N.D. N.D. N.D.

PAGE 39

29 Table 4. Day 30 GC-MS results of OCP concentrations in eggs (ng/g) taken during Days 1-4 (early clutches), 13-16 (mid clutches) and 27-30 (late clutches). N.D. is defined as not detected. Early Clutches Control Low Middle High Chlordanes N.D. 14.46 11.62 .88 DDE (metabolites) 157.9 108.43 75.58 4.44 Dieldrin 52.63 28.92 17.44 .89 Toxaphene N.D. N.D. N.D. N.D. Mid Clutches Control Low Middle High Chlordanes N.D. N.D. N.D. 21.36 DDE (metabolites) 119.41 73.17 245.9 106.76 Dieldrin 29.85 14.63 98.36 32.03 Toxaphene N.D. N.D. N.D. N.D. Late Clutches Control Low Middle High Chlordanes N.D. N.D. 5.22 10.06 DDE (metabolites) 42.61 57.84 46.96 110.56 Dieldrin 17.05 14.46 10.43 30.15 Toxaphene N.D. N.D. N.D. N.D. Table 5. Day 30 GC-MS results of OCP concentrations in male and female gonads (ng/g). N.D. is defined as not detected. control low medium high male female male female male female male female Chlordanes 17.54 7.0 28.16 26.0 126.58 23.0 132.35 70.77 DDE (metabolites) 61.4 20.0 126.76 182.18 746.84 224.22 779.41 549.91 Dieldrin 8.77 7.0 14.08 15.0 113.92 31.0 117.65 65.34 Toxaphene N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.

PAGE 40

CHAPTER 4 DISCUSSION The results of this study suggested that 30-day exposure to diets containing the OCPs chlordane, p,p-DDE, dieldrin, and toxaphene at varying doses can result in internal carcass and gonad accumulation. The accumulation of OCPs is then maternally transferred to the eggs of fathead minnows at various concentrations. These results are consistent with other studies that suggest maternal OCP transfer (Rauschenberger, et al. 2004; Johnson, 2005). The value of our study was that OCP concentrations in maternal tissues and egg yolks appear to be strongly correlated with one another. Our data suggests maternal exposure to OCPs adversely affects reproduction by lowering the percentage of spawning pairs, lowering the survival of larvae up to Day 14, decreases the number of eggs produced over time, and lowers clutch viability over time. In analyzing fathead minnow whole body tissue, gonadal tissue, and eggs, (Tables 1 through 3) we can see that numerous OCP analytes were detected in the flake feed, whole body tissues, gonads, and eggs. This suggests that when making predictions about possible effects to wildlife populations or human risk assessment, the composition of the toxicant is a crucial factor. One reason for this is that different xenobiotic compounds may inhibit or induce specific biotransformation enzymes (Rauschenberger, 2004). Because genetic variability exists among populations, it is possible that certain individuals or populations may lack the genetic ability to produce a biotransformation enzyme that is required for the detoxification of specific OCP analytes. Examination of Table 2 shows that a dose-response relationship was established within whole body 30

PAGE 41

31 burdens of fathead minnows. While examining gonadal tissues, we also saw a dose-response relationship (Table 5) with the one exception of low and medium treatment groups of chlordane. Rauschenberger (2005) showed that in alligators, an adipose-to-yolk concentration ratio was close to 1. This would suggest that OCPs reach equilibrium within adipose tissue, and that lipids and OCPs are mobilized and subsequently incorporated into developing yolks. However, as seen in Table 3 and Table 4, there was no evidence to suggest that a consistent amount of chlordane, p,p-DDE, dieldrin, or toxaphene was transferred to mature eggs. For this reason, the hypothesis that direct toxicity occurs to fathead minnow eggs in the natural environment and adversely affects fertilized egg development and/or survivorship of larvae was abandoned, and the nanoinjection portion of this thesis was terminated. Table 4 shows that over a 30-day period, while females were continually dosed, with the exception of toxaphene which was not detected in any samples except the flake and dieldrin which remained relatively constant from the early sampling period to the late sampling period, chlordane and p,p-DDE either decreased to a non-detectable amount or decreased to nearly 50% of the initial concentration. Xie and Klerks (2002) showed that least killifish (Heterandria Formosa) exposed to cadmium developed a resistance to toxicity within a six generation timeframe. It is possible that the fathead minnows are able to adapt, possibly by increasing activity or production of enzymes that transform OCPs from hydrophobic xenobiotics to hydrophilic metabolites that are excreted, thus decreasing the potential for adverse affects. When examining Figure 1, we can observe that there were significant differences detected among the control vs. high and middle treatments and although not significantly

PAGE 42

32 different, a 20% decrease between the control and low treatment groups. Previous studies have demonstrated that exposure to certain contaminants adversely affects mating behavior in cyprinids. Jones and Reynolds (1997), found that exposure to various contaminants affected mating behavior in such ways as: increases or decreases in mating displays, increased courtship duration, performance of male-like behavior of masculinized females, decreased nest-building ability, decreased offspring defense, or changes in division of parental care between sexes. It is also known that cyprinids secrete steroid hormones that act as pheromones during courtship. It is possible that OCPs may disrupt the activity of these pheromones causing a disruption in courtship behavior. Cyprinidae are known to have complex mating behaviors, thus the possibility exists that exposure to OCPs may adversely affect mating behavior and consequently decrease the ability for fathead minnow pairs to successfully spawn. Figures 3 and 4 show that the mean number of eggs hatched and clutch viability both show similar trends in the controls and treatment groups. While the low treatment group displayed an increased number of eggs hatched, and a higher clutch viability when compared to the control, the middle and high treatment groups showed a decrease in the number of eggs hatched, as well as clutch viability. Because OCP levels in the eggs of the fathead minnow were either non-detectable or inconsistently transferred from the adult female to the egg; conclusions can be drawn that there is not a correlation between maternally transferred OCPs to eggs and reduced mean number of eggs hatched and reduced clutch viability. In an attempt to explain the significant decreases in the middle and high treatment groups, it should be noted that the intermediate and highly exposed females continued to lay eggs that were not significantly different than the mean number

PAGE 43

33 of control eggs. The middle and high treatment females continually produce and deposit eggs, however, the ova appear to be unable to sequester the nutrients required to produce healthy and viable eggs. A lack of nutrients results in a decreased amount of energy, as well as, structural supplies that are available for developing embryos and possibly yolk sacs that larvae use as a source of nutrition for the first several days after hatching. This is an observation that has been speculated in other studies (Rauschenberger, 2004; Johnson, 2005) however, until the completion of this study, little evidence has been able to support this hypothesis. Although circulating sex steroids were not analyzed in this experiment, effects that these hormones have on reproductive success should be addressed. Gross et al. (2002) found that circulating concentrations of E 2 in female largemouth bass and 11-KT in male largemouth bass exposed to OCPs were on average1,500 pg/mL less than what was reported for pond-reared largemouth bass sampled in the same calendar year. The ability for any exogenous compound to bind a sex steroid hormone receptor and agonize and/or antagonize the action of an endogenous hormone can severely affect normal endocrine function. The reasoning for this is that normal estrogen or testosterone concentrations and actions are critical for development of gonads in both male and female fish (Johnson, 2005). It is possible that fathead minnows exposed to OCPs may alter the concentrations of these two circulating sex steroids thus reducing reproductive success. Vitellogenin is a protein synthesized by the liver and its uptake by growing oocytes and its storage as yolk serves as a nutrient source by developing embryos. During this period, extraovarian proteins are gathered, processed, and packaged into oocytes. Consequently, this period is of particular importance when considering maternal transfer

PAGE 44

34 by lipophilic proteins of OCPs to developing oocytes (Di Giulio and Tillitt, 1999). The possibility exists that because studies have shown that circulating vitellogenin acts as an important transport protein that binds lipophilic hormones, OCPs may bind to vitellogenin and agonize and/or antagonize vitellogenin receptors on gonads, thus impeding the deposition of yolk reserves, and therefore oocyte growth. Again, this is one explanation as to why decreased reproductive success is observed in my experiment while direct embryo toxicity is not. Favorable water temperature, salinity, food availability, as well as egg quality are critical to the survivorship, growth, and the metamorphosis to the juvenile stage of larval fish. Fast growth to fish larvae has been correlated to the presence of high plankton concentrations where larvae have adequate opportunities to capture prey. Other environmental factors that influence larval development include dissolved oxygen, turbidity, nutrients, water movement, and meteorological events. The first stage of larval development is the yolk sac phase. The yolk sac contains nutrition used by larval fish while it adapts to its new aquatic environment. Larvae absorb the yolk and will continue to grow and develop while they begin to look for their first prey, plankton. As larvae continue to develop, they begin to take on the appearance of juveniles and further develop the ability to swim and capture prey. Typically, fathead minnows can not swim for long periods of time as larvae, they can however, swim long enough to seek appropriate resting habitat. During the course of our experiment, every effort was made to eliminate any possibility of larvae mortality due to water quality parameters or insufficient food availability. At no time during the course of the experiment were hatched larvae exposed to harmful levels of dissolved oxygen, salinity, or temperature.

PAGE 45

35 Hatched larvae were also fed ad libitum adequate supplies of artemia. Because all obvious growth and developmental parameters were satisfied, it leads us to believe that the significant differences in the percent of larvae survived to Day 7 and Day 14 (Figures 5 and 6) were solely due to OCP exposure. While a dose-response relationship was not established for either Day 7 or Day 14 larvae survivorship, all three treatment groups for the percent of larvae survived to Day 7 were either lower or significantly lower when compared to the control, and all three treatment groups of the percent of larvae survive to Day 14 were significantly lower when compared to the control. While no teratological or morphometrical measurements were taken during the course of this experiment, further discussion should be made as to the specifics of the high mortality observed in this experiment. Most teratogenic contaminants are believed to be non-specific (Newman and Unger, 2003). According to Karnofskys law, any agent will be teratogenic if it is present at concentrations or intensities that produce cell toxicity (Bantle, 1995). Teratogens act by disrupting mitosis, interfering with transcription and translation, disturbing metabolism, and producing nutritional deficits (Weis and Weis, 1987). Consequences of these disruptions may include abnormal cell interactions, excessive growth, or cell death. Retardation of growth, adverse effects on the skeletal, musculature, circulatory, and optical system are the most common effects seen in larval fish exposed to toxicants. Skeletal problems in fish may include lateral curvature of the spine (scoliosis) or the extreme forward curvature of the spine (lordosis). Wies and Wies (1989), showed that the mummichog, (Fundulus heteroclitus) when exposed to 10 mg l -1 of Pb 2+ failed to uncurl its tail post hatch, essentially leaving the larval fish to certain death. Seplveda et

PAGE 46

36 al. (2000) found that largemouth bass larvae exposed to p,p-DDE and dieldrin showed yolk sac edema, and eye deformities including opaque corneas. Studies of largemouth bass larvae exposed to paper mill effluent also showed deformities including: increased head abnormalities, a decrease in length and weight, and a shortened vertebral column (Seplveda et al., 2003). Any or all of these abnormalities may have led to the decreased survival rates observed in my experiment. Gonadosomatic index (GSI) is defined as the ratio of the weight of the gonad to the weight of whole body. Although GSI is not specific to a particular mechanism of toxicant action, it is an endpoint commonly used in toxicological studies, and is a good predictor of reproductive success that may be linked to population level responses to contaminants. However, caution must be applied when using GSI to make a hypothesis concerning contaminant effects to individuals. Dependent upon the species of fish and the time of the year, an increase in GSI may be a result of responses to reproductive and/or environmental cues. Gross et al. (2000) found that gonads in largemouth bass began to mature in October and reached a considerable size (approximately 5% of fish body weight) by January, and continued to peak until February. Depending on the month or season of the year, GSI may not be an accurate endpoint for contaminant exposure. Also, fathead minnows are batch spawning fish. Consequently, their gonads undergo rapid cyclical changes over short periods of time (every few days) as successive batches of eggs or sperm are produced, and thus means the size of the gonads in breeding adults can vary considerably between individuals at any point in time (Harries et al., 2000). In my study, neither male nor female displayed significant differences in mean GSI (Figures 7 and 8). This is consistent with a study that exposed largemouth bass to 10.0 mg/pellet

PAGE 47

37 of p,p-DDE and 1.0 mg/pellet of dieldrin (Muller, 2003). My results are however, not consistent with a study of male guppies (Poecilia reticulata) who when fed a diet of 150ug of p,p-DDE for 30 consecutive days showed a decrease in GSI. Seplveda (2000) showed that there can be differences in GSI not only between various spawning seasons, but also between different habitats within the same area. Again, these inconsistencies with GSI should throw caution when using this tool as an endpoint to measure reproductive success or toxicant exposure. There were significant differences in the percent of females spawning and the number of spawns (Figure 9). Over a 30-day period, the percent of females spawning in the control went from 80% to 10% after the fifth spawn. The low treatment group went from 70% of females spawning to only 30% after the third spawn. Although the middle treatment group spawned the most in a 30-day period, six times, the percent of females spawning started at 40% and remained relatively constant throughout the 30-day period at 20%. The high treatment group started at 50% and by the fifth spawn dropped to only 10%. The frequency of spawnings in our control fish were slightly lower than what has been reported in other studies. Gale and Buynak (1982) reported that the mean number of spawns in their experiment was every 3.9 days. In contrast, in my study, the low treatment group spawned approximately every 8 days, the middle treatment group spawned approximately every 5 days, and the high treatment group spawned approximately every 6 days. Although the frequency of spawning was reduced in the control and treatment groups, two critical observations should be made. The first observation is that as previously mentioned, not only did treatment groups have reduced frequency in spawning, so did the control. Our data shows that over a 30-day period the

PAGE 48

38 percent of control females spawning went from 80% to 0%. This critical observation is in that numerous state, federal, and private institutions use the fathead minnow as a model in aquatic toxicity tests. Protocols should be established that limit the frequency a specific breeding pair of fathead minnows is used without adequate recovery time before employing them in continuous toxicity tests. The second observation that should be made is that as seen in Figure 10, altered frequency of spawns is also correlated with the mean number of eggs laid. This would suggest that in reference to a population of fathead minnows in a particular ecosystem, altered frequency of spawning would not be offset by an increase in fecundity. This is in contrast to a study where fathead minnows exposed to weak active endocrine mimics had a decrease in the number of spawnings, however, there was a reciprocal increase in the size of the egg batch (Harries et al., 2000). The mean number of eggs laid per spawn showed a lower number of eggs produced for treated versus controls (Figure 10). This figure also showed altered patterns across treatments. Although the control started with a low mean number of eggs laid, by the second spawn, the mean number of eggs laid per spawn decreased, and fish stopped spawning after the forth spawn within a 30-day period. The low treatment group showed a similar pattern with the most eggs laid during the first spawn, the mean of eggs laid tapering off until the third spawn when no more eggs were laid. Although the middle treatment group displayed a similar pattern, having the greatest number of eggs during the first spawn, then gradually tapering off, there was a lower mean number of eggs laid on the first spawn, however, this treatment group spawned the most times, six, out of all treatment groups. The fact that the middle treatment group continued to spawn for a

PAGE 49

39 longer period of time than other treatment groups, may indicate the fathead minnow has adapted to mid-exposure and although fewer eggs are being produced, the frequency of spawning is significantly longer. The high treatment group continued to show the same pattern of the most eggs being spawned early, then gradually tapering off until no further spawning took place. Although OCPs may affect the frequency of spawning as well as the mean number of eggs laid per spawn, it could also be possible that the differences observed in mean number of eggs is in part due to differences in the sizes of the fish. It is apparent however, that the largest clutch sizes take place early and gradually taper off in the control and treatment groups possibly due to the availability of mature oocytes. Control clutch viability varied from 55% to 90% throughout the number of times spawned within the 30 trial period. There appeared to be no distinguishable pattern within the percent ranges (Figure 11). The low treatment group did however, show a higher clutch viability toward the early stages of the experiment than the later stages. From this, one might assume that bioaccumulation of OCPs are decreasing clutch viability, however, the middle and high treatment groups show an increase and then decrease in clutch viability over the number of times spawned as well as the 30-day period. Although no obvious patterns across treatments were observed, all three treatment groups had significantly lower clutch viability percentages when compared to the control. This observation is similar to alligator eggs exposed to OCPs which also showed decreased clutch viability (Rauschenberg, 2004). This indicates decreased clutch viability is due to decreased egg quality associated with senescence. Recently, numerous studies have clearly established that various man-made and natural chemicals exist within aquatic environment that have the potential to mimic

PAGE 50

40 androgens, estrogens, or their antagonists. A number of EDCs bioaccumulate and/or result from environmental degradation or metabolism from their parent compound. In order to establish a cause-effect relationship, between exposure to EDCs and reproductive success and survivorship, it is necessary to conduct whole animal studies. Given the large number of chemicals that have the potential to be EDCs, as well as are regularly found within the aquatic environment, there is a need to develop a biological model that is practical to work with in a laboratory setting that is capable of demonstrating quantifiable reproductive parameters as well as other endocrine disruptive biomarkers such as vitellogenin induction. Other studies have demonstrated documented bioaccumulation of OCPs in the carcass and gonads of largemouth bass (Johnson, 2005), and endocrine modulation caused by OCPs (Muller, 2004). Seplveda et al. (2004) found alterations in endocrine function and increased developmental mortality in largemouth bass inhabiting the Emeralda Marsh. In another study, relative contributions of losses during in ovo development in alligators at impacted sites in Florida are lower clutch viability, higher rates of damaged eggs, higher rates of early embryo mortality, and higher rates of late embryo mortality, all of these were due to exposure to OCPs (Rauschenberger, 2004). While all of these studies displayed affects caused by exposure to OCPs, these were all captive studies that were both labor intensive and financially demanding. Hence, the need to develop a series of short-term, economical, laboratory tests using a biological model that shows effects of exposure to EDCs including fecundity, GSI, induction of vitellogenin and other circulating sex steroids (E 2 and 11-KT), and survivorship is needed to predict and assess potential impacts of EDCs either on larger individuals or on populations within ecosystems.

PAGE 51

41 Further research on the reproductive effects of p,p-DDE, dieldrin, chlordane, and toxaphene on fathead minnows could focus on the measurement of additional reproductive endpoints. For example, estradiol-17, 11-KT and vitellogenin would potentially add additional information concerning the development stages of oocytes and address the size and condition of gonads. Although male gonads were staged, assessing gamete viability in males would eliminate the possibility that EDCs are disrupting male reproductive potential. Nakayama et al. (2005) found that when Japanese medaka (Oryzias latipes) were exposed to tributyltin (TBT), the number of eggs laid remained relatively constant, however, the number of fertilized eggs decreased. Another study showed that as levels of TBT increased the percent of sperm that lacked flagellum or had a decrease in the volume of milt also increased (McAllister and Kime, 2003). Another factor that may affect OCP toxicity that was not addressed in my experiment is in that low temperatures have been associated with increased DDT toxicity in fish (Rattner and Heath, 2003). Fluctuation in water temperature, as well as fluctuations in OCP levels, mimicking hotspots may also provide invaluable insight into the correlation between OCP exposure and reduction in reproductive success.

PAGE 52

42 0102030405060708090100controllowmiddlehighTreatment GroupPercent * Figure 1. The percent of spawning pairs among treatment groups. Control, low, middle, and high treatment groups had 10 potential spawning fathead minnow pairs. Mean standard error results are shown. Significant differences between control middle and high treatment groups using One-way ANOVA (P<.05). Asterisks indicate differences in relation to controls. 050100150200250300350400450controllowmiddlehighTreatment GroupMean Number of Eggs Laid * Figure 2. The mean number of eggs laid per spawn among treatment groups. Control, low, middle, and high treatment groups had 10 potential spawning fathead minnow pairs. Mean standard error results are shown. Significant differences between low and control treatment groups, low and middle treatment groups using One-Way ANOVA (P<.05). Asterisks indicate differences in relation to controls.

PAGE 53

43 050100150200250300350controllowmiddlehighTreatment GroupMean Number of Hatched Eggs * Figure 3. The mean number of eggs hatched among treatment groups. Control, low, middle, and high treatment groups had 10 potential spawning fathead minnow pairs. Mean standard error results are shown. Significant differences between low and control treatment groups, low and high treatment groups, and low and middle treatment groups using One-Way ANOVA (P<.05). Asterisks indicate differences in relation to controls. 0102030405060708090100controllowmiddlehighTreatment GroupPercent of Clutch Viability * Figure 4. The percent of clutch viability among treatment groups. Control, low, middle, and high treatment groups had 10 potential spawning fathead minnow pairs. Mean standard error results are shown. Clutch viability = No. of eggs yielding a live hatchling / Fecundity x 100. Significant differences between low and middle treatment groups, low and high treatment groups, control and middle treatment groups, control and high treatment groups, and middle and low treatment groups using One-Way ANOVA (P<.05). Asterisks indicate differences in relation to controls.

PAGE 54

44 0510152025303540455055controllowmiddlehighTreatment GroupPercent of Larvae Survived * Figure 5. The percent of larvae survived to Day 7 among treatment groups. Control, low, middle, and high treatment groups had 10 potential spawning fathead minnow pairs. Mean standard error results are shown. Significant differences between control and high treatment groups and control and middle treatment groups using One-Way ANOVA (P<.05). Asterisks indicate differences in relation to controls. 051015202530354045controllowmiddlehighTreatment GroupPercent of Larvae Survived * * Figure 6. The percent of larvae survived to Day 14 among treatment groups. Control, low, middle, and high treatment groups had 10 potential spawning fathead minnow pairs. Mean standard error results are shown. Significant difference between control and middle treatment groups using One-Way ANOVA (P<.05). Asterisks indicate differences in relation to controls.

PAGE 55

45 00.20.40.60.811.21.41.6controllowmiddlehighTreatment GroupMale Mean GSI (g) Figure 7. Mean GSI among male treatment groups. Control, low, middle, and high treatment groups had 10 potential spawning fathead minnow pairs. Mean standard error results are shown. No significant differences using One-Way ANOVA (P<.05). 024681012141618controllowmiddlehighTreatment GroupFemale Mean GSI (g) Figure 8. Mean GSI among female treatment groups. Control, low, middle, and high treatment groups had 10 potential spawning fathead minnow pairs. Mean standard error results are shown. No significant differences using One-Way ANOVA (P<.05).

PAGE 56

46 0123456789101st2nd3rd4th5th6thNumber of Times SpawnedPercent control low mid high Figure 9. Percent of Females Spawning vs. Number of Spawns. Control, low, middle, and high treatment groups had 10 potential spawning fathead minnow pairs. Mean standard error results are shown. Note: Decreased number of spawning females for treated versus controls and altered patterns across treatments. 0501001502002503001st2nd3rd4th5th6thTotalNumber of Times SpawnedMean Number of Eggs Laid control low mid high Figure 10. Mean Number of Eggs Laid vs. Number of Times Spawned. Control, low, middle, and high treatment groups had 10 potential spawning fathead minnow pairs. Mean standard error results are shown. Note: Lower number of eggs produced for treated versus controls and altered patterns across treatments.

PAGE 57

47 01020304050607080901001st2nd3rd4th5th6thNumber of Times SpawnedPercent of Clutch Viability control low mid high Figure 11. Percent of Clutch Viability vs. Number of Times Spawned. Control, low, middle, and high treatment groups had 10 potential spawning fathead minnow pairs. Mean standard error results are shown. Note: Lower clutch viability produced for treated versus controls and altered patterns across treatments.

PAGE 58

CHAPTER 5 CONCLUSION Research from this study provides evidence that dietary exposure to a mixture of p,p-DDE, dieldrin, toxaphene, and chlordane bioaccumulate in maternal tissues and at inconsistent rates are transferred to developing eggs. Our data shows that maternal exposure to OCPs indicates endocrine disruption, and adversely affects reproduction by lowering the percentage of spawning pairs, lowering the survival of larvae up to Day 14, decreases the number of eggs produced over time, and lowers clutch viability over time. The fathead minnow could potentially be used as a biological model to assess effects of various contaminant exposures including pharmaceuticals, sewage effluent, paper mill effluent, and numerous heavy metals. Adult fathead minnows are omnivores, consuming a variety of resources as food, hence giving the fish an opportunity to accumulate numerous toxicants. My study has shown OCPs bioaccumulate in fathead minnow adipose and gonadal tissue and is maternally transferred at various concentrations to their eggs. Fathead minnows in my study responded similarly to largemouth bass and alligators that were orally exposed to various concentrations of OCPs, making the fathead minnow an eco-relevant model. However, unlike largemouth bass, fathead minnows are not seasonal spawners, multi-generation tests can easily be performed, and costs of performing these tests are greatly reduced in comparison with largemouth bass. The ease of laboratory handling, the low financial burden, as well as the ability of fathead minnows to bioaccumulate contaminants makes it a good model to 48

PAGE 59

49 examine potential reproductive effects, thus giving researchers the ability to extrapolate cause-effect relationships in higher order organisms. My data also concluded that although fathead minnows exposed to OCPs continued to produce and deposit eggs, the ova appear unable to sequester the proper nutrients required to produce healthy eggs. The conclusion can be made that bioaccumulation of OCPs in the female ovaries may cause lower egg quality prior to the induction of vitellogenesis, however, total protein or lipid analysis would further support this conclusion. Inconsistent amounts of OCPs were transferred to fathead minnow eggs, however, the relatively same endpoints were observed whether high quantities of OCPs were transferred or low quantities were transferred. This would suggest that the mechanism of action is maternal.

PAGE 60

LIST OF REFERENCES Ankley, G.T., Jensen, K.M., Kahl, M.D., Korte, J.J., Makynen, E.A., 2000. Description and evaluation of a short-term reproduction test with the fathead minnow (Pimephales promelas). Environ. Toxicol. Chem. Bantle, J. A. 1995. FETAX: a developmental toxicity assay using frog embryos. Fundamentals of Aquatic Toxicology: Effects, Environmental Fate, and Risk Assessment, 2 nd ed., Rand, G.M., Ed., Taylor and Francis, Washington, D.C. Bryan, T. E., Gildersleeve R. P., Wiard, R. P., 1989. Exposure of Japanese quail embryos to op-DDT has long term effects on reproductive behaviors, hematology and feather morphology. Teratol 39: 525-236. Cahn, A. R. 1927. An ecological study of southern Wisconsin fishes: the brook silversides (Labidesthes sicculus) and the cisco (Leucichthys artedi ) in their relations to the region. Illinois Biological Monographs 11:1-151. Coyle, E. E. 1930. The algal food of Pimephales promelas (fathead minnow). Ohio Journal of Science 30:23-35. (Not seen; cited in Scott and Crossman 1973.) Danzo, B. 1997. Environmental xenobiotics may disrupt normal endocrine function by interfering with the binding of physiological ligands to steroid receptors and binding proteins. Environmental Health Perspectives 105: 294. Darnerud, P. O., Sinjari, T., and Jonsson, C. J. 1996. Fetal uptake of coplanar polychlorinated biphenyl (PCB) congeners in mice. Pharmacological Toxicology 78: 187. Di Giulio, R. T., and Tillitt, D. E. 1997. Reproductive and Developmental Effects of Contaminants in Oviparous Vertebrates. SETAC. Pensacola, Florida, United States. Pp. 20-21. Flouriot, G., Pakdel, F., Ducouret, B., and Valotaire, Y. 1995. Influence of xenobiotics on rainbow trout liver estrogen receptor and vitellogenin gene expression. Journal of molecular endocrinology 15: 143. 50

PAGE 61

51 Foster, E. P., Fitzpatrick, M. S., Feist, G. W., Schreck, C. B., Yates, J., Spitsbergen, J. M., and Heidel, J. R. 2001. Plasma Androgen Correlation, EROD Induction, Reduced Condition Factor, and the Occurrence of Organochlorine Pollutants in Reproductively Immature White Sturgeon (Acipenser transmontanus) from the Colomia River, USA. Archives of Environmental Contamination and Toxicology 41: 182-191. Gale W. F. and Gerard B. L. 1982. Fecundity and Spawning Frequency of the Fathead MinnowFractional Spawner. Transactions of the American Fisheries Society. 111:35-40. Gallager, E. P., Gross, T. S., and Sheehy, K. M. 2001. Decreased glutathione S-transferase expression and activity and altered sex steroids in Lake Apopka brown bullheads (Ameriurus nibulosus). Aquatic Toxicology 55: 223-237. Gayle, W.F., Buynak, G.L. 1982. Transactions of the American Fisheries Society. Vol. 111: 35-40. Gildersleeve, R. P., Tilson, H., Mitchell, C., 1985. Injection of diethylstilbestrol on the first day of incubation affects morphology of sex glands and reproductive behavior of Japanese quail. Teratology 31: 101-109. Gleason, T. R. and Nacci, D. E. 2001. Risks of Endocrine disrupting compounds to wildlife: Extrapolating from effected individuals to population response. Human and Ecological Risk Assessment 7: 1042. Gross, T. S., Arnold, B. S., Seplveda, M. S., and McDonald, K. 2003. Endocrine disrupting chemicals and endocrine active agents. Pages 1033-1098 in D. J. Hoffman, B. A. Rattner, G. A. Burton, and J. Cairns, editors. Handbook of Ecotoxicology, 2 nd edition. Lewis Publishers, Boca Raton, Florida. Gross, T. S., Guillette, L. J., Percival, H. F., Masson, G. R., Matter, J. M., and Woodard, A. R. 1994. Contaminant-induced reproductive anomalies in Florida. Comparative Pathology Bulletin 26: 2-8. Gross, T. S., Seplveda, M. S., Wieser, C. M., Wiebe, J. J., Schoeb, T. R., and Denslow, N. D. 2002. Characterization of annual reproductive cycles for pond-reared Florida largemouth bass (Micropterus salmoides floridanus). Pages 205-212 in D. P. Phillip and M.S. Ridgeway, editors. Proceedings of the Black Bass 2000 Symposium. American Fisheries Society, Bethesda, Maryland. Harries, J. E., Runnalls, T., Hill, E., Harris, C. A., Maddix, S., Sumpter, J. P., and Tyler, C. R. 2000. Development of a Reproductive Performance Test for Endocrine Disrupting Chemicals Using Pair-Breeding Fathead Minnows (Pimephales Promelas). Environmental Science Technology 34: 3003-3011. Hoffman, D. J., Rattner, B. A., Burton, Jr., G. A., and Cairns, Jr., J. 2003. Handbook of Ecotoxicology. Second Ed. CRC Press LLC. Boca Raton, Florida, USA.

PAGE 62

52 Hori, S. H., Kodama, T., and Tanahashi, K. 1979. Induction of vitellogenin synthesis in goldfish by massive doses of androgens. General and Comparative Endocrinology 37: 306-320. Jansen, H. T., Cooke, P. S., Porcelli, J., Liu, T. C., and Hansen, L. G. 1993. Estrogenic and anti-estrogenic actions of PCBs in the female rat: In vitro and in vivo studies. Reproductive Toxicology 7: 237. Jenkins, R. E., Burkhead, N. M. 1993. Freshwater fishes of Virginia. Page 252. American Fisheries Society, Bethesda, Maryland. Jensen, K. M., Korte, J. J., Kahl, M. D., Pasha, M. S., and Ankley, G. T., 2001. Aspects of basic reproductive biology and endocrinology in the fathead minnow (Pimephales promelas). Comparative Biochemistry and Physiology Part C 128:127-141. Jobling, S., Nolan, M., Tyler, C. R., Brighty, G., and Sumpter, J., P. 1998. Widespread sexual disruption in wild fish, Environmental Science Technology 32: 2498-2506. Johnson, K. G. 2005. Dietary exposure to the organochlorine pesticides p,p-DDE and dieldrin and their effects on steroidogenesis and reproductive success in Florida largemouth bass (Micropterus salmoides floridanus). Masters Thesis. University of Florida, Gainesville, FL. Jones, J. C., Reynolds, J. K. 1997. Effects of pollution on reproductive behavior of fishes. Reviews in Fish Biology and Fisheries. Vol 7. 463-491. Kelce, W. R., Stone, C. R., Laws, S. C., Gray, L. E., Demppainen, J. A., and Wilson, E. M. 1995. Persistent DDT metabolite p,p-DDE is a potent androgen receptor antagonist. Nature 375(6532): 581-585. Marburger, J. E., Johnson, W. E., Gross, T. S. Gross, Douglass, D. R., and Di, J. 2002. Residual organochlorine pesticides in soils and fish from wetland restoration areas in central Florida, USA. Wetlands 22:705-711. Markus, H. C. 1934. Life history of the blackhead minnow (Pimephales promelas). Copeia 1934:116-122. Mayer, F. L. 1971. Dynamics of dieldrin in rainbow trout and effects on oxygen consumption. Diss. Abstr. Int. 32(1): 527-B. Miller, R. R. 1986.Composition and derivation of the freshwater fish fauna of Mexico. Anales de la Escurela Nacional de Ciencias Biologicas, Mexico 30:121-153. Mills, L., Gutjahr-Gobell, R. E., Haebler, R. A., Borsay Horowitz, D. J.,Jayaraman, S., Pruell, R. J., McKinney, R. A., Gardner, G. R., and Zaroogian, G.E. 2001. Effects of estrogenic (o,p-DDT; octylphenol) and anti-androgenic (p,p-DDE) chemicals on indicators of endocrine status in juvenile summer flounder (Paralichthys dentatus). Aquatic Toxicology 52: 157-176.

PAGE 63

53 Muller, J. K. 2003. An evaluation of dosing methods and effects of p,p-DDE and dieldrin in Florida largemouth bass (Micropterus salmoides floridanus). Masters Thesis. University of Florida, Gainesville, FL. Muller, J. K., Johnson, K. G., Sepulveda, M. S., Borgert, C., and Gross, T. S. 2004. Accumulation of dietary DDE and dieldrin by largemouth bass, Micropterus salmoides floridanus. Bulletin of Environmental Contamination and Toxicology 73: 1078-1085. Nelson, J.S. 1984. Fishes of the World, 2 nd edition. Wiley, New York. Newman, M. C. and Unger, M. A. 2003. Fundamentals of Ecotoxicology 2 nd Edition. CRC Press. Boca Raton, Florida. Okoumassoun, L.-E., Averill-Bates D., Gagne, F., Marion, M., and Denizeau, F. 2002. Assessing the estrogenic potential of organochlorine pesticides in primary cultures of male rainbow trout (Oncorhynchus mykiss) hepatocytes using vitellogenin as a biomarker. Toxicology 178: 193-207. Pearse, A. S. 1918. The food of the shore fishes of certain Wisconsin lakes. U.S Bureau of Fisheries Bulletin. Lewis Publishers 35: 246-292. Pelissero, C., Flouriot, G., Foucher, J. L., Bennetau, B., Dunogures, J., Le Gac, F., and Sumpter, J. P. 1993. Vitellogenin synthesis in cultured hepatocytes; an in vitro test for the estrogenic potency of chemicals. The Journal of Steroid Biochemistry and Molecular Biology 44: 263-272. Rattner, B. A. and Heath, A. G. 2003. Environmental factors affecting contaminant toxicity in aquatic and terrestrial vertebrates. In Handbook of Ecotoxicology: 679-699. Hoffman, D. J., Rattner, B. A., Burton, G. A. Jr. and Cairns, J. Jr. (Ed.). Boca Raton, FL USA Rauschenberger, H. R., Wiebe, J. J., Buckland J. E., Smith, T. J., Sepulveda, M. S., and Gross, T. S. 2004. Achieving environmentally relevant organochlorine pesticide concentrations in eggs through maternal exposure in Alligator mississippiensis. Marine Environmental Research: 851-856. Redding, M. J., and Patino, R. 1993. Reproductive physiology. Pages 503-534 in D. H. Evans, editor, The Physiology of Fishes. Marine Science Series. CRC Press, Boca Raton, Florida. Ree, G. E. and Payne J. F. 1997. Effecto to toxaphene on reproduction of fish. Chemosphere. 34(4): 855-867. Robins, C.R. 1991. Common and scientific names of fishes from the United States and Canada, 4 th edition. American Fisheries Society Special Publication 20. Rosner, W. 1990. The functions of corticosteroid-binding globulin and sex hormone-binding globulin: Recent advances. Endocrinology Review 11: 80.

PAGE 64

54 Russell, R. W., Frank, A. P., Gobas, C., and Haffner, G. D. 1999. Maternal ransfer and in ovo exposure of organochlorines in oviparous organisms: A model and field verification. Environmental Science Technology 33: 416-420. Ryffel, G. U. 1978. Synthesis of vitellogenin, an attractive model for investigating hormone-induced gene activation. Molecular and Cellular Endocrinology 12: 237-246. Scott, W. B. and Crossman E. J. 1973. Freshwater fishes of Canada. Bulletin of Fisheries Resources. Board, Canada. pp.184. Seplveda, M. S. 2000. Effects of Paper Mill Effluents on Health and Reproductive Success of Largemouth Bass (Micropterus salmoides): Field and Laboratory Studies. Doctoral Dissertation. University of Florida, Gainesville, FL. Shelby, M.D., Newbold, R. R., Tully, D. B., Chae, K., and Davis, V. L. 1996. Assessing environmental chemicals for estrogenicity using a combination of in vitro and in vivo assays. Environmental Health Perspectives 104: 1296-1300. Smith, H. M. 1945. The freshwater fishes of Siam, or Thailand. Bulletin of the United States National Museum 188:1-622. Starrett, W. C. 1950. Food relationships of the minnows of the Des Moines River, Iowa. Ecology 31:216-233. Stebbing, A.R.D. 1982. Hormesis: the stimulation of growth by low levels of inhibitors. Science Total Environment. Vol.22. pp 213-234. Sumpter, J. P. and Jobling, S. 1995. Vitellogenesis as a biomarker for estrogenic contamination of the aquatic environment. Environmental Health Perspectives, supplement 103: 173-178. Thomas, M. J. and Thomas, J. A. 2001. Hormone assays and endocrine function, in Principles and Methods of Toxicology, 4 th Ed., Taylor & Francis. Philadelphia, PA. U. S. EPA 1982. Users guide for conducting life-cycle chronic toxicity test with fathead minnows (Pimephales promelas). EPA/600/8-81-011. Duluth, MN. U.S. EPA 1987. Guidelines for the culture of fathead minnows Pimephales promelas for use in toxicology tests. EPA/600/3-87/001. Duluth, MN. U. S. EPA 1989. Pesticide assessment guidelines. Sub-division E, Hazard evaluation: Wildlife and aquatic organisms. EPA-540/09-82/024. Washington, D.C. U. S. EPA, 1991. Methods for measuring the acute toxicity of effluents and receiving waters to freshwater and marine organisms 4 th ed. In: Weber, C.I. (Ed.), EPA-600/4-90/027. Office of Research and Development, Environmental Monitoring Systems Laboratory, Cincinnati, OH.

PAGE 65

55 U.S. EPA, 1994. Short-term methods for estimating the chronic toxicity of effluents and receiving water to freshwater organisms, 3 rd edition In: Lewis, P.A., Klemm, D.J., Lazorchak, J.M., Norberg-King, J.J., Peltier, W.H., Heber, M.A.., (Eds), EPA/600/4-91/002. Office of Research and Development Environmental Monitoring Systems Laboratory, Cincinnati, OH. U.S. EPA, 1998. Endocrine Disruptor Screening and Testing Advisory Committee Report. Office of Prevention, Pesticides, and Toxic Substances, Washington, DC. Van Der Kraak, G., Chang, J. P., and Janz, D. M. 1998. Reproduction. Pages 465-488 in D. H. Evans, editor. The Physiology of Fishes. CRC press, Boca Raton, Florida. Wahli, W., Dawid, I. B., Ryffel, G. U., and Weber, R. 1981. Vitellogenesis and the vitellogenin gene family. Science 212: 298-304. Wallace, R. A. 1970. Studies on amphibian yolk Xenopus vitellogenin). Biochimica et Biophysica Acta 215: 176-183. Wallace, R. A., and Selman, K. 1981. Cellular and dynamic aspects of oocyte growth in teleosts. American Zoologist 21: 325-343. Weis, J.S. and Weis, P. 1987. Pollutants as developmental toxicants in aquatic organisms, Environmental Health Perspectives. Vol.71: pp 77-85. Weis, J.S. and Weis, P. 1989. Tolerance and stress in a polluted environment: the case of the mummichog, Fundulus heteroclitus., Bioscience. Vol.39: pp 89-95. Wells and Van Der Kraak, G. 2000. Differential binding of endogenous steroids and chemicals to androgen receptors in rainbow trout and goldfish. Environmental Toxicology and Chemistry 19: 2059-2065. Xie, L., and Klerks, P.L. 2002. Responses to selection for cadmium resistance in the least killifish (Heterandria Formosa). Environmental Toxicology and Chemistry. Vol. 22, No. 2, pp 313-320. Zeeman, M., Gifford, J. 1993. Ecological hazard evaluation and risk assessment under EPAs Toxic Substances Control Act (TSCA): an introduction. In Landis W. Hughes J, Lewis, M. editors. Environmental toxicology and risk assessment. Volume 1. Philadelphia PA: ASTM. STP 1179 p. 7-21.

PAGE 66

BIOGRAPHICAL SKETCH Dane H. Huge was born August 29, 1970. He attended Fredericktown High School in Fredericktown, Ohio, and graduated in 1989. Dane then served 5 years in the U.S. Navy as a photographer. Dane graduated from the University of Florida in 1999. Post graduation, Dane worked as a biological technician at the United States Geological Survey working on spawning behaviors of invasive and listed species of minnows. Dane then enrolled as a graduate student in spring 2004 under the guidance of Dr. Timothy S. Gross. He focused his work on reproductive effects of organochlorine pesticides on fathead minnows. Dane received his Master of Science degree from the Department of Physiological Sciences, College of Veterinary Medicine, at the University of Florida in May 2006. 56


Permanent Link: http://ufdc.ufl.edu/UFE0014367/00001

Material Information

Title: Organochlorine Pesticide Reproductive Effects in Fathead Minnows (Pimephales promelas): Comparison of Embryo and Maternal Exposure
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0014367:00001

Permanent Link: http://ufdc.ufl.edu/UFE0014367/00001

Material Information

Title: Organochlorine Pesticide Reproductive Effects in Fathead Minnows (Pimephales promelas): Comparison of Embryo and Maternal Exposure
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0014367:00001


This item has the following downloads:


Full Text











ORGANOCHLORINE PESTICIDE REPRODUCTIVE EFFECTS INT FATHEAD
MINTNOWS (Pimephales promela;s): COMPARISON OF EMBRYO AND
MATERNAL EXPOSURE














By

DANE HOLLAND HUGE


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2006
































Copyright 2006

by

Dane Holland Huge
















ACKNOWLEDGMENTS

I extend my gratitude to Dr. Timothy S. Gross for providing the resources and

guidance required for me to succeed as a graduate researcher and graduate student.

Special thanks go to all the staff at the United States Geological Survey-Florida

Integrated Science Center, Ecotoxicology Lab (Gainesville, Florida). Special thanks go

to Dr. Richard H. Rauschenberger, Dr. Maria S. Sepulveda, Carla Weiser, Janet

Scarborough, Travis Smith, and Jon Wiebe. I would also like to thank Dr. David Barber

for his invaluable assistance in my thesis revision. My research was supported from a

grant to Dr. Timothy S. Gross from the National Institutes of Environmental Health

Sciences.

I would also like to thank my friends, family, and especially my wife Tina who

supported me throughout my career as a graduate student. I would also like to thank my

grandfather (Edwin J. Kent), who exposed and explained to me the irreplaceable value of

our aquatic ecosystems.





















TABLE OF CONTENTS


page

ACKNOWLEDGMENT S ............_...... .............. iii...


LIST OF TABLES ............ ..... .__ ..............vi...


LI ST OF FIGURE S .............. .................... vii


AB STRAC T ................ .............. ix


CHAPTER


1 INTRODUCTION ................. ...............1.......... ......


Background ................... ............ ...............1.......
Selection of Animal Model ................. ...............3................
Fathead Minnow Reproductive Cycle .............. ...............4.....
OCP Characteristics................. .. ...........
OCP Exposure Responses in Fish............... ...............9..
Significance of this Study ................. ...............12................

2 MATERIALS AND METHODS .............. ...............14....


Experimental Animals .............. ...............14....
Chemicals and Dosing ................ ...............14........... ....
Pilot Study .............. .. .. ...............15.
Nanoinj section Experiment ................. ...............16........... ....
Maternal Transfer Experiment ............... ... .. .... ....... ............ ............1
Analysis of Fathead Minnow Tissues and Eggs for OCPs ................ ................ ... 18
Gonad Hi stol ogy ................. ................. 19..............
Statistical Analy sis............... ...............20

3 RE SULT S .............. ...............21....


Maternal Exposure ................. ...............21.................
Nano-Inj section Experiment ................. ...............24................


4 DI SCUS SSION ................. ...............3.. 0......... ....


5 CONCLU SION................ ..............4












LIST OF REFERENCES ................. ...............50................


BIOGRAPHICAL SKETCH .............. ...............56....












































































v

















LIST OF TABLES


Table page

1 Day 30 GC-MS results of flake feed OCP concentrations (ng/g). N.D. is
defined as not detected. ............. ...............28.....

2 Day 30 GC-MS results of female whole body burden OCP concentrations
(ng/g). N.D. is defined as not detected. .............. ...............28....

3 Day 30 GC-MS results of Egg OCP concentrations (ng/g). N.D. is defined
as not detected. ............. ...............28.....

4 Day 30 GC-MS results of OCP concentrations in eggs (ng/g) taken during
Days 1-4 (early clutches), 13-16 (mid clutches) and 27-30 (late clutches). N.D. is
defined as not detected. ............. ...............29.....

5 Day 30 GC-MS results of OCP concentrations in male and female gonads
(ng/g). N.D. is defined as not detected. .............. ...............29....

















LIST OF FIGURES


Figure pg

1 The percent of spawning pairs among treatment groups. Control, low,
middle, and high treatment groups had 10 potential spawning fathead minnow
pairs. Mean a standard error results are shown. Significant differences between
control middle and high treatment groups using One-way ANOVA (P<.05).
Asterisks indicate differences in relation to controls. ............. .....................4

2 The mean number of eggs laid per spawn among treatment groups. Control,
low, middle, and high treatment groups had 10 potential spawning fathead
minnow pairs. Mean a standard error results are shown. Significant differences
between low and control treatment groups, low and middle treatment groups
using One-Way ANOVA (P<.05). Asterisks indicate differences in relation to
control s. .............. ...............42....

3 The mean number of eggs hatched among treatment groups. Control, low,
middle, and high treatment groups had 10 potential spawning fathead minnow
pairs. Mean a standard error results are shown. Significant differences between
low and control treatment groups, low and high treatment groups, and low and
middle treatment groups using One-Way ANOVA (P<.05). Asterisks indicate
differences in relation to controls. .............. ...............43....

4 The percent of clutch viability among treatment groups. Control, low,
middle, and high treatment groups had 10 potential spawning fathead minnow
pairs. Mean a standard error results are shown. Clutch viability = No. of eggs
yielding a live hatchling / Fecundity x 100. Significant differences between low
and middle treatment groups, low and high treatment groups, control and middle
treatment groups, control and high treatment groups, and middle and low
treatment groups using One-Way ANOVA (P<.05). Asterisks indicate
differences in relation to controls. .............. ...............43....

5 The percent of larvae survived to Day 7 among treatment groups. Control,
low, middle, and high treatment groups had 10 potential spawning fathead
minnow pairs. Mean a standard error results are shown. Significant differences
between control and high treatment groups and control and middle treatment
groups using One-Way ANOVA (P<.05). Asterisks indicate differences in
relation to controls............... ...............44

6 The percent of larvae survived to Day 14 among treatment groups. Control,
low, middle, and high treatment groups had 10 potential spawning fathead










minnow pairs. Mean a standard error results are shown. Significant difference
between control and middle treatment groups using One-Way ANOVA (P<.05).
Asterisks indicate differences in relation to controls. ............. .....................4

7 Mean GSI among male treatment groups. Control, low, middle, and high
treatment groups had 10 potential spawning fathead minnow pairs. Mean &
standard error results are shown. No significant differences using One-Way
ANOVA (P<.05). ............. ...............45.....

8 Mean GSI among female treatment groups. Control, low, middle, and high
treatment groups had 10 potential spawning fathead minnow pairs. Mean &
standard error results are shown. No significant differences using One-Way
ANOVA (P<.05). ............. ...............45.....

9 Percent of Females Spawning vs. Number of Spawns. Control, low, middle,
and high treatment groups had 10 potential spawning fathead minnow pairs.
Mean a standard error results are shown. Note: Decreased number of spawning
females for treated versus controls and altered patterns across treatments. .............46

10 Mean Number of Eggs Laid vs. Number of Times Spawned. Control, low,
middle, and high treatment groups had 10 potential spawning fathead minnow
pairs. Mean a standard error results are shown. Note: Lower number of eggs
produced for treated versus controls and altered patterns across treatments. ..........46

11 Percent of Clutch Viability vs. Number of Times Spawned. Control, low,
middle, and high treatment groups had 10 potential spawning fathead minnow
pairs. Mean a standard error results are shown. Note: Lower clutch viability
produced for treated versus controls and altered patterns across treatments. ..........47
















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

ORGANOCHLORINE PESTICIDE REPRODUCTIVE EFFECTS INT FATHEAD
MINTNOWS (Pinsephales pronzela;s): COMPARISON OF EMBRYO AND
MATERNAL EXPOSURE

By

Dane Holland Huge

May 2006

Chair: Timothy S. Gross
Major Department: Veterinary Medicine

Previous studies from this laboratory have documented decreased reproductive

efficiency and altered hormone concentrations in wild populations of American alligators

(Alligator naississippiensis) and largemouth bass (M~icropterus salmoides) from sites

contaminated with organochlorine pesticides (OCPs). These bioaccumulate significantly

in fish. Our purpose was to determine whether adverse effects to reproductive fitness are

caused by OCP-induced effects on maternal reproductive physiology.

We investigated maternal transfer of the OCPs dichlorodiphenyldichloroethylene

(DDE), dieldrin, chlordane, and toxaphene from adult fathead minnows (Pinsephales

pronzela~s) to egg and larvae. Adult female fathead minnows were dosed at rates of high,

middle, and low. The high dose was a complex mixture comparable to concentrations

found in blue tilapia (Oreochronsis aurea) from a historically contaminated lake in

central Florida. The middle dose was 50% of the high dose, and the low dose was 25%

of the high dose. We recorded the frequency of spawns, the number of eggs laid per










spawn, the number of eggs hatched, and the number of larvae surviving to Day 14. At the

end of the dosing period, we made histology slides of male and female gonads to

determine stage of sexual maturation. We calculated GSI on male and female gonads. We

used GC-MS analysis to analyze concentration levels of OCPs in fathead minnow eggs,

male and female gonads, and whole body burdens. Statistical analysis was completed by

using a One-way ANOVA to compare treatment groups.

Significant differences among control and treatment groups were detected when

examining egg counts, the number of eggs hatched, clutch viability percentage,

survivorship percentage to Day 7, and survivorship percentage to Day 14. No significant

differences were detected between treatment groups when examining GSI. In conclusion,

laboratory populations of fathead minnows maternally exposed to OCPs spawn less

frequently, have a decreased hatch rate, and have decreased survivorship.















CHAPTER 1
INTTRODUCTION

Background

The group of chemicals known as organochlorine pesticides (OCPs) consists of

structurally diverse compounds used to control pests that have potential to damage

agricultural crops, and to serve as vectors for human diseases. Because of the chemical

stability, lipophilicity, and low water solubility of OCPs, these compounds and their

metabolites persist in the environment. The OCPs can bioaccumulate in vertebrate

species, causing negative effects in developmental and reproductive potential. OCPs

alter enzyme activities (such as Ca2 -ATPase and phosphokinase), and alter

electrophysical properties (such as K Na' ion exchange of nerve cell membranes), thus

affecting neural transmission. Because of their environmental persistence and their

ability to biomagnify in food webs, the U.S. Environmental Protection Agency (USEPA)

began to restrict (or in some cases, ban) the use of OCPs on agricultural lands between

1978 and 1983.

Growing concern has arisen that many environmental pollutants have the capability

to interfere with normal function of human and animal endocrine systems. This type of

pollutant is known as an endocrine disruptor. The endocrine system is composed of

numerous types of tissues and can briefly be described as any tissues or cells that release

chemical messengers or hormones that signal or trigger a physiological response from a

target tissue (Thomas and Thomas, 2001). The endocrine system is integrated with many

other biological functions and is widely dispersed throughout entire organisms. Because









of this, a wide range of toxicological responses may develop after exposure to endocrine

disrupting compounds (EDCs) (Newman and Unger, 2003). Responses may include

sexual developments, sexual differentiation, and success rates of reproduction.

Reproductive processes in fish and other species of wildlife are largely controlled by

complex hormonal pathways, thus giving endocrine disruptors the potential to target

reproductive organs.

The USEPA developed a tiered testing paradigm, and paralleled assays to identify

potential EDCs such as OCPs (USEPA, 1998). Some OCPs alter endocrine pathways

controlled by thyroid hormones, estrogens, and androgens. Recommendations for the

initial (Tier 1) screening assays include 3 assays using male or female rats at different life

stages, using the amphibian (Xenopus laevis) as metamorphosis test, and a short-term

reproduction test with the fathead minnow (Pimephales promela;s) (USEPA, 1994). In

general, Tier 1 screening assays include exposing fathead minnows to the chemical of

concern for up to 21 days. Post exposure, the survival, behavior, fecundity, and

secondary sexual characteristics are assessed. Fertility and early development of the Fl

generation may also be evaluated. At the end of the test, plasma concentrations of sex

steroids (P-estradiol, testosterone, 11-ketotestosterone [11-KT]) and vitellogenin (Vtg)

are measured. Gonadal status is also assessed using a gonadosomatic index (GSI) and

hi stop athol ogy .

Although a general reduction in use of OCPs has been observed, several field

studies suggest that OCPs adversely affect endocrine function in fish. Thus indicating

aquatic and semi-aquatic organisms are continuously being exposed to levels of toxicants

capable of altering reproductive parameters. Marburger et al. (2002) tested OCP levels in









soil from the Emeralda Marsh Conservation Area (on the north-east shore of Lake

Griffin, Florida, United States), a historically contaminated lake. The lake contained

concentrations of p,p '-DDE, dieldrin, and toxaphene over 3,000, 500, and 40,000 ng/g,

respectively. The same study showed concentrations of OCPs in largemouth bass

(M~icropterus salmoides) ovaries and fat reached levels of 4,000 and 17,000 ng/g

respectively, for total DDT derivatives, over 100 and 700 ng/g for dieldrin, and over

4,000 and 20,000 ng/g for toxaphene.

Selection of Animal Model

Numerous reasons exist for selecting the fathead minnow as a model species for

EDC screening (Ankley, 2000). The fathead minnow is a member of the Cyprinidae

family. Cyprinidae represent the largest Hish family in the world. Over 2,000 species of

Cyprinids or true minnows make up 25% of all freshwater fish and 9% of all Eish species

(Nelson, 1984). The Cyprinids are distributed in the fresh waters of North America,

Europe, Asia, Africa, and Australia. They exist in virtually every freshwater habitat

including swamps, sloughs, springs, ponds, lakes, large rivers, and tiny creeks. Although

considered a freshwater species, some Cyprinids have been known to frequent tidal fresh

and brackish water (Jenkins and Burkhead, 1994). North America contains approximately

295 described species of Cyprinids, 9 being exotic (Miller, 1996; Robins et al., 1991).

Although most minnows are commonly considered small, the Cyprinidae family contains

the smallest American minnow, the Mexican shiner (Notropis saladnis), reaching 150

mm total length (TL), as well as the endangered Colorado squawfish (Ptychocheihts

htcius) which is known to reach a TL of 1800 mm and weigh as much as 45 kg (Smith,

1945).










Specifically, the fathead minnow represents an excellent model for the recognition

of contaminant accumulation for numerous reasons. Adult fathead minnows are

omnivores. Different populations have shown that one ate primarily insects, another only

algae, another entirely detritus (Cahn 1927; Coyle, 1930 in Scott and Crossman, 1973;

Starrett 1950) and yet another consumed microcrustaceans, insects, algae, and detritus

(Pearse, 1918). Because of the fathead minnow' s range in diet, they possess a

moderately coiled gut which is an intermediate between the long and coiled gut of

detritivores and herbivores and the S-shaped gut found primarily in carnivores. Their

wide variety in diet insures they have the potential to eat, digest, and possibly

bioaccumulate environmental contaminants located in various levels of the food web.

Approximately 1 1,080 developmental and survival tests in support of regulatory

programs in North America and Europe (USEPA 1982, 1989, 1991, and 1994) have been

established, giving an extensive background in sub-lethal and lethal effects on fathead

minnows caused by agents and metabolites in numerous classes of environmental

toxicants.

Fathead Minnow Reproductive Cycle

Because the fathead minnow is widely distributed across the United States and

North America, specific times of maturation and spawning temperatures are difficult to

pinpoint. The fathead minnow is native to the central portion of North America (Scott

and Crossman, 1973). However, numerous populations exist in California, Arizona,

Texas, and into the New England states; all of which possess different maturation times

and spawning temperatures.

Generally, spawning occurs in water temperatures of 15 to 32 oC (Markus, 1934).

Because the population distribution of the fathead minnow ranges from southern Texas to









northern Minnesota, actual months of the year that the fathead minnow spawns will vary

tremendously. When photoperiod and temperature reach acceptable limits, males

develop dark vertical bands, turn a rusty brown, and develop white breeding tubercles

that are prominent on the tip of the snout and top of the head. Once a dominant male is

selected, he establishes a territory which he defends against other subordinate males in

the local vicinity. He will then clean the nesting site which may be the underside of a flat

rock or submerged vegetation, stones, logs or other acceptable substrates. Spawning

typically takes place between dawn and 10:00am. After engaging in courtship, the

female will deposit eggs onto the prepared surface of the selected substrate. Gale and

Gerard (1982) showed in a study that female fathead minnows laid between 9 and 1,136

eggs per session (clutch). They also showed that between May 22 and August 22 five

pairs of fish produced 16 to 26 clutches of eggs. Post fertilization, the male will assume

responsibility of nest guarding. Again, depending on water temperature, the eggs will

hatch into larvae in approximately six days and remain in the nesting area for several

more days until their yolk material is absorbed.

Along with environmental cues such as photoperiod and temperature, teleost fish

reproductive cycles are also regulated by endogenous hormonal cues (Gross et al., 2002).

A combination of these factors stimulate the hypothalamus of the fish to release

gonadotropin-releasing hormone (GnRH), norepinephrine (NE), as well as other

neuropeptides to stimulate the pituitary gland which releases the primary teleost

gonadotropins GTH-I and GTH-II (Van Der Kraak et al., 1998). These two

gonadotropins are similar to the mammalian lutenizing hormone (LH) and follicle

stimulating hormone (FSH) (Redding and Patino, 1993). GTH-I is the gonadotropin









associated with stimulation of events leading to vitellogenesis and spermiogenesis as well

as early gonadal development. GTH-II is involved in the stimulation of events that will

eventually lead to the final oocyte maturation and ovulation in females, as well as

spermiation in males. The primary sex steroids involved in regulation of gametogenesis

in the maj ority of male and female teleost fish are 1 1-KT and 17P-Estradiol. An increase

in plasma concentrations of these hormones has been shown to be associated with the

onset of seasonal reproductivity (Gross et al., 2002).

In females, an increase in estrogen (E2) lCVOIS within the blood stimulates the liver

to produce vitellogenin, a phosphoglycolipoprotein that serves as a precursor to yolk

production in oviparous vertebrates (Wahli et al., 1981). Vitellogenin is then released

into circulation where it travels to the gonad and is used as a nutrient source for

developing oocytes. Within the promoter region of the vitellogenin gene lies an estrogen

responsive element which is transcribed in response to an estrogen receptor (ER)

complex (Wahli et al., 1981). The surface of the oocyte contains vitellogenin receptors,

which once these receptors are occupied, the oocyte is cleaved into smaller yolk proteins.

The yolk proteins are embodied into yolk granules which will in turn constitute maj ority

of the mature oocytes. The yolk granules are stored and will serve as the nutrient source

for developing embryos (Wahli et al., 1981). Once oocytes have reached their properly

developed size, vitellogenesis stops, and the oocytes complete maturation. During this

maturation phase, follicles increase in size due to hydration, as well as collect additional

vital proteins. At the time of germinal vesicle breakdown, protein uptake ceases. Due to

hydration, the follicle volume continues to increase. It is at this time the cellular envelope

that surrounds an egg in preparation for ovulation known as the chorion, begins to









develop. The time of ovulation is species specific and will take place when follicles

reach a specific size (Wallace and Selman, 1981).

Although male and female fish contain the vitellogenin gene, concentrations of E2

normally only found in females are needed to produce measurable levels of vitellogenin

(Wallace, 1970; Ryffel, 1978; Hori et al., 1979). Presence of vitellogenin in males can

then be used as an indicator of estrogenic compound exposure (Pelissero et al., 1993;

Sumpter and Jobling, 1995; Shelby et al., 1996; Okoumassoun et al., 2002).

OCP Characteristics

OCPs are synthetic compounds that contain certain amounts of chlorine substituted

for hydrogen on a hydrocarbon backbone, also known as chlorinated hydrocarbons.

However, also included in this group are a few compounds such as dieldrin and

methoxychlor that have oxygen incorporated in their structure. Most OCPs do not

resemble naturally occurring organic compounds in their structure, hence, they degrade

slowly within their environment (Newman and Unger, 2003). Because OCPs have high

molecular weights and are non-polar, they have low water solubilities and are soluble in

lipids that are found within living organisms. The lipophilicity of OCPs causes

bioaccumulation and possibly biomagnification.

One of the first widely used OCPs was dichloro-diphenyl-trichloroethane (DDT).

DDT became a popular pesticide for numerous reasons. DDT has the capability to

control or kill a wide range of insect pests, it is persistent in the environment, and it has

low mammalian toxicity. DDT was used during World War II to control fleas, lice, flies,

and mosquitoes which are vectors for malaria and typhus transfer to servicemen and

civilian populations. Based on the success of DDT during World War II, DDT became

the pesticide of choice for agricultural and commercial use. Gildersleeve et al. (1985)









and Bryan et al. (1989) were able to demonstrate through laboratory studies that in ovo

treatment with DDT to Japanese quail resulted in female progeny that lay eggs without

shells on a regular basis, as well as, have reproductive tract malformations. These, along

with numerous other studies on exposure of DDT to wildlife led to the banning of DDT

by the Environmental Protection Agency (EPA) in the United States at the end of 1972.

DDT undergoes degradation in the environment to the metabolites DDD and DDE. Both

of these metabolites have a half life on the order of years. Most samples taken in the

environment today contain predominately the DDE and DDD isomers.

Another class of insecticides commonly found in the environment that act similar

to DDT are the Cyclodienes. Included in the Cyclodiene class are toxaphene, dieldrin,

and chlordane. One complex mixture of polychlorobornanes and camphenes is known as

toxaphene. The chlorination of technical camphene or a-pinase can consist of over 300

congeners (Di Giulio and Tillitt, 1997). Between 1972-1975, toxaphene was widely used

in the United States on cotton crops. Toxaphene was banned in the United States in

1982, however, each structurally different compound in the mixture will have a specific

set of chemical properties, and thus predicting its environmental fate proves difficult.

Dieldrin is a common name of an insecticide that was routinely used in the 1950's

to the early 1970's. It was used in agriculture for soil and seed treatment and in public

health to control disease vectors such as mosquitoes and tsetse flies. It was also used as a

sheep dip and has been used in the treatment of wood and woolen products. Most uses of

dieldrin were banned in 1975 and currently it is not produced or imported by the United

States. Studies have shown that oral uptake, as well as, exposure through fish gills can









adversely affect fish nitrogen content in the liver and reduce swimming speed at which

fish can maintain themselves (Mayer, 1971).

Chlordane is another insecticide within the Cyclodiene class that was used in the

United States between 1948 and 1988. Chlordane is not a single chemical, but is a

mixture of many related chemicals of which approximately 10 are maj or components

including trans-chlordane, cis-chlordane, heptachlor, and trans-nonachlor. Prior to 1978,

chlordane was used as a pesticide on agricultural crops. From 1983-1988, chlordane was

only approved to be used as a pesticide to control termites in homes. Because of

concerns over cancer risk and adverse affects in wildlife, the EPA cancelled the use of

chlordane on food crops and phased out other above-ground uses.

OCP Exposure Responses in Fish

Numerous studies have linked disturbances in reproductive function to exposure of

different classes of toxic chemicals (Jobling et al., 1998). However, when looking

specifically at OCPs, much research has been concentrated on endocrine disruption

(Rauschenberger et al., 2004; Ree and Payne, 1997; Russell et al. 1999; Gleason and

Nacci, 2001). It is thought that one area of reproductive success that is affected by OCP

exposure is circulating sex steroids. Studies have shown that abnormalities exist in

concentration levels of circulating sex steroids in fish exposed to OCPs. Johnson (2005)

found that Florida largemouth bass (M~icropterus salmoides florid anus),dd~~~dd~~ exposed to p,p '-

DDE and dieldrin for a 120-day period, led to depressed concentrations of plasma E2 foT

female largemouth bass, increases in 11-KT in female largemouth bass and a lack of

consistent increases and/or depressions of male largemouth bass E2 and 1 1-KT

concentrations. Female largemouth bass in another study (Muller, 2003) also showed

higher circulating E2 COncentrations when exposed to 2.5 mg of DDE compared to









control groups. Male largemouth bass when exposed to 5.0 mg of DDE also consistently

showed higher circulating levels of E2. Not only have laboratory studies shown altered

sex steroid hormones, but also fish captured from wild populations. Largemouth bass

collected from the Emeralda Marsh Conservation Area (EMCA) located on the north-east

shore of Lake Griffin, Florida, USA, demonstrated depressed levels of sex steroid

hormones 17P-estradiol and 1 1-KT concentrations that are possibly a result of a

disruption in the hypothalamus-pituitary-gonad axis which is responsible for stimulating

synthesis and secretion of sex steroid hormones (Gross et al., 2003).

Another example of altered circulating sex steroids was demonstrated by (Kelce et

al., 1995). They demonstrated that p,p '-DDE acts as a potent anti-androgen in rats by

binding to the androgen receptor (AR), preventing testosterone synthesis, and causing

demasculinization. This phenomenon has also been demonstrated in several fish species

including white sturgeon (Acipenser transmonrtrtrtrtanus)~r~r~r (Foster et al., 2001) and goldfish

(Cara~ssius auratus) (testes only), (Wells and Van Der Kraak, 2000).

Numerous mechanisms of action exist for EDCs and may interrupt multiple

pathways along the hypothalamic-pituitary-target-tissue axis. These interruptions may

disturb the transport, binding, release, biotransformation, elimination or normal synthesis

of natural hormones. EDCs may alter the hypothalamic-pituitary axis which could have a

cascading affect on the endocrine system downstream of the hypothalamus. EDCs may

interfere with neurotransmitters that control GnRH secretion resulting in decreased levels

of GnRH production, as well as a reduction in gonad size, and may be responsible for the

alterations in the concentrations of circulating sex steroids (Jansen et al., 1993). Specific

endocrine tissues synthesize hormones that are secreted into the bloodstream where they









are bound to proteins and transported to target tissues where they interact with receptors,

bring about responses, or may be metabolized or degraded. EDCs may block or enhance

the function of these hormones by interfering with one or several of these steps. EDCs

may cause synthesis failure of certain hormones or limit the uptake of critical precursors

to produce hormones. Additionally, EDCs may alter the rate at which hormones are

metabolized as is the case with the super family of enzymes, CYP 450 which are critical

in the synthesis and metabolism of steroid hormones. EDCs may also induce hormone-

like effects due to the alternating rates of degradation. EDCs may interfere with

hormones binding to transport proteins thus preventing delivery to target tissues

(Darnerud et al., 1996). Some EDCs have the ability to mimic estrogens or androgens.

These EDCs may bind to globulin proteins, thus displacing and possibly increasing the

elimination of endogenous circulating sex steroids (Rosner, 1990). EDCs may also have

the potential to bind to hormone receptors and either activate, (agonize), (Flouriot et al,

1995) or inhibit (antagonize) (Danzo, 1997) receptor function. Particular research

interest has been focused on EDCs and their ability to bind to estrogen receptors (ER).

Most ER are located in the nucleus of target cells. ER-DNA complexes interact with

chromosomal proteins and transcription factors in order to induce or inhibit transcriptions

of specific genes, thus enabling endocrine specific responses. It is possible for EDCs to

enhance or block the function of a hormone or endocrine target tissue by interfering with

one or several critical steps in the transcription process (Hoffman et al., 2003). Unlike

ER that have a specific E2 TOSponse element, EDCs that have androgenic activities may

exert broader effects than those attributed to a simple androgen mimic. Endocrine-

disrupting effects may also occur due to direct or indirect toxicities on specific target









tissues. Lipophilic EDCs such as OCPs will accumulate primarily in fatty tissues

(Raushenberger, 2004) such as gonads and the liver, potentially interfering with the

mobilization and synthesis of lipids, thus inhibiting specific endocrine related functions

such as vitellogenesis.

Significance of this Study

Numerous oviparous vertebrates become subjects for studies because they are

sensitive to adverse effects of chemical contaminants released into the environment,

highly visible by the public, or obtain interest by organizations because they are viewed

as limited natural resources. However, a species that attracts attention by the public or is

federally listed may not be considered appropriate for laboratory studies. It is crucial

when assessing risks to wildlife populations that not only must one understand

environmental exposures, but also dose-response relationships using measured endpoints.

Endpoints may be measured as lethal (mortality) or as sub-lethal effects such as adverse

changes in reproduction or development.

In many instances use of a surrogate species may be appropriate to assess possible

risks involved in exposure to chemical contaminants. Biological endpoints can be used to

help express potential for chemicals to cause negative ecological effects. Surrogate

species are often used to in laboratory tests to complete acute or chronic toxic effects for

specific chemicals (Zeeman and Gifford, 1997). Most surrogate species are selected

because they are easily cared for and cultured under laboratory settings and provide an

inexpensive and reliable assessment of acute and chronic toxicity of potential chemical

contaminants. Identifying potential ecological and physiological characteristics related to

the susceptibility to effects of contaminants, along with their exposure routes is important

in managing wildlife populations for the optimal use by humans (Rauschenberger, 2004).









The ability to develop the fathead minnow into a biological model would provide wildlife

managers a standard by which to measure ecological impacts brought about by

anthropogenic chemicals dispersed throughout aquatic environments.

The obj ectives of this study were:

1) to determine if OCPs are maternally transferred from the adult female fathead
minnow to her offspring;

2) to determine if maternal OCP exposure adversely affects hatch rate and larval
mortality; and

3) to determine if oral exposure of OCPs cause masculinization in female fathead
minnow gonads and/or feminization in the gonads of male fathead minnows.

Previous studies have shown that OCPs may act as endocrine system modulators

affecting reproductive success in numerous animal species (Gallager et al., 2001; Gross

et al., 1994; Mills et al., 2001; Muller et al., 2004). We hypothesized that oral exposure

to fathead minnows by a mixture of p,p '-DDE, dieldrin, toxaphene, and chlordane at

increasing concentrations would bioaccumulate in muscle tissue and in the gonads of

both male and female fathead minnows due in part to the highly lipophilic nature of these

compounds, and thus be maternally transferred to fathead minnow eggs. Exposure to the

OCP mixture would also demonstrate an enzyme inducing and/or estrogenic properties

that would directly or indirectly increase larval mortality, adversely affect clutch size and

frequency of spawns per fathead minnow pair, and would cause feminization or

masculinization of fathead minnow gonads.















CHAPTER 2
MATERIALS AND METHODS

Experimental Animals

Male and female fathead minnows of 5 to 7 months of age were obtained from

Aquatic Bio Systems Inc. in March 2005. The fish were transported to the USGS-FISC

laboratory where they were maintained in 10-gallon aquariums with a flow-through water

system supplied by on-sight well water and aeration. Upon arrival, all fish looked

healthy, disease free, and weighed between 2.20 and 4. 13 grams (g). Fish were fed a diet

of Ziegler "Prime Tropical 45-9 Flake Feed" ad libitum once per day. The fish were

allowed two weeks to acclimate to their new environment prior to dosing.

Water quality parameters measured included temperature, pH, and dissolved

oxygen. Water quality parameters remained within acceptable ranges for the duration of

the experiment. Temperature ranges were from 21.30 to 22.30 oC. Dissolved oxygen

ranges were from 5.30mg/L to 6.35mg/L. pH levels were from 7.90 to 8.70.

Chemicals and Dosing

The organochlorine pesticide 1,2,3,4,10,10O-hexachloro-6,7-epoxy-

1 ,4,4a,5,6,7,8,8a-octahydro-1 ,4,5,8-dimethanonaphthalene (dieldrin, Lot#77H3 578) was

obtained from Sigma (St. Louis, MO). The organochlorine pesticide 2,2-bis(4-

chlorophenyl)-1,1 -dichloroethylene (p,p '-DDE, Lot#0902KU) was obtained from Aldrich

Chemical Co. (Milwaukee, WI). The organochlorines chlordane (Lot#303-16B) and

toxaphene (Lot#302-125B) were purchased from Chem Service (West Chester, PA). The

chemicals were added to 1690.0 ml of Yelkin oil provided by Ziegler Bros., Inc. at the









following concentrations: dieldrin 5.0 mg, p,p '-DDE 12.0 mg, chlordane 10.0 mg,

toxaphene 95.0mg. The contaminated Yelkin oil was then mixed with 25.0 pounds (lbs.)

of "Prime Tropical Flake Feed" by Ziegler Bros., Inc.

Pilot Study

Prior to the conduction of the experiment, a pilot study was undertaken to establish

body burdens and possible lethal doses of the experimental fish, as well as contaminant

concentration amounts present in the treated and control feeds. The control and treated

feed was analyzed by the Center for Environmental and Human Toxicology, University

of Florida, Gainesville, Florida. Screen assays were performed to establish limits of

detection, as well as limits of quantification (Table 1).

When determining the amount of contaminants to add to the flake feed, an attempt

was made to achieve target parts per million (ppm) values comparable to what is seen in

blue tilapia (Oreochromis aureus) stocked at the north shore restoration area in Lake

Apopka, Florida, a historically hypereutrophic and contaminated lake (Pollman et al.,

1988). A biomagnification factor of 15 was used based on previous studies performed at

the USGS-FISC, Gainesville, Florida, laboratory.

Post feed analysis, the control and treated feed was mixed to establish a high,

middle, and low dose. One hundred twenty grams of control feed was added to a 64.0

ounce (oz.) clean plastic container. One hundred twenty grams of the high dose feed was

also added to a 64.0 oz. clean plastic container. The medium dose was established by

mixing 60.0 g of control feed with 60.0 g of treated feed in a 64oz. clean plastic

container. The low dose was established by mixing 90.0 g of control feed with 30.0 g of

treated feed in a 64.0 oz. clean plastic container. The middle and low doses were then

shook to ensure thorough mixing and stored in a refrigerator at 5 oC.









Five female fish of each treatment group and the control were placed in separate

10-gallon aquariums. Fish were then fed approximately 2.5% of their body weight once

per day for 30 consecutive days. At the end of 30 days, all Eish were separately wrapped

in foil, frozen, and delivered to the Center for Environmental and Human Toxicology,

University of Florida, for chemical analysis. During the analysis, 2 Eish from each

treatment group were individually assayed, and the remaining 3 were analyzed via a

composite assay with the exception of the low dose treatment group and control group

where each sustained one mortality. In this case, 2 individual fish from the low dose

treatment group and control group were analyzed and the remaining 2 Eish from each

group were analyzed via a composite assay. The results of the individual and composite

assays are located in Table 2.

Nanoinjection Experiment

A nanoinj section experiment was performed that involved four replicate trials. Each

trial contained Hyve treatments. Eggs were injected at a low, middle, and high dose rate at

5, 10, and 20 ppm of p,p '-DDE at a quantity of 83.5-116.9 micrograms. A control that

was not inj ected, as well as a vehicle control that was inj ected with the same quantity of

triolein was used. Eggs were inj ected with a Kanetec MB-B manipulator and pre-made

needles comprised of aluminosilicate 0.68-mm capillary tubes. Eggs were held in place

for injection by agar placed on microscope slides acting as a substrate. Once the eggs

were injected, they were placed in a flow-through incubator for hatching. The number of

hatched larvae, as well as the number of surviving larvae were monitored to Day 11.

Maternal Transfer Experiment

Ten female fathead minnows per treatment group and control were randomly

selected and placed in separate 10-gallon aquariums supplied by flow-through, on-sight









well water and aeration. Water quality parameters were measured, and remained within

acceptable levels throughout the experiment. Photoperiod was gradually increased from

an 8 light/16 dark hour day to a 12 light/12 dark hour day. Temperature was gradually

increased from 22 oC (A 1 oC) to 25 C (A 1 oC). The fish were dosed with the same feed

mixture as in the pilot study. Based upon the amount of food consumed by the fish

during the pilot study, a reduction was made in the amount of food provided to the fish to

approximately 2% of the fish' s body weight. The fish were then dosed for a consecutive

30 days.

At the end of the dosing period, male fish were separated from each other, and

randomly placed in 10-gallon aquariums. Female fish were then randomly paired with

the male fish. Each treatment and control groups contained 10 pairs of male and female

Hish. Water quality was monitored throughout the experiment and aquariums were

siphoned as needed. Control Hish were fed .181 .02 g per pair of Ziegler "Prime Tropical

45-9 Flake Feed" once per day; while the 3 treatment groups remained on their diet of the

previously mixed treated feed at .18+ .02 g per pair once per day.

Spawning substrates consisted of PVC pipe with a 4-inch diameter cut in half to 4

inches long. The substrates were inspected for the presence of eggs daily at

approximately 10:30am. Substrates that contained eggs were removed, and the eggs

counted under a bench magnifying glass. The substrates with eggs were then placed into

a 64 oz. plastic container with 1000 micron mesh attached to the sides in order to allow

water flow-through. The container was then placed into a flow-through water bath at

22.211 oC with a constant drip consisting of aerated well water. A 2-inch airstone










supplied heavy aeration into the container to prevent fungus from attaching to the eggs.

Egg substrates were replaced to aquaria.

Larvae began hatching 7 to 11 days after spawning. Hatched larvae were

transferred to a clean 64 oz. container and individually counted. This process was

repeated on Days 7 and 14 after hatch. A composite assay containing 0.2 g to 0.4 g of

eggs with all four treatment groups was performed to confirm that contaminants were

being maternally transferred. This assay was performed by the Center for Environmental

and Human Toxicology, University of Florida, Gainesville, Florida. The trial was

repeated dosing 40 female fathead minnows 7-9 months of age for 30 consecutive days.

At the end of the 30-day dosing period, female fathead minnows were paired with male

fathead minnows 7 to 9 months of age into individual 10-gallon aquariums with the same

flow-through water system. Each pair of fish was fed 0.4 g of control and treated feed

once daily for an additional 30 consecutive days. A spawning substrate constructed of a

4-inch long, 4-inch diameter PVC pipe cut in half was placed in the aquarium. Spawning

substrates were checked daily for the presence of eggs. Any eggs present were

individually counted and recorded. A composite sample containing 0.2 g to 0.4 g of the

control and 3 treatment group eggs were collected at the beginning, middle, and end of

the 30-day trial. The Center for Environmental and Human Toxicology, University of

Florida, Gainesville, Florida, performed an OCP screen on the eggs to affirm the presence

of contaminants.

Analysis of Fathead Minnow Tissues and Eggs for OCPs

Analysis was conducted by the Center for Environmental and Human Toxicology,

University of Florida. Briefly, the OCPs in a weighed, homogenized portion of sample

2.0 g were extracted into ethyl acetate. Sample clean-up included use of C18 and NH2









solid-phase extraction cartridges prior to analysis by GC-MS. Each analyte was

quantified against a standard curve having at least five points and a correlation coefficient

>0.995. For the flake feed analysis, the Est LOD-LOQ range was 0.3-7.5 (ng/g) for

chlordane, 0.1-7.5 (ng/g) for DDE metabolitess), 0.8-1.5 (ng/g) for dieldrin, and 63-1500

(ng/g) for toxaphene. Fathead minnow body burdens had an Est LOD-LOQ range of 0.3-

1.5 (ng/g) for chlordane, 0.1-1.5 (ng/g) for DDE metabolitess), 0.5-1.5 (ng/g) for dieldrin,

and 41-1500 (ng/g) for toxaphene. The fathead minnow gonads had an Est LOD-LOQ

range of 1.2-7.5 (ng/g) for chlordane, 0. 1-7.5 (ng/g) for DDE metabolitess), 0.4-1.5 (ng/g)

for dieldrin, and 42-1500 (ng/g) for toxaphene. Fathead minnow eggs had an Est LOD-

LOQ range of 0.2-1.5 (ng/g) for chlordane, 0. 1-1.5 (ng/g) for DDE metabolitess), 0.8-1.5

(ng/g) for dieldrin, and (ng/g) 42-1500 for toxaphene.

Gonad Histology

Tissue staining with hematoxylin and eosin (H&E), sectioning and slide mounting

was performed by Histology Tech Services (Gainesville, Florida). Ovaries were

observed under a light microscope at 40X and stage of sexual maturation was assigned

(Sepulveda, 2000).

Briefly, stage 1 ovaries are undeveloped with mostly primary phase follicles. Stage

2 ovaries are pre-vitellogenic with primary and secondary phase follicles, but have no

vitellogenic follicles. Stage 3 ovaries are early vitellogenic with some vitelline granules

in follicles of varying size and no fully developed eggs. Stage 4 ovaries are late

vitellogenic with a maj ority of follicles containing numerous vitelline granules and fully

developed eggs are present (Muller, 2003).

Stage 1 testes are non-spermatogenic, with an extremely thin germinal epithelium

and no sperm present. Stage 2 testes show low spermatogenic activity, with a thin










epithelium that contains scattered proliferation and maturation of spermatozoa. Stage 3

testes show moderate spermatogenic activity, thick germinal epithelium and diffuse to

moderate proliferation and maturation of sperm. Stage 4 testes show thick germinal

epithelium, high proliferation and maturation of sperm.

Statistical Analysis

All statistical analyses were performed using Statistical Analysis system (SAS)

Enterprise Reporter software version 2.6. The experimental design required statistical

analysis comparing each treatment group by a One-Way ANOVA. ANOVAs were

performed and significance was set at alpha = 0.05. Results are represented as means +

standard error.

A Tukeys Multiple Comparison test (P<0.05) was used to compare treatment

groups and the stage of the gonad in both male and female fathead minnows.















CHAPTER 3
RESULTS

Maternal Exposure

Maternal exposure to contaminant mixture led to a decrease in the percent of

spawning pairs in all three treatment groups when compared to the control. The percent

of females spawning per treatment group resulted in the following: Control group 80%,

Low treatment group 60%, Middle treatment group 40%, and High treatment group 50%.

Significant differences were observed between the control and the middle treatment

group and the control and the high treatment group when using a One-way ANOVA

(P<.05). The total number of potential spawning pairs for the control and treatment

groups were 10 pairs over a 30-day period.

Exposure to the contaminant mixture led to an increase in the mean number of eggs

laid per spawn in the low treatment group 350 vs. 220, and in the high treatment group

270 vs. 220, but a decrease in the number of eggs laid per spawn in the middle 190 vs

220. The mean and standard error of eggs laid for each treatment group is represented in

Figure 2. The mean number of eggs laid for the control group & standard error was

220.04 & 32.36. The mean number of eggs laid for the low treatment group was 351.67 &

41.16. The mean number of eggs laid for the middle treatment group was 188.73 &

34.41. The mean number of eggs laid for the high treatment group was 271.68 & 29.41.

The controls laid the most total number of eggs at 5501, the low treatment group laid the

second highest amount of eggs at 5275, the high treatment group laid the third most










number of eggs at 4890, and the middle treatment group laid the fewest number of total

eggs at 2831.

Exposure to the contaminant mixture led to an increase in the mean number of eggs

hatched in the low treatment group, but a decrease in the mean number of eggs hatched in

the middle and high treatment groups when compared to the control. This is the same

phenomenon witnessed in the mean number of eggs laid. The mean and standard error

number of hatched eggs for each treatment group is represented in Figure 3. The mean

number of hatched eggs for the control group and a standard error was 144.64 & 25.77.

The mean number of eggs hatched for the low treatment group was 265.60 & 31.98. The

mean number of eggs hatched for the middle treatment group was 69.67 & 21.66. The

mean number of eggs hatched for the high treatment group was 82.95 & 28.06.

Significant differences were detected among the low vs. mid treatment groups, low vs.

control treatment groups, and low vs. high treatment groups.

Exposure to the contaminant mixture also led to an increase in clutch viability for

the low treatment group and a decrease in clutch viability for the middle and high

treatment groups when compared to the control. This is the same phenomenon in

witnessed in the mean number of eggs laid and hatched. The mean and standard error of

clutch viability percentage is represented in Figure 3. The mean number of clutch

viability percentage for the control group was 64.98 & 6. 19. The mean number of clutch

viability percentage for the low treatment group was 76.39 & 3.72. The mean number of

clutch viability percentage for the middle treatment group was 30.64 & 9.71. The mean

number of clutch viability percentage for the high treatment group was 28.57 & 8.35.

Significant differences were detected among the low vs. middle treatment groups, low vs.










high treatment groups, control vs. middle treatment groups, and control vs. high treatment

groups.

Exposure to the contaminant mixture led to a decrease in the percent of larvae

surviving to Day 7 in all three treatment groups when compared to the control. The mean

and standard error of surviving larvae to Day 7 percentage is represented in Figure 4.

The mean and standard error of surviving larvae to Day 7 percentage in the control

treatment group was 43.59 & 6.98. The mean number of surviving larvae to Day 7

percentage in the low treatment group was 31.35 & 5.98. The mean number of surviving

larvae to Day 7 percentage in the middle treatment group was 8.35 & 4.91. The mean

number of surviving larvae to Day 7 percentage in the high treatment group was 15.71 &

7.09. Significant differences were detected in the control vs. middle treatment groups

and the control vs. high treatment groups.

Exposure to the contaminant mixture led to a decrease in the percent of larvae

surviving to Day 14 in all three treatment groups when compared to the control. The

mean and standard error of surviving larvae to Day 14 percentage is represented in Figure

5. The mean and standard error of surviving larvae to Day 14 percentage in the control

treatment group was 32.52 & 8.14. The mean number of surviving larvae to Day 14

percentage in the low treatment group was 39.67 & 9.62. The mean number of surviving

larvae to Day 14 percentage in the middle treatment group was 7.60 & 4.57. The mean

number of surviving larvae to Day 14 percentage in the high treatment group was 7.06 &

4.03. Significant differences were detected between the control vs. middle treatment

groups.










With the exception of the high treatment group in regards to female GSI, the mean

GSI for the low and middle remained approximately the same when compared to the

control, while the high treatment group increased. The mean and standard error for GSI

in the female control groups was 10.44 & 5.57. The mean and standard error for GSI in

the female low treatment group was 9.44 & 1.79. The mean and standard error for GSI in

the female middle treatment group was 9.50 & 1.99. The mean and standard for GSI in

the female high treatment group was 14. 12 & 1.63. Mean GSI for male and female

fathead minnows showed no effect.

With the exception of the middle treatment group in regards to male GSI, the mean

GSI for the low and high groups remained approximately the same when compared to the

control, while the middle treatment group decreased. The mean and standard error for

GSI in the male control treatment group was 1.36 & 0.79. The mean and standard error

for GSI in the male low treatment group was 1.33 & 0.15. The mean and standard error

for GSI in the male middle treatment group was 0.66 & 0.13. The mean and standard

error for GSI in the male high treatment group was 1.36 & 0.23. No significant

differences were observed among treatment groups or sex in regards to GSI.

Nanoinjection Experiment

Nanoinj section trials indicated that fathead minnow eggs inj ected with p,p '-DDE

were adversely affected by the chemical. Trials 1, 2, and 3 showed eggs injected with 20,

10, and 5 ppm of p,p '-DDE had lower hatch and survivorship when compared to control

eggs. Trial 4 showed hatch and survivorship to be higher in the p,p '-DDE inj ected eggs

than the controls. Overall, hatch and survivorship rates during this trial were lower than

previous trials.









The percent of hatched eggs for the control group was 81 with a standard error of a

10. The triolein group showed a percent of hatched eggs at 67 & 7. The treatment group

at 5 ppm of p,p '-DDE had a percent of hatched eggs at 49 & 17, the treatment group at 10

ppm had a percent of hatched eggs at 56 & 18, and the treatment group at 20 ppm had a

percent of hatched eggs at 54 & 2. The percent of hatched eggs when comparing

treatment groups to the controls showed no significant differences.

The percent of fry surviving to pre-swim-up stage for the control group was 68

with a standard error of f 11. The triolein group showed the percent of survived fry to

pre-swim up at 83 & 8. The treatment group at 5 ppm of p,p '-DDE had a percent of

survived fry to pre-swim up stage at 60 + 9, the treatment group at 10 ppm had a percent

of survived fry to pre-swim up stage at 56 & 18, and the treatment group at 20 ppm had a

percent of survived fry to pre-swim up stage at 54 & 2.

The percent of fry surviving to post-swim-up stage for the control group was 93

with a standard error of f 4. The triolein group showed the percent of survived fry to

post-swim up at 93 & 4. The treatment group at 5 ppm of p,p '-DDE had a percent of

survived fry to post-swim up stage at 90 & 6, the treatment group at 10 ppm had a percent

of survived fry to post-swim up stage at 88 & 6, and the treatment group at 20 ppm had a

percent of survived fry to post-swim up stage at 97 & 3.

The percent of survivorship to Day 30 for the control group was 51 with a standard

error of a 1 1. The triolein group showed the percent of survivorship to Day 30 at 5 1 & 6.

The treatment group at 5 ppm of p,p '-DDE had a percent of survivorship at 26 & 7, the

treatment group at 10 ppm had a percent of survivorship to Day 30 at 28 & 10, and the

treatment group at 20 ppm had a percent of survivorship at 31 & 9. Although I observed a









lower percentage of hatched larvae in fathead minnow eggs inj ected with p,p '-DDE, there

was not a decrease in survivorship.

Use of a Tukeys Multiple Comparison Test showed that there were no significant

differences in male or female fathead minnow gonads when comparing the stage of the

ovary and/or testis to the treatment group when the p value was declared at equal or lower

than 0.05. Only 1 sample collection was performed for both male and female fathead

minnows at the end of the 30-day exposure.

Control flake feed did exhibit some levels of OCP contaminants however, this is

expected due to the fact that the flake feed is comprised of wild-caught fish and shellfish

that naturally contain some levels of contaminants. The levels of OCPs in the

contaminated feed were within the selected range while attempting to achieve ppm values

comparable to blue tilapia (Oreochromis aureus) located at the north shore of Lake

Apopka, Florida, USA, with a biomagnification factor of 15 (Table 1).

Again, the controls of body burdens exhibited minimal levels of OCP

contaminants, while the treatment groups displayed a dose-response relationship in all

contaminants in the mixture (Table 2). When examining the levels of OCPs maternally

transferred to the eggs in treatment groups, it was demonstrated that individual

contaminants either did not transfer from the female to the egg or did not show a dose-

response pattern. In some cases, the amount of contaminant transferred to the control

group was indeed higher than those transferred to the treatment group (Table 3). Because

these patterns were unexpected, a second round of maternal transfer from dosed female

fathead minnows to eggs was performed to confirm our findings (Table 4). In early

clutches (Days 1-5), again, the controls showed a higher amount of contaminant was









transferred in the controls than in the treatment groups. In the middle clutches (Days 13-

17), there were no dose-response patterns observed with some contaminants being higher

in the controls than the treatment groups, some contaminants being higher in the medium

treatment groups, and some contaminants being the mid-range of the high treatment

groups. The late clutches (Days 25-30), displayed no obvious dose-response relationship

throughout all contaminants. While the chlordane did show a dose-response in the

control and treatment groups, p,p '-DDE, dieldrin, and toxaphene did not.

Gonad OCP concentrations displayed a dose-response relationship across all

contaminants with the only exception being the low treatment group of chlordane and the

medium treatment group of chlordane (Table 5).










Table 1. Day 30 GC-MS results of flake feed OCP concentrations (ng/g). N.D. is
defined as not detected.
Control Treated
Chlordanes 5.57 105.74

DDE metabolitess) 44.61 528.67

Dieldrin N.D. 200.0

Toxaphene 3850.37 6447.13


Table 2. Day 30 GC-MS results of female whole body burden OCP concentrations
(ng/g). N.D. is defined as not detected.
Control Low Middle High
Chlordane 3.54 8.5 21.07 40.07

DDE metabolitess) 7.07 93.44 102.42 206.57

Dieldrin N.D. 6.8 15.06 33.92

Toxaphene N.D. N.D. N.D. N.D.


Table 3. Day 30 GC-MS results of Egg OCP concentrations (ng/g). N.D. is defined as
not detected.
Control Low Middle High

Chlordane N.D. N.D. N.D. 2.79

DDE metabolitess) 298.11 117.19 79.3 103.26

Dieldrin 3.77 N.D. N.D. 2.79

Toxaphene N.D. N.D. N.D. N.D.





Chlordanes
DDE metabolitess)
Dieldrin
Toxaphene


Table 5. Day 30 GC-MS results of OCP concentrations in male and female gonads
(ng/g). N.D. is defined as not detected.
control low medium high
male female male female male female male female
Chlordanes 17.54 7.0 28.16 26.0 126.58 23.0 132.35 70.77
DDE metabolitess) 61.4 20.0 126.76 182.18 746.84 224.22 779.41 549.91
Dieldrin 8.77 7.0 14.08 15.0 113.92 31.0 117.65 65.34
Toxaphene N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.


Table 4. Day 30 GC-MS results of OCP concentrations in eggs (ng/g) taken during Days
1-4 (early clutches), 13-16 (mid clutches) and 27-30 (late clutches). N.D. is
defined as not detected.
Early Clutches


Control
N.D.
157.9
52.63
N.D.



Control
N.D.
119.41
29.85
N.D.



Control
N.D.
42.61
17.05
N.D.


Low
14.46
108.43
28.92
N.D.



Low
N.D.
73.17
14.63
N.D.


Middle
11.62
75.58
17.44
N.D.



Middle
N.D.
245.9
98.36
N.D.



Middle
5.22
46.96
10.43
N.D.


High
.88
4.44
.89
N.D.


Mid Clutches


High
21.36
106.76
32.03
N.D.



High
10.06
110.56
30.15
N.D.


Chlordanes
DDE metabolitess)
Dieldrin
Toxaphene


Late Clutches


Low
N.D.
57.84
14.46
N.D.


Chlordanes
DDE metabolitess)
Dieldrin
Toxaphene















CHAPTER 4
DISCUSSION

The results of this study suggested that 30-day exposure to diets containing the

OCPs chlordane, p,p'-DDE, dieldrin, and toxaphene at varying doses can result in

internal carcass and gonad accumulation. The accumulation of OCPs is then maternally

transferred to the eggs of fathead minnows at various concentrations. These results are

consistent with other studies that suggest maternal OCP transfer (Rauschenberger, et al.

2004; Johnson, 2005). The value of our study was that OCP concentrations in maternal

tissues and egg yolks appear to be strongly correlated with one another. Our data

suggests maternal exposure to OCPs adversely affects reproduction by lowering the

percentage of spawning pairs, lowering the survival of larvae up to Day 14, decreases the

number of eggs produced over time, and lowers clutch viability over time.

In analyzing fathead minnow whole body tissue, gonadal tissue, and eggs, (Tables

1 through 3) we can see that numerous OCP analytes were detected in the flake feed,

whole body tissues, gonads, and eggs. This suggests that when making predictions about

possible effects to wildlife populations or human risk assessment, the composition of the

toxicant is a crucial factor. One reason for this is that different xenobiotic compounds

may inhibit or induce specific biotransformation enzymes (Rauschenberger, 2004).

Because genetic variability exists among populations, it is possible that certain

individuals or populations may lack the genetic ability to produce a biotransformation

enzyme that is required for the detoxification of specific OCP analytes. Examination of

Table 2 shows that a dose-response relationship was established within whole body









burdens of fathead minnows. While examining gonadal tissues, we also saw a dose-

response relationship (Table 5) with the one exception of low and medium treatment

groups of chlordane. Rauschenberger (2005) showed that in alligators, an adipose-to-

yolk concentration ratio was close to 1. This would suggest that OCPs reach equilibrium

within adipose tissue, and that lipids and OCPs are mobilized and subsequently

incorporated into developing yolks. However, as seen in Table 3 and Table 4, there was

no evidence to suggest that a consistent amount of chlordane, p,p '-DDE, dieldrin, or

toxaphene was transferred to mature eggs. For this reason, the hypothesis that direct

toxicity occurs to fathead minnow eggs in the natural environment and adversely affects

fertilized egg development and/or survivorship of larvae was abandoned, and the

nanoinjection portion of this thesis was terminated. Table 4 shows that over a 30-day

period, while females were continually dosed, with the exception of toxaphene which was

not detected in any samples except the flake and dieldrin which remained relatively

constant from the early sampling period to the late sampling period, chlordane and p,p '-

DDE either decreased to a non-detectable amount or decreased to nearly 50% of the

initial concentration. Xie and Klerks (2002) showed that least killifish (Heterandria

Formosa) exposed to cadmium developed a resistance to toxicity within a six generation

timeframe. It is possible that the fathead minnows are able to adapt, possibly by

increasing activity or production of enzymes that transform OCPs from hydrophobic

xenobiotics to hydrophilic metabolites that are excreted, thus decreasing the potential for

adverse affects.

When examining Figure 1, we can observe that there were significant differences

detected among the control vs. high and middle treatments and although not significantly









different, a 20% decrease between the control and low treatment groups. Previous

studies have demonstrated that exposure to certain contaminants adversely affects mating

behavior in cyprinids. Jones and Reynolds (1997), found that exposure to various

contaminants affected mating behavior in such ways as: increases or decreases in mating

displays, increased courtship duration, performance of male-like behavior of

masculinized females, decreased nest-building ability, decreased offspring defense, or

changes in division of parental care between sexes. It is also known that cyprinids

secrete steroid hormones that act as pheromones during courtship. It is possible that

OCPs may disrupt the activity of these pheromones causing a disruption in courtship

behavior. Cyprinidae are known to have complex mating behaviors, thus the possibility

exists that exposure to OCPs may adversely affect mating behavior and consequently

decrease the ability for fathead minnow pairs to successfully spawn.

Figures 3 and 4 show that the mean number of eggs hatched and clutch viability

both show similar trends in the controls and treatment groups. While the low treatment

group displayed an increased number of eggs hatched, and a higher clutch viability when

compared to the control, the middle and high treatment groups showed a decrease in the

number of eggs hatched, as well as clutch viability. Because OCP levels in the eggs of

the fathead minnow were either non-detectable or inconsistently transferred from the

adult female to the egg; conclusions can be drawn that there is not a correlation between

maternally transferred OCPs to eggs and reduced mean number of eggs hatched and

reduced clutch viability. In an attempt to explain the significant decreases in the middle

and high treatment groups, it should be noted that the intermediate and highly exposed

females continued to lay eggs that were not significantly different than the mean number









of control eggs. The middle and high treatment females continually produce and deposit

eggs, however, the ova appear to be unable to sequester the nutrients required to produce

healthy and viable eggs. A lack of nutrients results in a decreased amount of energy, as

well as, structural supplies that are available for developing embryos and possibly yolk

sacs that larvae use as a source of nutrition for the first several days after hatching. This

is an observation that has been speculated in other studies (Rauschenberger, 2004;

Johnson, 2005) however, until the completion of this study, little evidence has been able

to support this hypothesis.

Although circulating sex steroids were not analyzed in this experiment, effects that

these hormones have on reproductive success should be addressed. Gross et al. (2002)

found that circulating concentrations of E2 in female largemouth bass and 1 1-KT in male

largemouth bass exposed to OCPs were on averagel,500 pg/mL less than what was

reported for pond-reared largemouth bass sampled in the same calendar year. The ability

for any exogenous compound to bind a sex steroid hormone receptor and agonize and/or

antagonize the action of an endogenous hormone can severely affect normal endocrine

function. The reasoning for this is that normal estrogen or testosterone concentrations

and actions are critical for development of gonads in both male and female fish (Johnson,

2005). It is possible that fathead minnows exposed to OCPs may alter the concentrations

of these two circulating sex steroids thus reducing reproductive success.

Vitellogenin is a protein synthesized by the liver and its uptake by growing oocytes

and its storage as yolk serves as a nutrient source by developing embryos. During this

period, extraovarian proteins are gathered, processed, and packaged into oocytes.

Consequently, this period is of particular importance when considering maternal transfer










by lipophilic proteins of OCPs to developing oocytes (Di Giulio and Tillitt, 1999). The

possibility exists that because studies have shown that circulating vitellogenin acts as an

important transport protein that binds lipophilic hormones, OCPs may bind to

vitellogenin and agonize and/or antagonize vitellogenin receptors on gonads, thus

impeding the deposition of yolk reserves, and therefore oocyte growth. Again, this is one

explanation as to why decreased reproductive success is observed in my experiment

while direct embryo toxicity is not.

Favorable water temperature, salinity, food availability, as well as egg quality are

critical to the survivorship, growth, and the metamorphosis to the juvenile stage of larval

Eish. Fast growth to fish larvae has been correlated to the presence of high plankton

concentrations where larvae have adequate opportunities to capture prey. Other

environmental factors that influence larval development include dissolved oxygen,

turbidity, nutrients, water movement, and meteorological events. The first stage of larval

development is the yolk sac phase. The yolk sac contains nutrition used by larval Eish

while it adapts to its new aquatic environment. Larvae absorb the yolk and will continue

to grow and develop while they begin to look for their first prey, plankton. As larvae

continue to develop, they begin to take on the appearance of juveniles and further

develop the ability to swim and capture prey. Typically, fathead minnows can not swim

for long periods of time as larvae, they can however, swim long enough to seek

appropriate resting habitat. During the course of our experiment, every effort was made

to eliminate any possibility of larvae mortality due to water quality parameters or

insufficient food availability. At no time during the course of the experiment were

hatched larvae exposed to harmful levels of dissolved oxygen, salinity, or temperature.









Hatched larvae were also fed ad libitum adequate supplies of artemia. Because all

obvious growth and developmental parameters were satisfied, it leads us to believe that

the significant differences in the percent of larvae survived to Day 7 and Day 14 (Figures

5 and 6) were solely due to OCP exposure. While a dose-response relationship was not

established for either Day 7 or Day 14 larvae survivorship, all three treatment groups for

the percent of larvae survived to Day 7 were either lower or significantly lower when

compared to the control, and all three treatment groups of the percent of larvae survive to

Day 14 were significantly lower when compared to the control.

While no teratological or morphometrical measurements were taken during the

course of this experiment, further discussion should be made as to the specifies of the

high mortality observed in this experiment. Most teratogenic contaminants are believed

to be non-specific (Newman and Unger, 2003). According to Karnofsky's law, any agent

will be teratogenic if it is present at concentrations or intensities that produce cell toxicity

(Bantle, 1995). Teratogens act by disrupting mitosis, interfering with transcription and

translation, disturbing metabolism, and producing nutritional deficits (Weis and Weis,

1987). Consequences of these disruptions may include abnormal cell interactions,

excessive growth, or cell death.

Retardation of growth, adverse effects on the skeletal, musculature, circulatory, and

optical system are the most common effects seen in larval fish exposed to toxicants.

Skeletal problems in fish may include lateral curvature of the spine (scoliosis) or the

extreme forward curvature of the spine lordosiss). Wies and Wies (1989), showed that

the mummichog, (Funduhus heteroclitus) when exposed to 10 mg I~ of Pb2+ failed to

uncurl it' s tail post hatch, essentially leaving the larval fish to certain death. Sepulveda et










al. (2000) found that largemouth bass larvae exposed to p,p '-DDE and dieldrin showed

yolk sac edema, and eye deformities including opaque corneas. Studies of largemouth

bass larvae exposed to paper mill effluent also showed deformities including: increased

head abnormalities, a decrease in length and weight, and a shortened vertebral column

(Sepulveda et al., 2003). Any or all of these abnormalities may have led to the decreased

survival rates observed in my experiment.

Gonadosomatic index (GSI) is defined as the ratio of the weight of the gonad to the

weight of whole body. Although GSI is not specific to a particular mechanism of

toxicant action, it is an endpoint commonly used in toxicological studies, and is a good

predictor of reproductive success that may be linked to population level responses to

contaminants. However, caution must be applied when using GSI to make a hypothesis

concerning contaminant effects to individuals. Dependent upon the species of fish and

the time of the year, an increase in GSI may be a result of responses to reproductive

and/or environmental cues. Gross et al. (2000) found that gonads in largemouth bass

began to mature in October and reached a considerable size (approximately 5% of fish

body weight) by January, and continued to peak until February. Depending on the month

or season of the year, GSI may not be an accurate endpoint for contaminant exposure.

Also, fathead minnows are batch spawning fish. Consequently, their gonads undergo

rapid cyclical changes over short periods of time (every few days) as successive batches

of eggs or sperm are produced, and thus means the size of the gonads in breeding adults

can vary considerably between individuals at any point in time (Harries et al., 2000). In

my study, neither male nor female displayed significant differences in mean GSI (Figures

7 and 8). This is consistent with a study that exposed largemouth bass to 10.0 mg/pellet










of p,p '-DDE and 1.0 mg/pellet of dieldrin (Muller, 2003). My results are however, not

consistent with a study of male guppies (Poecilia reticulata) who when fed a diet of

150ug of p,p '-DDE for 30 consecutive days showed a decrease in GSI. Sepulveda (2000)

showed that there can be differences in GSI not only between various spawning seasons,

but also between different habitats within the same area. Again, these inconsistencies

with GSI should throw caution when using this tool as an endpoint to measure

reproductive success or toxicant exposure.

There were significant differences in the percent of females spawning and the

number of spawns (Figure 9). Over a 30-day period, the percent of females spawning in

the control went from 80% to 10% after the fifth spawn. The low treatment group went

from 70% of females spawning to only 30% after the third spawn. Although the middle

treatment group spawned the most in a 30-day period, six times, the percent of females

spawning started at 40% and remained relatively constant throughout the 30-day period at

20%. The high treatment group started at 50% and by the fifth spawn dropped to only

10%. The frequency of spawnings in our control Eish were slightly lower than what has

been reported in other studies. Gale and Buynak (1982) reported that the mean number

of spawns in their experiment was every 3.9 days. In contrast, in my study, the low

treatment group spawned approximately every 8 days, the middle treatment group

spawned approximately every 5 days, and the high treatment group spawned

approximately every 6 days. Although the frequency of spawning was reduced in the

control and treatment groups, two critical observations should be made. The first

observation is that as previously mentioned, not only did treatment groups have reduced

frequency in spawning, so did the control. Our data shows that over a 30-day period the










percent of control females spawning went from 80% to 0%. This critical observation is

in that numerous state, federal, and private institutions use the fathead minnow as a

model in aquatic toxicity tests. Protocols should be established that limit the frequency a

specific breeding pair of fathead minnows is used without adequate recovery time before

employing them in continuous toxicity tests. The second observation that should be

made is that as seen in Figure 10, altered frequency of spawns is also correlated with the

mean number of eggs laid. This would suggest that in reference to a population of

fathead minnows in a particular ecosystem, altered frequency of spawning would not be

offset by an increase in fecundity. This is in contrast to a study where fathead minnows

exposed to weak active endocrine mimics had a decrease in the number of spawnings,

however, there was a reciprocal increase in the size of the egg batch (Harries et al.,

2000).

The mean number of eggs laid per spawn showed a lower number of eggs produced

for treated versus controls (Figure 10). This figure also showed altered patterns across

treatments. Although the control started with a low mean number of eggs laid, by the

second spawn, the mean number of eggs laid per spawn decreased, and fish stopped

spawning after the forth spawn within a 30-day period. The low treatment group showed

a similar pattern with the most eggs laid during the first spawn, the mean of eggs laid

tapering off until the third spawn when no more eggs were laid. Although the middle

treatment group displayed a similar pattern, having the greatest number of eggs during

the first spawn, then gradually tapering off, there was a lower mean number of eggs laid

on the first spawn, however, this treatment group spawned the most times, six, out of all

treatment groups. The fact that the middle treatment group continued to spawn for a









longer period of time than other treatment groups, may indicate the fathead minnow has

adapted to mid-exposure and although fewer eggs are being produced, the frequency of

spawning is significantly longer. The high treatment group continued to show the same

pattern of the most eggs being spawned early, then gradually tapering off until no further

spawning took place. Although OCPs may affect the frequency of spawning as well as

the mean number of eggs laid per spawn, it could also be possible that the differences

observed in mean number of eggs is in part due to differences in the sizes of the fish. It is

apparent however, that the largest clutch sizes take place early and gradually taper off in

the control and treatment groups possibly due to the availability of mature oocytes.

Control clutch viability varied from 55% to 90% throughout the number of times

spawned within the 30 trial period. There appeared to be no distinguishable pattern

within the percent ranges (Figure 11). The low treatment group did however, show a

higher clutch viability toward the early stages of the experiment than the later stages.

From this, one might assume that bioaccumulation of OCPs are decreasing clutch

viability, however, the middle and high treatment groups show an increase and then

decrease in clutch viability over the number of times spawned as well as the 30-day

period. Although no obvious patterns across treatments were observed, all three treatment

groups had significantly lower clutch viability percentages when compared to the control.

This observation is similar to alligator eggs exposed to OCPs which also showed

decreased clutch viability (Rauschenberg, 2004). This indicates decreased clutch

viability is due to decreased egg quality associated with senescence.

Recently, numerous studies have clearly established that various man-made and

natural chemicals exist within aquatic environment that have the potential to mimic









androgens, estrogens, or their antagonists. A number of EDCs bioaccumulate and/or

result from environmental degradation or metabolism from their parent compound. In

order to establish a cause-effect relationship, between exposure to EDCs and reproductive

success and survivorship, it is necessary to conduct whole animal studies. Given the

large number of chemicals that have the potential to be EDCs, as well as are regularly

found within the aquatic environment, there is a need to develop a biological model that

is practical to work with in a laboratory setting that is capable of demonstrating

quantifiable reproductive parameters as well as other endocrine disruptive biomarkers

such as vitellogenin induction. Other studies have demonstrated documented

bioaccumulation of OCPs in the carcass and gonads of largemouth bass (Johnson, 2005),

and endocrine modulation caused by OCPs (Muller, 2004). Sepulveda et al. (2004)

found alterations in endocrine function and increased developmental mortality in

largemouth bass inhabiting the Emeralda Marsh. In another study, relative contributions

of losses during in ovo development in alligators at impacted sites in Florida are lower

clutch viability, higher rates of damaged eggs, higher rates of early embryo mortality, and

higher rates of late embryo mortality, all of these were due to exposure to OCPs

(Rauschenberger, 2004). While all of these studies displayed affects caused by exposure

to OCPs, these were all captive studies that were both labor intensive and financially

demanding. Hence, the need to develop a series of short-term, economical, laboratory

tests using a biological model that shows effects of exposure to EDCs including

fecundity, GSI, induction of vitellogenin and other circulating sex steroids (E2 and 11-

KT), and survivorship is needed to predict and assess potential impacts of EDCs either on

larger individuals or on populations within ecosystems.









Further research on the reproductive effects of p,p '-DDE, dieldrin, chlordane, and

toxaphene on fathead minnows could focus on the measurement of additional

reproductive endpoints. For example, estradiol-17P, 11-KT and vitellogenin would

potentially add additional information concerning the development stages of oocytes and

address the size and condition of gonads. Although male gonads were staged, assessing

gamete viability in males would eliminate the possibility that EDCs are disrupting male

reproductive potential. Nakayama et al. (2005) found that when Japanese medaka

(Oryzia~s latipes) were exposed to tributyltin (TBT), the number of eggs laid remained

relatively constant, however, the number of fertilized eggs decreased. Another study

showed that as levels of TBT increased the percent of sperm that lacked flagellum or had

a decrease in the volume of milt also increased (McAllister and Kime, 2003). Another

factor that may affect OCP toxicity that was not addressed in my experiment is in that

low temperatures have been associated with increased DDT toxicity in fish (Rattner and

Heath, 2003). Fluctuation in water temperature, as well as fluctuations in OCP levels,

mimicking "hotspots" may also provide invaluable insight into the correlation between

OCP exposure and reduction in reproductive success.

























30-

20-


10


control low


middle high


Treatment Group

Figure 1. The percent of spawning pairs among treatment groups. Control, low, middle,
and high treatment groups had 10 potential spawning fathead minnow pairs.
Mean a standard error results are shown. Significant differences between
control middle and high treatment groups using One-way ANOVA (P<.05).
Asterisks indicate differences in relation to controls.


control low


middle
Treatment Group


Figure 2. The mean number of eggs laid per spawn among treatment groups. Control,
low, middle, and high treatment groups had 10 potential spawning fathead
minnow pairs. Mean a standard error results are shown. Significant
differences between low and control treatment groups, low and middle
treatment groups using One-Way ANOVA (P<.05). Asterisks indicate
differences in relation to controls.













I)En


300




1 50


1 00


d 50



50


control low


middle high


Treatment Group


Figure 3. The mean number of eggs hatched among treatment groups. Control, low,
middle, and high treatment groups had 10 potential spawning fathead minnow

pairs. Mean a standard error results are shown. Significant differences
between low and control treatment groups, low and high treatment groups,
and low and middle treatment groups using One-Way ANOVA (P<.05).
Asterisks indicate differences in relation to controls.


100

90

80
-"
70
-

S60

S50

40
-

S30
-


0-


control low


middle
Treatment Group


Figure 4. The percent of clutch viability among treatment groups. Control, low, middle,
and high treatment groups had 10 potential spawning fathead minnow pairs.
Mean a standard error results are shown. Clutch viability = No. of eggs

yielding a live hatchling / Fecundity x 100. Significant differences between
low and middle treatment groups, low and high treatment groups, control and
middle treatment groups, control and high treatment groups, and middle and
low treatment groups using One-Way ANOVA (P<.05). Asterisks indicate
differences in relation to controls.































control low


middle high


Treatment Group

Figure 5. The percent of larvae survived to Day 7 among treatment groups. Control,
low, middle, and high treatment groups had 10 potential spawning fathead
minnow pairs. Mean a standard error results are shown. Significant
differences between control and high treatment groups and control and middle
treatment groups using One-Way ANOVA (P<.05). Asterisks indicate
differences in relation to controls.


*30

%25

S20
o

15


01


control low


middle
Treatment Group


Figure 6. The percent of larvae survived to Day 14 among treatment groups. Control,
low, middle, and high treatment groups had 10 potential spawning fathead
minnow pairs. Mean a standard error results are shown. Significant
difference between control and middle treatment groups using One-Way
ANOVA (P<.05). Asterisks indicate differences in relation to controls.




































control low middle high
Treatment Group


Figure 7. Mean GSI among male treatment groups. Control, low, middle, and high
treatment groups had 10 potential spawning fathead minnow pairs. Mean &
standard error results are shown. No significant differences using One-Way
ANOVA (P<.05).


18

16

14

S12

10
-



S6
LL.

4

2


-
0 -!-


control low


middle


Treatme nt Group


Figure 8. Mean GSI among female treatment groups. Control, low, middle, and high
treatment groups had 10 potential spawning fathead minnow pairs. Mean &
standard error results are shown. No significant differences using One-Way
ANOVA (P<.05).











10




6 -O control
H ow
mid
I high





1st 2nd 3rd 4th 5th 6th
Number of Times Spawned

Figure 9. Percent of Females Spawning vs. Number of Spawns. Control, low, middle,
and high treatment groups had 10 potential spawning fathead minnow pairs.
Mean a standard error results are shown. Note: Decreased number of
spawning females for treated versus controls and altered patterns across
treatments.


300

'5250

En 200100 -
w Control
5 Iow
e I Mmid
E" Ohigh





1st 2nd 3rd 4th 5th 6th Total
Number of Times Spawned



Figure 10. Mean Number of Eggs Laid vs. Number of Times Spawned. Control, low,
middle, and high treatment groups had 10 potential spawning fathead minnow
pairs. Mean a standard error results are shown. Note: Lower number of eggs
produced for treated versus controls and altered patterns across treatments.












100
900
=80
.- 70 --

S60 -- O control
l ow
S 50 -
O mid
40 -
o~ High


S20 ----- -
10 ---- -


1st 2nd 3rd 4th 5th 6th

Number of Times Spawned



Figure 11. Percent of Clutch Viability vs. Number of Times Spawned. Control, low,
middle, and high treatment groups had 10 potential spawning fathead minnow
pairs. Mean + standard error results are shown. Note: Lower clutch viability
produced for treated versus controls and altered patterns across treatments.















CHAPTER 5
CONCLUSION

Research from this study provides evidence that dietary exposure to a mixture of

p,p'-DDE, dieldrin, toxaphene, and chlordane bioaccumulate in maternal tissues and at

inconsistent rates are transferred to developing eggs. Our data shows that maternal

exposure to OCPs indicates endocrine disruption, and adversely affects reproduction by

lowering the percentage of spawning pairs, lowering the survival of larvae up to Day 14,

decreases the number of eggs produced over time, and lowers clutch viability over time.

The fathead minnow could potentially be used as a biological model to assess

effects of various contaminant exposures including pharmaceuticals, sewage effluent,

paper mill effluent, and numerous heavy metals. Adult fathead minnows are omnivores,

consuming a variety of resources as food, hence giving the fish an opportunity to

accumulate numerous toxicants. My study has shown OCPs bioaccumulate in fathead

minnow adipose and gonadal tissue and is maternally transferred at various

concentrations to their eggs. Fathead minnows in my study responded similarly to

largemouth bass and alligators that were orally exposed to various concentrations of

OCPs, making the fathead minnow an eco-relevant model. However, unlike largemouth

bass, fathead minnows are not seasonal spawners, multi-generation tests can easily be

performed, and costs of performing these tests are greatly reduced in comparison with

largemouth bass. The ease of laboratory handling, the low financial burden, as well as

the ability of fathead minnows to bioaccumulate contaminants makes it a good model to










examine potential reproductive effects, thus giving researchers the ability to extrapolate

cause-effect relationships in higher order organisms.

My data also concluded that although fathead minnows exposed to OCPs continued

to produce and deposit eggs, the ova appear unable to sequester the proper nutrients

required to produce healthy eggs. The conclusion can be made that bioaccumulation of

OCPs in the female ovaries may cause lower egg quality prior to the induction of

vitellogenesis, however, total protein or lipid analysis would further support this

conclusion. Inconsistent amounts of OCPs were transferred to fathead minnow eggs,

however, the relatively same endpoints were observed whether high quantities of OCPs

were transferred or low quantities were transferred. This would suggest that the

mechanism of action is maternal.
















LIST OF REFERENCES


Ankley, G.T., Jensen, K.M., Kahl, M.D., Korte, J.J., Makynen, E.A., 2000. Description
and evaluation of a short-term reproduction test with the fathead minnow
(Pinsephales pronzela;s). Environ. Toxicol. Chem.

Bantle, J. A. 1995. FETAX: a developmental toxicity assay using frog embryos.
Fundamentals of Aquatic Toxicology: Effects, Environmental Fate, and Risk
Assessment, 2nd ed., Rand, G.M., Ed., Taylor and Francis, Washington, D.C.

Bryan, T. E., Gildersleeve R. P., Wiard, R. P., 1989. Exposure of Japanese quail
embryos to o 'p-'DDT has long term effects on reproductive behaviors,
hematology and feather morphology. Teratol 39: 525-236.

Cahn, A. R. 1927. An ecological study of southern Wisconsin fishes: the brook
silversides (7ablid e~\lik1\ siccuhts) and the cisco (7 men it Irhy1 a\rtedi ) in their
relations to the region. Illinois Biological Monographs 11:1-151.

Coyle, E. E. 1930. The algal food of Pinsephales pronzela;s (fathead minnow). Ohio
Journal of Science 30:23-35. (Not seen; cited in Scott and Crossman 1973.)

Danzo, B. 1997. Environmental xenobiotics may disrupt normal endocrine function by
interfering with the binding of physiological ligands to steroid receptors and
binding proteins. Environmental Health Perspectives 105: 294.

Darnerud, P. O., Sinjari, T., and Jonsson, C. J. 1996. Fetal uptake of coplanar
polychlorinated biphenyl (PCB) congeners in mice. Pharmacological Toxicology
78: 187.

Di Giulio, R. T., and Tillitt, D. E. 1997. Reproductive and Developmental Effects of
Contaminants in Oviparous Vertebrates. SETAC. Pensacola, Florida, United
States. Pp. 20-21.

Flouriot, G., Pakdel, F., Ducouret, B., and Valotaire, Y. 1995. Influence of xenobiotics
on rainbow trout liver estrogen receptor and vitellogenin gene expression. Journal
of molecular endocrinology 15: 143.










Foster, E. P., Fitzpatrick, M. S., Feist, G. W., Schreck, C. B., Yates, J., Spitsbergen, J.
M., and Heidel, J. R. 2001. Plasma Androgen Correlation, EROD Induction,
Reduced Condition Factor, and the Occurrence of Organochlorine Pollutants in
Reproductively Immature White Sturgeon (Acipenser transmontanus) from the
Colomia River, USA. Archives of Environmental Contamination and Toxicology
41: 182-191.

Gale W. F. and Gerard B. L. 1982. Fecundity and Spawning Frequency of the Fathead
Minnow--Fractional Spawner. Transactions of the American Fisheries Society.
111:35-40.

Gallager, E. P., Gross, T. S., and Sheehy, K. M. 2001. Decreased glutathione S-
transferase expression and activity and altered sex steroids in Lake Apopka brown
bullheads (Ameriurus nibulosus). Aquatic Toxicology 55: 223-237.

Gayle, W.F., Buynak, G.L. 1982. Transactions of the American Fisheries Society. Vol.
111: 35-40.

Gildersleeve, R. P., Tilson, H., Mitchell, C., 1985. Injection of diethylstilbestrol on the
first day of incubation affects morphology of sex glands and reproductive behavior
of Japanese quail. Teratology 31: 101-109.

Gleason, T. R. and Nacci, D. E. 2001. Risks of Endocrine disrupting compounds to
wildlife: Extrapolating from effected individuals to population response. Human
and Ecological Risk Assessment 7: 1042.

Gross, T. S., Arnold, B. S., Sepluveda, M. S., and McDonald, K. 2003. Endocrine
disrupting chemicals and endocrine active agents. Pages 1033-1098 in D. J.
Hoffman, B. A. Rattner, G. A. Burton, and J. Cairns, editors. Handbook of
Ecotoxicology, 2nd edition. Lewis Publishers, Boca Raton, Florida.

Gross, T. S., Guillette, L. J., Percival, H. F., Masson, G. R., Matter, J. M., and Woodard,
A. R. 1994. Contaminant-induced reproductive anomalies in Florida. Comparative
Pathology Bulletin 26: 2-8.

Gross, T. S., Sepulveda, M. S., Wieser, C. M., Wiebe, J. J., Schoeb, T. R., and Denslow,
N. D. 2002. Characterization of annual reproductive cycles for pond-reared Florida
largemouth bass (M~icropterus salmoides florid anus).d~~~dd~~dd Pages 205-212 in D. P.
Phillip and M. S. Ridgeway, editors. Proceedings of the Black Bass 2000
Symposium. American Fisheries Society, Bethesda, Maryland.

Harries, J. E., Runnalls, T., Hill, E., Harris, C. A., Maddix, S., Sumpter, J. P., and Tyler,
C. R. 2000. Development of a Reproductive Performance Test for Endocrine
Disrupting Chemicals Using Pair-Breeding Fathead Minnows (Pimephales
Promela~s). Environmental Science Technology 34: 3003-3011.

Hoffman, D. J., Rattner, B. A., Burton, Jr., G. A., and Cairns, Jr., J. 2003. Handbook of
Ecotoxicology. Second Ed. CRC Press LLC. Boca Raton, Florida, USA.










Hori, S. H., Kodama, T., and Tanahashi, K. 1979. Induction of vitellogenin synthesis in
goldfish by massive doses of androgens. General and Comparative Endocrinology
37: 306-320.

Jansen, H. T., Cooke, P. S., Porcelli, J., Liu, T. C., and Hansen, L. G. 1993. Estrogenic
and anti-estrogenic actions of PCBs in the female rat: In vitro and in vivo studies.
Reproductive Toxicology 7: 237.

Jenkins, R. E., Burkhead, N. M. 1993. Freshwater fishes of Virginia. Page 252.
American Fisheries Society, Bethesda, Maryland.

Jensen, K. M., Korte, J. J., Kahl, M. D., Pasha, M. S., and Ankley, G. T., 2001. Aspects
of basic reproductive biology and endocrinology in the fathead minnow
(Pimephales promela~s). Comparative Biochemistry and Physiology Part C
128:127-141.

Jobling, S., Nolan, M., Tyler, C. R., Brighty, G., and Sumpter, J., P. 1998. Widespread
sexual disruption in wild fish, Environmental Science Technology 32: 2498-2506.

Johnson, K. G. 2005. Dietary exposure to the organochlorine pesticides p,p '-DDE and
dieldrin and their effects on steroidogenesis and reproductive success in Florida
largemouth b ass (M~icropterus salmoides florid anus)~ddd~~ddd~~. Master' s The si s. University
of Florida, Gainesville, FL.

Jones, J. C., Reynolds, J. K. 1997. Effects of pollution on reproductive behavior of
fishes. Reviews in Fish Biology and Fisheries. Vol 7. 463-491.

Kelce, W. R., Stone, C. R., Laws, S. C., Gray, L. E., Demppainen, J. A., and Wilson, E.
M. 1995. Persistent DDT metabolite p,p '-DDE is a potent androgen receptor
antagonist. Nature 375(6532): 581-585.

Marburger, J. E., Johnson, W. E., Gross, T. S. Gross, Douglass, D. R., and Di, J. 2002.
Residual organochlorine pesticides in soils and fish from wetland restoration areas
in central Florida, USA. Wetlands 22:705-711.

Markus, H. C. 1934. Life history of the blackhead minnow (Pimephales promela;s).
Copeia 1934:116-122.

Mayer, F. L. 1971. Dynamics of dieldrin in rainbow trout and effects on oxygen
consumption. Diss. Abstr. Int. 32(1): 527-B.

Miller, R. R. 1986.Composition and derivation of the freshwater fish fauna of Mexico.
Anales de la Escurela Nacional de Ciencias Biologicas, Mexico 30: 121-153. Mills,
L., Gutjahr-Gobell, R. E., Haebler, R. A., Borsay Horowitz, D. J.,Jayaraman, S.,
Pruell, R. J., McKinney, R. A., Gardner, G. R., and Zaroogian, G.E. 2001. Effects
of estrogenic (o,p '-DDT; octylphenol) and anti-androgenic (p,p '-DDE) chemicals
on indicators of endocrine status in juvenile summer flounder (Paid irhibys
dentatus). Aquatic Toxicology 52: 157-176.










Muller, J. K. 2003. An evaluation of dosing methods and effects of p,p '-DDE and
di eldrin in Flori da largemouth b ass (M~icropterus salmoides florid anus)~ddd~~ddd~~. Master' s
Thesis. University of Florida, Gainesville, FL.

Muller, J. K., Johnson, K. G., Sepulveda, M. S., Borgert, C., and Gross, T. S. 2004.
Accumulation of dietary DDE and dieldrin by largemouth bass, M~icropterus
salmoieloides flori~dd~ddanus. Bulletin of Environmental Contamination and Toxicology
73: 1078-1085.

Nelson, J.S. 1984. Fishes of the World, 2nd edition. Wiley, New York.

Newman, M. C. and Unger, M. A. 2003. Fundamentals of Ecotoxicology 2nd Edition.
CRC Press. Boca Raton, Florida.

Okoumassoun, L.-E., Averill-Bates D., Gagne, F., Marion, M., and Denizeau, F. 2002.
Assessing the estrogenic potential of organochlorine pesticides in primary cultures
of male rainbow trout (Oncorhynchus mykiss) hepatocytes using vitellogenin as a
biomarker. Toxicology 178: 193-207. Pearse, A. S. 1918. The food of the shore
fishes of certain Wisconsin lakes. U.S Bureau of Fisheries Bulletin. Lewis
Publishers 35: 246-292.

Pelissero, C., Flouriot, G., Foucher, J. L., Bennetau, B., Dunogures, J., Le Gac, F., and
Sumpter, J. P. 1993. Vitellogenin synthesis in cultured hepatocytes; an in vitro test
for the estrogenic potency of chemicals. The Journal of Steroid Biochemistry and
Molecular Biology 44: 263-272.

Rattner, B. A. and Heath, A. G. 2003. Environmental factors affecting contaminant
toxicity in aquatic and terrestrial vertebrates. In Handbook of Ecotoxicology: 679-
699. Hoffman, D. J., Rattner, B. A., Burton, G. A. Jr. and Cairns, J. Jr. (Ed.). Boca
Raton, FL USA

Rauschenberger, H. R., Wiebe, J. J., Buckland J. E., Smith, T. J., Sepulveda, M. S., and
Gross, T. S. 2004. Achieving environmentally relevant organochlorine pesticide
concentrations in eggs through maternal exposure in Alligator mississippiensis.
Marine Environmental Research: 851-856.

Redding, M. J., and Patino, R. 1993. Reproductive physiology. Pages 503-534 in D. H.
Evans, editor, The Physiology of Fishes. Marine Science Series. CRC Press, Boca
Raton, Florida.

Ree, G. E. and Payne J. F. 1997. Effecto to toxaphene on reproduction of Esh.
Chemosphere. 34(4): 855-867.

Robins, C.R. 1991. Common and scientific names of Eishes from the United States and
Canada, 4th edition. American Fisheries Society Special Publication 20.

Rosner, W. 1990. The functions of corticosteroid-binding globulin and sex hormone-
binding globulin: Recent advances. Endocrinology Review 11: 80.










Russell, R. W., Frank, A. P., Gobas, C., and Haffner, G. D. 1999. Maternal transfer and
in ovo exposure of organochlorines in oviparous organisms: A model and field
verification. Environmental Science Technology 33: 416-420.

Ryffel, G. U. 1978. Synthesis of vitellogenin, an attractive model for investigating
hormone-induced gene activation. Molecular and Cellular Endocrinology 12: 237-
246.

Scott, W. B. and Crossman E. J. 1973. Freshwater fishes of Canada. Bulletin of
Fisheries Resources. Board, Canada. pp.184.

Sepulveda, M. S. 2000. Effects of Paper Mill Effluents on Health and Reproductive
Success of Largemouth Bass (Micropterus salmoides): Field and Laboratory
Studies. Doctoral Dissertation. University of Florida, Gainesville, FL.

Shelby, M.D., Newbold, R. R., Tully, D. B., Chae, K., and Davis, V. L. 1996. Assessing
environmental chemicals for estrogenicity using a combination of in vitro and in
vivo assays. Environmental Health Perspectives 104: 1296-1300.

Smith, H. M. 1945. The freshwater fishes of Siam, or Thailand. Bulletin of the United
States National Museum 188:1-622.

Starrett, W. C. 1950. Food relationships of the minnows of the Des Moines River, Iowa.
Ecology 31:216-233.

Stebbing, A.R.D. 1982. Hormesis: the stimulation of growth by low levels of inhibitors.
Science Total Environment. Vol.22. pp 213-234.

Sumpter, J. P. and Jobling, S. 1995. Vitellogenesis as a biomarker for estrogenic
contamination of the aquatic environment. Environmental Health Perspectives,
supplement 103: 173-178.

Thomas, M. J. and Thomas, J. A. 2001. Hormone assays and endocrine function, in
Principles and Methods of Toxicology, 4th Ed., Taylor & Francis. Philadelphia, PA.

U. S. EPA 1982. User' s guide for conducting life-cycle chronic toxicity test with fathead
minnows (Pinsephales pronzela;s). EPA/600/8-81-011. Duluth, MN.

U.S. EPA 1987. Guidelines for the culture of fathead minnows Pinsephales pronzela~s for
use in toxicology tests. EPA/600/3-87/001. Duluth, MN.

U. S. EPA 1989. Pesticide assessment guidelines. Sub-division E, Hazard evaluation:
Wildlife and aquatic organisms. EPA-540/09-82/024. Washington, D.C.

U. S. EPA, 1991. Methods for measuring the acute toxicity of effluents and receiving
waters to freshwater and marine organisms 4th ed. In: Weber, C.I. (Ed.), EPA-
600/4-90/027. Office of Research and Development, Environmental Monitoring
Systems Laboratory, Cincinnati, OH.










U.S. EPA, 1994. Short-term methods for estimating the chronic toxicity of effluents and
receiving water to freshwater organisms, 3rd edition In: Lewis, P.A., Klemm, D.J.,
Lazorchak, J.M., Norberg-King, J.J., Peltier, W.H., Heber, M.A.., (Eds),
EPA/600/4-91/002. Offce of Research and Development Environmental
Monitoring Systems Laboratory, Cincinnati, OH.

U.S. EPA, 1998. Endocrine Disruptor Screening and Testing Advisory Committee
Report. Office of Prevention, Pesticides, and Toxic Substances, Washington, DC.

Van Der Kraak, G., Chang, J. P., and Janz, D. M. 1998. Reproduction. Pages 465-488
in D. H. Evans, editor. The Physiology of Fishes. CRC press, Boca Raton, Florida.

Wahli, W., Dawid, I. B., Ryffel, G. U., and Weber, R. 1981. Vitellogenesis and the
vitellogenin gene family. Science 212: 298-304.

Wallace, R. A. 1970. Studies on amphibian yolk Xenopus vitellogenin). Biochimica et
Biophysica Acta 215: 176-183.

Wallace, R. A., and Selman, K. 1981. Cellular and dynamic aspects of oocyte growth in
teleosts. American Zoologist 21: 325-343.

Weis, J.S. and Weis, P. 1987. Pollutants as developmental toxicants in aquatic
organisms, Environmental Health Perspectives. Vol.71: pp 77-85.

Weis, J.S. and Weis, P. 1989. Tolerance and stress in a polluted environment: the case
of the mummichog, Fundulus heteroclitus., Bioscience. Vol.39: pp 89-95.

Wells and Van Der Kraak, G. 2000. Differential binding of endogenous steroids and
chemicals to androgen receptors in rainbow trout and goldfish. Environmental
Toxicology and Chemistry 19: 2059-2065.

Xie, L., and Klerks, P.L. 2002. Responses to selection for cadmium resistance in the
least killifish (Heterandria Formosa). Environmental Toxicology and Chemistry.
Vol. 22, No. 2, pp 313-320.

Zeeman, M., Gifford, J. 1993. Ecological hazard evaluation and risk assessment under
EPA's Toxic Substances Control Act (TSCA): an introduction. In Landis W.
Hughes J, Lewis, M. editors. Environmental toxicology and risk assessment.
Volume 1. Philadelphia PA: ASTM. STP 1179 p. 7-21.
















BIOGRAPHICAL SKETCH

Dane H. Huge was born August 29, 1970. He attended Fredericktown High School

in Fredericktown, Ohio, and graduated in 1989. Dane then served 5 years in the U.S.

Navy as a photographer. Dane graduated from the University of Florida in 1999. Post

graduation, Dane worked as a biological technician at the United States Geological

Survey working on spawning behaviors of invasive and listed species of minnows. Dane

then enrolled as a graduate student in spring 2004 under the guidance of Dr. Timothy S.

Gross. He focused his work on reproductive effects of organochlorine pesticides on

fathead minnows. Dane received his Master of Science degree from the Department of

Physiological Sciences, College of Veterinary Medicine, at the University of Florida in

May 2006.