<%BANNER%>

Developmental Mortality in American Alligators (Alligator mississippiensis) Exposed to Organochlorine Pesticides

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 E20110114_AAAADC INGEST_TIME 2011-01-14T15:41:33Z PACKAGE UFE0008223_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES
FILE SIZE 73074 DFID F20110114_AABWKL ORIGIN DEPOSITOR PATH rauschenberger_r_Page_162.jpg GLOBAL false PRESERVATION BIT MESSAGE_DIGEST ALGORITHM MD5
a0e756c45a38a3044be1080693d7460a
SHA-1
82bac1be0d7a85114bb13fb8b1de1562a7360255
1946 F20110114_AABVAH rauschenberger_r_Page_034.txt
91ec9ac254d033cf837c35be33e989d7
99d6ff3f893e14bc0a446fd6207ce938bae36793
23371 F20110114_AABUQZ rauschenberger_r_Page_160.QC.jpg
6dde6346f9a73846b39c0bd81478be67
c73b7e2f7c6e69fbd1fe6ca57add7747f80ed1ae
73186 F20110114_AABVNS rauschenberger_r_Page_179.jpg
4e71c8e56403fabfaeb14cfd55eae02c
4e64367c86a437cc8d209be2afa6e1334d84237a
73910 F20110114_AABWKM rauschenberger_r_Page_165.jpg
4b231823546d580de035408da73a69e9
46c78d1332b5b0bbc6f2265f39f59ef5d1043d94
1053954 F20110114_AABVAI rauschenberger_r_Page_031.tif
ab7494c68ea1c1d00601179ab7c22e40
38aff1f52cb4b9d67baa07821c0c17468f947d17
F20110114_AABVNT rauschenberger_r_Page_214.tif
663231579f0d0430243b862efaba1911
b771368678b0efaecbfb811a375c987a49fe8b4f
70826 F20110114_AABWKN rauschenberger_r_Page_166.jpg
96249bb89e8431edb283f62df37a692e
63f7950f4330e3965bf4a6cad3a3f3613e39fe57
103829 F20110114_AABVAJ rauschenberger_r_Page_144.jp2
ee60ff4699822ea4a3e002e53be7b142
d2a01079049da2d7181989bb1cbc2165a668de11
F20110114_AABVNU rauschenberger_r_Page_025.tif
d9e18d43916f85bc6885ef9d33a6f18c
03eeb22b60fe82f21e7c4246dde39c44bc06e6c2
69485 F20110114_AABWKO rauschenberger_r_Page_170.jpg
85a6ec544bea508cd5fc9eda8a31d695
f5e36188087c9285bcc53a67a4c25fad9ee2366b
F20110114_AABVAK rauschenberger_r_Page_215.tif
efa684d5d0e37560e677c4513dc39833
ffc5f83148140e034a798599946274f87340e0c0
82913 F20110114_AABVNV rauschenberger_r_Page_061.jp2
8e7b303183b89a44ccd367fc1b9e2200
748b406203f4c7612a290abb3a9fb138b2d8de05
73588 F20110114_AABWKP rauschenberger_r_Page_181.jpg
4fec7d2c8575ff54e19d80e8a9c5f53c
bbdec24b089395b8f16749310a9e693ac0d7a9aa
90852 F20110114_AABVAL rauschenberger_r_Page_017.jp2
c5735ae4b269938be31c47974b43dbd4
d918a7d2d3f1827d4be5180536de2978141d1975
37928 F20110114_AABVNW rauschenberger_r_Page_183.jp2
a089aea653c1fb73a9fc0531ac12dfdd
57245cfd50fd927e64be24be4e202b2625937dee
48473 F20110114_AABWKQ rauschenberger_r_Page_182.jpg
1bf39b4a0404082c29e64ba8d40ba0c3
d9864f951b45397e248667a48c852202e118a98a
2950 F20110114_AABVAM rauschenberger_r_Page_158thm.jpg
fc549e71f4c9fe3ff11b353563b86152
3655dc8b8061543980b07f6d5ce7bdbb2f03b38f
24576 F20110114_AABVNX rauschenberger_r_Page_083.QC.jpg
babbacdc837a57b103569d729301da2a
d05a932f61dad3941ef7a3a91367fc42a8ab46b3
68641 F20110114_AABWKR rauschenberger_r_Page_187.jpg
cd01bc50182892c1c06ae5c94ffdf6b3
0989d4500f9bbd9b168e623c2511d27c3692e6cf
74397 F20110114_AABVAN rauschenberger_r_Page_086.jpg
c6ad8ce63584bf7081a62eb4e52282ac
aefe6bcdaa17c880520d0158ad99312289d8a197
6743 F20110114_AABVNY rauschenberger_r_Page_048thm.jpg
fa3dbee6b60d3def842ec2befe8b952a
6aed15d655d088c8db780074d5177c53a1cac4ed
20873 F20110114_AABWKS rauschenberger_r_Page_191.jpg
7714bcc259a1c31045681046b91fdcce
bf0846ad13b8dc0df00a8ef7db94b8e6ea821d62
22510 F20110114_AABVAO rauschenberger_r_Page_171.QC.jpg
853d24c581cd6c84369349dc7e85f960
1ce54f24d2b7fd14de73486b232be42044c0629d
F20110114_AABVNZ rauschenberger_r_Page_158.tif
20c66c3442292b505a372b15cb62f3af
68e8ac0ace818727ee3620e6e226be257dd9c196
22519 F20110114_AABWKT rauschenberger_r_Page_194.jpg
7c2f43b5291de0b07d452f023e3a2eb9
7f5fa1be73c4072d74c9cc64fd02f1bd327778e7
9339 F20110114_AABVAP rauschenberger_r_Page_113.QC.jpg
417ee1a634c610bd34269df61e2ab863
29350778337472fb639d94d83dc54f6cb6e02b38
72198 F20110114_AABWKU rauschenberger_r_Page_199.jpg
248acf483c0cd17977286ea37aeabb78
149fd0f8cd21004ae08e4d12929c9f0da29d17f3
11128 F20110114_AABUWA rauschenberger_r_Page_060.QC.jpg
112b2aabf3143f2f60d5ea8065a3ff54
70e70d0e34af1723e18584bd1850113b84be4acc
98334 F20110114_AABVAQ rauschenberger_r_Page_008.pro
e3a0eca7883e972b60959be481cfd6fe
34f3e4418358494684a74d9cf1a19517b1782577
68009 F20110114_AABWKV rauschenberger_r_Page_203.jpg
ceb47cf1dfb7febd4c3b2eb4b3c0cdc4
849df185d056d4cd9ca667a4b8a79e43e8277797
1051956 F20110114_AABUWB rauschenberger_r_Page_007.jp2
66dfb59ddc5c1084091f35e26f9d1ebd
e31543c2c0971ee1fb4c2f29d791268e8eef62d8
90496 F20110114_AABVAR rauschenberger_r_Page_229.jpg
2c3ee72d7960fda934e16bd22435893c
2724743cf0b5a3952cd2bc1cdab92db7026a30eb
75884 F20110114_AABWKW rauschenberger_r_Page_206.jpg
74b3c09a159feeafa8c6575b242c7804
22da33a33b3544d0f9f0e54575c94486e9b1f19f
F20110114_AABUWC rauschenberger_r_Page_002.tif
ce89b9393f711ab22151c2a80cc39124
6c2f1ea04ab33faad600dd7f3dce55a00e9844d9
6837 F20110114_AABVAS rauschenberger_r_Page_052thm.jpg
7a8492aabaecb0c597d12dc35a8f3dee
0f411681ebcb294a1f790c9bd9787de415fec233
62300 F20110114_AABWKX rauschenberger_r_Page_208.jpg
eac2bcbea48669ba75d2d125a9e1df20
5ba97a63e0c6d219a266980e5f0de646d40768f3
F20110114_AABUWD rauschenberger_r_Page_220.tif
33b5d861e0f5435ed6505739fb77e9e4
2401e32ea9ba7329c168ca6cb75f3f98b42a1b00
6668 F20110114_AABVAT rauschenberger_r_Page_106thm.jpg
180bb9c106a3ce2d26b737765971a0f8
d8d5fffad9ca9393bbf349995022e4e3880eeb25
73682 F20110114_AABWKY rauschenberger_r_Page_214.jpg
5e57282ea97e1a618777649a205ae71e
18612fc536856b1ad4531476387e933ee1d3b168
1054428 F20110114_AABUWE rauschenberger_r_Page_138.tif
29f1c97e5272dc9f8cb4e34227bd44d1
d7de26ba94c16b039ce547d0f9f8ef6950ceab88
6131 F20110114_AABVAU rauschenberger_r_Page_039thm.jpg
dc5395a4dce0228c1cb0001d4d7df220
9118d702162ad485badbe37a4d61b785c3c2659d
72016 F20110114_AABWKZ rauschenberger_r_Page_216.jpg
972fda8cebf1c261895074fc553e0b54
9c4580d09e9fd7579bb9df290699dad64e6fdeda
74939 F20110114_AABUWF rauschenberger_r_Page_142.jp2
74f51ea1c3a65e1d3e478a254c970ebc
cef5db16cc879a846fb8727e33a6838b461adc0c
1949 F20110114_AABUWG rauschenberger_r_Page_215.txt
ad493d43211b04c14856cad412f3c859
8487906448344c6a8a9c9ca17a7e2140eada2e93
74431 F20110114_AABVAV rauschenberger_r_Page_119.jpg
4410342e226cc708d6b2fe834ea3af1b
3ca2897a4671b131907b6a208fe005a49232bd02
27786 F20110114_AABUWH rauschenberger_r_Page_013.QC.jpg
5576d6ae8d41f42333a26ff3d8f6a371
6d4583f91094783d8d4ea7a6de72716a13a7a048
F20110114_AABVAW rauschenberger_r_Page_086.tif
01fd29dee6160afd7aecbfc859a033ca
3cfe2aed77091eacd13a1eeaed0137081b065c22
70719 F20110114_AABVTA rauschenberger_r_Page_178.jpg
7873925339152c32d4eee6557c2a8dfc
41a559cb5d117c6c96b91169b7e6f08ed5d537f7
48478 F20110114_AABUWI rauschenberger_r_Page_062.jp2
d3180ad3d3b476f258f5c184d6c243b8
2f919b831dfc9d1317450051e23d7f6781d031fa
1894 F20110114_AABVAX rauschenberger_r_Page_105.txt
e5c6e5e08ded16a9a425e02ceeef2f04
ba0ab943769eda4f3468d046c5ebfe1c26e4d3df
2072 F20110114_AABVTB rauschenberger_r_Page_086.txt
75ab2e4c33b6e617ea070279aa00fbe4
eb14d6a1aa6b89b0724a95da07e9f75687dfd9d6
48113 F20110114_AABUWJ rauschenberger_r_Page_189.jpg
6f98b9910e758d24c89ef44284496b7d
f4c4fcc9d945184971153d931d01bbe3faebc999
73274 F20110114_AABVAY rauschenberger_r_Page_081.jpg
c68db5c88255c8ff04489f3fc402c6b6
7307debafade7b244a0b0162141cc307b3f9ed36
22438 F20110114_AABVTC rauschenberger_r_Page_041.QC.jpg
e7c132ae77200fe754e5f972b7575d4b
cc2c29fc15180eef60b60425e2e2050ee96ce3b7
108762 F20110114_AABUWK rauschenberger_r_Page_108.jp2
4bfc7f0107fb743b671acaee5cbeb9e4
34c520e2890100ea2953ea475a6157e21155c4b6
46929 F20110114_AABVAZ rauschenberger_r_Page_123.pro
be3592f5343badcc48f9df1d59fb7a0b
df445425d9740679dff2fd994e21b9e1e433fceb
48488 F20110114_AABVTD rauschenberger_r_Page_133.pro
9190b418c49ee3ba83b9f00be63514b3
c2d19e6446ef0f3a9edf490961c5e7528404863e
7960 F20110114_AABUWL rauschenberger_r_Page_067.QC.jpg
8dc322e5af916d817b8e08e2e9aed997
750dff2171ceda227e35872d7cc3b12a13b8c0ce
21283 F20110114_AABVTE rauschenberger_r_Page_181.QC.jpg
a7f2058d8ed8a126b7509f86f452a6b1
94e50069b0f65b5ad4557675983c577546651055
107130 F20110114_AABVTF rauschenberger_r_Page_044.jp2
5351869b6391cfea8b4f65d195a21bea
8a62963ad665a10e588630abf6d2e1cd7b85359a
112724 F20110114_AABUWM rauschenberger_r_Page_164.jp2
1b7588d7fc10f0fd059ed3364f20c822
208030c470544815064882a86064e571bf940d43
41213 F20110114_AABVTG rauschenberger_r_Page_017.pro
bc114c181602b4435a0ebcc6b71d0250
caad591b6ab2724b4a96c4dec9e3eedbce7dc97a
48682 F20110114_AABWQA rauschenberger_r_Page_168.pro
83a85ab5b903839957fda8782f2cc9f2
c73787dfb19086d620f0ea1f1e7bfb872936a8af
F20110114_AABUWN rauschenberger_r_Page_187.tif
dc6712e50d3557498b1188344df6e060
0c7d42b71b4c56f694e671b1d17ccccda43fcae3
2026 F20110114_AABVTH rauschenberger_r_Page_205.txt
7c12711c327dbaf2463672210a5f89ae
9324efb3e38ef2937ff982fda7139ea6132232c6
48653 F20110114_AABWQB rauschenberger_r_Page_174.pro
e9b0abe1276cb121c95d682104ad8974
a0e89d39bc4b3e3ec533fa472271a496280f20f7
F20110114_AABUWO rauschenberger_r_Page_023.tif
55480dce2b042bc17f1b2ba19ccd27b7
cd0cf53de6ccbc0406a958cd53a6b9fe54c2bcf5
50573 F20110114_AABWQC rauschenberger_r_Page_177.pro
b27a3a27b1b69021f32a0554675db579
74e4484d207664d04598db8a46f9de0f789987cc
7904 F20110114_AABUWP rauschenberger_r_Page_156.QC.jpg
0a586bf1785221ad182318ba5052553b
ec5856b7dc13d62b9431532069cbcbaa23098cd2
49482 F20110114_AABVTI rauschenberger_r_Page_033.pro
a563ed555352b1e8d2c29ddd0ab352e2
c39b6fd83d6f5ecc53314202a9b50930aec6f128
40170 F20110114_AABWQD rauschenberger_r_Page_182.pro
39d5b4eb9525f8b82335d94bee53fc37
f8c03fcb5f6d7c1bf347b044d8e8dca1fdc97bdc
5606 F20110114_AABUWQ rauschenberger_r_Page_017thm.jpg
c327408219cf5d454891b2608f646462
34307c1c20b73cda91b167015e6923da0b2e0eb4
6366 F20110114_AABVTJ rauschenberger_r_Page_107thm.jpg
0fe79b6c3e13cafef60e7dc5106fc018
f4f013d6587e669d77e70895ac2161689f336cd0
108888 F20110114_AABUWR rauschenberger_r_Page_103.jp2
8ba6b31108d2ac6cb198846006e933be
23213df064abd0412bac4e1a440b6e535a60fcd1
22976 F20110114_AABVTK rauschenberger_r_Page_116.QC.jpg
8e8b9bf3c9b5bdbb11709d4d22c87e9b
f38cf0dacd44dc47ce27c68af30639ed539424c4
26282 F20110114_AABWQE rauschenberger_r_Page_185.pro
4784515efe5d3328f92c9450eab283cc
9378b0fa1c2dc8cf9bae60982e808c5656dae7f7
22909 F20110114_AABVGA rauschenberger_r_Page_166.QC.jpg
53279a5c5ec445ebaa3729d3686fdaa7
89767d3f18eda389def4745c7dd6b21249c82de0
22262 F20110114_AABUWS rauschenberger_r_Page_096.QC.jpg
12dbc05a81e71827253e1ff121c0da71
118925dca5ce81c8fb18f6169d7d294a6545ef07
3515 F20110114_AABVTL rauschenberger_r_Page_061thm.jpg
d4845fa0cf01025f424059d103fe159d
a6d7a7bf26db378f2bb697eed3c4ffe6bdb254cc
7891 F20110114_AABWQF rauschenberger_r_Page_197.pro
3e2de50b7d48f33d116a1fae4f94915d
a65b23aff8a263083ddadb451e137df872577f97
11004 F20110114_AABVGB rauschenberger_r_Page_155.pro
c094fa0d4860e33d5ad42ef22dd474a2
d1726a83a71c839c194d756166370c9a812f922b
F20110114_AABUWT rauschenberger_r_Page_113.tif
34272eed6fcf745301974d45cf45aec3
ab3c18ee104bfcd8768f225c949e5397d3415026
22543 F20110114_AABVTM rauschenberger_r_Page_033.QC.jpg
bd702f5ab8a16af241bd5b1ed0888020
c3c4abdf09cbe1a1edc82c61008ebe5143a369d7
41918 F20110114_AABWQG rauschenberger_r_Page_198.pro
40ff798b928e83c08eeeaf4b65c97598
5cfa6ef9493061724bd5baf51e1d9be7d236b39d
74694 F20110114_AABVGC rauschenberger_r_Page_221.jpg
dbd7b90875a9b0d191832553cacadf15
e0bf1e98793e92ea496d19815fab542a655eb4f2
6542 F20110114_AABUWU rauschenberger_r_Page_214thm.jpg
2d939ba102c896194e4e9081dd602e58
dd8cf65c5cf80b801d6255e4ae2e1794199d258a
1988 F20110114_AABVTN rauschenberger_r_Page_222.txt
5d90d8dcd75768877aacbf7ba8461792
5d9feaf86cc9edede19e400ad534115dcfc831ea
51818 F20110114_AABWQH rauschenberger_r_Page_201.pro
3920de9b7b09563123b2be799b5eb890
3d7c9da7e5536699202575a61f5a8763d6f263e4
67922 F20110114_AABVGD rauschenberger_r_Page_046.jpg
05d8b9fe19dc97b9b4cf33a6d465a4c5
5fa63385d4dfae1f09914c267f253a6b8d10cdad
6742 F20110114_AABUWV rauschenberger_r_Page_083thm.jpg
e062aabd489df635b24fac25052b3051
0f085dfc3da5fe6f9609b118c4d1044d5aa118f9
70835 F20110114_AABVTO rauschenberger_r_Page_213.jpg
c55406d89e205a95b1f7207c003bb109
3800a0d298572994864c7e14712292cbcba9df02
53029 F20110114_AABWQI rauschenberger_r_Page_206.pro
2940044122e11abdbcdb0eeb7b069dfc
db442a54ebda650f4563b950c0b0db8cf55d2022
70419 F20110114_AABVGE rauschenberger_r_Page_174.jpg
16654790b793ef4b61f79f86370964e1
56b1ede9ce3744cdac647ddbdaba7e017bd62889
45549 F20110114_AABUWW rauschenberger_r_Page_115.pro
a5eb4ad55b22275a60ae60f52a327636
5e8ffdea882b128d04f19af94bfb8d2821e98462
33728 F20110114_AABVTP rauschenberger_r_Page_189.pro
b9d714f665c0b4fe90e659689ca03698
cddcdbe7c3c014c0df85dd0b561368be5c739c3f
49947 F20110114_AABWQJ rauschenberger_r_Page_217.pro
25aecf8d2f0e444b39815c1d84cd0d6f
138054fdb29bd3d52f0ac9e68046dcc811532170
2132 F20110114_AABVGF rauschenberger_r_Page_083.txt
9d02daefb7bbee2f498c11c722b8616a
0a9afedaee9ae4fbf5a0b53d6ae4b07671b5d380
F20110114_AABUWX rauschenberger_r_Page_097.tif
f9577410621fff306ff63e799199b020
a98d5ed553d1e94b2a61e7cca1a3bebeed047205
12229 F20110114_AABVTQ rauschenberger_r_Page_059.QC.jpg
2b5b1f415e590da176bf806a98866efc
d9e4201f7fc9e968a23f46332342e8e77f30532f
61975 F20110114_AABWQK rauschenberger_r_Page_230.pro
754041c1cc82f7482c9f81bba9f99c52
999a245cab70d7d0619f9c597d9481be8a80ae94
24148 F20110114_AABVGG rauschenberger_r_Page_054.QC.jpg
ace68008ee97f83e7a1d1d8a08f2bb0d
1a197037bce939925867aab5b04511dce705ca3b
86663 F20110114_AABUWY rauschenberger_r_Page_208.jp2
93803831d73ec81e5c17de9f179de731
6e53cd5a61b72cd4302297b4103fbb0d44c44c4b
2497 F20110114_AABVTR rauschenberger_r_Page_143thm.jpg
b41921e107cec2ae2d72ff8ca6678c79
cba8c62761a5bc8e044ee8122a0fdb0d77f32fa7
33827 F20110114_AABWQL rauschenberger_r_Page_233.pro
d1632aa1df50f38ec5d6980a8fc7e3db
0914adcbbeeb26052aa1bd3d26bf315271cc75d8
114888 F20110114_AABVGH rauschenberger_r_Page_086.jp2
bb0309dbd6f8a95353166e69c3d2d721
aa279c20c22db0514e2b8827ab3735b9f13ccd19
71303 F20110114_AABUWZ rauschenberger_r_Page_103.jpg
0e62d2098613c4bed46940678599ccc5
e414df9ec007a442e1ae1eb48878cd0963666ee6
4704 F20110114_AABWDA rauschenberger_r_Page_007.txt
69abb0aa628f73792cd11f9210a691db
504c6a49b7661649efd364077ab45078a2dba119
6307 F20110114_AABVTS rauschenberger_r_Page_025thm.jpg
fa3365f216d4fc54dc73246c8f753260
eb27bc20388100628bd5c6a36dce21bc63ed9875
136 F20110114_AABWQM rauschenberger_r_Page_002.txt
d22950e9121cc4ddfd0e478726adc842
0c4c18aef637e294df570eadd424339577de90c7
F20110114_AABVGI rauschenberger_r_Page_211.tif
de344cd05c5b30f971b43780f9b070f9
83dbd6753e49385a56d41317025950e7dfc2080d
72354 F20110114_AABWDB rauschenberger_r_Page_104.jpg
0e4759c61789ce613f7111b6d4285215
6e595ecf98a01846625554f022c94417e5c34a9b
64256 F20110114_AABVTT rauschenberger_r_Page_225.pro
267ac395737287504a06ba1c3443fd51
4f148949db64c68698911f03789756e23ff810f7
3966 F20110114_AABWQN rauschenberger_r_Page_008.txt
aa602412791051bc9b4954a620f470c8
ad1072ff40af5de06e433024bbbcb3db91ae476d
2860 F20110114_AABVGJ rauschenberger_r_Page_209thm.jpg
9dfca454e4be95753059a48bbedacd78
c463527e13fab5b687706b6da58728fd658fd424
28403 F20110114_AABWDC rauschenberger_r_Page_010.QC.jpg
684fc3b6d784a34230ad3c4106e6b332
d10529ac2d95bb628cc37430b20987d8f9e5aefa
23136 F20110114_AABVTU rauschenberger_r_Page_105.QC.jpg
06717dfbd14c85b93ba453bd7400206a
8274087ce534079a153770add77a83e169b7b112
3378 F20110114_AABWQO rauschenberger_r_Page_013.txt
a7215929c7674a5593c2e8eb960b33ff
9f2149d8899f93bc8d7cdd6f1d2b7410930963ea
38386 F20110114_AABVGK rauschenberger_r_Page_063.jp2
7f68046cae989d5f48bd96708cf4d20d
dd2b694e602c3bef4af7f61b0e85c2e32c729241
42465 F20110114_AABWDD rauschenberger_r_Page_169.pro
13aea0e15e6d2a2dfb01ba918589bbc8
43c4b877a520877ae501129e122ccee56ef8ab19
49662 F20110114_AABVTV rauschenberger_r_Page_029.pro
f3199603d40d157fafec96b2ebc672d6
317adc9d4f29cb2268d0906dc428f0600af143f2
201 F20110114_AABWQP rauschenberger_r_Page_014.txt
58f67baba9b284e96fdc5f195787dd31
58d70326f58b9545fe5bbb574579d308718fb111
1051934 F20110114_AABVGL rauschenberger_r_Page_013.jp2
1b5928f6c92bcd320a1d67005943d5bf
8aabfa54b609d03a1d8dbc034ec1328602c7ca4a
F20110114_AABWDE rauschenberger_r_Page_019.tif
a88cfdf9a4bf5f5083cb3fcfcf70908d
753f8a9c31b1fbf6b60198e7d54261544cdd1f58
45042 F20110114_AABVTW rauschenberger_r_Page_039.pro
bc7609c12b60d4de9b9ba91ba31cdcac
f82ffa56e127ab6604e03b19b13554ca7f99a064
2095 F20110114_AABWQQ rauschenberger_r_Page_019.txt
bc193e33915e762b8e8f7e462c004572
1aa1982b1bc995dbaafdfdebb179459b6bb1ec86
23125 F20110114_AABVGM rauschenberger_r_Page_218.QC.jpg
d80080e2f69856d41a8f80dffd8a6478
d9506f4507bff46cda370f5f608d58a6e3d2e6d5
F20110114_AABWDF rauschenberger_r_Page_203.tif
a52fce0a77d5662abdd00db83cc423ef
4f50d4f821e2ec4c7bdb28152712ff5e8e51dd18
51223 F20110114_AABVTX rauschenberger_r_Page_144.pro
85998ec9e94c55f85050db857fcd9334
5c67db6717ea0d23bbb16c8b294d03e80fa24b39
1930 F20110114_AABWQR rauschenberger_r_Page_025.txt
df50a0a67142c2e08f7fa2d8f3ad9f42
89a79d920997e209fa1a8d42e347132554ac3983
F20110114_AABVGN rauschenberger_r_Page_018.tif
8de76ce1d2623ae30852edbddcc69ddb
a071c0f2a89ffd4d7249694c306ca004f061bd85
17421 F20110114_AABWDG rauschenberger_r_Page_232.QC.jpg
b5424e195cc0e1918fa575747e0b3759
b4ab805341ed856c45c9369aef0f37c3c8226625
1835 F20110114_AABVTY rauschenberger_r_Page_004.txt
2bc55300e81a2bbc8fe73b94c5a7ef55
0e7261f03274602b7d5bca0070e880c4d0707b66
76098 F20110114_AABVGO rauschenberger_r_Page_147.jp2
4bf9aefe5007a8d136d1be51531943b9
9794af0d20e9b0066072562faefbfdeaa3be9918
481 F20110114_AABWDH rauschenberger_r_Page_001.txt
428ee306fafa83ff721a727c39956d8d
dc6fde64608eb9907eedbd09f212f8011878bd95
6526 F20110114_AABVTZ rauschenberger_r_Page_121thm.jpg
2d207eef951d8ca52001d5d044c3e6ec
d359dce8db6a168c290994e60ec99ec078b35692
1895 F20110114_AABWQS rauschenberger_r_Page_035.txt
4e4b43fd805330d682af1a970ed1b748
c56aff69d8d72aee6c540c2907731afdcce7e092
6458 F20110114_AABVGP rauschenberger_r_Page_029thm.jpg
2200f4612c56a31ad307c3b0598fcba2
cf22273c32753413c6742020fcdb7343310ca08a
2436 F20110114_AABWDI rauschenberger_r_Page_037thm.jpg
0bc7489db75d0779a220fd4cdcd3bdef
93d996f53db91da3c39674ba504f78e2fb290246
1913 F20110114_AABWQT rauschenberger_r_Page_043.txt
6e524b769fb73fea3b6529dc3b156041
ae09326974c73dae645f660c7cc449464384ce41
38913 F20110114_AABVGQ rauschenberger_r_Page_015.pro
369b82dc5b5ca5c7ba665a7367d2f6a2
0d450ccb62d415f832d0daaad45a923cb8bb14f8
57757 F20110114_AABWDJ rauschenberger_r_Page_232.jpg
2cf885327d7f0c5284610e949ffd486b
ad6fff538e1d30adde0f7fdee061329b3375f595
2062 F20110114_AABWQU rauschenberger_r_Page_048.txt
dfa49cefff27619e9949a6e326dff0e3
ab98771cf4cec4cdb34e0f03b107abd164ab5efb
7801 F20110114_AABVGR rauschenberger_r_Page_157.QC.jpg
062ac64aae591bac0872bd38b913eb4d
fd7e59d23de6319b33f856d79aa8e1b8a121cdb8
2727 F20110114_AABWDK rauschenberger_r_Page_068thm.jpg
5cf88de05c122ad791f6c6f6a07875c8
98ef37390c17eadf45b755883f34b59f8a7226cf
2019 F20110114_AABWQV rauschenberger_r_Page_054.txt
10817a41be6380f31f99be6c6d128d98
1e2ae2e3bf4fc843ee7c620c571e96bce4639c46
74333 F20110114_AABVGS rauschenberger_r_Page_129.jpg
095cb634ba6d73f78ca04870a6588127
e445e6080e50dd68b2f964210bd1d8492c671b33
F20110114_AABWDL rauschenberger_r_Page_103.tif
109788ef012a9573d150b69440bb3a4d
e12bc9b68d1860e03c44531e32be6019758336c5
1331 F20110114_AABWQW rauschenberger_r_Page_058.txt
6799629a5f4742fb63c585e5a7e4e8d4
1236d7efe3936cecd0b1057348e7a85bee89535b
6594 F20110114_AABVGT rauschenberger_r_Page_128thm.jpg
4135868ec4dc7a715787817f69a9a500
f7c75c261103c4842ab27b4af07f9cf7316fece2
73066 F20110114_AABWDM rauschenberger_r_Page_047.jpg
d2569d5e166fb884d89387454fd8d65d
d4363b4530f0a62153f787ebbbe3751fbdb8c193
2024 F20110114_AABWQX rauschenberger_r_Page_072.txt
50ae79faec95b31c4646cee0031e86e6
1fe1bb39d947211789414152e30fe344e10a9dcb
8059 F20110114_AABVGU rauschenberger_r_Page_068.QC.jpg
ed4bd6d83a4b69f957e020d3a728412e
aad54041e79b834dc469a692da236b92eedd7406
5981 F20110114_AABWDN rauschenberger_r_Page_012thm.jpg
bc28d98ae58a26107c3c3c8119aa26e3
1c43c6123a81813b3f8f2d940c171583cea25648
1997 F20110114_AABWQY rauschenberger_r_Page_074.txt
03aefd870644e6ac7d65b1c7f7ddc85f
a358f6f858f83ee7a0fddc025381a6c029ad9dca
109797 F20110114_AABVGV rauschenberger_r_Page_084.jp2
aeee6de6f8be054b01ba138637992975
089ab5e6c3b7f97ace7a1e800944611101c45874
6413 F20110114_AABWDO rauschenberger_r_Page_173thm.jpg
6c71dc36cae166a29f7b552412b1650c
117553f41aff8270641afecba5df033a0ce705cc
1935 F20110114_AABWQZ rauschenberger_r_Page_075.txt
bfb6b96bc168a29938845063b6978001
973b306ece3f3968782b373e18c26effcc37afe9
37933 F20110114_AABVGW rauschenberger_r_Page_142.pro
e71c50b12d7b928a500ef29a360ecaa8
d4d0afb293bb42bfe7d92c49c8d8ef9fbd067276
52233 F20110114_AABWDP rauschenberger_r_Page_052.pro
4e234d42a774114906e6ab4fdef597a5
476511f07dc0d3635265c211311cc4a268187c3a
755 F20110114_AABVGX rauschenberger_r_Page_093.txt
660a8ff20394c875b8393ca7fda4b483
ba41ce923ea3ce06e46c33440929407ad5025a8b
72559 F20110114_AABVZA rauschenberger_r_Page_018.jpg
dcb87e8ef3daa72f2a91d0fbdcf95390
156191e349e72ca2c38c2c117c10e7d514ba0bb0
24178 F20110114_AABWDQ rauschenberger_r_Page_122.QC.jpg
14507396cc4385e451838a6c6821cd90
4c2d261e79f6749689e0f25115617b934735d4d0
108122 F20110114_AABVGY rauschenberger_r_Page_085.jp2
b443b058c3f2ca1478868a83de3b6c9d
3e914b85b812bf957de08230ddd64b6bbfbc6298
25000 F20110114_AABVZB rauschenberger_r_Page_156.jpg
870364dd0f0df95bd397325f2702a5f6
2f10f97e77f6fcdcbdaa8e0101e337250451df39
F20110114_AABWDR rauschenberger_r_Page_135.txt
7c7afc9fb76e7d9011d9425a2fa28ee8
fe186506adc99d09cb916b383301661f111270f2
17999 F20110114_AABVGZ rauschenberger_r_Page_113.pro
abbdf9ba28570c3c7f18155c7af2ddec
38842031c285119d16cb90d42e60e7158f826ddb
15790 F20110114_AABVZC rauschenberger_r_Page_210.QC.jpg
eaa294a8667ea22709dff202699e7c02
a5ddd98c3bcbb61cd891c40cbe7210d23a0889f1
1881 F20110114_AABWDS rauschenberger_r_Page_027.txt
aaf020cecbac23b569e553ef2e96ccf4
68a8c701f09aa0ce0c3e459b8f252a1cc7ed5049
111713 F20110114_AABVZD rauschenberger_r_Page_072.jp2
ae384004b936f8574e6f63a178b07936
49b3dd12d83e11ab24f790eca9c7f0b42524187e
129798 F20110114_AABWDT rauschenberger_r_Page_230.jp2
6382185ffb6c397f9ecf6e161bb31f56
74a7f853c73d14ae5c46e3669e0edfac4e438250
1613 F20110114_AABVZE rauschenberger_r_Page_091.txt
927a3576b1e2da6457d44b29fc52d2f7
4342feaed56b2be4c8edec74b14d8f5a563615ca
22534 F20110114_AABWDU rauschenberger_r_Page_173.QC.jpg
ea7dd75a922a9afb361287d8dd7e4f75
e36a0d4d818540d003a047f286b07fc1d285a9ae
14147 F20110114_AABUPA rauschenberger_r_Page_143.pro
a17bf25999968d4b099c22a7b7566ef8
f4e38fde39d2d41009f4487978d870205859205c
8374 F20110114_AABVZF rauschenberger_r_Page_003.pro
6beb13b8fc792f4c25da1ba121bf3d08
434fe740612481a4574faf471bd7cb17cb5dd5f4
47375 F20110114_AABWDV rauschenberger_r_Page_041.pro
c7560ee1229d76ae36b477a40fed335e
0cc4fc8ecffb592e241fc6f53e7618d95bb1859c
106836 F20110114_AABUPB rauschenberger_r_Page_107.jp2
e8d20b4357fac4b05d0dec8f8956f966
c4bfd0dbb36b606dd441d0400ec0eab88dbe993d
F20110114_AABVZG rauschenberger_r_Page_039.tif
a1c5caa017e75e4bd6e0fb17ab29b23c
cc2e27eb38d22fa1b2369a02e3f929dd75d71944
366 F20110114_AABWDW rauschenberger_r_Page_194.txt
9917a38ce612dc2310e372a4b73cdfa8
40952fec32d7cddd79135a3df1109dcf060c61a7
53552 F20110114_AABUPC rauschenberger_r_Page_057.jp2
3cd411abd63991c5575a38df14d59b8d
1ed8c75acb9a74f204374c29e80719e7deee7de6
22370 F20110114_AABVZH rauschenberger_r_Page_009.QC.jpg
bf43c48564f398ae75ac9615d69aef8c
787b23afade9489529992ea49e03c231736edca7
6599 F20110114_AABWWA rauschenberger_r_Page_200thm.jpg
f6614e27848783732d32a279e11768f0
e888d4301507650586a17d531d9c796f70b28c32
102022 F20110114_AABUPD rauschenberger_r_Page_120.jp2
79cb9036bec7d083753425842227def1
0594d12bba789d6231d5022dab016f1454fdbcb9
46169 F20110114_AABVZI rauschenberger_r_Page_016.pro
9cb50b1cf238827ab1671136bd01f234
5265876710691bf5fe79c98317c3b2a323435425
F20110114_AABWDX rauschenberger_r_Page_091.tif
37bc57d6381ffbb03c55d9388440a22b
537c75f21e140b7d12daa8885bac79e23214b4c8
6658 F20110114_AABWWB rauschenberger_r_Page_201thm.jpg
2ab56712fba9c478f3a8fe00a3b64bff
1cf355f7e2b81bbd46ef0ce9330e4755572c5908
F20110114_AABUPE rauschenberger_r_Page_148.tif
aa6cf424f3c667f0317a6e5c19f6410b
ef0a770e655feb4fd2fb42dce6bd9113c7c3ecc7
6699 F20110114_AABVZJ rauschenberger_r_Page_225thm.jpg
dabd9e46947bb793f80745033b1aaadd
47f4d8a6e1e901c6f10cabec4f104e8189d87d7e
F20110114_AABWDY rauschenberger_r_Page_061.tif
aa194026759a81134f84943b12bdf411
f81828af5fb4435476b712fb5e5c5e9c1810be04
6655 F20110114_AABWWC rauschenberger_r_Page_202thm.jpg
9282ccb3159742f8a0f51f1727a85f1c
1a71d687171eacc76b927432049ddd241e7c6dea
107121 F20110114_AABVZK rauschenberger_r_Page_053.jp2
349537097159668895271fe9794a341f
c1fb27019d1b2ac852f8cb9fa1e86ce0fd6d2a96
F20110114_AABWDZ rauschenberger_r_Page_116.tif
30355dd05b826ec1a5273b0c14c48a66
b9f00ba0769bebf8e392147ebe63daa9531e9d8a
6195 F20110114_AABWWD rauschenberger_r_Page_203thm.jpg
8d0000e27539194ee52430fcd4119707
4824179de3f6be41e9d45b9891569c4b262b11b0
F20110114_AABUPF rauschenberger_r_Page_209.tif
a84520a348a6f1aae0f8a08868a61967
6e79da234ea13266abef1573483d975ac29fbcff
F20110114_AABVMA rauschenberger_r_Page_124.tif
05359b835d5f74a97220ae7c05755805
cb85952bcd7a71728c45b399bcd8826fdf0cd29d
F20110114_AABVZL rauschenberger_r_Page_050.txt
2fc3ad1a002ead4b18cc4a34e2e2312b
3eda1b2c98a24f90078ecb113a84d3c194689f00
6559 F20110114_AABWWE rauschenberger_r_Page_204thm.jpg
c3dadf297a3d2abb9391aefd87862ea6
fc3eac6882ce95bc8841bde635f2f6f47cad4321
21236 F20110114_AABUPG rauschenberger_r_Page_070.QC.jpg
0c59f42f095b763aaa0c247474c07185
6d48251bc54c262d403a427991c745ff8cab522b
7258 F20110114_AABVZM rauschenberger_r_Page_010thm.jpg
80efd33050963c0a3b6a8f0a61caf50b
3173cf9f3ac56428387080288a1aa2daeac14555
17801 F20110114_AABWWF rauschenberger_r_Page_208.QC.jpg
304ca5110db9499a161698a1de9519db
171db6574c34a09e1e5ecb2d25578b4c64569752
110897 F20110114_AABUPH rauschenberger_r_Page_049.jp2
cca1f0bc014379c6f39e8c6f5087fe9f
de131327ce2f1cc8b490c4cb1335584059e11833
50749 F20110114_AABVMB rauschenberger_r_Page_172.pro
8d7baf5460f0208b0ce90021ed33c3af
726633404de86800ab30ea5f7b69aa65f99699cf
23707 F20110114_AABVZN rauschenberger_r_Page_001.jpg
0d9afdc925661581301851ec4bab7ce9
459fa629ba337c65862215edbc3b891855cdfe80
5579 F20110114_AABWWG rauschenberger_r_Page_208thm.jpg
045c8d91f130f94e7e77f2ec1b7e188c
b18aafeb608c643f9ca898a65dcbab9069f77025
15504 F20110114_AABUPI rauschenberger_r_Page_151.QC.jpg
dbed93d2d955770818c7c73961cefd9e
57f2cf03c8b4e63f1e8cd1ae0d5121d698faeed5
6221 F20110114_AABVMC rauschenberger_r_Page_159thm.jpg
f1f8cc74c2bca1dda123e89c2b148093
4a89e5831fedad44809a9642e3c515497cc5d7b6
5701 F20110114_AABWWH rauschenberger_r_Page_212thm.jpg
7f3a57c48baa3cc925fa8d2e6d300217
a526538d12bd90cb57f9ed79416741c75c20cd09
101599 F20110114_AABUPJ rauschenberger_r_Page_224.jp2
8e27301a338e2d58c6d34f1a75712132
7ab0d51429bb217e99a91e1120853bce4168a829
6262 F20110114_AABVMD rauschenberger_r_Page_032thm.jpg
3bf2bfda7730a51ce9f92ae74e7be2d3
f5cc54c60009052a23fc486baabc6c8e06874767
F20110114_AABVZO rauschenberger_r_Page_071.tif
d338048281e22a49eeacfd57df359edf
f2484e16f1714688203c306a1387f9e9cfa6ae85
23030 F20110114_AABWWI rauschenberger_r_Page_215.QC.jpg
da89bd9d4e9a69b6be39c6aa2ad30eb2
89b877cd45f3fa186c59a78434bfcd9d3b350540
105356 F20110114_AABUPK rauschenberger_r_Page_025.jp2
85e6c9a0910a3412af30d68374fa5272
a89ea88523618d9ee6af220a5225594e787f0005
6252 F20110114_AABVME rauschenberger_r_Page_009thm.jpg
a4567116885a7c3be27bc180f1dd4d26
9f7448e746c043940b76286e113f8981113d9451
2882 F20110114_AABVZP rauschenberger_r_Page_113thm.jpg
2bea9ae5f24a2e1ca0445d52b1f14f00
0d252e76e6e0a3b9ca7825501848e9003416a5be
6492 F20110114_AABWWJ rauschenberger_r_Page_217thm.jpg
6160dfe468e508b49aa03e85e64042a7
843a2b2e737319d9fa848af917e3cbec4c60e4aa
2175 F20110114_AABUPL rauschenberger_r_Page_146.txt
fdfdb3ee98453403f63374499d96c6a2
5f1b103d9f6413c38244dda1a0ad5b0ae3784bd8
39926 F20110114_AABVMF rauschenberger_r_Page_208.pro
22ca4ccc3a4e32739480ac558d7c55a2
f5bc1bbbc32eb36ace09a575f0c0562d65c70da5
F20110114_AABVZQ rauschenberger_r_Page_069.tif
ef96b4674ee6243b4808ecb6fe138efb
d745c22cc562556713e53481712bf0fa4932189e
71452 F20110114_AABUPM rauschenberger_r_Page_049.jpg
c7a5a1373465f0094b09b931e50f55d8
a1e77000ddab9d65558a98bc5ca1ef816262d145
35591 F20110114_AABVMG rauschenberger_r_Page_151.pro
e9d45b52b57fc8fd5b4450c159d383c2
4300bbd3ba816ed1e193e540b829db62a3cb22ee
22059 F20110114_AABVZR rauschenberger_r_Page_016.QC.jpg
850bb041192b4cba83a72bd2ddd33e27
303ba8a108488a44f7a47261e88ca4f88271c81d
6633 F20110114_AABWWK rauschenberger_r_Page_221thm.jpg
702b978e3d80663ba4ad7d66852afff8
349b638c7338ec7758839ac98a39390879213f0a
2009 F20110114_AABUPN rauschenberger_r_Page_162.txt
930b16a1475174e47dc0cf5506aaa5b1
0328579c539feb0b02099000a0a3babf88cbf35e
F20110114_AABVMH rauschenberger_r_Page_060.tif
d820ea250303e982e6bb1b436000f699
256cfccf75f1415ed0e447a8b5ea7e3c85658ee7
36712 F20110114_AABWJA rauschenberger_r_Page_011.jpg
458964157288139ff95e9afe343bdcfb
db76a9034c17acd35e4cd43052afbde53bd42d81
771 F20110114_AABVZS rauschenberger_r_Page_068.txt
340b70cfa4c275521d58a9ed40f83311
0f67db860f0184714defcf65cd436ca8c6fe828f
3101 F20110114_AABWWL rauschenberger_r_Page_223thm.jpg
d9eeec485831bb185f9a1c47ed6766fd
ed630c708a0f5e488fca2fec6ebe700d6dad724f
83278 F20110114_AABUPO rauschenberger_r_Page_006.pro
218d0aea6688af5b7af35bed3670d7dd
e69494c9e3ac0136f2a226d68238c9898d9c3258
F20110114_AABVMI rauschenberger_r_Page_198.tif
9515e30039e98078e11865440efd3481
cd2c733fd284af1a599b995e8bc8f5acdc712b6d
60618 F20110114_AABWJB rauschenberger_r_Page_015.jpg
3e786bfb76ee0b2752dd59f511ade999
afccdad6ca4644abc31685f4f7523e4173177a5d
6293 F20110114_AABVZT rauschenberger_r_Page_116thm.jpg
c46f66a0e0d632f446b4ec85baddcc26
2ff9ab817b29e06dd43e9e5ffd73e2196f10ed52
20878 F20110114_AABWWM rauschenberger_r_Page_224.QC.jpg
27e42dba8f85b59dbf34f81534a86fd0
6938e102014c08bdd7d4986ded294120a9d964d5
48579 F20110114_AABUPP rauschenberger_r_Page_025.pro
a7256a7c349ba8e056cab8d3e75c1a3f
c5659fd19ae4a5adcc1beeb6069904c737ad5e86
1833 F20110114_AABVMJ rauschenberger_r_Page_039.txt
dbcb7d54f25cdce3ca193a7207125733
61812e8619dcf817caa3f3305dc6251b419c8273
68537 F20110114_AABWJC rauschenberger_r_Page_020.jpg
38f4b8811a18d9872047550dbc1cd4ff
3d520a4c5eb4a94adf688aea25d1ad1f76b88017
112299 F20110114_AABVZU rauschenberger_r_Page_179.jp2
cb104bd8d8cf6625bc23de4aab54aa48
49c61a8638317da678980c0dec79377bd9c9cb2d
7093 F20110114_AABWWN rauschenberger_r_Page_226thm.jpg
cbb7a7c651640bb6075473db5a21848c
a442961846d6f84c343c360d19ae079f98c9c1fe
1978 F20110114_AABUPQ rauschenberger_r_Page_182.txt
3b89c91750cf0075249d9f912703c6c9
4cde5c2379d91eb42026fc3d31b58f833b898628
111576 F20110114_AABVMK rauschenberger_r_Page_102.jp2
d95d039658c2e3c675bde21ffc1b5315
def3a8a4e13ab40ff43c155876cbb6f3f46bf2ac
70683 F20110114_AABWJD rauschenberger_r_Page_021.jpg
157fe8745c45a4ccdbba205b17e68fd1
99ee4ef0b8452cd53f0503fa6955b91bb4320e2e
46836 F20110114_AABVZV rauschenberger_r_Page_020.pro
20a5f76538e1b34f1a69769687abe1cb
16bce71c58dad0398ebc5fddc3e61fbc0e347b52
26391 F20110114_AABWWO rauschenberger_r_Page_227.QC.jpg
6b904e43ed46f5b39e189011ebbf4d64
29ae063cb5ebadae3d9bc7fefd93af8a9a4d383d
67236 F20110114_AABUPR rauschenberger_r_Page_092.jp2
82f5a86876fefa79af761922f582efa4
507e720011dd5447567292708a21b70fa7fa3540
21932 F20110114_AABVML rauschenberger_r_Page_193.jpg
3054fcfe08bd59336dd31e73d29e3004
8dfe468aa27e2573cbf9aa30361077bac2d38807
74155 F20110114_AABWJE rauschenberger_r_Page_024.jpg
2edb21c85cc662e24c3f68c295cbc86c
25ae21b10b13b36bc53e7bd9b9d208af517dcd43
F20110114_AABVZW rauschenberger_r_Page_221.txt
68296beb9f6b03be73ce0afeb1e10a84
19fdf34e8b39bb209f05a3040ce8b1c414cc131d
26295 F20110114_AABWWP rauschenberger_r_Page_228.QC.jpg
c1bc25b56202684154c7771e1a1f5629
c57efed041b1067779d52932d5c864b22840bd64
F20110114_AABUPS rauschenberger_r_Page_099.tif
9ec152c306025b230eec97c12ea60ddf
fbaea5c513c51af8e1a7c118bb67b6ac0e486f22
2136 F20110114_AABVMM rauschenberger_r_Page_080.txt
d008c9a4f8094cb21efceb2fc29b3e71
4cf1b9f74ec5ce9efc3fdd99bd69c9673284280f
67962 F20110114_AABWJF rauschenberger_r_Page_027.jpg
4f2214a1011a1dea675250a84142e2d8
23c87c3a794595717da9c8480018112dcd39632a
1974 F20110114_AABVZX rauschenberger_r_Page_029.txt
57193ade98c8becae99b4f8b97df65d6
73fa1e485e68dc229e10b5f8b2367d8927fc7a3b
6596 F20110114_AABWWQ rauschenberger_r_Page_230thm.jpg
522807ff6ad97632f1775c05fb679da7
67436cf6e54039a23c85277793db3cb35d269dda
69674 F20110114_AABUPT rauschenberger_r_Page_159.jpg
3d0294d3ccc151cfed6c4141a39572c1
98ca0674b91bac87e5660e7ef7d113dad64f6c3c
F20110114_AABVMN rauschenberger_r_Page_201.tif
8a082bd71e8cf1c367eb0651999fa857
8747cb6f7ee0783605a4ade51a2d2f64bebf1e0e
71245 F20110114_AABWJG rauschenberger_r_Page_034.jpg
f6ad87f9b2fedc636ae93dbcf6530450
446d5e2fcea8ee5c076fdaac082b3e658dfa7166
101503 F20110114_AABVZY rauschenberger_r_Page_039.jp2
d26b942d0e050f7082861a2fd8a0499b
2f9aac0010743425dc429fa5a102c266f7a7a1ba
25559 F20110114_AABWWR rauschenberger_r_Page_231.QC.jpg
a784772edfd0bf3fced46821a90800be
37e5b73e82436d0270d5b0a33efa98217b954ba8
114770 F20110114_AABUPU rauschenberger_r_Page_019.jp2
75f55917243f03dcc3de98fd38c252d3
2292325da97c1c6322c42e373fba0501f9dfa597
93529 F20110114_AABVMO rauschenberger_r_Page_228.jpg
6a37f6d44cfb6abe67a43dd0ac4ceadd
7b324ac90a6006b829f00d543bf01d281208c300
74506 F20110114_AABWJH rauschenberger_r_Page_036.jpg
4a81187c159cf00c7970f3cc820dbacd
681efecff1b8192b95f686028cc62772f6c1966f
1866 F20110114_AABVZZ rauschenberger_r_Page_020.txt
025cd124d268e7997a785e8d9973490a
9da4b18f38c7504d31ccf7b06ebf874f46e0b559
276487 F20110114_AABWWS UFE0008223_00001.mets FULL
661eb4c70cef9ed6b6507ff5909cfd6f
0f93f213acd1035bdc18c74ffceedb267a52d293
18271 F20110114_AABUPV rauschenberger_r_Page_183.pro
db02eb52a3d56b9bf5a5b5850d607bee
c40df59db604f12ed4fb2e74b85fe1b35d5efce8
2017 F20110114_AABVMP rauschenberger_r_Page_214.txt
f336e6e52c8f62016c53bf206e8b7f71
14a2e139984da137c0207a7c0fbe9626f4d5cae8
75150 F20110114_AABWJI rauschenberger_r_Page_054.jpg
a3767c4b6ff4083a849351e149e1f01b
8be8c02161e96b0bbf9c02a450fde997342d6fe0
25353 F20110114_AABUPW rauschenberger_r_Page_126.QC.jpg
142bb5059337e2b6a6a1fe70c96583aa
e887bf129db95448dbea0b74da2fea97d22cd8db
6066 F20110114_AABVMQ rauschenberger_r_Page_093.QC.jpg
4ef99e6f05c8895f9ec317b41ba88e4d
372b386ffa5478e263bc4a54a3de9e5f08675fc4
15166 F20110114_AABWJJ rauschenberger_r_Page_056.jpg
61379bc8d8245c8760f82aac1db928c3
a59a38dd753bab2d69178a8975840072a8defc20
886 F20110114_AABUPX rauschenberger_r_Page_156.txt
4d02fb9af53fbddd76d248cd158cf378
7e1827154a0065323fdb368e4d9e317307f05fcf
39024 F20110114_AABVMR rauschenberger_r_Page_038.jpg
2ae69aa8f90de263d5a1fa3ca2a4a773
ec0d700907c131b53d1c4bfdedff99b92a7a225b
38305 F20110114_AABWJK rauschenberger_r_Page_057.jpg
6815fbf5d7e61b736e4d38a98922b7fb
7b223a7ae9c34b2f213b52a81d0ff8b09fdffe0c
23756 F20110114_AABUPY rauschenberger_r_Page_217.QC.jpg
5f3f3b949fb7357eb7b7eb9733d02795
d7b02cd9c1bd9305c98178afdd00a3ed2a32bb8c
70113 F20110114_AABVMS rauschenberger_r_Page_107.jpg
996c2531b978c62e463a13025b349c7c
03b037ccc2de52ae90afaf7c9937c0f7262da6b8
40501 F20110114_AABWJL rauschenberger_r_Page_061.jpg
30d1c4de6e97af3202596ff47762acac
867d9b8f80560357809ce1cedc90771caa7fe07c
5326 F20110114_AABUPZ rauschenberger_r_Page_015thm.jpg
7866299aa99287e6fe0e4e258710938f
792bc03ee529c3781ae163919888f777cb577301
6486 F20110114_AABVMT rauschenberger_r_Page_215thm.jpg
fd15d8c458d723e879d9b658572edaff
5831a9cffdac6b8c908b513bc72bafac7c67d388
26420 F20110114_AABWJM rauschenberger_r_Page_066.jpg
ccab0edb44041583fe4a110f55a4a335
9766278a051149435c0cb16153336ccfad7bcc4f
69664 F20110114_AABVMU rauschenberger_r_Page_022.jpg
d82b985cacb0ec2a27b1dfde62490c63
642b1fbe8c897e755bcaff02a02b087d56942484
25773 F20110114_AABWJN rauschenberger_r_Page_067.jpg
d50535ebdfd41c5ab7372988d442a2c7
42bce1270d3899bb7267c4bf3842322addbde7ce
1906 F20110114_AABVMV rauschenberger_r_Page_171.txt
6f94de6f68d82fb43b683e7fe2d250b2
330d349cc39903a100422cbd360bb4991f9f862f
70572 F20110114_AABWJO rauschenberger_r_Page_071.jpg
090f90dd7be8da90eb980c354279b1d0
dd55007dae8fdd2d086e5fb76ce778df60131f34
25033 F20110114_AABVMW rauschenberger_r_Page_037.jpg
95da539db739740ff5e8f58ad30ff340
ba8eba4b29c8ecb4cac1f8bd9638862f742f78e2
73027 F20110114_AABWJP rauschenberger_r_Page_072.jpg
ab573bf3cf766bf52bc49d93569393a7
00cc4a3f8d01162204175a095383c4be9a933770
F20110114_AABVMX rauschenberger_r_Page_058.tif
2c4c6a41d84543bc2a57b63738de54c2
8873ce5bda0b7efe560ddd03b921cb01593304b7
69410 F20110114_AABWJQ rauschenberger_r_Page_076.jpg
9dd110fc5314bef0fca54f4f78e4bccb
5d00b1956a7c8d8451505e46ba4226ae18daf154
41968 F20110114_AABVMY rauschenberger_r_Page_140.jpg
92a6218693c50c1a0f06073921a40886
863819aa6b62bf5983016b978626e84162cdf137
71747 F20110114_AABWJR rauschenberger_r_Page_078.jpg
56a189c2bc39a8781bd87f4d3e2b3be1
72af9d3ae1888a387fb2443f34bda9f1a72b823c
25271604 F20110114_AABVMZ rauschenberger_r_Page_114.tif
8249f457c52ba2bc56aa7dd5b80b098e
c7445c3ec955a73f272bdf1087518e0d69838d10
54148 F20110114_AABWJS rauschenberger_r_Page_088.jpg
762bd7d32dd608f333f5c9fd05a9d97a
d28ca67698b1a665d32ca90121da4b6eca97d7c9
20547 F20110114_AABWJT rauschenberger_r_Page_093.jpg
d8751ca616d58a5b3d007a0a05d9ab6a
88f1373468390bceb2a30022448f8b3e257032de
69552 F20110114_AABWJU rauschenberger_r_Page_096.jpg
9228cd4188d4ad30fc537272e232d97f
cfdab9cb957ca39636b9902f0cbf37b482574584
2056 F20110114_AABUVA rauschenberger_r_Page_052.txt
bbaaeb432602195b2b9e444841f48441
3b92d62802cb2e11e67da299e7e77acca96796ff
73836 F20110114_AABWJV rauschenberger_r_Page_099.jpg
3d64e72903eec85b4bbb7df67e973762
48af4ae8d78425f516e673fa3dea89049a462470
68896 F20110114_AABUVB rauschenberger_r_Page_044.jpg
b9713c1f32a87bfc3b5af40bfb396feb
fb4de8e63e3fc2cd2f4cf13f7a47f3166eb7df24
71247 F20110114_AABWJW rauschenberger_r_Page_116.jpg
7992c9c4116bde69519b51a4c9e2e0c2
b667bdd35d20a4d30762eeddea88cac69c9a12a4
106844 F20110114_AABUVC rauschenberger_r_Page_173.jp2
28a4e5defcaa20f2526f3eeae8f04235
9a5e66568f358f5f516c9de8b95a24747b951213
73384 F20110114_AABWJX rauschenberger_r_Page_117.jpg
c34116b773c3aa1b382221c5aaeffc3f
815f44dc8547e651afea51c635665477f4dcfeae
2649 F20110114_AABUVD rauschenberger_r_Page_156thm.jpg
64efae0dd3a6fc538079c76a9b261523
ca4c7f2645615b2c6b7eb5836305d179c4b90048
72802 F20110114_AABWJY rauschenberger_r_Page_121.jpg
6a146f06a91b0d729c737636ae780af5
d2cceb4216c00d1fbe3ef53c86ec44a0ec156858
94151 F20110114_AABUVE rauschenberger_r_Page_059.jp2
aa2aec32dc1bbf27a7b9376d7f5105c3
d01b6bd3fa610eb5ea4d5cf549305fe35cd29cf1
73742 F20110114_AABWJZ rauschenberger_r_Page_122.jpg
45160e37705c8e883a7ef86c56bcacc8
5ac9a72f341031503c3ff0eb531e4db319ea530b
103587 F20110114_AABUVF rauschenberger_r_Page_096.jp2
8c03c69d768e6c8b1afcb364e42ef69e
636de0bbcbdde9baecd180fb51df7611a30f1af9
49351 F20110114_AABUVG rauschenberger_r_Page_215.pro
5f1ece31dd406c9e3b99b4543be1c91a
41a5facaaecd1cb7bc0ac2ad68fd444ba4ed08dd
47610 F20110114_AABUVH rauschenberger_r_Page_181.pro
4e580f0fd3d131325ff607590a0ec093
0ab9bdeb4a32342006cbec276e4b7caf74379576
1890 F20110114_AABVSA rauschenberger_r_Page_096.txt
4d258238c1f7e91da4ca1e808e33ab72
474863e45d0923d29bc2e763879b78563a26b462
70903 F20110114_AABUVI rauschenberger_r_Page_168.jpg
ca1a6f1c00a1c5a3dfde59b1a31be084
41f592a04122959176e2a90756c83d741b9e6c4e
110234 F20110114_AABVSB rauschenberger_r_Page_099.jp2
7e15d21185ce8393928fd1310e3b2d65
538f42f0bc513a2754678197e6203d5cb24a97de
66675 F20110114_AABUVJ rauschenberger_r_Page_028.jpg
a166e29331401e6785c996b8ef79b591
9432d700cdfcdcc9017a75a72521bd38b6991bbb
49194 F20110114_AABVSC rauschenberger_r_Page_173.pro
e182be506f219a2db3e925ac42ac7451
7baf236a096a41337328f9fb27dacc6a554a51a9
110741 F20110114_AABUVK rauschenberger_r_Page_104.jp2
f5f83ad805ad0d4b61e3770672a2dab7
975de6382f71b8dc03d5d8ca6686aa74c3d38d4e
23086 F20110114_AABVSD rauschenberger_r_Page_057.pro
70e58a90fa06158f521a4bbaed4aef3b
111d304edbd6636d4e29a789f3b220c28e42a075
107799 F20110114_AABVSE rauschenberger_r_Page_133.jp2
771983e8ecb4719258ea9abca99fe683
41f6fc1017cac5291efe1a9bb75f29bca168ff1b
5800 F20110114_AABUVL rauschenberger_r_Page_008thm.jpg
7b18ae862e30c6bff333e92808760d85
e8e238d8f71fae69ce78ae50ff30f9cd76400688
1333 F20110114_AABVSF rauschenberger_r_Page_147.txt
ea75c4c5a609123e95e43b732e397ffa
2dfb6fdb8acdf1ac9be1035f5d2524ecdfdb0be6
F20110114_AABUVM rauschenberger_r_Page_126.tif
1d9f5d8784ecec944d4aa41176347065
9caa6038b2b836ea40097500396167a9449b9e3e
107322 F20110114_AABVSG rauschenberger_r_Page_029.jp2
a2303d973d6e23f14c21e67c5ac0d1fd
7b0f8fedd3246f41c2d392b5a7e319c5fe12a9ce
52238 F20110114_AABWPA rauschenberger_r_Page_048.pro
6e90f287ab3c6efd65ea360761af32bb
1cb5393e8252626c4de7a0ff7206158777789de9
104914 F20110114_AABUVN rauschenberger_r_Page_118.jp2
e0b30d8d0b67c60d7280176bb11a5327
6c11def4b5e09c61c50cb68fcb8c726b87db8629
36094 F20110114_AABWPB rauschenberger_r_Page_060.pro
22179fc0499efd2ecbf3e6623efb2918
26dcdadb05c68b09e8178efb5a8b120eb6f8c953
24186 F20110114_AABUVO rauschenberger_r_Page_201.QC.jpg
8fb37fd2ceb8bb242c2031373341d5ac
37ffe81959925995448334d7a5afa079d92c0341
74301 F20110114_AABVSH rauschenberger_r_Page_127.jpg
9a0236d7cf749ba34b0000ff7b0afdfa
639fa92cf2ceda8d91131a791f52a82bd205e38c
40506 F20110114_AABWPC rauschenberger_r_Page_061.pro
7642745ca578093524f84fc1131b8414
a12cd0cb23b7b03b52ab078ad5ce1d583782147c
22999 F20110114_AABUVP rauschenberger_r_Page_012.QC.jpg
7ee5f609cf23cf08c9d290f96ff15daa
14984f9e1a54d5b6826eece9ba13664d2cea7289
F20110114_AABVSI rauschenberger_r_Page_155.tif
0bf0ca834e25f3e5a172818ba7089d3f
96faa1981aa1fa5baaac53648a53a9f7f163a314
52578 F20110114_AABUVQ rauschenberger_r_Page_233.jpg
9edd4bf241d9da6691c075f850cd7a0d
7315c73f3205a63a42f4ffdd453d0a084c60d73c
6577 F20110114_AABVSJ rauschenberger_r_Page_044thm.jpg
09a4d78ac29ff9b8f3a00e51fb527bc7
b081163b70c1a9bc143272e3281efa4c80b65a1e
10306 F20110114_AABWPD rauschenberger_r_Page_066.pro
7c8cdeae8c9239b616a94307868ac964
b203825785161e314d1007b41eae0083a17ba2bb
F20110114_AABUVR rauschenberger_r_Page_219.tif
3a9b92dba7fb7abc2ff7a4768c9c14f9
13b1c6579bf41367c70b5cb2c8bb925d01ff19bb
618 F20110114_AABVSK rauschenberger_r_Page_143.txt
698e95471420f84c2d28b26e85b7bb36
737ac4a762070423d01a29f51e7af88927f41276
9485 F20110114_AABWPE rauschenberger_r_Page_067.pro
34cd700c72adae0c91b999b39945cc58
433c474358f5a14c41129670fde25f1346c8edd0
14975 F20110114_AABVFA rauschenberger_r_Page_156.pro
6b07c3d8501bbe3fcecede5ce64ad199
527063a7a9d8506d4d00e934d5580cddc3a2a569
F20110114_AABUVS rauschenberger_r_Page_199.tif
2fc28f715b3a1a98b1d9c2936d52fd7e
7c9df181836421526b48358d89a45ecf903a4fc5
1953 F20110114_AABVSL rauschenberger_r_Page_049.txt
45e13f071a41925acfcb7c26e6302910
864a9dc95933d49884fbea2daa1b451894a8d5cf
9923 F20110114_AABWPF rauschenberger_r_Page_069.pro
4755d2dc07d5d68be508b77bd3d24e52
d0e199a55540fab72bf130ce65c56d88f5152c71
25930 F20110114_AABVFB rauschenberger_r_Page_148.pro
4257e25bcb389e8c160abfa303ced0d1
0c8b1d37e7c3ec3d1b1d0d669ec2d682863626ea
49193 F20110114_AABUVT rauschenberger_r_Page_097.pro
66a992dcf946aae771446ef299f7827e
beb4069f7074af8e13115691881fad76e43b9c99
109392 F20110114_AABVSM rauschenberger_r_Page_098.jp2
cc265bef92d2fce3d1c5838cfce9606f
29edc45676ea9d605be8cacc78550a28c427745f
51182 F20110114_AABWPG rauschenberger_r_Page_081.pro
aba22c7bd87c9ce6cb07a9f247c29656
e9172171d645f1c7cafa941d9970816e9d21c642
22460 F20110114_AABVFC rauschenberger_r_Page_195.jpg
291c9261ce2fcc9d6869f8bfc7a439d8
9202186c37ba5b42766d004c21981a2d8b5814bc
51530 F20110114_AABUVU rauschenberger_r_Page_204.pro
6f8affd702db2c7c24c0b1e2c650efcc
a9ed1cb8d97adbdac989763f1db1f573a329f5fb
50795 F20110114_AABVSN rauschenberger_r_Page_211.jp2
b8293bd93bcb5f1f9a5972ef8ceaffc5
b654f4f64d5786dd1e9285f8a2d8daeab710b33f
19548 F20110114_AABWPH rauschenberger_r_Page_093.pro
2a8a8df149881d985553fc450305582d
6c999e57453480f740e2068ee75f2232f5edf0a9
563 F20110114_AABVFD rauschenberger_r_Page_037.txt
9c1a41c4400509c2f06bf4c817d377d3
8d9e83b92d05c0f45e60d121faa293576f9bc8cf
1966 F20110114_AABUVV rauschenberger_r_Page_217.txt
c909fba7a59a25b85cbbb7aa2c3a0dc1
5b3534ed7afdd60c71b101853a580cd87220f2f1
72535 F20110114_AABVSO rauschenberger_r_Page_131.jpg
2377d79a08522646ffe9dc01f25a9ab8
6df875b3119dd8a4cb03fcade5d664c093c3f40b
17065 F20110114_AABWPI rauschenberger_r_Page_095.pro
8f81f2e55a27c1d5c2d6afc4057026c8
ad6fbacad7dc7de07c6d26bfe8e185e2977039d2
F20110114_AABVFE rauschenberger_r_Page_188.tif
042ff2c0488ace57da7b921e7da8a975
e9ba908c6d540965e2c4d2b23d492d8053e4fea9
50796 F20110114_AABUVW rauschenberger_r_Page_200.pro
f7833a849b0c4080945f645d5a91bfcc
85691fda59474836132b554ee3c1b36a8fab2e34
23965 F20110114_AABVSP rauschenberger_r_Page_202.QC.jpg
bee5a08126027e3c814e44e94bfef9e1
a582f42fcae6ac63afc3263ad0f6f48280041e21
46415 F20110114_AABWPJ rauschenberger_r_Page_096.pro
d0cc639bbe1613b64993a4455f7f55cc
b8be522d87b72ccadb318fcee827c252c8e35797
F20110114_AABVFF rauschenberger_r_Page_224.tif
531fb235dd7c58b654014f393194b348
817a4163c0b63d9a50e94c4b1100cd922f35e261
49127 F20110114_AABUVX rauschenberger_r_Page_103.pro
2bd78cb0b37c66c0c7d61b715058d703
f727e4d3d92958bc93545b8701642fc0bef04dff
37904 F20110114_AABVSQ rauschenberger_r_Page_211.jpg
828224f925cea6fbd3e683125c0ee903
89570fa734e4ed9a15f0dc81170e41d19200f1b1
49721 F20110114_AABWPK rauschenberger_r_Page_101.pro
872ca2ae6d240ced9f5d87df0cbdfb30
6a8e2908a7f729ac51638872788330ec5f612bf9
F20110114_AABVFG rauschenberger_r_Page_202.txt
bbd840547df6436e76ae8045343729aa
d4b5bff1d3a9fb4aa2ba8021fa84289f83d08adb
103048 F20110114_AABUVY rauschenberger_r_Page_123.jp2
828e7f5b2e9f3c8eea7046ee558d36c6
74f7798fc9e52bf1a3e1b9da1ea864c3cfcecd9c
49577 F20110114_AABVSR rauschenberger_r_Page_049.pro
61efd730c0572b0e762b43e52a046905
1ebe4cda907462dd98cce010577c6009799cb26e
48785 F20110114_AABWPL rauschenberger_r_Page_107.pro
d5a92ee7b520e008edcd95718c037072
068d1b3c2cb2439443bd8d93c6227b5cd240864b
71038 F20110114_AABVFH rauschenberger_r_Page_173.jpg
b9b22f5200b904aaae5d303d0265abb4
8f0bf2c8c2a5721b0039150c95d5d99811997c30
3411 F20110114_AABUVZ rauschenberger_r_Page_211thm.jpg
115d5da7b31c7e476fbbafbba81bf815
c1112b2401c40ccb453e7a768cccb0a010746b6f
36026 F20110114_AABWCA rauschenberger_r_Page_088.pro
207030aba205bf99d853d2c6e4d45aa1
e95826c2b0ec970ac001ddae08344819ae649e13
6381 F20110114_AABVSS rauschenberger_r_Page_021thm.jpg
a94cea528c76fde954621c5f001b69b1
d957e4f74ab0c9aafac441dc25d1e48bc2d483a9
50040 F20110114_AABWPM rauschenberger_r_Page_117.pro
58e16ef879ac6a0522e87e9e91caae4c
efe97a62d7323a7811f3fcaebf81b591dd3d6830
1951 F20110114_AABVFI rauschenberger_r_Page_033.txt
26a58f3935cbd7646337b5e6f12c05ac
15eeaa95fccc39c48d488f81f3b0911756a938fc
24233 F20110114_AABWCB rauschenberger_r_Page_087.QC.jpg
3f201096ff93be40c9883a38f97be26d
0a8a7cb3934c9bfc1ce3e1f9c562f3c1817d0129
45553 F20110114_AABVST rauschenberger_r_Page_140.pro
20f05ba985426782c3f15ddbd35c0a04
f440b8be28b13bcbf12792e13acce76564705204
47509 F20110114_AABWPN rauschenberger_r_Page_118.pro
0b45636def49e59733c9a0f32be47662
370952000fdf7cd0a3f2d5e41306210973d8cd96
1970 F20110114_AABVFJ rauschenberger_r_Page_065.txt
82529d64885118abc0c9cb7f0d694402
ba15313268ea9d25fdaecac453169e8c028130e0
43769 F20110114_AABWCC rauschenberger_r_Page_093.jp2
91486dba9bdadaf444add4fd18e74757
0f589c5ce6b1da788e214c3c2371613d29ede9f0
1874 F20110114_AABVSU rauschenberger_r_Page_073.txt
032db7ea55c8f1cb040f5c3fad361a7f
0012fb5c9fcd26c7309fc917db56c11e598f4083
46937 F20110114_AABWPO rauschenberger_r_Page_125.pro
112acaed0cba28555ce3f97cae0c5cec
70e9a1476ea86c69af957d712d3b4486fc7d31bc
3462 F20110114_AABVFK rauschenberger_r_Page_006.txt
24cf8677876cf98eb838e4ec7862f952
82cd08502d79bf2988ff5f4348c200cafcc6bef1
5798 F20110114_AABWCD rauschenberger_r_Page_070thm.jpg
0b90756ec54f5c7c08a2cbe6bfd2d89d
5ca326b1cd4cb42d7096380bd80dad92b839e844
51103 F20110114_AABVSV rauschenberger_r_Page_202.pro
be54df8de97d7bb0b7bb3079ee15fb92
f8c7b9ed89b66634221312d26574887a052054ac
49803 F20110114_AABWPP rauschenberger_r_Page_129.pro
956efcdeb3fb69f968150a9e048dc45f
e92c3c0d6909e5ad04d406b19653816dd260e0bf
23948 F20110114_AABVFL rauschenberger_r_Page_117.QC.jpg
9332721331178fcf9758fa76f4ccb942
416c86e774635b0d21078f440df6bc8ed6fc14c9
24594 F20110114_AABWCE rauschenberger_r_Page_042.QC.jpg
73eb699f5ee5d131d826db74d7e4c647
3ab70488f8b13531d85e5b279b4e2e5ffadc5a05
F20110114_AABVSW rauschenberger_r_Page_052.tif
cbcbcfdbfc1fe99f2d08b15f20badb12
438b6ccdbe61192754fd24962b5386e2bdf49b90
46821 F20110114_AABWPQ rauschenberger_r_Page_131.pro
1eab11d60e1522aee1c78641cf1373ac
d1df2f80f7c0777cb1d7d05c706fb0b4d9923ea2
5090 F20110114_AABVFM rauschenberger_r_Page_142thm.jpg
174177efa75644f1937d88d082d0cdf2
8fb2390da37cd46f8d7825817db480b264d57f7a
8683 F20110114_AABWCF rauschenberger_r_Page_209.QC.jpg
86e8d6b12d46527e70b0b9b9ea49daaf
9b4e9787edc126f0b61b49763523803e6dc9cc7e
1279 F20110114_AABVSX rauschenberger_r_Page_038.pro
fc25ff6f6130cc355b772dee31a3d51b
19fcc34b0e0c0302b28e0f0e2e8d6b14c3a1bc49
46647 F20110114_AABWPR rauschenberger_r_Page_134.pro
89bd34c00d1e3af015e2a66ac7799fa6
833736c8e39ec22d36b1fce9a00b5ed3e8cfc9ed
65072 F20110114_AABVFN rauschenberger_r_Page_231.pro
98130c68068a7c95d6c91312892d9830
341ebfdbab5524a727d97a379dd91130f1ce5e2d
116277 F20110114_AABWCG rauschenberger_r_Page_083.jp2
58447b0349fc29da0a8f2119bb9098fa
b7f6cf95cbef6a83273fabb1fb17bae077bb8a1f
6437 F20110114_AABVSY rauschenberger_r_Page_130thm.jpg
9b2f40e5bf4d1150aa8fae4bb71213e8
19dc3092cc3af5149f0d99e3feb6c4df2b67dcf9
56861 F20110114_AABWPS rauschenberger_r_Page_145.pro
accdb37fa8672c45e54bd99a95c5a294
2a0c87025478fb14d6e71f0e6134f23b489a60d4
F20110114_AABVFO rauschenberger_r_Page_181.tif
93d9f1f155bffbbd073fa00afb3fee94
77a71bd3a570153cf62f2fb4a14e781511974eae
97445 F20110114_AABWCH rauschenberger_r_Page_030.jp2
07939d33536608e42f1b37c294a10a1a
6604b477810bd61931db45b7e28f4d754ef29177
F20110114_AABVSZ rauschenberger_r_Page_006.jp2
2f77418008c6fc0c83f17a8af497a653
5c3d344707738820b6e37c5b7e42bf725f28bed1
49505 F20110114_AABWPT rauschenberger_r_Page_146.pro
594e691bf84b494190c07514a7266280
01b90b7b5dee7fa7f0006af5b0f7294768f061d3
15686 F20110114_AABVFP rauschenberger_r_Page_114.QC.jpg
d846f44c6a0133699368c1e9519c81c9
e3b3fbdd1fd9c3c759961f5efaaad0d025aefc0f
45658 F20110114_AABWCI rauschenberger_r_Page_138.pro
b6d203ebf417a52eb3e28645c973ad1a
87288e29b704ce703e1209980497077961b1024b
16244 F20110114_AABWPU rauschenberger_r_Page_150.pro
b0cf1d8810084308dbd5fd1e615f4945
363a642656e4d26262127527098d9fad923add42
6427 F20110114_AABVFQ rauschenberger_r_Page_104thm.jpg
ed540c1a22f2a8f676fb0583d9591b11
7f4f16424d74a472aa18dcfe84c146d0644fb574
108272 F20110114_AABWCJ rauschenberger_r_Page_218.jp2
661b3e96663053b7d800d5a4e98e1894
003be52dc49799a6ad3233e8e9e80ec0a4e31cbf
12749 F20110114_AABWPV rauschenberger_r_Page_152.pro
014de769484d0a6f38668d96879fefb3
242fce500a9a5dc036bbd09a5d06b3b47fff7d6e
F20110114_AABVFR rauschenberger_r_Page_154.tif
16fdb225874bece05e6fbfa818e8e2e8
b2eab81e2e2f3932c679aef6149c7aade501cb91
71347 F20110114_AABWCK rauschenberger_r_Page_033.jpg
8738ba30f4c2319a9bf6bd85059f88bf
91616bede08274d65db868774868f5f1cfe83793
10999 F20110114_AABWPW rauschenberger_r_Page_157.pro
923eac79dc3267f390ce1e5139833d0e
481f7e931a26e826d022210a79fb60d51749d2b1
23703 F20110114_AABVFS rauschenberger_r_Page_084.QC.jpg
6ece2cf6d1cba00e02fda28824ee0c3b
c12f04c4ecbbf38653182394824d3e7e7613da74
23308 F20110114_AABWCL rauschenberger_r_Page_075.QC.jpg
0a858400a9540fa220e6441ee9da5bb6
e056aec9400b6e645c5fc712919a03d62bb6080b
50228 F20110114_AABWPX rauschenberger_r_Page_161.pro
acd965e46bc4662e9bab010989e2fe8f
fc1f25f08a14a79e0d2a4cfb5f9427b82452c55d
40578 F20110114_AABVFT rauschenberger_r_Page_232.pro
beb94eef1864fdbb622f69b02460ab8d
078861c27912b1eb85262e240bd50ebf1f524f9d
72144 F20110114_AABWCM rauschenberger_r_Page_065.jp2
3c1c618d794d7d9fe513fdf4d0450b37
83190b1273baecccc90881fa571c602815e98002
50784 F20110114_AABWPY rauschenberger_r_Page_164.pro
cc8c1b895984fd085339557ac18f2c9c
5754c7426197c1a8bf5bd7a62e14f0a496c9c450
72647 F20110114_AABVFU rauschenberger_r_Page_160.jpg
c4c85228c2a3a397d0ebd69ead145bdf
d0fcf7744359274489b8b64af8d525e4ad09a765
1998 F20110114_AABWCN rauschenberger_r_Page_200.txt
b28cd2a8f91901e3899acb6ebd0dd049
5028ed682ffd0672ea3d5e401849b14104238660
49379 F20110114_AABWPZ rauschenberger_r_Page_166.pro
481dd685be6420a69cb795cc24a23e3b
c3d39202b5793d42289e158b5996ccc57d1dc613
F20110114_AABVFV rauschenberger_r_Page_175.txt
d125aa26acb0f840ac0a4fd22a971c11
f2a86c5998232da197e11278a78437dce4bfa7b8
F20110114_AABWCO rauschenberger_r_Page_160.txt
0dceb0f5576eae955ec6bb682cd9c4d5
ba6ac7c5244ab345e985d7b5d95684097fcab2d8
68263 F20110114_AABVFW rauschenberger_r_Page_023.jpg
2c493584f39bdd13969fed628bad60cb
9f85878d8800b028c043052684485a07765b07dd
39031 F20110114_AABWCP rauschenberger_r_Page_058.jpg
affe5cc087ac63800f4eda93d9945539
59ce43bc99389a8412184a202898ffe823f2e7ca
F20110114_AABVFX rauschenberger_r_Page_136.tif
f23a71a1710a015675375994e198c446
ccf7d3de5434f2b44275372ac043dbcb0ff67d94
F20110114_AABVYA rauschenberger_r_Page_144.tif
96b8624dcfb7e3fa941dfd119de30955
5fb71e84c76355f8fe5e651e9dd1fb4e7846389b
23529 F20110114_AABWCQ rauschenberger_r_Page_036.QC.jpg
06a371935f6d4de59cb2642a7152c663
e51d22535ac3cf7af1e6f879de5b43ea4f63f3af
46678 F20110114_AABVFY rauschenberger_r_Page_023.pro
9a66979fc02446fe7a7cb6db4e1901a6
e108b8f561243ccc257b7a841f32b0563b6e751e
70687 F20110114_AABVYB rauschenberger_r_Page_075.jpg
aa0e43bafa784c49b45a244b8dd977bc
429a20bcf37795528fbeb00c562633c9cc6cdca9
845 F20110114_AABWCR rauschenberger_r_Page_066.txt
503383e604d93e55dd7cc8ca42cc2325
4112b2f165eb6eb9482b5c9ae5566133fa6e0514
1521 F20110114_AABVFZ rauschenberger_r_Page_002.pro
cc5b5ef5740d68152d786f8e903a2f43
4d33744f18b8562949e94bf63af9781c4a167dd9
24426 F20110114_AABVYC rauschenberger_r_Page_129.QC.jpg
7a6aacf44e1e09a8df1e39532dd37c3f
1a36c6669a49eaf72ceb1b4081e20aabde980d23
6516 F20110114_AABWCS rauschenberger_r_Page_133thm.jpg
85a155d0b8928bfdb5cd9ff2d3e9d906
d4d69652bfd567a0814f93b898b252eb541c3596
52042 F20110114_AABVYD rauschenberger_r_Page_163.pro
eeeac5b1c3971f95807218315756b0e6
f716cb60e231bd97c63290585f99c8ec446acbbc
110927 F20110114_AABWCT rauschenberger_r_Page_117.jp2
7d81d9f434a581c53eec0126577422a0
0f17e251059e9ab1fedf4041f4cdf388f1cd6506
1588 F20110114_AABVYE rauschenberger_r_Page_207thm.jpg
fda4492237b659c53e6d28e664e7191d
4d53aef0eac3beca61ee24226f48ef6157f584ff
8617 F20110114_AABWCU rauschenberger_r_Page_155.QC.jpg
b234ea1874d558c30c30a4f3c66b892e
0589324dce9809104059112eab06ffdd729465c3
F20110114_AABUOA rauschenberger_r_Page_041.txt
7aec512534f7131caa201bcbf10a0d63
a2c87ccb539bd013334b7d241cd31494ba088580
F20110114_AABVYF rauschenberger_r_Page_090.tif
057113ec96f16a4ad2b754c1f512e0b1
c6c5809e6a3704b790e269bdc918e59af7785fdc
68780 F20110114_AABWCV rauschenberger_r_Page_123.jpg
9b2d6c6621d4bc678509ffcb21aa36fe
a5337248e089cffa40a9afd58543dc44c00eda3d
F20110114_AABUOB rauschenberger_r_Page_090.txt
d434907f099793976a91b58cad556530
2fbec9337be76ec04f90bcfcbbf3579d9489e5e6
F20110114_AABVYG rauschenberger_r_Page_115.tif
baa138fb730eb5afa3e7f743600e070a
9425cb8365845d9d393d2485a1c2d5000d2dfd8f
19083 F20110114_AABUOC rauschenberger_r_Page_005.pro
761d12abdc7732826f6c7144e4efde09
26d5466d26fadff3d74bfc899d87434b729385f1
F20110114_AABVYH rauschenberger_r_Page_041.tif
1493dc76cb79b48d0f3a1bc023176fd3
b612a4c5f6c6d550a17e09ebb4dc70a91ad2ff48
F20110114_AABWCW rauschenberger_r_Page_050.tif
c8cd7e8bffc737492b4e023f58c634b6
059419572e8834b85cf7687ed0644f0af117384d
6524 F20110114_AABWVA rauschenberger_r_Page_162thm.jpg
2aa74905b6c8cfdbae68a27cdaaf34fa
55ed4d3b1168275440b337964fea728e0e4ac9e2
9809 F20110114_AABUOD rauschenberger_r_Page_207.jp2
30ff74c9ad3f411f20eeff153d02d853
abb8e432468524f08e861ba69c28eb705a555d6a
1699 F20110114_AABVYI rauschenberger_r_Page_015.txt
1e937a645c549f20a177dfccc9452164
80e6431aca0988f606f4fdbd46eeec63a0486b25
1986 F20110114_AABWCX rauschenberger_r_Page_024.txt
23384ff9ccffc537cb8aae83c4e34d1a
6cde3f382f3e2638f5f9180b818b079ba4d00f98
24739 F20110114_AABWVB rauschenberger_r_Page_163.QC.jpg
427e9c39f3a6f642037314b112d29839
71ea3de6cf498fce7eb577cbf2e46870b6d89bbc
8413 F20110114_AABVYJ rauschenberger_r_Page_066.QC.jpg
8ef4f6915edf0b1575ba323095f1822e
1bb2be5f7c16f3d9c38305f66327245bfd5f690e
2834 F20110114_AABWCY rauschenberger_r_Page_152thm.jpg
473b27c63d2a5308485a5b62404f7e2d
88e2b5ea7f73b20d1263ffe0913f617e2efe867c
24175 F20110114_AABWVC rauschenberger_r_Page_165.QC.jpg
83a435b770d63266d8f5b880a33e306a
cec34537137497d2bc068eddde9476858a6ca711
49723 F20110114_AABUOE rauschenberger_r_Page_216.pro
17f30583fe7461b4e22074797286fb72
b15a7d05c7b4057e69a6b68d5f730653211fd7f0
F20110114_AABVYK rauschenberger_r_Page_206.tif
6f5ddab497ccb7a893cd87a4de1b1a9a
6e50f24a11fb353d2a4020c26521ef87a3335918
31022 F20110114_AABWCZ rauschenberger_r_Page_092.jpg
d70034647c3f216bbc209323950b9269
6f254405ff295dce89fc4fbac8639f45a7141db0
6436 F20110114_AABWVD rauschenberger_r_Page_166thm.jpg
9b0db0b89c57f366002239f9f2f4a7b3
ae359e51f399835f2e47ffc3548268beb63bfd7e
152 F20110114_AABUOF rauschenberger_r_Page_038.txt
fdbabe3419187ea190a72233228ef452
18fe52e709e6a24dfe0a99e697b03b64928e4733
F20110114_AABVYL rauschenberger_r_Page_178.txt
e5272d6e669a7549a1fb0a40979f5127
607f6384a18fc8fa49e90453862c67834ae4a6ac
22715 F20110114_AABWVE rauschenberger_r_Page_170.QC.jpg
0d3cd9f5a1ec80d3bbf3966ed4176573
3c271db288244b72eda62c873c29beadac3ba398
48812 F20110114_AABUOG rauschenberger_r_Page_130.pro
f63db342c7973e34d735646600343fa6
91bd544ac198179f3cfec492d14bcd602a7252f5
75539 F20110114_AABVLA rauschenberger_r_Page_019.jpg
3d986a1f5382abb6eda8de01b9954313
b3d803588c08c1b21ba366ff1a3bb54a5e2b3261
3349 F20110114_AABVYM rauschenberger_r_Page_060thm.jpg
2352dfb0b0137193be876df7fb1ac9b5
f60456c5a810f5b4869ffd7da08c68c60bbfb5b3
23061 F20110114_AABWVF rauschenberger_r_Page_174.QC.jpg
759fc716be9f1e899f49fd1384026017
184b784fa7fa11a695d5fd451332bc9e08e52d0d
4229 F20110114_AABUOH rauschenberger_r_Page_210thm.jpg
319a909ee1f97cb6245fa6611027edd3
e29b3b05acb8a65ea50656424a56b8ecea0d465a
107500 F20110114_AABVLB rauschenberger_r_Page_128.jp2
2508b289e3b0011dc78dea9627c5e577
b4a92b022c93c84ba52b33d3315aac232e842360
6289 F20110114_AABWVG rauschenberger_r_Page_174thm.jpg
e46a50ac02022806468992c43411cae6
cc56b973e01f07ef6f0e5b68343d10e162e5fb10
23616 F20110114_AABUOI rauschenberger_r_Page_128.QC.jpg
17fafb626877d6be8ac4883ac703977a
853bf23ea5b6e89645b336ee43ba6c4ebc118c8b
10555 F20110114_AABVLC rauschenberger_r_Page_068.pro
42b5f5f08610134c36e8e4d8c06307e1
5cb1e98050cdb59122d2305306118edf394ae274
F20110114_AABVYN rauschenberger_r_Page_077.tif
df6189e2a39bbf2c9e756805c7aecd14
995f13d130919d9bc1e2afbfbb62696ef72c75d1
23106 F20110114_AABWVH rauschenberger_r_Page_175.QC.jpg
199e0668ca6e7da8a063a1d90c889ec0
b9136cf9f5af5ca1ac6e79b76ee568a5d0042530
2010 F20110114_AABUOJ rauschenberger_r_Page_082.txt
c9a45b62db3ca2d1365c2ced1a3ed56d
52c6780a6c021be1763f7c3edb921a2c216523cf
2011 F20110114_AABVLD rauschenberger_r_Page_084.txt
a81f0cf76215af174c7c053f74da324b
5a3edf9948734c9a6a710f446b63d64515504a3b
70622 F20110114_AABVYO rauschenberger_r_Page_097.jpg
418e58c14cab239da718accff50fb67d
2d7ab767c4a4b308b9c5f161cd33fd57f9252021
6533 F20110114_AABWVI rauschenberger_r_Page_176thm.jpg
8e3d1a6f55ec757d43395da000385f80
801706670a92d7753a0177126095b52b34263fbc
384 F20110114_AABUOK rauschenberger_r_Page_003.txt
0d208178dcfb6739cff4f6bb8aa196e9
0ac37a01baa63421fdd626070aec03c79f44ec53
24253 F20110114_AABVLE rauschenberger_r_Page_221.QC.jpg
602f0c1cd86af9646c1e9c48187619c2
d5ed29cf53463ecdb460c66e16abf280b64f0327
1697 F20110114_AABVYP rauschenberger_r_Page_169.txt
b719732b884434caeebce56e81f7c9d3
c1261469fc2ea389f8272ba684dc5772a4dd6dc1
35835 F20110114_AABUOL rauschenberger_r_Page_114.pro
2f60c4e72e173b9532334ced92d53942
a19b4583476c7220a797fa03b4cb751c2700d068
F20110114_AABVLF rauschenberger_r_Page_119.tif
54406acb00aa74c58da6c448e33f61bb
e91142be775e26e53766366448603ec319aa01a8
2665 F20110114_AABVYQ rauschenberger_r_Page_197thm.jpg
59828cceb7f02bbd5f683a0064b92ffe
3ec2957573771d28c024d9dd411470c799523696
6415 F20110114_AABWVJ rauschenberger_r_Page_178thm.jpg
d89123ad8e295025ea8ef27cd84fc53d
f48cd15fca8fe5081bd8d0fba0b7052d9adbacf1
1757 F20110114_AABUOM rauschenberger_r_Page_070.txt
970a021a46fe6ecc0e975d2a017b92fe
6cf9311674393392ca2211f1ce718eb5edb08cee
F20110114_AABVLG rauschenberger_r_Page_189.tif
c63014a7ec9dfada7d9acbfe2ed44707
e8d07dab721ae9a8a46df8a59895f58b24b5b317
80744 F20110114_AABVYR rauschenberger_r_Page_109.jp2
3fe1e8f89acbbdda570527b1a1e56f01
4054a3382b9283bf25f99c40c32f6f75ed165403
6531 F20110114_AABWVK rauschenberger_r_Page_179thm.jpg
90cab2ccc1b73ba1250986b074021b17
c9d9fee756c3ac9f5acc699877a4cb48ffb162f7
89006 F20110114_AABUON rauschenberger_r_Page_140.jp2
f5e2f24f335171f72de9e910f097fad2
4b4a1558d99d831f99e9c68a36fb6559c20d4957
97969 F20110114_AABVLH rauschenberger_r_Page_004.jp2
a448c9462e2549b404d41e7645252b24
44b6f508f8c3f3b2c1c352bfca610cee6d1b1481
3973 F20110114_AABWIA rauschenberger_r_Page_207.QC.jpg
0e52ca406ea9369ec83b4b420993967f
77f05cb419f473879ff5ea5a455a2fd4d041f0a5
6234 F20110114_AABVYS rauschenberger_r_Page_007thm.jpg
3b40168f745e96b1472d94a915208048
32f1e5497869c770a3619446f414b3e4f789a92f
17645 F20110114_AABWVL rauschenberger_r_Page_180.QC.jpg
9ab09abe8a5dbcc05c94c9f91583aae9
61a09193ac6196a6440a8e6a469de0b9026e3efe
F20110114_AABUOO rauschenberger_r_Page_130.tif
93203d11ca37af582b168bd691de91a4
51e69f3fdffeee45c13e2b7925f07bd05124786d
2662 F20110114_AABVLI rauschenberger_r_Page_228.txt
dc730b621ceb64579a40f10a54925415
3fe607ebcb115aada54bc6cd96b8f61ad0209f53
1235 F20110114_AABWIB rauschenberger_r_Page_185.txt
de3019c57087ef8f0a9b84bbb83e8326
7209092c1fa69291b33e8d80c23d60d0fdce5b42
F20110114_AABVYT rauschenberger_r_Page_003.tif
2684d6ddd8fb0af5dfce59dadddf2a9b
f15b910d686caecc0b13f10da963a06c220f9e94
5447 F20110114_AABWVM rauschenberger_r_Page_181thm.jpg
53542d553a104fbfbda3e32b1c85f5ff
3b0909b41b26bad405d62301115882888ec95f02
5163 F20110114_AABUOP rauschenberger_r_Page_056.QC.jpg
24a1e21acdfaad90f7fdf609c72ebc6d
1ef7186bec45b7fd22b282a5afbea35b7e1ec8fc
1220 F20110114_AABVLJ rauschenberger_r_Page_149.txt
cfc2533b987023c8bf63ff5a29941c66
82a83caa0ea1e00340746e4f59476e5f56702e8f
47246 F20110114_AABWIC rauschenberger_r_Page_027.pro
8e6d49f134ba7c57669f4d6e9b4f75a5
ebe1be190b43c30f368f8436087d07f18072da94
F20110114_AABVYU rauschenberger_r_Page_159.tif
48db26dd33b6141ed990b671b159cffa
315b8177c70994629c121d2ada4a6e7bf290614c
2632 F20110114_AABWVN rauschenberger_r_Page_183thm.jpg
415dc68ce621f5282aad74553e350f7b
2f64ff4aab01b36c0c598a3b379b87daab76d135
801 F20110114_AABUOQ rauschenberger_r_Page_113.txt
c75006b30db19a97081c59babc28fd11
58b67304e79f8a35160bc8f8051f6cf8338ed6f9
F20110114_AABVLK rauschenberger_r_Page_127.txt
6f0cc23850c3247e99f9656198c27146
7122006ea23890ea77ddac6ee5fe031799abf8a2
72831 F20110114_AABWID rauschenberger_r_Page_055.jpg
8f1b44f57b8d9532e2a958883ee6afc1
dc812c87b0ea4e1b0684e2a9300b54a4eab1c92a
23439 F20110114_AABVYV rauschenberger_r_Page_199.QC.jpg
7eb9cb95582e261bf7994ea94cee1412
7e573a17da393b245566ea528a76cb92b3431055
13994 F20110114_AABWVO rauschenberger_r_Page_184.QC.jpg
7d00bdca15bbd813fd127a381d5f21c2
8b9494ac378a2620c33a6aa3cf531041f4b5dd59
29791 F20110114_AABUOR rauschenberger_r_Page_223.jpg
567a3fc290fc8c7c3c941ddbdc9551a5
f222375fd287c3aa3453eb3b58879098445b86de
22327 F20110114_AABVLL rauschenberger_r_Page_032.QC.jpg
845a0c45900f7a46afce640f6178f8f9
485b5f8d0fc3659f0e44b87e857a5867b5af76ac
1051943 F20110114_AABWIE rauschenberger_r_Page_010.jp2
bf568b172cf1b4facf826faa7fb80065
370ebf741170628ed91828eac7baa0e2845350c5
6549 F20110114_AABVYW rauschenberger_r_Page_071thm.jpg
80c4477095675c4c36807d9e70363168
f3918c93f536e48ebaed0d8fe847563452bce479
4101 F20110114_AABWVP rauschenberger_r_Page_184thm.jpg
e991520b957be5e4e0d33350d97a6f62
b45d57a016c26838b303db0fc62f40ae9cd915d2
22947 F20110114_AABUOS rauschenberger_r_Page_197.jpg
0d9ec4f4e5484362fd5aaf1587131abf
95a705785654c3c04021da31c6075be75fcbd8e0
27995 F20110114_AABVLM rauschenberger_r_Page_069.jp2
bc85e8ec6381ef10c3bd32e17b7fcf25
0a9cb0d1d782af027ce6e602ae0e454faf5a08cd
F20110114_AABWIF rauschenberger_r_Page_020.tif
c8159de5291be6b1187d0effea135e2c
8631e1b2f7d1e2b9e5a28f8d8cab81fe7111c329
F20110114_AABVYX rauschenberger_r_Page_004.tif
3097485a09474fedd85be9d3cfc339a5
561de321f91448e476debe6c080edb18412574d2
3405 F20110114_AABWVQ rauschenberger_r_Page_185thm.jpg
c8e38fa58ae34971e3db861f02146671
a22b29cf6e547cf54c718756e9b52b7a0785690b
3888 F20110114_AABUOT rauschenberger_r_Page_092.txt
f3dce11f7baf2147dfbf62e4c37da058
0ed638e4908dec56147262a4f81deb2653c6c6f7
102367 F20110114_AABVLN rauschenberger_r_Page_051.jp2
9d4e489ce6a81850a95949fba4719f76
127bd15db42f8b374bc3b94cf8f2a4f6e0f7fd05
F20110114_AABWIG rauschenberger_r_Page_213.tif
8e1159ddd91a78891cde7c178dcca9e7
3af2db27176a161d40c040bdf2ed054585c3ea50
79511 F20110114_AABVYY rauschenberger_r_Page_090.jp2
edfc4a5c83a520bd736da2683f6166de
28b7d54a739a91a349479c1a0e8ab29818c24e44
18322 F20110114_AABWVR rauschenberger_r_Page_186.QC.jpg
4d218c6d88e8c7f302135d24cab8955a
8acb663e703202038d5dfb61015b93763f65b728
49978 F20110114_AABUOU rauschenberger_r_Page_218.pro
6853288fedd1a00d88f32975421c3e5f
3bc5ccfdf79096361cb031b6789672384f4f5ea8
11381 F20110114_AABVLO rauschenberger_r_Page_058.QC.jpg
d76f82895011979a008b56da94e16530
ef37619d36c83c35ee0dfb9f42c59d46fb8c652f
7777 F20110114_AABWIH rauschenberger_r_Page_153.QC.jpg
6c7927a56a98ccd3c32419c350762178
5923b5e16ca9c18e0f59e375b5e27040a4b92ab3
F20110114_AABVYZ rauschenberger_r_Page_074thm.jpg
d1d94f6c3fb6a9b31005217e2ab9444f
202a04395e9b880bea5f36d711c403a5a90cd916
4157 F20110114_AABWVS rauschenberger_r_Page_189thm.jpg
2091885bfca05cb467739a50cbd9ae39
50bca61908e382f89dbb644e755becceb88624e2
113327 F20110114_AABUOV rauschenberger_r_Page_220.jp2
6b7833c7c0da566f971665cc7af3cead
e9d00373302b3407854b1a93159df33254e46954
21821 F20110114_AABVLP rauschenberger_r_Page_004.QC.jpg
7bd9c5403e89d53a2c07683a4075f23c
fd53390f9a940d72b3a93cf064d4bb941ee47d6b
72541 F20110114_AABWII rauschenberger_r_Page_172.jpg
59d018810d78a41bba413b3820b84d0c
901255a4504af61f26acf2fe9629c2c32668b9bd
14977 F20110114_AABWVT rauschenberger_r_Page_190.QC.jpg
9957b7670a8572077a84c20de56bf033
f645adc0064727299b8801b094fbd2cb70874af3
1990 F20110114_AABUOW rauschenberger_r_Page_199.txt
e0873ec29fd41d81b1b1e34a6b81a162
c654e5d64cf823ae711ba23c63c1500711410083
113522 F20110114_AABVLQ rauschenberger_r_Page_214.jp2
48c59f9ee2f5d78ab5312504f2e9f73c
787c4bedbcd2f223400782cfe95613329eab3242
65569 F20110114_AABWIJ rauschenberger_r_Page_198.jpg
976b140efc9df30a433a41049fb4bdde
af67efc7fe3cf9954cff1adcb5569c4b35b80c2b
4252 F20110114_AABWVU rauschenberger_r_Page_190thm.jpg
575c887f2b67fff4652c1e41eb4c10ad
06715fc34e89ec088cb059b0107bc71a849bf55c
100281 F20110114_AABUOX rauschenberger_r_Page_028.jp2
095a743df736905a72a9bfe904c5e8c3
ae76f9eeb9a3edb5eebb1b340267b558631ac8f1
43046 F20110114_AABVLR rauschenberger_r_Page_138.jpg
2038b51e4daee314fac107a5467dfe49
9b3c080c44ff4969931b867b1990d6dada05ecc3
74804 F20110114_AABWIK rauschenberger_r_Page_200.jpg
30cfdbd8011c0490f6a7a1ebfda35af7
9897f676591be71f45dd43d87b864ddfbf88954e
2514 F20110114_AABWVV rauschenberger_r_Page_192thm.jpg
5bc96877267e28eebf3541e751c3cf67
acaa79629d9a6ba0d32a58e971544f6955cf9d6e
7121 F20110114_AABUOY rauschenberger_r_Page_001.QC.jpg
975102b678ed4c2cf34e00676fad50e9
63daefbc5daba45bf69c6eaf5baacdbd4dd8b2e7
141672 F20110114_AABVLS rauschenberger_r_Page_226.jp2
25231e649cd480351ba73d794809c9f9
6d4b27f484d5d5221d1e98b21d1a362d470000cb
28952 F20110114_AABWIL rauschenberger_r_Page_154.jp2
c928827831c62080841e983283ce4073
f0efd15b580e322ad3e3402b20ba6b1ea6439c16
7069 F20110114_AABWVW rauschenberger_r_Page_193.QC.jpg
8e516804d5133d7aa1d678b6445fbd3e
6d32cee89b06723411831cf38cff8f9aaa975b8c
113809 F20110114_AABUOZ rauschenberger_r_Page_201.jp2
a75cb7bf0128e5603f599667352ac37b
3b0aa590d02bc27ddc3101c4ec3ea7e5a616ce50
103179 F20110114_AABVLT rauschenberger_r_Page_020.jp2
3ec73e04bfd9227574f43415e5f3bd4f
4859f40d5f3f4d472d8ecebd24d4b62ba4bd14e2
1934 F20110114_AABWIM rauschenberger_r_Page_085.txt
3e0e040b1154cf8b3c4b7e71ed7a02f1
c3c452ace5b84779fffcf82bb60364d417e65928
2490 F20110114_AABWVX rauschenberger_r_Page_195thm.jpg
1b19cff5496af1f089b6b687b7cead2b
2329be0cd84d27b4ff2f9e9040c5f332613fafe6
46872 F20110114_AABVLU rauschenberger_r_Page_105.pro
b0aab49ab999116b57d8c51bf0bb84e3
eff652e42a1d3e988e0c95b6bcc17824141c0acc
23504 F20110114_AABWIN rauschenberger_r_Page_076.QC.jpg
3f383d9356699ad4b52011e33c0c4f49
9f6e933872e0ec45c7f013d76efb6aeb87111d31
20863 F20110114_AABWVY rauschenberger_r_Page_198.QC.jpg
827cc715fadecb9abecde678c30408b7
b9616698af2712476328ed4a3dd75aacdfb46f4d
75151 F20110114_AABVLV rauschenberger_r_Page_087.jpg
cd8bae7d8ecf6a79ab4292b2f5efa2b4
7c4ed125678462f44fe9459ed7e03a33d706b638
49450 F20110114_AABWIO rauschenberger_r_Page_127.pro
1f1a5babc16102ed438597ff9b58c536
7d939ece6433c9b46a5943d4803cd6414cbdbcfb
5914 F20110114_AABWVZ rauschenberger_r_Page_198thm.jpg
dbeed5d690aa3972a36fe60f8c518584
da27fa3822094c77904fc2776a04e7602ea10da6
3526 F20110114_AABVLW rauschenberger_r_Page_148thm.jpg
48b53d2ab6ed272f4356919a4bbb8ff0
c6faec530182f62730c35319fc2dcf116cb1ad82
64427 F20110114_AABWIP rauschenberger_r_Page_091.jp2
336afc2aad0447df5ae8267da1c4d3b0
d6cc5bbe8d6e6d1f9402a2bff5effbf27cd93b8e
1898 F20110114_AABVLX rauschenberger_r_Page_114.txt
885f43f99d2e301be4478c17eabc090d
7fe86277762a6470cf61bd1a298353fa968d95c5
107904 F20110114_AABWIQ rauschenberger_r_Page_116.jp2
1f0f8c01410d2c549772ba45fdf82498
15973d2af73941983ad98a59ef331dbc183a36c0
1051985 F20110114_AABVLY rauschenberger_r_Page_150.jp2
c70e256150e8f649bd2b65bd6cc9dbf2
a91a332e43d383cfb7d4991e3a7e156ae34a1842
20193 F20110114_AABWIR rauschenberger_r_Page_188.jpg
539fcd707618c101a6752dee80990a9b
e9d6b98198d09ad5322c85e323f12cb302eb8a94
F20110114_AABVLZ rauschenberger_r_Page_197.tif
4cdfa9e1d8b929a6d4bfbe1482a057d4
f3f3c94ecf2c51ff8135213fae4cddc010e3f796
8423998 F20110114_AABWIS rauschenberger_r_Page_194.tif
b1700535f7f5ef0a26588a9217ef7629
c5ca26b3e147dd6b864eced331fa8fd01f94ec43
359222 F20110114_AABWIT UFE0008223_00001.xml
2964a089bd0fb5c747ec07b0155241b0
2ad4c09a8527cf240e765ebaa490eec61a922796
28122 F20110114_AABUUA rauschenberger_r_Page_209.jpg
b3f607ead4cedf09a12692a658265900
9588e09d5000f2fd0955245dc2420636c091570c
49620 F20110114_AABUUB rauschenberger_r_Page_077.pro
a552a1706562c126a6ae6bf5af2810b9
a848a8015f5828f8866e2d2d4a2b9da21cc270d4
33038 F20110114_AABWIW rauschenberger_r_Page_005.jpg
83bbe8599885c38f4a80627f22860846
b22055eea35c8d6ee6c788a37917248a9b2e4d9a
2022 F20110114_AABUUC rauschenberger_r_Page_126.txt
66b6698237fdf4e10c718fc20b42d8cb
0acddf246ce56fb6728289e981f11f971df8b066
102999 F20110114_AABWIX rauschenberger_r_Page_007.jpg
2f3f8d9d73ebaaf33b1d677eed45a588
08f491495a0afe63df917a8ab860ae0b7fa6cae1
838 F20110114_AABUUD rauschenberger_r_Page_183.txt
8dd83b1ba1aeaa2a944d5e9087c91d84
28d8ce2376d699b458789bfad3317b89b73c4ddf
90337 F20110114_AABWIY rauschenberger_r_Page_008.jpg
e9b007edf4e5679e9ae8b58bb3d33d68
a668ac3a80d27dad8fcd5038d4767466db4b30aa
30744 F20110114_AABUUE rauschenberger_r_Page_158.jp2
7c662f08767ec9b33713e943a7cdda33
45fc8ed187ee455b5c13cb583c5a32fa0ae87387
104465 F20110114_AABWIZ rauschenberger_r_Page_010.jpg
c74763c21a088a5b6cf00d55b8d4bf25
e86384190f1d226a0dffff65e822d4c0cbacec63
46871 F20110114_AABUUF rauschenberger_r_Page_170.pro
e6644ee91292d33d097f1b6ed4e3b123
692a58e8d2bae8ba523f6d46479903dd0d348c72
51036 F20110114_AABUUG rauschenberger_r_Page_102.pro
dc90d182ee3b2c2831ce61d05fe829f6
b9d55b8d74743ff50e68afb969f250e01af0406f
5523 F20110114_AABUUH rauschenberger_r_Page_186thm.jpg
cd4fbdcb14f1b0bb7b89aee54538fc18
bbae3ef5079123addc70e7b5e64d84fc407f7a6b
81162 F20110114_AABVRA rauschenberger_r_Page_088.jp2
3212febe31b02e2453ded0bbe9e9ffb4
31f1d19026162e3326ce536e40c63f5406460ba4
70231 F20110114_AABUUI rauschenberger_r_Page_043.jpg
b53806a83090559453c77b0e4dbfd89d
cf076c712ca839c2748a0a4c65cb9f7d8cc64419
51343 F20110114_AABVRB rauschenberger_r_Page_072.pro
0ecd5f751a24cbe00ad37814f0ea0668
45d4f4617e41dd811ce94e46523903a73ebabc92
2064 F20110114_AABUUJ rauschenberger_r_Page_106.txt
3a31f05eeba435e3e37059a67ec8c577
4b5b8fb42634e5258b41a24417446ed1c0ab88d8
F20110114_AABVRC rauschenberger_r_Page_100thm.jpg
bcf999eeec52a40b5e64a6d3e688b61e
bae5f896cf4523ee00b5c3a1be6aec7b8b0cd241
2741 F20110114_AABVRD rauschenberger_r_Page_157thm.jpg
2b06a20912003391f53c878a5bec363e
0dbcd08f652fd74193cf3fd6fb5f9d38269fa250
76671 F20110114_AABUUK rauschenberger_r_Page_060.jp2
62388dac2855428d7ed9292e72a74808
20004551884e84358b1d975cb0b29cede61456a6
40221 F20110114_AABVRE rauschenberger_r_Page_111.pro
94a19cb8f9ba762e74db363b92b7a167
49d729fc9d0701438e11461dcdea12a1a487221b
F20110114_AABUUL rauschenberger_r_Page_205.tif
53a33af287f009e883b288ffdf13b17a
58e5816b463841ef4759325adb1e4b4489583605
22315 F20110114_AABVRF rauschenberger_r_Page_062.pro
ebf151ffb01898980a1d9d829cb71e94
7d60ce1db1cf6b7133b91ace67eb61c51520e7a0
979 F20110114_AABUUM rauschenberger_r_Page_158.txt
1445167ffabf6263f43e632ca9eca620
2ca233429261e68946a0c034c13da4af9632c15c
F20110114_AABWOA rauschenberger_r_Page_161.tif
765f0a5c447bf67de49392560b171230
afde3c13760df19d14e79f537cb25a6718337d3a
69415 F20110114_AABUUN rauschenberger_r_Page_025.jpg
bf5a8db8dcfd8d32c0c0b82c7a9bc41b
b308a0e5b888bb77579758dabb09c6ac674d3e8a
1818 F20110114_AABVRG rauschenberger_r_Page_120.txt
c8321c520bef83b99e582da5a17e690d
bfd98c369dd41d0c3ca9e11d81d4b2438ccac33d
F20110114_AABWOB rauschenberger_r_Page_165.tif
33e1ad90ab9010c6898765b27661fce1
6877f85a1da4d2ca082f583972688d11c89bc9bc
71383 F20110114_AABUUO rauschenberger_r_Page_215.jpg
f441cb91397b88ad6df1ae41b12d8518
e97532c02eec8be1fe1050adc4555e9fefa3ec51
52174 F20110114_AABVRH rauschenberger_r_Page_179.pro
a72dc610de3202a9c6759b6d59036d00
f92203e0b51e631bd0f650a6f03e48a52e0d4833
23567 F20110114_AABUUP rauschenberger_r_Page_081.QC.jpg
5022c17b02310d51b6963cc8ca1b9379
9895e9669d7ba85e865e95e5192303767b2f5118
F20110114_AABVRI rauschenberger_r_Page_156.tif
ebf7beb67f3f2a9a1926a91ad82bce1f
11292ac0f4692a4d5c4e29a9a722af70cce5450e
F20110114_AABWOC rauschenberger_r_Page_166.tif
8bcfefd1299b4ad73838dfcf168f6836
49717d13838eb1984a7ff6ec2fb7f6710548ff32
101016 F20110114_AABUUQ rauschenberger_r_Page_027.jp2
93524c5fe76d7a310e46eb5e9c749d5b
2595d925f39c12a1604c3a7c2312d191773ac2ae
1915 F20110114_AABVRJ rauschenberger_r_Page_044.txt
1becc8210cae7b486ed694e8689a0b0a
9f7a2e71f15f0d06d43962a75a6f9ebf471cea6d
F20110114_AABWOD rauschenberger_r_Page_167.tif
ca167ff49da64d22d0d1e1c49a84b48d
9aa57dbaa300ea1bb0980593dc37a5cdad42ade9
6505 F20110114_AABUUR rauschenberger_r_Page_002.jp2
44f4fadbf4eae4a0dea79985904227fc
b2d6e035759d4c0cc2b66c1f3ea9d8713f9327ff
6432 F20110114_AABVRK rauschenberger_r_Page_097thm.jpg
541aa35e807ebc3abe0c64f7a4be575d
a55b0833725e61ad71a40549f41f53876b2438ee
F20110114_AABWOE rauschenberger_r_Page_168.tif
8b6f079cee06d1c463054aee3f6441da
b41fe2ee98136216813ce2ecea8ea23903882446
F20110114_AABVEA rauschenberger_r_Page_195.tif
fd19406d153cd327574bd4a0c29e09d8
37c92804cbe63799cc23e6245a15d88a94d2e89e
1964 F20110114_AABUUS rauschenberger_r_Page_108.txt
abec4891211e6f9cecce22a1e77d4e77
a30593ac0be449b23c195b194fab1ef2e60913be
64562 F20110114_AABVRL rauschenberger_r_Page_070.jpg
652cd23bd9164a017ca28eeecb04aa55
f9af8fb681bac51a90abf99ae468f588a3fe0582
F20110114_AABWOF rauschenberger_r_Page_171.tif
e5711e82a532d40e671f6bcc3db8b1a8
358f5009de962505020de47b45dea7966e847b96
1904 F20110114_AABVEB rauschenberger_r_Page_040.txt
e67b5aa24c0f84e6aa7ee991ea364548
ea2407ac35f021e33f1678261d2fe86346d50eea
95865 F20110114_AABUUT rauschenberger_r_Page_137.jp2
3231f6bb2fc31f89cfb2d76700b42a98
417e19913d00740359d91a8cc1e4774c9523c7a1
1919 F20110114_AABVRM rauschenberger_r_Page_167.txt
78fb95a8fad39a14926759bdb4441bdb
049f8636613262cfe004b478b4be8f7d6c5d6559
F20110114_AABWOG rauschenberger_r_Page_176.tif
c2603e92e57de6688cc1c3123883056b
bfcf251f2fbf8aac79c2d8fef646f0428fc43239
48481 F20110114_AABVEC rauschenberger_r_Page_044.pro
af5c99aa8efec28b880f10b91a4e05e1
323f8c2cb65410bf8d78b7d09ead475cba702a1f
19852 F20110114_AABUUU rauschenberger_r_Page_187.QC.jpg
3c0803b80a948e02722634f9638f8c57
c4e5cce764f1712ded9acfbeaa8914a1908a6399
F20110114_AABVRN rauschenberger_r_Page_162.tif
4049c127dfd8a0d69ee46d5655a06893
5aea4ff9cacdb7a97832cc80ca132824e51ac926
F20110114_AABWOH rauschenberger_r_Page_178.tif
473adf7bade83ffd19c70f458625ccc5
7aa81506b15b370c69c3cd539d96035ea25a19ab
F20110114_AABVED rauschenberger_r_Page_100.tif
c13d082ab65babff6e188d87df643d47
e9949357766eb44e8645c502672cb56319af7878
76372 F20110114_AABUUV rauschenberger_r_Page_233.jp2
a3462e85c76e196031f3d009dc4d250e
680939f7f40289df502d703a0e5d426987bf4523
49795 F20110114_AABVRO rauschenberger_r_Page_099.pro
af5dd82d0e73839efa3b9c50238bc4e7
2cef490674db3d634e8459ecce21869070fb429b
F20110114_AABWOI rauschenberger_r_Page_180.tif
167ca89bcf46b4acafe51683300a7962
80016915ad7031ae63a24955b0dd8b538fdf3e55
2043 F20110114_AABVEE rauschenberger_r_Page_163.txt
406e5e0358f8e7eecfe65ff9a1a035ef
3c23ba8078a711eee561d630d66bcb858043be6a
7501 F20110114_AABUUW rauschenberger_r_Page_192.pro
e67eb5304a302530b79b65bd3197ec07
8fcaa108aa87dd165989c1db43b19d1782ef0f06
F20110114_AABVRP rauschenberger_r_Page_057.tif
bd593140366c5a18566405d058d4ba11
e12fbed593bdb07dcb2a9e0fe7ec372ad97879a8
F20110114_AABWOJ rauschenberger_r_Page_190.tif
be9c5f8b1b7712047e28d41160cc0b42
e65dbee88caf3cc31f92f2ba7d98448746a6e442
42028 F20110114_AABVEF rauschenberger_r_Page_095.jpg
3e897c53763b636066bb365d4ce2bc1d
a55f1f76fbcc66afba634314090d55f9a67b7d04
47354 F20110114_AABUUX rauschenberger_r_Page_224.pro
f80e0a398ca69a223e5721d5fb8197e9
7f5f2dc9bdfdeda0c2c54fd4c42ccb8918edb27d
F20110114_AABVRQ rauschenberger_r_Page_136thm.jpg
4e6ea774106fb3ee8e6355ab465cbb87
a04f22ca5d605cd86dfcf613d23747488f748a26
F20110114_AABWOK rauschenberger_r_Page_196.tif
5327e8dd4b6c3670b0d34c1b3c9e9455
f6432ea9b14d6b7fff9e324f141a62d48e55316d
9124 F20110114_AABVEG rauschenberger_r_Page_063.QC.jpg
c037150362e8acfbb31259f0b7e84e5a
10fe56fe97036f68370ae4ecc2b534907b6eb29e
74515 F20110114_AABUUY rauschenberger_r_Page_052.jpg
712d197ef2c8c88805c3958d61a63770
b85569215428d62b58c5e1da925d5f053d21c157
23723 F20110114_AABVRR rauschenberger_r_Page_121.QC.jpg
54f2cc92ecbc83c012a9a1c34ae91635
6046755e81a94cd6b97b4c4bccacb80318d96803
F20110114_AABWOL rauschenberger_r_Page_208.tif
63d80d3c1c157f92a50c18c5d843cb3e
c70b6e1cc29da377e1161ff425ce202b96b3ccb1
F20110114_AABVEH rauschenberger_r_Page_038.tif
e3da00791b2a837c3b549b9ebb508508
f39b269502633c0d7b2b72f9825f43db3681c569
23354 F20110114_AABUUZ rauschenberger_r_Page_177.QC.jpg
4955dfd740f4021c5e521ac8f3631ae1
0b7fc853c3faa3c7fc418fba831b76f851231d3e
1959 F20110114_AABWBA rauschenberger_r_Page_121.txt
c9d0e00f1031cafe8c54a60bbdc416d3
08b439b6438878e868545414fb5b18b2be0847f4
23692 F20110114_AABVRS rauschenberger_r_Page_161.QC.jpg
6aafb1462c667bd939251d3efad5d331
254bc65b14148e157c1b82cc2b53de9b0b6d9fb8
F20110114_AABWOM rauschenberger_r_Page_210.tif
e1593aa9fbf3970079332510c824370f
5daf7f13cb6cc80453618de089df4b4abc2789ee
48720 F20110114_AABVEI rauschenberger_r_Page_116.pro
286e03375729cf22d915027207c692b4
cbc56393ced08747935fa79fb8745b27faa54ffd
114307 F20110114_AABWBB rauschenberger_r_Page_036.jp2
8cc1a60e46d3359b99e9b4a5fb1406a1
a07fe8fc427197319021bcb269cce1f06d625837
2007 F20110114_AABVRT rauschenberger_r_Page_079.txt
47c9d5822d59ba74d18a20025d0c0f32
f125944021cc1f8b7aa723df3b433110e9eea8be
F20110114_AABWON rauschenberger_r_Page_212.tif
81291f314d163eac77ddca1415a5ab1e
810f515cb3aa3fb61499707cc0adcfcd858f5043
30604 F20110114_AABVEJ rauschenberger_r_Page_037.jp2
4edfaf9dff42951b9ddb984bb243ef1d
fc9bb7b33d8b20a4dabe46ce070904bc621f199d
23473 F20110114_AABWBC rauschenberger_r_Page_178.QC.jpg
f796ab15c253b69ab2a4564c9c633665
a832c21d997faf623426c85d976653974143c80c
24218 F20110114_AABVRU rauschenberger_r_Page_079.QC.jpg
fe9dd180025be657256d36226969ae8a
a185329d15d3455d021bd985f601ea7d73ebdd5c
F20110114_AABWOO rauschenberger_r_Page_222.tif
33932ad9dfe77464e27032cbc3807069
6709a93e50645f1916140a5435ad1b8c3cb6559f
54486 F20110114_AABVEK rauschenberger_r_Page_080.pro
effa35cfa4e873ce875efe7fedd1ffac
be19d8a3d7e283a65b037aefa500a13f218cd7a6
9862 F20110114_AABWBD rauschenberger_r_Page_223.QC.jpg
e7cb1044dc4f01d79ca7c7a13f1fcb65
dfb353e823b974e314b8942b11c42a66a4396884
6460 F20110114_AABVRV rauschenberger_r_Page_028thm.jpg
43d8c3fe30a67d6c6b4d16c25ce67760
e782a3ec003a400535bb116f088ac0055399ce8c
F20110114_AABWOP rauschenberger_r_Page_226.tif
5ead904de8683f4d5ecd3b10c96cd761
951c39e44f877fde4286b9b3fa2381f41e21a921
50687 F20110114_AABVEL rauschenberger_r_Page_074.pro
049739126af13f335b272174f2b40206
2ab4c899ad9ebe8b214fb1ec4b16863edf0413b8
809 F20110114_AABWBE rauschenberger_r_Page_153.txt
12d1581803a303e044818d63e47794ef
f9ca3d683175743df7cfeef1cab76c0d7347138e
30352 F20110114_AABVRW rauschenberger_r_Page_190.pro
0d1c871f82b5244a77d66a7b9d27be76
cef84ad14c0f33cdeb01287cff133699b8fb770f
F20110114_AABWOQ rauschenberger_r_Page_229.tif
bd1cc9ece3e3699321660902b210efaf
64e5991bfd488e97ff3291e08a0e4998daaf96eb
F20110114_AABVEM rauschenberger_r_Page_170.tif
f8c70731b53081665444cada33c6ab46
842b92ace306c5f1f68eaf6b9fd60a329cb0b018
72402 F20110114_AABWBF rauschenberger_r_Page_128.jpg
66bffdbfc1ca940af9c061baaab59382
64d07613b14bdac00207aa9de3c34805fb80a101
1903 F20110114_AABVRX rauschenberger_r_Page_125.txt
a65f6fb48e537351b542fdd0b6e50dfc
aeba58ad43c27b062c2a18b01b4e30449a969d62
F20110114_AABWOR rauschenberger_r_Page_233.tif
f8474897ca802e97b35e93d09ed12561
63f45ec1069c8524ce5088927ea43dcb4120690e
22058 F20110114_AABVEN rauschenberger_r_Page_159.QC.jpg
abf75da8a7f22790ee88d0c17e50c143
d42eb940590a9679acf0cd44ff42d9a4affbcfdf
23456 F20110114_AABWBG rauschenberger_r_Page_168.QC.jpg
80a205c7894652f2ba9578ef3839cfd2
0d84b94c21b430d119df20a06a32c698e6f3f571
1843 F20110114_AABVRY rauschenberger_r_Page_203.txt
5ad83892cbb06f4039453c3aa714ce43
f19583f36efdfcb28b639362c801361b6dbb8bab
45883 F20110114_AABWOS rauschenberger_r_Page_004.pro
88e54c9ae2f7f17fbd514f31970184d0
95da4203ec909a215422718f328875c5011a1630
F20110114_AABVEO rauschenberger_r_Page_010.tif
304d53ba28e0740a441ca1b9ed8eb6a4
effcbcaa11495ac16f7909ad57f396b51b35c6e8
26205 F20110114_AABWBH rauschenberger_r_Page_158.jpg
0aea5c9ef9d4764d7a29e39b2e0ead25
3b5c0886059d1bd7004d3481e0b024e74fb8d8c1
25694 F20110114_AABVRZ rauschenberger_r_Page_068.jpg
fe414c2e808bdad50ff6f8a654dd8d1b
55d4fef7e2cd82feb981ad32c9c1dc29bd59b627
84163 F20110114_AABWOT rauschenberger_r_Page_013.pro
e6d38945044c3004fad537cc0d7c01f9
438c3547331f880ec77ba52c072bd34b3aafb239
113871 F20110114_AABVEP rauschenberger_r_Page_184.jp2
df4f85148477161299c5c5a96c34741f
f1b4a6af2d320d38b081cc4d629a5acac8652d6b
3196 F20110114_AABWBI rauschenberger_r_Page_149thm.jpg
52f491b74c90bcdfa1c3c4205c3fc51d
b9cbcb582e850ea703af5823b6934c836cde4ad5
48591 F20110114_AABWOU rauschenberger_r_Page_022.pro
d4e9fa04f38d9319a40c2f5ceca9c200
34690484ef4dca0b8eaa8f48f0a38c16a94f27a1
F20110114_AABVEQ rauschenberger_r_Page_092.tif
dae7088ab69705e8183b3e965d528b51
921ad7080dc86e29ece5d7bd7e419a8405e23ac5
23368 F20110114_AABWBJ rauschenberger_r_Page_082.QC.jpg
a701e205318d0f7bcde4c7b054965273
ac766812026f75c934dff01746ff4e9375b9ab1e
47812 F20110114_AABWOV rauschenberger_r_Page_026.pro
1d773c738757ea7ce4fff2c370eea1f0
1f591fba5b315285b8fc949dbcc2e895d01ac5b1
23493 F20110114_AABVER rauschenberger_r_Page_044.QC.jpg
146939ba1f06d11e4fd6ea9700990821
c736cb2ee8994405cc0dddbd8736e377dc34c08c
946 F20110114_AABWBK rauschenberger_r_Page_057.txt
416fe124fe0694cdaca34d4ecc933575
c26a653f2b06cec543fecbdadc0d60e3f6126380
44912 F20110114_AABWOW rauschenberger_r_Page_030.pro
49a1c55ad7a88782761e3623d707e50a
ddb857e98dcbe35605386aa999ea7dee12a94851
137229 F20110114_AABVES rauschenberger_r_Page_227.jp2
c81d7516287851ab3f3a5a61961aed1f
0592bd0401ad7d203ad8bdaa8783310574f9084c
6693 F20110114_AABWBL rauschenberger_r_Page_129thm.jpg
930d3b678ffaddf8d8e9199a6e6988a7
e565bd78a01f08d23b7d0066c2b973126ea25d82
46504 F20110114_AABWOX rauschenberger_r_Page_032.pro
4e91142dd899b9c98476f4e4d42d7049
4d9f3afb2d419342d65c65c5f8942cb2cc5c0b98
53023 F20110114_AABVET rauschenberger_r_Page_065.jpg
ad55f17946339455aacb62e07c1d51a3
f868a457cb25235759ab3c123139459ae9d0c822
7050 F20110114_AABWBM rauschenberger_r_Page_150thm.jpg
c826bbaa4eb7bfc0bc8dc51e214a8859
d36759d5a6de300842eb8a1ac5640e064001bc4f
47203 F20110114_AABWOY rauschenberger_r_Page_040.pro
44f275347787feded706789168ca737c
86efeba9887a1665dc421584b4afb40cee7f0103
11118 F20110114_AABVEU rauschenberger_r_Page_110.QC.jpg
52c1614a0da4c7699ec7cd585fbf0e9a
d096657efe5446c87aab8f3c8a6bca04152c9d07
112385 F20110114_AABWBN rauschenberger_r_Page_204.jp2
0d9e53c31b01e795257809355dea5a43
a6e12fd259d890801a4d65148d5b61c3c39ea6d7
47568 F20110114_AABWOZ rauschenberger_r_Page_046.pro
4c64bc59d6c8b8811d56ab669da2bbfa
ae2303b7cb3083188ded424cac0ce3b8d52e935f
11314 F20110114_AABVEV rauschenberger_r_Page_154.pro
e31385779d2d678d9a527f96aa10bf96
2cb1a458bdbd5256c894d88f4d0b0923b026df7d
6685 F20110114_AABWBO rauschenberger_r_Page_072thm.jpg
5effc5bb867bd8a9d8e9ca5d40a32dfe
75ecf6a5a286e4a20c5b355cef1558b9561dcc54
107745 F20110114_AABVEW rauschenberger_r_Page_178.jp2
43652c5156250ac15575efb802daa5a6
37bc42c6925f5a62d25184574d58d6b07795a456
46455 F20110114_AABWBP rauschenberger_r_Page_190.jpg
b725d11f7920da7428a6f425c2e47173
9eeab5f15ba24214647dfc0bb933950644bd0ba2
F20110114_AABVEX rauschenberger_r_Page_056.tif
72e3b65535787cba7381eca96212a5fb
8044eb9b3d56dc41997b7c38b248b9f468629018
3186 F20110114_AABVXA rauschenberger_r_Page_063thm.jpg
cbae25185b843cfd90e8511f7ce4515d
e5b1d634bbd1913f7a63df33af334242130956cf
759 F20110114_AABWBQ rauschenberger_r_Page_005.txt
0effca2803c88c3bab2ab3a77edbeb9c
65735971eccbf68daeaecc803b290f416168a83a
1893 F20110114_AABVEY rauschenberger_r_Page_132.txt
5b538b254920c391435f5c395cfa829f
e8fe9479d4bdedd50540b581eaf0c5096e58e750
8524 F20110114_AABVXB rauschenberger_r_Page_191.pro
7e4dc82b295100e1043e22f311c45184
dac1d43ac11aaa033e3a0a5661af13468a566a93
49467 F20110114_AABWBR rauschenberger_r_Page_135.pro
70a21706b800cfc3d804565f1edbb1de
7c8fcc09a3286a3bbaacc1994ca25dff610049c1
5157 F20110114_AABVXC rauschenberger_r_Page_006thm.jpg
fb677a9ddecafd802905adc9c8700eac
f627c678ee86ebdfe7f402009deaf00676415483
35705 F20110114_AABWBS rauschenberger_r_Page_060.jpg
2c8d75a0ba74c336f2c341c5acd69c2b
35b569c13299c3a766fe599a1b2be32303ec87fa
2865 F20110114_AABVEZ rauschenberger_r_Page_155thm.jpg
cac83ce7314725eae96787b754f35f88
557a939550f1dfb8166b31f5262046a3c9667454
F20110114_AABVXD rauschenberger_r_Page_174.tif
a98dfa7e240d43ba37c3a3bb0eb0063c
51c810e8e31ccd6bb61cc7b99123d59182ba9de6
2628 F20110114_AABWBT rauschenberger_r_Page_231.txt
f4c01220482c3902a417f89aface13b0
2337cf93691a51a810e6849495961cde5beed901
6411 F20110114_AABVXE rauschenberger_r_Page_172thm.jpg
ace0d36d04785980d1109fb112701a45
625c64e04f38772d1b81711d7272619ed1acc098
109828 F20110114_AABWBU rauschenberger_r_Page_127.jp2
1ac09ee06892f9e23950f285e9585e84
e2963a8c7690b182ee4833aceb823101f4c7332d
30466 F20110114_AABUNA rauschenberger_r_Page_067.jp2
1fe4ea7c1a59d8b3180d168abaa909e8
f3d1f2dfbb91d1d7be5d0d318f66f3e91798d0e5
6585 F20110114_AABVXF rauschenberger_r_Page_105thm.jpg
913d213ec42cf81fd483fe70cbb599c2
db2334a3996db3ac2ff6e5d8a1363ce97ba59352
3154 F20110114_AABUNB rauschenberger_r_Page_089thm.jpg
9323f42c8b99a261e717ce3ac12e5ee4
beb24c05af423709f6f7bcfb77bbe4bef2d3678e
F20110114_AABVXG rauschenberger_r_Page_157.tif
8b137d334e4575442ae6ab3a8317fe20
4f3877274556eb12ed002d05fc87b3a969741c0f
3218 F20110114_AABWBV rauschenberger_r_Page_092thm.jpg
98c4489aa20f4e4061821684564092d2
f5aa91043fa7036bfc993fb40096f7c94a6e430f
6590 F20110114_AABUNC rauschenberger_r_Page_164thm.jpg
3cb99f771b921bc00a2d86c4577eb6e9
205190c5e51a5f2fcae0a922ebec0cd43bbe190b
2726 F20110114_AABVXH rauschenberger_r_Page_094.txt
374e5267177b43a7fa9169d61c224515
a09ae51f662b106bb09016b1c841f40827ce9fe0
26958 F20110114_AABWBW rauschenberger_r_Page_226.QC.jpg
80eef28d6b6df7f27864697316121349
ac9a95f1825a5c96c6c8557534bd8d400fadb518
4734 F20110114_AABWUA rauschenberger_r_Page_109thm.jpg
2e8cb568f855eb05e85327347b330dfd
0052bf30053e4793909318e345c2c53cc8c8738d
3577 F20110114_AABVXI rauschenberger_r_Page_146thm.jpg
9ca6a137edbc07046ca635f11d0cf7af
08146c64bd34bd054a549fd636c67eb12977aef2
5961 F20110114_AABWBX rauschenberger_r_Page_169thm.jpg
13a329319f5697acbae5ff11b4b6fdaa
85d6e67f82ee3658a5cd06cce7424767c8ae4bf8
3456 F20110114_AABWUB rauschenberger_r_Page_110thm.jpg
be7317d2e9263d206c563607d639950a
2b97b8d9cd86b92ed080037630535260bd609a5a
58243 F20110114_AABUND rauschenberger_r_Page_151.jpg
865c2332c3a552e51147d5740902745d
9600d7f40eb0a00c1ca2d712520d1a07ddafe127
48310 F20110114_AABVXJ rauschenberger_r_Page_136.pro
b3d3ffa5fd32ce4c4a0bd276cebb9e8a
097c9c064edaf040a3506f21fdbeac1b521e2b44
90491 F20110114_AABWBY rauschenberger_r_Page_227.jpg
25b59ee03e2c6dbef1dd44d870f7a821
e29e6351763abfba35672b6b32be803125d31264
3684 F20110114_AABWUC rauschenberger_r_Page_111thm.jpg
61ff3e0936cf0a747bb0b54faddc66fc
99871d05febfc84c37bbfa7133665357534ecc64
55662 F20110114_AABUNE rauschenberger_r_Page_095.jp2
eab662ca3fd128148dce0a89848ecc96
d3c0c9f2988aeef91cea4b9a717acdb56c9cc7f8
24691 F20110114_AABVXK rauschenberger_r_Page_069.jpg
01fc348915931fa1170ae66f32e70083
bbd6468196700fce63e2c83e5a66b358170620fb
6718 F20110114_AABWBZ rauschenberger_r_Page_192.QC.jpg
7de83319d80f5f137286a2e44f911bff
eab96ed2e72a64b188605dc699c7f9d0b5e9114d
4912 F20110114_AABWUD rauschenberger_r_Page_112.QC.jpg
9291412439f53c8dba50ebb8482a0b51
1f25bf1b13d53ebff42366925b095be9e77b5fe5
F20110114_AABUNF rauschenberger_r_Page_074.tif
5cf19fa90973f3e7e9c09730fd26ac0d
933c39f258502499c5ca864565a201d2bf33113d
73259 F20110114_AABVXL rauschenberger_r_Page_204.jpg
537db37d25c7874dcd11f6b4d408f91c
f7b68566c2898dd80e0654ac93cd71370081020f
21690 F20110114_AABWUE rauschenberger_r_Page_115.QC.jpg
ce8971e78ff7c484d5e3d96246b66b8c
2a183f73e6d2360e69e0530d881aa0416727e533
3982 F20110114_AABUNG rauschenberger_r_Page_182thm.jpg
2a25ccdbb4d23d0c0c04d7173edeabbe
b59cf16e84e0b1d885b47951f05ea6bc217b295f
16467 F20110114_AABVKA rauschenberger_r_Page_142.QC.jpg
347a83868fe9d9eb6f099ec94f018b3d
06ed08fa36342b776c82075c6c2f3aee03b76af0
6349 F20110114_AABWUF rauschenberger_r_Page_118thm.jpg
c3542bed53f94d1d3062927635cb0ca9
1de658144c259c6d418ad5204047c179d6716289
F20110114_AABUNH rauschenberger_r_Page_169.tif
d79ad2a9c4ab1191f1feebc8910ac14b
11667bdbadd3a3f0aa06f45564ff9baddf9d8e5f
F20110114_AABVKB rauschenberger_r_Page_028.tif
1d62c6290c319447c262351ae2860705
7e04b7114fdaf57b641504f3c2b08ff34d33c6ae
F20110114_AABVXM rauschenberger_r_Page_202.jpg
479387afc32ebede4c89efcaa1057163
4057b3ad4d2dd7207d1077c1988bbfd387286f1e
6615 F20110114_AABWUG rauschenberger_r_Page_119thm.jpg
70e1b44cf73079feeec8e67f3652ec6c
edc4a68b3273fa1a571af35b4a38463c8f5e71a3
72731 F20110114_AABUNI rauschenberger_r_Page_176.jpg
cf1a0052e939a481d2e02c33a3a936e0
9a315051edfee56fe9320ff592ded36ce7be9746
F20110114_AABVKC rauschenberger_r_Page_151.tif
2f7140324d68137b84e1df97a0e505b4
f828fb413bf39011904bdeea061eda1d56b8ace2
49082 F20110114_AABVXN rauschenberger_r_Page_178.pro
8f005245b332df455c1d8c7efda8a9fd
af4df537a1fba770049ee17b4db2a58f9b3d51ff
6136 F20110114_AABWUH rauschenberger_r_Page_120thm.jpg
b806d7f126c641b245b705b5aba48973
46fe900c9c4161fbb0a90443445ecd4f087a455f
F20110114_AABUNJ rauschenberger_r_Page_045.tif
b3f9db85d93b45935002ff4517bedaa9
cf9431dc7aed6e3a700aa49bc84093bc74ddaba4
6375 F20110114_AABVKD rauschenberger_r_Page_026thm.jpg
4f7509483736fdc45afc095677196581
66003f65af73cdec87996477ac25848b2449f789
68037 F20110114_AABVXO rauschenberger_r_Page_039.jpg
437481a5b4cc500d9451812d740c0c94
56daa34587ec7e33354e33db57abddfec471b814
2520 F20110114_AABUNK rauschenberger_r_Page_230.txt
aa273723ff37453d497fbbee1dc83da3
c903570cb323a2c853fd0a490686f6a157411279
75050 F20110114_AABVKE rauschenberger_r_Page_042.jpg
d265bdc3ff7a389a7c81a869abf97fae
e99435327017ee7a6eb94be87c47e4a888419848
27607 F20110114_AABVXP rauschenberger_r_Page_001.jp2
c5339f28754d5faf9aa5242a61428a24
7ea385695af3d1ada19648c7a45b60012f6903e7
6749 F20110114_AABWUI rauschenberger_r_Page_122thm.jpg
f630b2a32d6da47e15b8652e02a6f5b8
c8d3aa2808ca367e1617739b2c8b3a19aeddbe33
50330 F20110114_AABUNL rauschenberger_r_Page_199.pro
285db2dd15014ed68492556bf966ad30
b9335f42d054e2600431653ec5d7119566445739
13730 F20110114_AABVKF rauschenberger_r_Page_189.QC.jpg
d95645a0ef60e54ad8d01fbdb3d85695
0f4a1b4bc1a2725c9a822162f3cfd3dc9eff1d3d
23755 F20110114_AABVXQ rauschenberger_r_Page_018.QC.jpg
ce9a3d074e8777bd3c419c99abd181e7
7426566743b39613505513bf8c6a02ff43ba35e0
6370 F20110114_AABWUJ rauschenberger_r_Page_123thm.jpg
c88154389392c055948250a3ea393267
1e8dbc4528e9a7016efbbde3a77daf63d4aa5fa7
15316 F20110114_AABUNM rauschenberger_r_Page_147.QC.jpg
e34acdf593904f89f77a4293f877480f
1d8f9645672a0d5045f2a70ac64f2827daf2d00c
25412 F20110114_AABVKG rauschenberger_r_Page_153.jpg
fb6f4de7a04e606e33ac27e5aa1c2717
f263a0d303bdb35c40b1b4abdbc53eec4d165302
79324 F20110114_AABVXR rauschenberger_r_Page_180.jp2
49d156c5c4a3dd90ed26782982a55308
1bd51cc9c27b3e746a629f910a64a231450c206c
6508 F20110114_AABWUK rauschenberger_r_Page_124thm.jpg
64319aa3ff04b26e00c2d4ce42ef9ea3
3fd59d6052473bb0e6ab019d8100ae8145ec58c3
47955 F20110114_AABUNN rauschenberger_r_Page_050.pro
af186824575b2681c1e505c6853c6968
488420945edfbceef9b5e4b683ac10f6326ce19d
105172 F20110114_AABVKH rauschenberger_r_Page_131.jp2
c525360d51b1eab9d0f85d7979c89fc0
f165018bf9c16f492ffde7a9f4e4a4b9c2f0ef18
F20110114_AABWHA rauschenberger_r_Page_173.tif
67c65fd12c3a33200646b02ba8f9d3bf
70484a29f7b4c4df19b538b964e8d1c312f4084b
51437 F20110114_AABVXS rauschenberger_r_Page_205.pro
49bcb26520d960c99b215d9bc5320df3
9370964f13c118a1e905e8f92fc1033146ee4566
22538 F20110114_AABWUL rauschenberger_r_Page_125.QC.jpg
646d2dc46cc52461c8240a90749e6fed
0bfa238b9d1a90c7c16fa72b955baec39ff577fb
31339 F20110114_AABUNO rauschenberger_r_Page_113.jpg
88ba438bef0178906b2cc65daa68f6a2
94f37cff3082cd988a9f06d79c53db375fde2aca
111129 F20110114_AABVKI rauschenberger_r_Page_124.jp2
a39c4dd5f0691907a3a7885d8475d91a
e82062d659fcbad61affe969f728d4c6be0ea3d7
F20110114_AABWHB rauschenberger_r_Page_141thm.jpg
bcbf6ad650fd4c91bee332ed68cd4d7a
aeb21091def216027321f1238746d6cd19e30314
49297 F20110114_AABVXT rauschenberger_r_Page_021.pro
f0af84f389f70058ac10b1b14246afe9
02b4f65acaedb3cf4dc961ab94b47808f8777d73
24195 F20110114_AABWUM rauschenberger_r_Page_127.QC.jpg
348c6f6d67230757431e84c71ed49924
ece07691eead171e2634bcf17901cf2b322ca8e0
9223 F20110114_AABUNP rauschenberger_r_Page_001.pro
886113a0875438180f3026c6e93f57a2
9fdd5c5d52b0668a795a4eeba3c044b458a42ebb
2627 F20110114_AABVKJ rauschenberger_r_Page_229.txt
416aef7fbc6d3c13b73cf2944efc7a6e
efb604f706a425e16bf7e623fb92286da6728d47
22342 F20110114_AABWHC rauschenberger_r_Page_123.QC.jpg
de86512b2c527101a9db1dbe3f88648e
2e6717469d7c558c26bd23f5bf6c2081e7880871
F20110114_AABVXU rauschenberger_r_Page_054.tif
dd820c78e1880dfdc18c9108660a79ee
3f45fc978737248aa9959331d2f105058fe0dca5
6675 F20110114_AABWUN rauschenberger_r_Page_127thm.jpg
20a2970820c881fd4596d8bac9892ec9
c5f0cd37cd1bead6b73ea5de29ed43887157aa4c
F20110114_AABUNQ rauschenberger_r_Page_007.tif
1aab889eea8bc1a286a94ffaf63248fe
50d1a14242d4499c74cd22b6db9d868162df6d4a
F20110114_AABVKK rauschenberger_r_Page_127.tif
a7c95f8481b700fc758085dfe0278de4
865571d71ee8af9b2f98bfab2db3c1c62097961a
104092 F20110114_AABWHD rauschenberger_r_Page_159.jp2
0309a0c9f712aab27fc7fec3ce2fffb2
a1d9647ea69b683d1fc24dfb1fb71e1e8c8982a7
2074 F20110114_AABVXV rauschenberger_r_Page_201.txt
6cbec81f173e967dac8cc4b37c0fda4b
7b7ba71f1268477a0310220a11d77650138e99c5
23738 F20110114_AABWUO rauschenberger_r_Page_131.QC.jpg
6c3220eebb23c068f22083c256850497
28c49ad104a0eef0594b8537294ca2bd90d754c9
72025 F20110114_AABUNR rauschenberger_r_Page_074.jpg
870c91f5370bca1bb61c8798aa873d2b
f586df9871b320e4c544a8eb50508c8868ee2349
52078 F20110114_AABVKL rauschenberger_r_Page_220.pro
578fd72cdc60a663f35a4241728b46db
33661b498a70a97b42d4dfd1f5b27a471043fab6
43453 F20110114_AABWHE rauschenberger_r_Page_141.pro
431b8f510b574ba4eba62e401d9beea4
e4734336371c977a2170f360c59afed8c3de8da9
42257 F20110114_AABVXW rauschenberger_r_Page_059.jpg
1842c82dc56bd5f3f8dffabbbe596a17
379a7961a9613b63eae933e9357c2a26da255572
6656 F20110114_AABWUP rauschenberger_r_Page_131thm.jpg
892d22b3bd27d2161b3cba03559f4b36
e5e65f7b0280bec35eefde90b64328f54254b1ef
11828 F20110114_AABUNS rauschenberger_r_Page_211.QC.jpg
03ac30bebf83e0d5f3115182063e748f
0315ca804855eb0b094636ee5ee56886af6259fa
24818 F20110114_AABVKM rauschenberger_r_Page_062.jpg
21ede6d607a44d320b4d05fea99ccd09
3674e3ae5e6106b4d2830047ecc43715e956788e
1646 F20110114_AABWHF rauschenberger_r_Page_189.txt
afe01a51bfebbec3ca58e63dc88e86e2
77d8a63f1b30b044d0bfe3ef4446b835d6844734
F20110114_AABVXX rauschenberger_r_Page_106.tif
b536032f4953d24f3f7988b27996eea8
7b133a3112aabdeb56f048659df2d08578001c8f
23526 F20110114_AABWUQ rauschenberger_r_Page_132.QC.jpg
1c10692890c19085e8a897619bbabc0b
edc1a0ae583cbb601d4f815e6c12cac54fa11d1c
4316 F20110114_AABUNT rauschenberger_r_Page_095thm.jpg
f586adacf49336a6630a14628e48d8ff
e5e88e51231213b68899310a21d04ca1a4a736da
66326 F20110114_AABVKN rauschenberger_r_Page_012.pro
58d8d623147b1067c415aa470a4c48df
d5632fcfab215874e5eae9310c2b9c200b1b8630
7303 F20110114_AABWHG rauschenberger_r_Page_195.QC.jpg
914144b1ba6b4b7204367b93b61e3ec9
923780f0837db2ea2b3179bdfd660ee2cfd7e6da
9871 F20110114_AABVXY rauschenberger_r_Page_188.pro
736f600290b1e56345a755390c4de4b8
8e014489ba23472a9bcc8b54e90bdd385e6568e2
22812 F20110114_AABWUR rauschenberger_r_Page_133.QC.jpg
dd4eac25b1397980c6de7bd5e37988be
442980ed03c08d69f48e72c450765802da8978a2
111686 F20110114_AABUNU rauschenberger_r_Page_119.jp2
57ac8021f9849d3aee472d8fb3d04089
784f5e5128ed2564d237487d182a85cb4cca09a1
191341 F20110114_AABVKO rauschenberger_r_Page_193.jp2
606f344e4bae565859cdedeb1ee0ee16
751eea6e6c0798aca28cef684b64bada8cc4f0af
24000 F20110114_AABWHH rauschenberger_r_Page_049.QC.jpg
86740b39679fd9c7f016f60c77f1b04e
dfc06e6b3979caeaf03db8faceb4249d0973e019
106403 F20110114_AABVXZ rauschenberger_r_Page_050.jp2
e445052635b88c045ae7d7de4fabf6ed
7f9c11e1b980f1ef5e3601a57d00f43a6f1b0f45
23137 F20110114_AABWUS rauschenberger_r_Page_136.QC.jpg
26a9fb2911db1c2f4f5b4b1cf136d5c4
b8e47bd1ff206dcd0e3862a01d6cf13710924532
109877 F20110114_AABUNV rauschenberger_r_Page_160.jp2
b36e4931365aa52e53850b743ec059a2
baf56caf5af29cebf8397549b669ac921e618d5d
2030 F20110114_AABVKP rauschenberger_r_Page_042.txt
433f81c765d03247d3c697347c379ddd
c0fc0382960be2d2159555c8f24fc78a713195ae
3429 F20110114_AABWHI rauschenberger_r_Page_058thm.jpg
d95133a8df8236ae1d2c19a56ee91842
2eaa0dec9648c1cb2c51e6a7700fd6fc96b16397
21023 F20110114_AABWUT rauschenberger_r_Page_137.QC.jpg
cf3bdacc87b647e86061748dc1a9be68
4b8067fbbfa9be0895b413986df0d77949f78047
2859 F20110114_AABUNW rauschenberger_r_Page_066thm.jpg
ca2e6b86992ceb9fe95c0ae778d2f7fc
d57d8b79c085519f900b7f795358f65fe367085c
64525 F20110114_AABVKQ rauschenberger_r_Page_212.jpg
2083f899cd4bfb6baaf14345efd9cd1f
6b2fa38123b946fbd33411d99b16d9712402263f
71103 F20110114_AABWHJ rauschenberger_r_Page_133.jpg
aec219ac5790041f60ba61203e289b40
f24c7c8ddc30084bd559ebbff16c4251ef93bdb7
7850 F20110114_AABWUU rauschenberger_r_Page_143.QC.jpg
f843c174f2621c03a35b3474474b6cee
2c4c5fd068fe7651764cb75892642ad1f3c07d83
25841 F20110114_AABUNX rauschenberger_r_Page_155.jpg
ba2ba777917720a4f996695ecc76e616
8254144df77146aa8299b5d1bbf984df3e8f8e15
52882 F20110114_AABVKR rauschenberger_r_Page_086.pro
44a5371732049d0ecb36711d5d620c6b
e92adca4959b609d8ff50266fc2ae5e86f3d4894
23611 F20110114_AABWHK rauschenberger_r_Page_055.QC.jpg
f10e62aadfd9cfe274e8432a12e46d63
e2f2250830eb06c7a83948732e40f6135b9b8e61
13539 F20110114_AABWUV rauschenberger_r_Page_145.QC.jpg
200b6180d08bc2cd3a8253aede58cf40
9bbd1312ae6b5d87d80969f26e0619cffa886d64
2504 F20110114_AABUNY rauschenberger_r_Page_191thm.jpg
8b1981abb79d6b5c6c60b6aa62cd3b7b
90799c488e1edcf8fbce76ce8d9b1710753f2602
25608 F20110114_AABVKS rauschenberger_r_Page_110.pro
509ea4a402f0f82e03a5c20c3d89ea80
ba486ac8843a8b607ed54893125663967581f9d5
76259 F20110114_AABWHL rauschenberger_r_Page_048.jpg
d8b1d91d4ea082fb29ce86f2a6397f92
00d5265833d5f038d93c309b482f0fe82c7e019b
11350 F20110114_AABWUW rauschenberger_r_Page_148.QC.jpg
c8681a330cdcf1c90e3ab370c044730f
3baf4580795171af784a85f07e5129b07227bae8
71107 F20110114_AABUNZ rauschenberger_r_Page_009.pro
d5a44a4cfc3517a397bb3ea8fb836474
14a7359c73653e2b10eb1df9fe2975dd9f5a0101
2002 F20110114_AABVKT rauschenberger_r_Page_055.txt
96544370af35d82af47d1b0a88bcdca2
fff347b18a75f3c8491770a64f4836c338c3b5ae
6784 F20110114_AABWHM rauschenberger_r_Page_126thm.jpg
ce69b58ab91d3ffefd788e1902d4a978
22c600f6262a1ec02479794ec1d057e713dce5e9
7603 F20110114_AABWUX rauschenberger_r_Page_152.QC.jpg
a09f368544e5dbaf0accd0b6b125aec4
f9811ca13598e8533905acf69b67b6d9302c2733
F20110114_AABVKU rauschenberger_r_Page_053.tif
1b10041b837456b9818e118cda1b51b6
f47650f9392a5a0091c4f360a9a70cc2b863898e
110332 F20110114_AABWHN rauschenberger_r_Page_139.jp2
daaa154879423cd3b7a835f1ea855a97
5a943fd0d3905c01c591859a326882b1da023174
F20110114_AABWUY rauschenberger_r_Page_154thm.jpg
35168371d66d20143d6a7a1ff6356030
4a8f4e7dcbe104cf16fb1e8c22d06d5e4302e506
F20110114_AABVKV rauschenberger_r_Page_073.tif
be6e8d6dad22c19c2f373a2256136191
10781e65d6dba9f84b122288bccb6a2d8224c4d3
24444 F20110114_AABWHO rauschenberger_r_Page_048.QC.jpg
b08ce1121127c71d7ffa7628e168df2f
a617a55e676739cda94a26f1d3e4dea645f00274
8584 F20110114_AABWUZ rauschenberger_r_Page_158.QC.jpg
a37a6bee2627e4794b25538ec31284de
4015a0a8fea3080ef487e1306641a4783b339c6d
2590 F20110114_AABVKW rauschenberger_r_Page_193thm.jpg
c15bee58f1111605f74f3b08cf26430d
2754d14004b0c61d52784670c5a475c6783e7e68
25321 F20110114_AABWHP rauschenberger_r_Page_154.jpg
b7b32e8560cf4fc10641b6f9f3ad0884
437697cfbb04caeb2b1f99909d7643ca173f4ab9
110890 F20110114_AABVKX rauschenberger_r_Page_081.jp2
bb007d01593f632bd235e8a04eaa257e
6bda074f9000b3cb01f2b838f260dcd792f959d8
48972 F20110114_AABWHQ rauschenberger_r_Page_076.pro
b3526bf6df3e0ed0370ab018e97cb6a3
0590a6dd7fe4a6036d3e0f25d3999a65fc667d43
F20110114_AABVKY rauschenberger_r_Page_208.txt
ee766e297e1a40a00948c578ccf95e38
53b7f628bffd60961902dd327c371e5eb9b5a35b
105594 F20110114_AABWHR rauschenberger_r_Page_022.jp2
574eb11ff4c6b863c784eaceb39c40eb
54efcc88c948bd1d1e449d8f51da7ccd1468a319
104592 F20110114_AABVKZ rauschenberger_r_Page_041.jp2
e7dfcf9ed4e791693643e1bfbfbcde2c
a7ae0377ac112246cfae03088d99602a25769444
102602 F20110114_AABWHS rauschenberger_r_Page_013.jpg
04a5328fb3a19f3f8624d8c6fb6d7f75
41519de408b0d052b83495741412ee64e769cec7
1688 F20110114_AABWHT rauschenberger_r_Page_111.txt
427c33b2a6816af2747d8e12bc16b553
4e7d59b94e8b8b41b38c977c9a2bb5959f819937
6522 F20110114_AABWHU rauschenberger_r_Page_082thm.jpg
c05ceebe25c31e570c1f38008dc3b3ed
e3c71c99c4ad999dbfbcf9fa9d4ebfc9f0192f83
35169 F20110114_AABUTA rauschenberger_r_Page_209.jp2
e40e051d97ed3c122ed6cacc214294c5
5fdb1cf6c6aae2aada943575e7cf8895a0e82100
1976 F20110114_AABWHV rauschenberger_r_Page_071.txt
8f6b91596e39c708b3dde67c14c73746
3ac79386b94c65d40285614635b99cf9b726773c
42952 F20110114_AABUTB rauschenberger_r_Page_137.pro
d9c4873cab07850994b463a1e8d85cc5
b4f7932f092fb6348efddd029370899b51eb394f
48396 F20110114_AABWHW rauschenberger_r_Page_053.pro
d3288ab6f6f4f62bdaa41281d0210290
b87f7a7512e0c4b5b112afd8612a879b88e60fa2
1177 F20110114_AABUTC rauschenberger_r_Page_110.txt
79a259353a52ef4d2a9bda2f93f12e4b
1dc5ea569ad33471bde8cf2e23838ced360d876e
53584 F20110114_AABWHX rauschenberger_r_Page_180.jpg
18ccf07bc8378b8975eccc1220ab5603
3500619ddc313ce6553b766158a2dc9aebb04e5d
52824 F20110114_AABUTD rauschenberger_r_Page_221.pro
321ca074ccb0680f4ae50b236c06829a
19e49f91fce125bd07e066255eebb362ce7b9c1c
6619 F20110114_AABWHY rauschenberger_r_Page_219thm.jpg
dc904990a6e2fdfdbb60a3728b85cad2
cab1bdbea42ae7d9711f288ea111b97a71cc6355
F20110114_AABUTE rauschenberger_r_Page_072.tif
84f257962a6273be3f758686fb1fcf6f
d02f7524a60f06514057dedc0bddba7bbd97ff36
48792 F20110114_AABWHZ rauschenberger_r_Page_085.pro
4d8d9fdf20b2d0f4399cf69869283afa
d2d155ade190bbdd5ac84d8e0dc2b5ecd3602ecf
3088 F20110114_AABUTF rauschenberger_r_Page_011thm.jpg
4bf15ff30776e2ee886f905ecf4d2f9f
4cf94a61353f9e4cda084fa87885405eba95e5dc
F20110114_AABUTG rauschenberger_r_Page_131.tif
69734ebe28caf11aeaf19c305509682b
ba4f72adfd4dd06074a03ca2af562861842efa51
22540 F20110114_AABUTH rauschenberger_r_Page_020.QC.jpg
fb74abab20757bfd0a2e6c28c5076d87
6cbb021bc4120e1d12e84ee20abb6294c75ea42f
994 F20110114_AABVQA rauschenberger_r_Page_063.txt
3958337929ff7d399e4b4e695518e0a5
5946611621e5735acef07ad21bb85e50f099e67f
54369 F20110114_AABUTI rauschenberger_r_Page_210.jpg
c78136175a7cfaa56271d5764994fe17
430a5f969a533f870d29ecb879998af193b3ab51
1416 F20110114_AABVQB rauschenberger_r_Page_190.txt
e258caeb085a846662c28b23df8d7175
f540201e33e4c7b24d2a73eec943c9eac93d9005
69084 F20110114_AABVQC rauschenberger_r_Page_041.jpg
adc1ed0bf3cf704670218ba296e312a6
43a16652db0736d512053fbc6c06b689a42ffa64
23093 F20110114_AABUTJ rauschenberger_r_Page_034.QC.jpg
7f62a193a20c7752129a109f6057be0b
7d1b5e2ee4d8d8b78170185b57b037e14e1fb3c0
2575 F20110114_AABVQD rauschenberger_r_Page_196thm.jpg
0f36c8e731219b2a3163c29eec9b8a38
96cb60b1978debfba709abe00ad5870d6ff2e559
6383 F20110114_AABUTK rauschenberger_r_Page_020thm.jpg
e32eeee5f4fdcf807e267564e47530c8
bf4ed64f5828d6e6c11b576c04a5c104db2680b2
6546 F20110114_AABVQE rauschenberger_r_Page_024thm.jpg
295cc3c33f0f2ea46f94259616dfe94c
9e23db4d6943b44433cc2382c15267d70d47e33b
F20110114_AABUTL rauschenberger_r_Page_199thm.jpg
620c4e72bc1c7d5a73099f014b3ca3bd
b8557bedcd46517416d95240e7c711fb0bb0b880
54230 F20110114_AABUTM rauschenberger_r_Page_147.jpg
b50c30c3995fd51b3e6cd3e81270c111
68efe3fdd9d2e425912bdb6d26719c517457ab51
70347 F20110114_AABVQF rauschenberger_r_Page_035.jpg
5b0b3ad77d67cdd450a6e28b27aa4f0e
5fc8728b06b581bf6617cebe1094db94f69f58ab
F20110114_AABWNA rauschenberger_r_Page_042.tif
5ed715debe5c112d2d66e957cf0024f3
a69da1f5edfb68ac440ad1ae04fbbe2c82b30eeb
22279 F20110114_AABUTN rauschenberger_r_Page_191.jp2
b1a91614e33877ef032cee509e2b923d
cae595ef4ec37ee1c894fabff7630990a6ce1123
44980 F20110114_AABVQG rauschenberger_r_Page_144.jpg
13851054cd4901fdc2914d75993b450b
00793dd97bcdd5d7ce6bfba1cbe631b5afaf9cee
69083 F20110114_AABUTO rauschenberger_r_Page_184.pro
250271b119b7cf40483a637d95c5b7e3
80cbeadf378de6f7f8548a15f657360562290f09
F20110114_AABVQH rauschenberger_r_Page_070.tif
a0256bebbb7ca66419b0cadbbac9e85b
789c27a283ca46e5c5f9c66b3d2423832a7ebdc6
F20110114_AABWNB rauschenberger_r_Page_044.tif
87aeca4c5f3e6061cce8491e53d3b9f5
a058f20b2b361cc63f9fe82cb04267d3c70d42e1
36531 F20110114_AABUTP rauschenberger_r_Page_065.pro
b0b49ca3d66cd832ffcf66a09cf4d063
e64539c772c64436a7c096a156ce200ccb262e58
104647 F20110114_AABVQI rauschenberger_r_Page_125.jp2
a6b3f068683a142b43f7ac34433e91e1
23544607f0d4f43cc58e653b7d9c3af3f3165995
F20110114_AABWNC rauschenberger_r_Page_059.tif
46beedff093290e75d2e2c84d01b45cb
fc6dd2160c41e8822c7d2a0c5b8ece8b0baabedb
111401 F20110114_AABUTQ rauschenberger_r_Page_202.jp2
615bdc259eca9b8116de70b6428760d3
bb79859367d50ccb8efdd58e40deb0fef0ac2375
35957 F20110114_AABVQJ rauschenberger_r_Page_064.jp2
bc1ba6d6ac2a3b0d7df54974573c7330
f5cdd0b0e181e1b6d2cc7340d5e00b39838009c4
F20110114_AABWND rauschenberger_r_Page_066.tif
2df474a53235d99fecf44d1bd3cf42b1
1179d15b41087c9afb7cb8efe10266b8822fe185
F20110114_AABUTR rauschenberger_r_Page_193.tif
7ab127dc0c85eeb9472ee6e24f78b008
d3410433a03eeb1042ec50f80dcf921f3e853a77
F20110114_AABVQK rauschenberger_r_Page_184.tif
e18c080ab81779a05f33762fac531df9
9f4ca46a31240ea75f60bba0e26230c1a5792c7f
F20110114_AABWNE rauschenberger_r_Page_068.tif
a1507a47069eed73b460e88ef663fca1
b2c56fa029c4e2f598d8b599e7e913ae834f8e9f
24022 F20110114_AABVDA rauschenberger_r_Page_176.QC.jpg
cffe8f51168492822cdfb82829881434
8227997dcc395441fa1b578e8645cfc026842cc3
103309 F20110114_AABUTS rauschenberger_r_Page_170.jp2
f99b7c9836a514927f9c277f5a2d4041
bac8e3191ce2f7435b12091fe5584f4956481099
23890 F20110114_AABVQL rauschenberger_r_Page_102.QC.jpg
f1a3122ee5900e703381838cb2019007
3e318c9ba6045a88253a0422b4b5aff38837fc67
F20110114_AABWNF rauschenberger_r_Page_075.tif
5f0b4c35aee501e0329e1be01f903506
c52aaee679f2fbf9069a3805596145c98ceff6b6
23626 F20110114_AABVDB rauschenberger_r_Page_074.QC.jpg
63d2fb5452b99301f448fe5a94108f46
a8673317bdb5412ee6404c131832d6834d4becd1
F20110114_AABUTT rauschenberger_r_Page_146.tif
519283d28cc7f13a2caff2c837fd4075
e99a0214c73dd8a4874cf0cd5774fc9b5e947e1c
51294 F20110114_AABVQM rauschenberger_r_Page_187.pro
f701512000d2404745a307e844824fa8
c1841b2e1b165b46b531dc165254272837e6feb6
F20110114_AABWNG rauschenberger_r_Page_082.tif
ff2e2c9648ab2064c56cc3c4b5920238
c70834c1db2894df9fe6186fa5f5d5faea3dc319
1933 F20110114_AABVDC rauschenberger_r_Page_130.txt
b2e4a95e3bec257a478ee654c3fc41a8
47415079fa92871330fefac3e909548901efb545
82589 F20110114_AABUTU rauschenberger_r_Page_010.pro
1fda005552fcb858eefc4b3d9d015e64
ba3d096df70c45df40d85b7b2bed2c1b98dba8e4
112993 F20110114_AABVQN rauschenberger_r_Page_024.jp2
e724b177f8f864b1e0eaf401168c31c7
a06d3672ddf3a57e2e51f5fef903dfa406ff3876
F20110114_AABWNH rauschenberger_r_Page_088.tif
69d84fd11511be3a874462e78d4e5bf6
05de422b4c2802d3e28bbd00ff778ca3f186310e
1931 F20110114_AABVDD rauschenberger_r_Page_022.txt
2ed50c58fd7dadde28bff9e579448a9c
d3e42fe1e1cd60148e7e226f30245c0544a2fc07
48780 F20110114_AABUTV rauschenberger_r_Page_167.pro
398d07cede538f0bf5f40b0a4f3edecf
17ad0bf38cc05d82caaf0f3c16be139819693ac8
1656 F20110114_AABVQO rauschenberger_r_Page_232.txt
d2de9fb3516e2ad3e844903be39dc567
99e0e9222b36c06a9049ead38364bfb4fba6cac3
F20110114_AABWNI rauschenberger_r_Page_093.tif
438256f721b1be5281a7214b7eb4b204
6f72244a1e04c47526ff55c244d02c9c7c2d3f90
F20110114_AABVDE rauschenberger_r_Page_128.txt
21dade6f63da1d4e176e8b20bf74143a
d258056e59439f914a925b6407d89ce7d8493ca9
1838 F20110114_AABUTW rauschenberger_r_Page_032.txt
e0294143e086de49eedf45867aa0f83c
02ed50ca473ec71de2c7ab2965cdc7f34519f24a
F20110114_AABVQP rauschenberger_r_Page_150.tif
90e3f61fee5ad060ebc835d1157b90a4
acdf2fb025bb522b17246e5b49da5890a52c6fb3
F20110114_AABWNJ rauschenberger_r_Page_105.tif
f47bdcf73574e721b486e5e74ce6717d
0e850430cb8befb6aae69224900ecca1754bee1c
11579 F20110114_AABVDF rauschenberger_r_Page_141.QC.jpg
aede4d13892e7833d0e44fe7d55be0e7
07e641aab343d51a8c91f03d290d993b0c2124ad
F20110114_AABUTX rauschenberger_r_Page_101.tif
9d7d5dffcbc9b85953c30a21fe6218d0
ee36281193c441fd4002bf167384d82659773d55
74796 F20110114_AABVQQ rauschenberger_r_Page_220.jpg
cc90105538f85fd57108295533365ff7
fa57eea40ba20adc12511cb6bcc1030f1a22b062
F20110114_AABWNK rauschenberger_r_Page_107.tif
9d9f5603c1545d3b65fac55325ed085a
817cfbe9dfb539ea59c36241b2400beac100514e
F20110114_AABVDG rauschenberger_r_Page_046.tif
013a4d653b2c45da06ab4cd2da0e3f83
9c9f615c8f65f4afc78d81e6f431d235fa7beaf4
22938 F20110114_AABUTY rauschenberger_r_Page_077.QC.jpg
f50f667597d02622c6c16fc9521a18d5
d8b1dcb058a533a2807acd93d87f83a19203d278
29515 F20110114_AABVQR rauschenberger_r_Page_152.jp2
bde10224a341da3cbf013826f6010e1d
2797de45e37442f22e47fc7a00322fd5ab481d92
F20110114_AABWNL rauschenberger_r_Page_108.tif
2391a0b4cdaf45ec86c1468feaccc09c
e9589ab519398e25ac65c6ecc2706d631751a5bc
67288 F20110114_AABVDH rauschenberger_r_Page_016.jpg
638cbee5e3ef53210ac26f0a30b348e5
481011d126bc197fcbb178cddae66e0aaf0e68af
25344 F20110114_AABUTZ rauschenberger_r_Page_230.QC.jpg
b3972e63fb05d6df0258f3e8cdb4442e
06eafad2b59e0d3d263e12d4740882bba17c590e
25613 F20110114_AABWAA rauschenberger_r_Page_157.jpg
dcb0c546818fe5f9908ebca6f3645ffe
985cadbe6d3269fac21d1da56d4c6a4107b158c6
2687 F20110114_AABVQS rauschenberger_r_Page_012.txt
af6c6c404394e763668f5b88822c8800
048211b481bb8ea93f1971890aad3d6cfffa5044
F20110114_AABWNM rauschenberger_r_Page_110.tif
4d3a56e0348292ab14fc2cc2c36683e0
93413a81e2f1ce1b13d8f457fcc4ebbb9df2aa3e
6358 F20110114_AABVDI rauschenberger_r_Page_023thm.jpg
099906c3989c6eb84e0339c036956273
10c650a778dd918afbba1a8c8cdb0fe30cc620de
32975 F20110114_AABWAB rauschenberger_r_Page_147.pro
d943353882dcd3507460e3676626d756
c4d6469adc0e6107279e00850ad9b498604b0026
1049 F20110114_AABVQT rauschenberger_r_Page_062.txt
d4d237c6149e38c2335c980d0a0b974b
838d93f1b130f8fd27ac6c836670f3e8f53f7c1e
F20110114_AABWNN rauschenberger_r_Page_118.tif
5bc592f3fafcac514810e1130c048273
61704f74e94ab13847b161e9dae79ec96f877ab7
23019 F20110114_AABVDJ rauschenberger_r_Page_043.QC.jpg
33990383ef36b3fc116fba1a04d0d7d6
83bc31f59e7f08b4c4b251c218d32d1506007c50
6613 F20110114_AABWAC rauschenberger_r_Page_079thm.jpg
ebc35765b612c0f555c07f6a1bdc1c52
eb0173f3c9a40d79965240c5caab581800cd01b5
1051984 F20110114_AABVQU rauschenberger_r_Page_012.jp2
137a851ce998f27db827da6929f2aeb8
9dff3e16b4c4e38443702a665d2295046bd1050b
F20110114_AABWNO rauschenberger_r_Page_122.tif
4db96f67c5c381014f513e97f200aba6
c913e24f29f0be69f20ba4364fa4423438bf0b59
74654 F20110114_AABVDK rauschenberger_r_Page_106.jpg
9fad8c025506ba88053a81cd36318c60
a72af4b3f683e2be1236f56e008fc54544ecdf26
114670 F20110114_AABWAD rauschenberger_r_Page_126.jp2
4b27caaf59477f99a3323d2a3708f9d6
04dd5fa5e2b87a8a26df550238025cef01d6e3d2
17329 F20110114_AABVQV rauschenberger_r_Page_233.QC.jpg
fc5ed0b11c1ddf7bed2f183f508ebd25
fd9a61ea95ceefae6ad84d35626c5d00af7c084d
F20110114_AABWNP rauschenberger_r_Page_123.tif
9629d27711c20125742200870c5f911d
f60af6668598079bfbba5118d87599d971c695f1
105797 F20110114_AABVDL rauschenberger_r_Page_073.jp2
7750881fa589f171524c93aaf5402810
2cac19bbb909cdcaabaa6fa47bdb21f57b304151
73328 F20110114_AABWAE rauschenberger_r_Page_219.jpg
ad6b70ac4bd67af7a6bae6d76853cfdb
7215892f68804ce087db6b54a5394a22c1019b21
F20110114_AABVQW rauschenberger_r_Page_095.tif
97365dffb06b1187fcb05eeabd892299
4865c4e38ca469412be66c1b686c8d9ca8ff7ca8
F20110114_AABWNQ rauschenberger_r_Page_129.tif
ec31a4593b7e0f39bad22b0e6bd4b154
3909f746b8b75c2cb5d01547ec738f476298f1b9
501 F20110114_AABVDM rauschenberger_r_Page_188.txt
8750d99ad3c3f23196016d394e43a983
cdfcbf4e4c1a7ad3d702dded7b436d4512d0c9eb
21948 F20110114_AABWAF rauschenberger_r_Page_149.pro
c7c80372f6ab19cc065d402c428cbbd1
0ee1aaa8a16a2719cbad8a34d3b65061ca7a567e
38050 F20110114_AABVQX rauschenberger_r_Page_110.jpg
37317e7d319f2e094a401dbd3a876a40
aee61c621edebcb80ac868d07c2f35d2f18e44dd
F20110114_AABWNR rauschenberger_r_Page_132.tif
e2bb3362ab3f9d295694a048afa944ab
32239f116bfb7d582366e76cb51b03ec5488d465
F20110114_AABVDN rauschenberger_r_Page_094.tif
ed0963ee0fa74b547777233e6ba6be57
64d11bb75e4d67bcbc72399e28a9a6f8325c3656
1899 F20110114_AABWAG rauschenberger_r_Page_026.txt
7f3a27342e362ab69333087afd1758bb
c720d6c8f5f560acd7c0f238305436e1448f8b99
49850 F20110114_AABVQY rauschenberger_r_Page_108.pro
40a1ef5b48281ec032dd39354a4b270a
94da6df73942ad5a1b6c6a458d182646b38fb1d9
F20110114_AABWNS rauschenberger_r_Page_133.tif
392a701f9c5d3928b6e2b53bd8d6dc74
b24a115595d6ddcbb138270b562613caeb5510b7
53024 F20110114_AABVDO rauschenberger_r_Page_087.pro
12a96542b72e1bb951403872c054df11
46571b9234779a29ee401db9bd8fa9ee6c362364
25099 F20110114_AABWAH rauschenberger_r_Page_089.pro
dfa9c05318b0107ef51900ab99508264
d3dd1345e2f386794755cfbd2b705abf1f00b188
43562 F20110114_AABVQZ rauschenberger_r_Page_212.pro
9271d14f061e712c0ea4377932f9eb65
f91f63b8f5499e6eed5a27ad155e5c1ac186b8f6
F20110114_AABWNT rauschenberger_r_Page_139.tif
211a2cea3a2027edcdc2fd50f8c17f49
f3c2791e6ddcdeb0199f1569ba434075ff394d1a
22227 F20110114_AABVDP rauschenberger_r_Page_120.QC.jpg
acb61bc108c04eeb50565ae50e326cac
6d1f68faae3fb901ff68834f7090667e7606137c
F20110114_AABWAI rauschenberger_r_Page_026.tif
414148ef7f376d0c676063a9aeeee726
29135412e0a1ab83252078c0f2462745605b5102
F20110114_AABWNU rauschenberger_r_Page_142.tif
1df4b012e8a3e63fbff2455fd34b0209
9417ecf31a0e57024dc3eb714a0959ba1dbb04ac
4710 F20110114_AABUZA rauschenberger_r_Page_014.QC.jpg
618c46037d61ed8c7384acac436939d1
597d48126c86952668f1142e892781829babf205
33630 F20110114_AABVDQ rauschenberger_r_Page_112.jp2
47251754602a3edfec58c18c8c783961
ed3b9b0a28606a4e1b54ea82ea31515deb1938fc
5840 F20110114_AABWAJ rauschenberger_r_Page_137thm.jpg
7e9b60c0235698232b67ae419e44ccc3
ac83865d426c82fd9adf656d89d0e18660b42bbb
F20110114_AABWNV rauschenberger_r_Page_143.tif
a31e396c5674be04c876d1e48f1e97ac
b88a48fcb05bd3a7c8fd76aeb3908973783bbd33
32350 F20110114_AABUZB rauschenberger_r_Page_066.jp2
b70b75dce64419e32354ac70c1167e91
9e8f575d102f27b90c55f9fc26ca3c41c7a7d3b4
F20110114_AABVDR rauschenberger_r_Page_033.tif
02cab1599c2deb5e972528ac2dd81a30
bc62ad85ac93299ea1aebae6474a46809de9db73
68948 F20110114_AABWAK rauschenberger_r_Page_118.jpg
2b28507838b3aaab7d30441bd1ef82c9
8d52dd807c21db0ea4c10ab81169db7d61dd9b60
F20110114_AABWNW rauschenberger_r_Page_145.tif
7a35a43de91ce155ed9646ac67903135
e2794c03ab6ee28d536867696971040f6112af96
49240 F20110114_AABUZC rauschenberger_r_Page_071.pro
4f11f94e19d7de24a6a512f2e6d0ab12
31e646fc1149d14837d4229c7ed9426eb5a971ca
583343 F20110114_AABVDS rauschenberger_r_Page_038.jp2
9912bc5bfb74bc8855fb95ce1b913bd8
b6ad56fbc9e3f72f23878ec6cd6c23fec593776e
F20110114_AABWAL rauschenberger_r_Page_200.tif
48e6b1cdda6a935469b338eefe1737c6
eb225d9fd3ebd9edb3d01e379718a52444337854
F20110114_AABWNX rauschenberger_r_Page_147.tif
dbad09cc07a09b9d2ac32d115e6b0c88
295cf4a080b4a9ce94d3ca9c23819e4f102232af
1851 F20110114_AABUZD rauschenberger_r_Page_159.txt
e81e962b29f8eda023743d8a6205a4e4
c1d112b5501f44ec22363b624211846fb4928e7c
F20110114_AABVDT rauschenberger_r_Page_117.tif
8f9a655b4ab4187f3440c2b4d80e07d2
7a1ac8bd2a6e6a59e54f2e7f522892182ea8017a
50438 F20110114_AABWAM rauschenberger_r_Page_165.pro
3a4ae54efb2b366801be1e478b647e5a
1196f925a9ad1891a9aecb58e81374eb84e48440
F20110114_AABWNY rauschenberger_r_Page_149.tif
db2ca702c736ced84b18f13e9e1d1313
a4f8fdb0839a2393e7f58151e6c672547532e8a6
110879 F20110114_AABUZE rauschenberger_r_Page_219.jp2
7aa9311063f66d32b4b44361648d1c90
d91f9d49d5e8f8a01bc105a1980271aa599c6dc0
6484 F20110114_AABVDU rauschenberger_r_Page_222thm.jpg
8004a0ff615aa415b078f9923602294d
d58b9e91d9ea122b9595ece2dcb5460768c69961
F20110114_AABWAN rauschenberger_r_Page_109.tif
9be2f6ab0540c8bdceff6609737a5cb7
602d51cef3bdd8c61b9361aadde20dee01c38636
F20110114_AABWNZ rauschenberger_r_Page_153.tif
7c6c7be28c5459c17c9d5c9cfadc1fd1
05165b485ecbe7a28d0611463424d49e0ae050ad
74814 F20110114_AABUZF rauschenberger_r_Page_163.jpg
3783c32262ae4e878bee7ecad78d69b8
9341aa9957bf20fe836748979419fa5b75caa22e
2863 F20110114_AABVDV rauschenberger_r_Page_009.txt
5ce7f89be9d827f107d9129e0ecc8c41
f1cf818d948e2bae1ad1f9f364017952a2d941bb
58080 F20110114_AABWAO rauschenberger_r_Page_189.jp2
477c1de390cf02ee644184e5e6248c71
50dbc9d34c168fc5fa4b8243f5c5940e498462d5
23451 F20110114_AABUZG rauschenberger_r_Page_085.QC.jpg
83eec8e768b513305a07bb4db8e58bed
26ef951774f5593eda1b852d1beebbb168ba723e
50462 F20110114_AABVDW rauschenberger_r_Page_222.pro
fdc87a855d59d68d1794ca398a073b28
c0396acf47684cea9e96aba5f56363a021d7af5c
6634 F20110114_AABWAP rauschenberger_r_Page_081thm.jpg
c98a2d1d0ca370d3216d4d5e1afa3903
3802caa8ad5a567a7fc73371f32c8bb5cc382c9d
23963 F20110114_AABUZH rauschenberger_r_Page_072.QC.jpg
4d860128546679664ff40b7243a5c27f
aee3e068192931c3049a7481bcd15de8183272f9
F20110114_AABVDX rauschenberger_r_Page_125.tif
dcd7e4298e351e35d5e01e49710e09b5
ba17614df0a05ec73c8a4e4904d47a3c31cba40e
F20110114_AABVWA rauschenberger_r_Page_063.tif
22a799539828767dd075bede1a8c5f90
ec0cddd9c8254b2c4ad7bfc1d3ab4f0aa6bc1c09
48141 F20110114_AABWAQ rauschenberger_r_Page_171.pro
bad6adc14d65faa92c3ecc209b3f16ec
d2e57c53c89d14a3cb9a7ea8a0d6bc98b9251f99
45095 F20110114_AABUZI rauschenberger_r_Page_159.pro
8c793ee8a4bd92b803f52da5dca6550b
e2ba8ccca90a1861444b7120720b59ea3dd5097c
F20110114_AABVWB rauschenberger_r_Page_065.tif
c6bab0419a16f4361081f76382b6b163
b7e63ce7e92e6948934bf2116e666cf2c06ba890
22565 F20110114_AABWAR rauschenberger_r_Page_027.QC.jpg
02bf5816092dc7bd17c37af7b3b25e56
5207c5b69d0fe7f6ae713df6b10458f8354f1448
45754 F20110114_AABUZJ rauschenberger_r_Page_051.pro
e776a2abef2ba8dbbcf91d41211a45d7
2982791b0ebd80f99566dd4500b35834728863b2
67501 F20110114_AABVDY rauschenberger_r_Page_120.jpg
d4226b70285cc7e59e16b8e07d652cab
5e091db2b83814f9d696487b66b71b241bb16cb4
104645 F20110114_AABVWC rauschenberger_r_Page_134.jp2
986106a60f5df8922a16676140949314
6fbf5417b3729ee4b56cfaed0331584a5e3adede
6723 F20110114_AABWAS rauschenberger_r_Page_206thm.jpg
30b8bf6d7832bc9db512a3ae4e140631
d27c9e09796f2b7c51158653ba5e621f3c5d8e3c
6560 F20110114_AABUZK rauschenberger_r_Page_175thm.jpg
f46dbad167bd21e8568af5d4c3ac67c5
77e0e7c6be5bdf37818797ac6e86b1c0ec15d68f
638 F20110114_AABVDZ rauschenberger_r_Page_196.txt
42abbfa4349f4be01e514afd2b837740
3122cf129ea5ce77a4a373f041cd36be3a29caca
64366 F20110114_AABVWD rauschenberger_r_Page_229.pro
ed4894970afe3bdd5e0c76eebde79c85
1fc76e3e102d1d3ae282d8f86ce1f1b5611ff620
6501 F20110114_AABWAT rauschenberger_r_Page_218thm.jpg
fa6f0b1b5e460c1fbcca2f914ee7fa57
941d26b84f459ac05755dc6a2b2b591fca6cf7d5
F20110114_AABUZL rauschenberger_r_Page_006.tif
62c1589ae213ed433374aae8119dbfe8
2a00879e826376914fdc9e3766a20831314c4328
F20110114_AABVWE rauschenberger_r_Page_175.tif
fb5b3ed0eac1e775b3d1ca238a6657b9
37185d5cd08010564d07bb1ffcc7b729718fbc53
2046 F20110114_AABUMA rauschenberger_r_Page_179.txt
f9208efdaf0e0f37f4973d335644725f
93693265e916d304745cda8f418444202bc56f45
2047 F20110114_AABUZM rauschenberger_r_Page_081.txt
aa1b188f55311add30e408cd6d945d1b
658d70a26bfd9df8538982fd0f31c9bee599bc27
F20110114_AABVWF rauschenberger_r_Page_016.txt
c8b77a99b39c75f1b7732d75faaa9fc2
c8b2654b5c77cc342f787d9a0ca3a378aebf6db0
2706 F20110114_AABWAU rauschenberger_r_Page_227.txt
f1a998d301a273ccd7b1e771c4c79866
ecd42d42b4a72001d5e51a1d4f9c55a8681a1e8c
19695 F20110114_AABUMB rauschenberger_r_Page_017.QC.jpg
ee11f1c8c6905eee29388e7c0ff6a2f7
578c045340452e903b8026bbb6c8098995135afa
51250 F20110114_AABUZN rauschenberger_r_Page_082.pro
00baf013986e8d280f5ab5de8d7b2ca2
ab1a5da9d71688e59a8842fce0ae55ad4ab90b06
50896 F20110114_AABVWG rauschenberger_r_Page_124.pro
9c17358daa0778b84c982c98823520ed
726bdf0c377b57fd4b5c9e0f0ea72ea845ca03e6
22419 F20110114_AABWAV rauschenberger_r_Page_134.QC.jpg
c2b6b9084e0b575401510cfaf8982dc8
6559d7312e0b35c57f4be787801b78f698be7186
2650 F20110114_AABUZO rauschenberger_r_Page_069thm.jpg
c0312b41a93300b082eb606412601fbc
3621923506ad25e89ce0a66f0694418d537ff979
1955 F20110114_AABVWH rauschenberger_r_Page_021.txt
f3bc02a06367e783bf4f833852397455
330f3818ccda609dcaa078e000339ee905275134
35886 F20110114_AABWAW rauschenberger_r_Page_091.pro
20e8679f0787a0b91105c410cbcc6329
852136a97ecf76edd1446b69ec3440c1ac051fc9
6557 F20110114_AABWTA rauschenberger_r_Page_045thm.jpg
eb200a835e62f91824fcf65f335ad67e
93bf286cdaa9e5608e2f9911981fb2b2c03a090c
3339 F20110114_AABUMC rauschenberger_r_Page_090thm.jpg
2f2523f5253df67a413ce20af69cfa6c
1c73e4c3df42ccf194a34c22f75f2308a85447e9
26156 F20110114_AABVWI rauschenberger_r_Page_143.jpg
2bf8f5e113051a8c76cccd71feb72734
5eef55347db329433629ebaedb974e09e872467d
115929 F20110114_AABWAX rauschenberger_r_Page_007.pro
ba4b6422085c0407bb4e06e956416729
7e35bbd7825b78516deb7111d96316e6d6d4881c
22592 F20110114_AABWTB rauschenberger_r_Page_046.QC.jpg
62c9c8df9eeff9d09861fb4dfe27d5df
f1e0a9bae9ce9a49fe2e61ab64f67c841e2247c0
16295 F20110114_AABUMD rauschenberger_r_Page_112.jpg
81fd5c17dc6eee71c7f7efc852c658f6
19feae258ba424e8722cd20788d1a45296bc65f5
88653 F20110114_AABUZP rauschenberger_r_Page_232.jp2
da2532a5d9029e8abf960e8b79ff8b35
5d59295ad78e4ec2ea23f27fe568491043e63ddd
6404 F20110114_AABVWJ rauschenberger_r_Page_033thm.jpg
0bca45cf29efe6e72b93f2271a530d03
dbbdf906d34a92220fb90a39569c8808826065b2
1917 F20110114_AABWAY rauschenberger_r_Page_118.txt
85dc109a3ae4668b92c0e5ba61c713d7
7aa061def7fcc78d0a805a0bfeb72e86288abc94
21767 F20110114_AABWTC rauschenberger_r_Page_051.QC.jpg
9e5d91e0400e9ffdae11731e973831dc
e5dd42bb41d4be490a9ca19d48aad1b6a4efaaa6
7922 F20110114_AABUME rauschenberger_r_Page_069.QC.jpg
8979c227b1e9f2a18807120772ef3605
0b2ba4ae7f24c356c2c2787be5c4f7021193fd94
6192 F20110114_AABUZQ rauschenberger_r_Page_027thm.jpg
3523111ccba663a0e57f3f55c49205e9
3f303de00a30517cd864f7d01c980c53fd176cda
62504 F20110114_AABVWK rauschenberger_r_Page_092.pro
05516badac178bc476753f4472eed5b9
6fcc53c5c89f91465e752b09fd3fcdc25c52503d
1589 F20110114_AABWAZ rauschenberger_r_Page_109.txt
6c3f5961b23cdc60eadeda4a96e65b2b
6c206b47edb0f160c182e10d1d4337d59a088f95
6631 F20110114_AABWTD rauschenberger_r_Page_054thm.jpg
cb87d8d438326da419caa9f574073f12
270cfc5ef0e631e5b225634f11c02a084d25afa2
104656 F20110114_AABUMF rauschenberger_r_Page_171.jp2
96ea3c16ba44727a99f6d237d598b0d7
a06317780002586c6bc3efd2bba3b33af399f86b
1389 F20110114_AABUZR rauschenberger_r_Page_233.txt
0ea8054d1629c8c368ba822b143241af
de15ef432e890162e37bec9be72311b0ab18a89e
11714 F20110114_AABWTE rauschenberger_r_Page_057.QC.jpg
6581b6e1c345a4d5a4f2d41b208972fd
05199ee31a0ef85712ce92aeb0f351632fc9a66c
8993 F20110114_AABUMG rauschenberger_r_Page_195.pro
c38d3610c017ad6e4b4d3c0b2161bb62
346898a95db003eb7d64bb30eca2628a095a921c
15689 F20110114_AABVJA rauschenberger_r_Page_014.jpg
73da21bd26a24751d11caade56684534
4dc6c857fbdd69538588e9b0c13b6355a030cf28
110932 F20110114_AABUZS rauschenberger_r_Page_216.jp2
68e90f02c580001462c1a9d800e964d9
f2f5d9ea7e3d663508b231054278a1ad55896cf8
24566 F20110114_AABVWL rauschenberger_r_Page_206.QC.jpg
917667b97d721447b4407290c0e74aa9
af2585f8f52facc6d47143e7acd53b1551452cd4
9045 F20110114_AABWTF rauschenberger_r_Page_064.QC.jpg
6c9f9f64db04cf24c337afa4efa02c8b
fce66a392b55a836a73484418d812bf6a1d6f103
F20110114_AABUMH rauschenberger_r_Page_112.tif
7a03d580526e2bdb3439da537f8d024e
3216b89352b05f4965b92da3175a66f6fb6334b6
48621 F20110114_AABVJB rauschenberger_r_Page_043.pro
5441c90b01ebc6628a52eb23136ad5aa
2bc10891b37ee062b1d576c3368a85de67ba1d09
F20110114_AABUZT rauschenberger_r_Page_027.tif
697c8e801f01c88be2c5ed932180d810
9d2553b7fe3594296b812d5af15497a6c06b8f9a
61154 F20110114_AABVWM rauschenberger_r_Page_017.jpg
c8e1bf058392f39f82d11549f88a9752
d203c33e83560670be160a29900fe36d372469d6
15082 F20110114_AABWTG rauschenberger_r_Page_065.QC.jpg
28483be469e9b378a388d583499040f4
67c66247bbd3750194ef2212d97ed1058397ca32
6593 F20110114_AABUMI rauschenberger_r_Page_047thm.jpg
b5c5e50b7282126d9de5b21bc60ec9f2
031b442f65a03030654ed562eabc8002406b1f83
66849 F20110114_AABVJC rauschenberger_r_Page_227.pro
747884415116b388d3b92fd0e511560b
295f6b7640b06a50b10f0f2f23d11cd5e8f2f42f
27982 F20110114_AABUZU rauschenberger_r_Page_058.pro
d43e7df77f8fbbb35f06b1bf68350985
41fe5075e607e7476c86810a92a7b5ff739da80f
2293 F20110114_AABVWN rauschenberger_r_Page_194thm.jpg
50040339e9aa4356ab62dd9d95d5b842
995afe37d8e875eb0fdc782e39000387a9f8f7b5
1889 F20110114_AABUMJ rauschenberger_r_Page_059.txt
9b3d3a4353b436e2b584e355de050f93
d3fcfa4de1db059c550461ea3bca5c74a08cbe96
F20110114_AABVJD rauschenberger_r_Page_121.tif
6106e018a545d1780d44a7e02c2139a6
320018818df5f0acedb6a9ac2828161606f94af9
F20110114_AABUZV rauschenberger_r_Page_012.tif
9b7d21ac320f0ddefdce1aacb3faa5ea
e68e13b80ca08e04a019d472d281a5affa838a1c
F20110114_AABVWO rauschenberger_r_Page_177.tif
f572c1d38f07f118a2b1166d8cd91bda
cec4e10aa3f006941d35db466233a6074921d81d
4158 F20110114_AABWTH rauschenberger_r_Page_065thm.jpg
f6f52b8f5a8a0bdf33c0da6cdced0a7c
e4e56010b199baad1868305709d150591961d257
107465 F20110114_AABUMK rauschenberger_r_Page_105.jp2
98c250033db6136691b637ae4067ce5e
68a73e16e06c872fe02cd100255a7591b44f11c1
F20110114_AABVJE rauschenberger_r_Page_036thm.jpg
52cd8da1e13a16bb9cb1abc8222a3912
b5ecc65a39b41ba1d232fd7343b8e37f6e949250
23946 F20110114_AABUZW rauschenberger_r_Page_205.QC.jpg
0893f4f00047282252fca4107a49dcd0
660c6219f9db3390b035edd4c95d842903e3c18d
1431 F20110114_AABVWP rauschenberger_r_Page_002thm.jpg
154e9fa55c438e9f136e068f0d4be36c
0de3fd66f0293ff38d88c799831769f2117f5d35
23225 F20110114_AABWTI rauschenberger_r_Page_071.QC.jpg
112d5c1f5bc0502b5832a1c97d9fc092
d8911053f53d3bfd5e2a3b8b9b2e0f4f8cc5191e
84446 F20110114_AABUML rauschenberger_r_Page_186.jp2
4a158b78e136ab2e6e8c30dcf4498dce
1f9a3f3ad41a3ff445f456114c4cb542a2175266
35595 F20110114_AABVJF rauschenberger_r_Page_180.pro
b493cc41e86a2fb7d4d844290f5d3ff5
f2b850b719c68e03d6862e69f2f50b5e1bfd28bd
112307 F20110114_AABUZX rauschenberger_r_Page_045.jp2
055708c84551e7ab0cd41f01a0490611
7c0c43a92dd7da8df8915016854ec8f8794edc0b
22556 F20110114_AABVWQ rauschenberger_r_Page_188.jp2
369ee8f47cf6f6010c2f84600202a462
92b845a13ac96ff46103e196912de2dd96cdbb2d
F20110114_AABWTJ rauschenberger_r_Page_073thm.jpg
c65b9a023fe8267e6f5451f5c69bb0d0
1339f1a0cb9f0e5514a50bfc65012e61f7f9d4ee
24158 F20110114_AABUMM rauschenberger_r_Page_214.QC.jpg
09ff5a84d4ce08368130f4122e642e88
70660e5e6fef8f529235e439dd983f24148a4a06
1995 F20110114_AABVJG rauschenberger_r_Page_100.txt
3a1be8c4937530bd8be876c12ee48057
d46cef0add0f2e8e16912ff5f26a3f88d11e211e
24060 F20110114_AABUZY rauschenberger_r_Page_130.QC.jpg
fac6d0be516081a12d8b04427789f38d
1f550bc9e511839e56ec1205aa6a4cdf0e3e31e5
52340 F20110114_AABVWR rauschenberger_r_Page_036.pro
34c2169123acf20d7ed7362cf2fe05ca
e0108e066cc0e3d7bec3a4117c95b7217f03640c
23312 F20110114_AABWTK rauschenberger_r_Page_078.QC.jpg
f08aa6c02deacab75ca0447be288e984
6860253a5417c63b12542b32bc4b080408940a7f
1980 F20110114_AABUMN rauschenberger_r_Page_098.txt
d0dd1557e86938d069747b14a48b15cd
017cd893d3b91538963657a386bd013b1772cc83
F20110114_AABVJH rauschenberger_r_Page_076.tif
5864a389cb45053b19bf5dd06a95007f
b5bd276a61edd2058ed4bfc2132200a6a1a11395
F20110114_AABUZZ rauschenberger_r_Page_216.tif
f0d60a30a7732713c32dae79470557bc
343a1ff1316007a8becbaa34c455f8763eaf875f
6149 F20110114_AABWGA rauschenberger_r_Page_115thm.jpg
42c884a25a29025404d858d11bb00284
8b97d52535db5ddcc3830b7414bf7fbaf6eed0e2
23462 F20110114_AABVWS rauschenberger_r_Page_219.QC.jpg
f7459f5332ddfd080fae2dfaae4f2acf
3a5395eed305221aa7e220271a4eed30eae6dca3
6553 F20110114_AABWTL rauschenberger_r_Page_078thm.jpg
09cc8ebf2255f5d50cae34f523eb963d
059041c865f254dc22099812ca914fcc79aa6228
114769 F20110114_AABUMO rauschenberger_r_Page_106.jp2
e9aa06712a74d857bd27438707db8371
2c1a0867eff33d0ab7e58809ae40bd12a72cea69
50943 F20110114_AABVJI rauschenberger_r_Page_122.pro
f9125c844cbffbdca8c6f45455d59cde
0ca055e5ad9d69ffddcc8b89d06d9c0f0f0f10bf
24007 F20110114_AABWGB rauschenberger_r_Page_179.QC.jpg
71882d05a9417f06f3ca24f3afd41b99
e11cc8b6fb4e69d943b16fde09311ef81c17c938
F20110114_AABVWT rauschenberger_r_Page_185.tif
50e8f79a473161ec3e11d0d70763ab9a
ba489445420cb86a434626a1fbaad540ef579dea
25577 F20110114_AABWTM rauschenberger_r_Page_080.QC.jpg
2a58ccef9d21b00828bad1f6bdfd4c74
3ee8dd7e72a410b961d2dabaa8f36a7ab65a2c7b
F20110114_AABUMP rauschenberger_r_Page_016.tif
de12455a6c69531c733d2170cf229160
f3dfb28bdfc334802de8ac7a74ba126afa87c7fe
6865 F20110114_AABVJJ rauschenberger_r_Page_228thm.jpg
267ba7743e90f48f821a297527b67d2a
1484e0c40346a6471ae469c1702a5a52f8b2989b
23034 F20110114_AABWGC rauschenberger_r_Page_135.QC.jpg
f740eb7078f697566ddfbe8cccf93d58
7da90b5dc5dc6a40a4be962e9b4551cd565b641b
73819 F20110114_AABVWU rauschenberger_r_Page_100.jpg
3f10f826739537ce1ee56fb18666398e
e65e17b8c2b677b3b8c75a8c32904022b8f7e906
6886 F20110114_AABWTN rauschenberger_r_Page_080thm.jpg
0d625453ec9542e0a72044566b0f2ddc
bedfb3bc7186569b921cec6b13c0c626b926859c
21127 F20110114_AABUMQ rauschenberger_r_Page_030.QC.jpg
a05b05f0b7e49f6c5b6060e769e23281
60b6d2578a65613627c4551d69ed5aef1fa70e9e
2656 F20110114_AABVJK rauschenberger_r_Page_139.txt
b5d22e452850ea38c74258309e5ec140
6049d51366b4b39f1a85c404cb9604ea583dfd76
13915 F20110114_AABWGD rauschenberger_r_Page_182.QC.jpg
e6d78ca3f69a843bc837a0d0e988c6a4
1de5176c1c0d18d49af58b739d79a65b136627a8
19160 F20110114_AABVWV rauschenberger_r_Page_003.jp2
a4c5d463160d0ac93e8a2c68261867cf
35771f25921fb1e10cc0ab9b1d35c1362e708ac6
F20110114_AABWTO rauschenberger_r_Page_084thm.jpg
24e03bb85a1316377170675f3de0a802
de64f216d34c589776a963330724bb5e3877a456
135908 F20110114_AABUMR rauschenberger_r_Page_228.jp2
6bcc26ec0031b1e4d61d47f9f27402a2
f1479bfe13030553e977f8e4fe383b8073c35ed4
36962 F20110114_AABVJL rauschenberger_r_Page_210.pro
453c1cea42f42816fd9c73245f74d149
67a2362f8cb9efba11746ea31b7e9a7d728da5dd
22499 F20110114_AABWGE rauschenberger_r_Page_035.QC.jpg
345bf10180ec220dc3ce315e61ef64fe
b1d2301aec9408953e210d4cc04bf59c468c56dc
6507 F20110114_AABVWW rauschenberger_r_Page_132thm.jpg
90b612404e5b648ebeb200cb1605474f
5a6d3b2a024a2fb02a331a1ba18312969b98d5bd
24070 F20110114_AABWTP rauschenberger_r_Page_086.QC.jpg
ff53009dba425e3c4e8cce105fe98427
92e09e6a8de08286171b92e2503d158cec3cdd26
781974 F20110114_AABUMS rauschenberger_r_Page_151.jp2
69cda91af4b127f17705c444e03f91be
5c62327ec02597b1a14f8321f44e7cc4e8f6881a
1067056 F20110114_AABVJM rauschenberger_r.pdf
d4fdba8d4b9bb1a7169c7ce1ce1dabf0
70d7158fc1037d4dd673ba7ee121dc4c32d4c0c5
110423 F20110114_AABWGF rauschenberger_r_Page_176.jp2
2af30d8a994de44368e90ac747d1dc8e
bde7913b037916480643e4bad2a0c0354bfc0565
48867 F20110114_AABVWX rauschenberger_r_Page_145.jpg
46005651001d4db8162bdb94f1c66190
5db1eaf48b21e617e8e55f33c2fc048b4b22a6da
6616 F20110114_AABWTQ rauschenberger_r_Page_086thm.jpg
133f4eaa82a623d9aab374a53373c72a
d56ab5ab4ebb9c983555ee98b405532217d30429
3871 F20110114_AABUMT rauschenberger_r_Page_145thm.jpg
b817d66d72ebb1ec32be32d4192d3c62
dd63fe157be05329fe0513129e46498cfa2dcf66
1925 F20110114_AABVJN rauschenberger_r_Page_138.txt
4c917441451cf25c7526dabf30e56bce
76cee5f73d7b242ad3b9df92386362adc82c699f
23038 F20110114_AABWGG rauschenberger_r_Page_213.QC.jpg
a1c8e68fdf3518002ea7eb8aa8444281
f90644574b949fb783813e005ca1b7a2945333bd
6591 F20110114_AABVWY rauschenberger_r_Page_165thm.jpg
49e1c76a0463c24a3c51a478d975bd71
a14bcdb9630cfc39c44b52232f23fea00d7c2332
10723 F20110114_AABWTR rauschenberger_r_Page_090.QC.jpg
539705c7566efb28075e2a324952487a
e5cfad12a4e1e8fa86f094e66bad488a26bbfccf
51056 F20110114_AABUMU rauschenberger_r_Page_162.pro
ea650a7dc34f3d2accbee36bcbd1c393
ef39d3bb83621a8240aef500f7b8de115bbbf547
51213 F20110114_AABVJO rauschenberger_r_Page_126.pro
33d1942f75f4adc94720045cda865826
b27381b937629309e0729b8d0d3ddd7a5f7c0a7a
114587 F20110114_AABWGH rauschenberger_r_Page_163.jp2
4a41ad15c50cc1574267655a5bae81d4
937d09709119ec8d71452f7c454c9dbd1029b97b
6703 F20110114_AABVWZ rauschenberger_r_Page_161thm.jpg
821ad3faf884d616d0fd36bb21006f03
3fcf07f0eb402ba52bdc81632fa07a3a8fa8c497
21642 F20110114_AABWTS rauschenberger_r_Page_094.QC.jpg
0b42aa003908509cae21c30ed9e9b70d
43e8a305aa06a8cc03fdcf7bd7b7aa208d246dd6
1811 F20110114_AABUMV rauschenberger_r_Page_051.txt
8a6d2edda2df06bc8076feeb6246be03
bfc257f583e8b0bdacd30e3071c2e3675d49ee38
F20110114_AABVJP rauschenberger_r_Page_034.tif
103c6f04ef152391fedd934f5b6ca13a
f10d2345ef04baac1a7816103f694eb28c6f22bc
F20110114_AABWGI rauschenberger_r_Page_047.tif
b746c2d7ca1b7be954cfb8f1d59d11ee
1ce3a7c24e4fe79be854a11b5913424202d93587
6154 F20110114_AABWTT rauschenberger_r_Page_096thm.jpg
15d5848459f891466ceb5ab29bd9e3d3
a4ae0d792f97a9227d3e90d427dd65f72c83711b
1854 F20110114_AABUMW rauschenberger_r_Page_123.txt
2d99aa1294402a88699264ddae370fb3
56209cb7a463253be6c8d1009b5770b508ecbf39
F20110114_AABVJQ rauschenberger_r_Page_046.txt
2265bb4df7a8237f7864a203d266e31c
58ee4c4f37c9539be03f87eeb299a86eca9e593b
F20110114_AABWGJ rauschenberger_r_Page_182.tif
be530d3fab4c81471d21f650f50004d4
1495fa4b258fe8adfaaf4a081b929f61249a233f
F20110114_AABWTU rauschenberger_r_Page_098thm.jpg
dd91b0d03981ff917970d44666c9d82e
9a0fdde1c5eba829dfb62ada7d0f2e80c74b84bf
7106 F20110114_AABUMX rauschenberger_r_Page_227thm.jpg
7c94295fc297c4dc3848c4ce5e6465af
5de30bab8cd806b600cbf63ce3b2d7690680d3eb
51843 F20110114_AABVJR rauschenberger_r_Page_045.pro
7467a1e40bef5878dec8ebadf530f26a
f86e0a96fcb8241b2711180cac6280ad32bacd5a
F20110114_AABWGK rauschenberger_r_Page_207.tif
2ef1312b0d4c51e5a78daec9f1767dd2
eb18d7bcf51d3e9f0b13f19d005369ec009dfd07
23961 F20110114_AABWTV rauschenberger_r_Page_099.QC.jpg
2f0bd40946f06cbbb434c5c0f187524b
c74eb3b07456fc8d97fd4576d0d529bad9673fc7
48281 F20110114_AABUMY rauschenberger_r_Page_078.pro
012b5ae4c0459750cf96de6c638ef131
202bcc2094e647be14af421ecd503cad7993d3b1
50443 F20110114_AABVJS rauschenberger_r_Page_047.pro
cb48826aa0ef4fc025a8f65f1c56a757
80dc8484346ff4ee2d67984f18da90679a0fa5aa
1996 F20110114_AABWGL rauschenberger_r_Page_047.txt
63b674e9529987d51594ec7a56871d71
7b7e5b1ce45c0996903edc551e543cb4bccac651
6752 F20110114_AABWTW rauschenberger_r_Page_099thm.jpg
93a413e76d330bdceb5bb7ca107ccc41
606a7aadcc96624791e15bba673e5cd693784743
F20110114_AABUMZ rauschenberger_r_Page_152.tif
4991ec308e0a7e44824558ecfb87da62
3c5f880dd4eba9b57af22e86a66f36332113fe47
12024 F20110114_AABVJT rauschenberger_r_Page_111.QC.jpg
f8284edf522e317be5a4d95906ecdc09
564f4a53b17b2887934d80bf7effc36c02fee10f
F20110114_AABWGM rauschenberger_r_Page_220thm.jpg
6782a7744cef7441ad4348fc0d717eca
99f7338e01758444b6df94e13555abefb83694f1
24379 F20110114_AABWTX rauschenberger_r_Page_106.QC.jpg
37950b9407f6bd24c3dce30267cc4bb1
e45d956eb94643180e0d4100f060d659748c3ada
74922 F20110114_AABVJU rauschenberger_r_Page_210.jp2
041dad4fa47265f90b591a9dd4a14562
770333c6fff565be2e2f7f1041ecb05f9cdaf48d
23105 F20110114_AABWGN rauschenberger_r_Page_103.QC.jpg
4cb3bfe2f0b111877a65aa0a6fb67cf6
f04e749b207cebcbfb0131d07d2dc68333291414
22893 F20110114_AABWTY rauschenberger_r_Page_107.QC.jpg
12de6f1e9dd6a933a8bff98cbd522fbf
2fbc4e1de882161e1f93e8b9374226a5b885e356
23199 F20110114_AABVJV rauschenberger_r_Page_029.QC.jpg
692c7d9cd2a27222fab56d72cbce6782
6aafc36aa57d464a2407fd52e7caae0bf9b8545c
F20110114_AABWGO rauschenberger_r_Page_137.tif
6b10f3fb432a27c09494014abffd662e
ad565c1fd94807e674b1c12a30d248bd7de29895
6329 F20110114_AABWTZ rauschenberger_r_Page_108thm.jpg
9f7324b5596048a1db7e55ad5de7ae1b
3a02ea32f7170e0f988bf83c47888fcd8b71c725
6205 F20110114_AABVJW rauschenberger_r_Page_188.QC.jpg
6ae1436352979846ff2a04452ef59453
75257d70de6d3297d22ebf43e3b0230f41655429
11804 F20110114_AABWGP rauschenberger_r_Page_061.QC.jpg
810dbf776185cd02fdcb3656ebd28fea
c958ce28ebe7e189574e01390f3078232fd32da9
F20110114_AABVJX rauschenberger_r_Page_163.tif
83e4b622e9198abca820111c65dca1a1
215451bda11eabce4d6e6c6660e002b36b4f371b
2425 F20110114_AABWGQ rauschenberger_r_Page_145.txt
45615cc2297464f0d3885b16d7b82ddc
eedad680fde9218785ecbf1fd66b6ce5e074557a
48987 F20110114_AABVJY rauschenberger_r_Page_175.pro
912fe8c90731fc3223dcf82ea304655d
8b08db6a7c1ac86fa5d88400f855d82a8599e468
5613 F20110114_AABWGR rauschenberger_r_Page_224thm.jpg
b662cbf9d8666aa6e230d8d99b141217
623ac5c7db34e154cd4769ccbdc5a540fee1ef43
69345 F20110114_AABVJZ rauschenberger_r_Page_032.jpg
aab39fb4aaf8d5592d85975195c551fe
46ef9deddc3e6a1443841fcd82d92fcc27a3d607
6523 F20110114_AABWGS rauschenberger_r_Page_101thm.jpg
6a40604fd23523f31a8ed24377ba8714
73f8102c0d62cce48c32aa4705259273b284da06
24346 F20110114_AABWGT rauschenberger_r_Page_200.QC.jpg
4563ad77f944b8c10aec9789a267ed3a
3dc8fdd37cb3b578ddf035dda0cf23078b7fd51f
F20110114_AABWGU rauschenberger_r_Page_117.txt
cdcb59589aa23ad668d5b0cab1a3c0cb
4d59716aa8de81ea71ad0cfd7fb0ed96e53db9fb
F20110114_AABUSA rauschenberger_r_Page_024.tif
64ee51502218fc145d3a8a7735e0b6aa
cd8d9086130d615d2afd045c97a9e2ebbf91d215
36671 F20110114_AABWGV rauschenberger_r_Page_109.pro
a0584eaafe64c7f3b6a9f2f3654dddc1
18a0c62698782b492a92593a638a9274529cf435
70057 F20110114_AABUSB rauschenberger_r_Page_226.pro
160a0646e53b1810776015dfa48099fe
822d554ab5678ae8458ee345bb3e5d8f04500ba2
F20110114_AABWGW rauschenberger_r_Page_227.tif
7376f06ae747d503f0cede2eecd6ea17
2992a38b05d9f779c69d68f2fe81e64d8440db54
64851 F20110114_AABUSC rauschenberger_r_Page_182.jp2
70ca5fa69cb7154d21444b9f678d7ccd
491b619054f6377ed9f12f7de9944fdfa42be02b
6140 F20110114_AABWGX rauschenberger_r_Page_004thm.jpg
6a64570158d0fb0f70663f8f48c26f4c
8c13d7bac9115f8cc9580ef30087848e58166e89
2994 F20110114_AABUSD rauschenberger_r_Page_091thm.jpg
67fc041cdafe9286865020f9462f4cf4
8d038c8441a2584df6e9960797247239512edac5
F20110114_AABWGY rauschenberger_r_Page_077thm.jpg
a23b65747dbfac17153446da98ed50a3
60682af827c00dde524057df64b4d644aa255a0b
6543 F20110114_AABUSE rauschenberger_r_Page_076thm.jpg
f1632700c561e5c8f4a30c2ddc61285d
3e8bd359b3b730a3ee1ccc9b45c71fec3f5e2062
74226 F20110114_AABWGZ rauschenberger_r_Page_164.jpg
24e1124e2a3d492facf44de3bd56c7d8
8dcb932210a4f27af39d5019edcb14898533cb38
F20110114_AABUSF rauschenberger_r_Page_176.txt
2c0da7614fe1a56bad6ff66f6e030f3e
ee5dc9c2ab901707e23df61187ea9844d1578454
88326 F20110114_AABUSG rauschenberger_r_Page_012.jpg
b3943068d6f777bf8e03ca563825b347
49aee8572f450cf0fe969c092e8e7e75734c9017
955 F20110114_AABUSH rauschenberger_r_Page_157.txt
afee4638d676c14fc0a2b92db7debecb
e7a1b4f1e3625c2cdc3305c7761190eca1cafa91
49657 F20110114_AABVPA rauschenberger_r_Page_139.jpg
b2dc5763c0c4db855878105109a414a7
93be8850b637daebcb261b3c007ae4acdc952a0a
52615 F20110114_AABVPB rauschenberger_r_Page_106.pro
2ec9ba66af5bbfcdc5e941b0891ed661
06ad662b8414d2de70212941424e3a90d603b73a
81583 F20110114_AABUSI rauschenberger_r_Page_006.jpg
cb069b297add5cb5934f59752b9b644f
823029e04818ca9c4aa0296255da68e514f5453c
F20110114_AABVPC rauschenberger_r_Page_064.tif
80bab9ed8edfa2a3108fddecc563d9d3
f3aa5dc3c2d75443d1d006c63c716746aeff8f1b
16507 F20110114_AABUSJ rauschenberger_r_Page_063.pro
7fa94dc9e2edf2e290853f8bf8d73c4f
0a5fda70119226a7f4319417f5ef6eb467f54f99
6684 F20110114_AABVPD rauschenberger_r_Page_042thm.jpg
b04f6f91a76ce273298c7477369975ca
d8e64bf69edf561ed399538c2b08b191930b69a8
6356 F20110114_AABUSK rauschenberger_r_Page_177thm.jpg
832abe340ee62e22de0a10c015266aa2
e9613c4ff6e8f2fc0dfcc02cead8c4c6bab81532
6343 F20110114_AABUSL rauschenberger_r_Page_075thm.jpg
7e10160097c648bdf0bc96e8a8a650c3
1688ebd201d77443f2c0cf36bbee62367638361c
F20110114_AABVPE rauschenberger_r_Page_040.tif
4ee89e0000640943e3ea97c6fa826147
3bead8be40cff35638eecb3ea9aaa8894955936b
51322 F20110114_AABUSM rauschenberger_r_Page_058.jp2
3e5817b0c13bf49cf40932584eac71e4
f01cc0380bd0c6a4d7a04ea629a9754c2b5cb871
2107 F20110114_AABVPF rauschenberger_r_Page_036.txt
0175d8387180ab8bb3b8b55e0422d7f3
ddc05d4d24f09cdf48c18d8ad4907b8896852ae8
102823 F20110114_AABUSN rauschenberger_r_Page_203.jp2
45d0acc71d883b28188d888c7f3b1466
7fdaefbd8b18fd39688566f01c49c0fab0df85bf
1848 F20110114_AABVPG rauschenberger_r_Page_131.txt
a49d9c5b8b4d11d0d9958b3b0c088a95
36ff0a2d8d9d46512d4217e2a3010317532121bd
110968 F20110114_AABWMA rauschenberger_r_Page_130.jp2
a89ee51982169a9ec01bcba76887e2af
0d2e693cfb324c82524ca09149b29b13ca87d69b
114255 F20110114_AABUSO rauschenberger_r_Page_052.jp2
10216b6ab148ab2e65ec31cb78301ec5
7b4743ec29b14430c63f30b29d4c59cb1541f033
22772 F20110114_AABVPH rauschenberger_r_Page_050.QC.jpg
09871e59c763267171734306e65355ed
0f766015ce343d78773df6bbadbb7bd892582b93
109817 F20110114_AABWMB rauschenberger_r_Page_135.jp2
240c642870d04894f05da859ff2ec055
ef0d5d14942c42895882f9f96611f60928270aae
58518 F20110114_AABUSP rauschenberger_r_Page_114.jpg
f8281224e40c95810a14b6d8046db1b3
bdc772c3263ef4dc18c8e049d1b69896d796b08f
73619 F20110114_AABVPI rauschenberger_r_Page_205.jpg
aff403637c056e3a4e9f0131e26e98f3
7f2d52ca9282aa6870550a1504e897a136ea2967
93351 F20110114_AABWMC rauschenberger_r_Page_138.jp2
c674326e3e7f3bccbdeb02e7a15913fc
ed6e3cb2c995f650e1bb6fe81c1fa162f0af94b3
1956 F20110114_AABUSQ rauschenberger_r_Page_101.txt
f571b11f7c0e69b5d13e2127a02784a9
75a5376869855daf92821ca28bf102ecde2d3066
F20110114_AABVPJ rauschenberger_r_Page_029.tif
3cc201b0b199e4a78fe5336536172f91
c7362fd466d9732bf48cd23bd07bdebcaf1b670d
113478 F20110114_AABWMD rauschenberger_r_Page_145.jp2
5820a79a84646a0c372de7334ea49af6
7dbdd0510cb58be2867b6302203f6a6c5d0466cd
46910 F20110114_AABUSR rauschenberger_r_Page_149.jp2
0586156bf5f864d41e0ba285094847ae
f00e23861260be00d77894e394d9128d2334ae26
F20110114_AABVPK rauschenberger_r_Page_049.tif
fdfda43f7bfe97f3fa59b3a14ce7d4ec
367248c1b6753822fde0995eb273b8feb9b0f7e2
53065 F20110114_AABWME rauschenberger_r_Page_148.jp2
f02f695c0a1a89f5bbf1f0b4eb0046f8
ed8f4c8fd7fcf9f34317a682fbc535d3629dfa83
77857 F20110114_AABVCA rauschenberger_r_Page_080.jpg
2b34a0d2ea6eb2e69f49dac820560604
0d714999d0d10c188ee59a4a491e8feb71570933
12903 F20110114_AABUSS rauschenberger_r_Page_138.QC.jpg
9ad29b110da202bb3e9595fd691431fb
dfae93f837ed706b7fb7500522e2da2f4ac315a8
3710 F20110114_AABVPL rauschenberger_r_Page_140thm.jpg
26bd6f0a0cb9431d8d9b19acab5690df
a19e4ec8b0fdb258cd8432cdc90588ce763ea884
29650 F20110114_AABWMF rauschenberger_r_Page_153.jp2
aaab3ba1bbfb7c0571dac5979e5d6c58
222ab7a183ea562d10ef7941e12b443cd043b65e
5630 F20110114_AABVCB rauschenberger_r_Page_094thm.jpg
bfa8fd6b006cb11b2540070d4b17454c
69933af5153c1b12234eb8e2cbc41c8f03ef4a03
23046 F20110114_AABUST rauschenberger_r_Page_073.QC.jpg
38a48e34d1925d097844b635e6613dd9
22276a85065e28d6d49e06a0cc4ed65b358e0753
6053 F20110114_AABVPM rauschenberger_r_Page_016thm.jpg
eb4b9a635e4dbaf4f6847fe947a73926
b8140ae06069646ceb2d6faa894e81f75ef4f3b5
29802 F20110114_AABWMG rauschenberger_r_Page_155.jp2
72d2ae1255f1c35c2d40f0f9e5b489db
76e8746c3c22878e48740f8c3a204bef4de8ff3b
911407 F20110114_AABVCC rauschenberger_r_Page_011.jp2
95ed411dedaf5eae665217867a4cf27d
f6348893404cb2567ec4217dc5b9a9f52deb31d2
8167 F20110114_AABUSU rauschenberger_r_Page_194.pro
b90ccca0dc823547e19207fa1194a836
df178193bd304753b2da2059b6891744f5b19370
F20110114_AABVPN rauschenberger_r_Page_040thm.jpg
9512c2e3d830ab4ec4f05e10a65e0d0f
5c8be2899b16e1fa05c1f42bbe6640989d74c5a7
27632 F20110114_AABWMH rauschenberger_r_Page_156.jp2
bbaac1e8d6554e08cb90e1006ea0f1e1
32d97430be911109f8b5cbfc7e3c71223f034328
110653 F20110114_AABVCD rauschenberger_r_Page_047.jp2
1a0aa56fb7e337de1ce5df8d0e4b0092
bce77e157c49bf7eafb90f96427f88dc091e078a
20533 F20110114_AABUSV rauschenberger_r_Page_212.QC.jpg
a55ec1eb504b0ccec81f30218049a52f
fa19d254eb6baeebc39a94353cfcd89b15c266df
1892 F20110114_AABVPO rauschenberger_r_Page_061.txt
b1cee3fbd9e2c39e536786db39ba1fdc
e83b4bc18d484bd73953a7c67a7ffb14482081e5
111339 F20110114_AABWMI rauschenberger_r_Page_162.jp2
703ac586d5fb7573ca07c4ff9b32dcc7
a1f68caa292669668652bbcc90bb13f66ef7122a
7358 F20110114_AABVCE rauschenberger_r_Page_193.pro
3f8e8457aa0cb7507f61e6f4341d6892
5bfa1c249b233afd7001c0d9867d1f67461d9ca9
49915 F20110114_AABUSW rauschenberger_r_Page_098.pro
2ed9f24d9af3a90a599b3e2873f7f545
94f957b9758e2eae2d9fd1ea29e316c4a1362afd
133684 F20110114_AABVPP rauschenberger_r_Page_229.jp2
116291349b9598f5f190d354d58579c1
b9b638e0877d3d09b35110398885fffdfec35d37
111848 F20110114_AABWMJ rauschenberger_r_Page_165.jp2
bb6daef1e9dc302136527573199f18d1
c4d6a939f0a25066056d4fe1526346220d762e6a
1971 F20110114_AABVCF rauschenberger_r_Page_099.txt
8ec0adb5ae8ce0264f80da057c5bd6a9
5c8b599df96d076912868a5d7c5a8c0e885b0d63
F20110114_AABUSX rauschenberger_r_Page_222.jpg
139420615298856409dcb55f31e365a7
2cd153f68bb23a513395d66655d4af63c85c989b
7546 F20110114_AABVPQ rauschenberger_r_Page_154.QC.jpg
4ba9704e31522fa66254e744c0d09196
e55ce1bff684a82290c61395b65a10fa27d0c1b6
108664 F20110114_AABWMK rauschenberger_r_Page_175.jp2
664a6195c6acf677d8797dc653129b13
e50057018088fb181bce66a513f0957ad53a560f
3524 F20110114_AABVCG rauschenberger_r_Page_059thm.jpg
f3503999e05527e06a683b6fbe462655
5f19482d358686c062a3a179f72066fb9b41b106
71545 F20110114_AABUSY rauschenberger_r_Page_105.jpg
5be1031948528f91fa03c5464b90135b
79351a0dda2d0e85fe56275db6fa1ae5229a092d
87606 F20110114_AABVPR rauschenberger_r_Page_225.jpg
94682f2cd80795f00bf62a758fe3f63b
5873d4076f37cafcc9bbd1e552e0b57a3030f668
190757 F20110114_AABWML rauschenberger_r_Page_192.jp2
4e3cf0602deee1b60cd489df3c77705a
e6b99b99621419386d1a85c62fa62092035b84fc
1629 F20110114_AABVCH rauschenberger_r_Page_014thm.jpg
adc48a61200b15fb94db02764d9e69a9
a511a758227cac2b487c10bf1049a8621cf23664
38521 F20110114_AABUSZ rauschenberger_r_Page_111.jpg
ecc3937be0bcc7c876ebcf5e6ef644cf
1f8b765d82f2bb724531848b579c862fc6f5b69f
107526 F20110114_AABVPS rauschenberger_r_Page_215.jp2
6679cd11b9d06ace356923211455f3c7
3ae7e4780e9f608b245f763c6e5c420de38fbb61
208706 F20110114_AABWMM rauschenberger_r_Page_194.jp2
2c9c7504348779b9f741d5f264e21025
f2045437b10b53ea50c7729689b6aeb50ae8c9f7
46944 F20110114_AABVCI rauschenberger_r_Page_089.jp2
da8f8ad706d553549d1289b7d436289d
ea0ade145b05868a6379d880f975a4478bfd0249
6139 F20110114_AABVPT rauschenberger_r_Page_051thm.jpg
910cb1fc7aeb9252a37747d80caca6b1
3c69a3963a100b5e6e36f398bae67db43679e1d6
110437 F20110114_AABWMN rauschenberger_r_Page_199.jp2
3305507884fcf7be1fd6fffc7c453f58
7fea8d0fa519336a7d8fb3bae5a34f048b98823c
10865 F20110114_AABVCJ rauschenberger_r_Page_002.jpg
53a7d214d0842b532947d138388a8f67
89b60049a11a2a7d727209ff2161beefd091b3d9
1928 F20110114_AABVPU rauschenberger_r_Page_076.txt
d57566c38f483fc22d8fd5700fb78db5
1fc8d35bbd9e2bb9c6472aa548c8e48945a6c155
96213 F20110114_AABWMO rauschenberger_r_Page_212.jp2
3a9bc8913d77ddb1bd60dbe0808547bf
8ada20a5fefe1445d6e143e6329247c283a55739
75270 F20110114_AABVCK rauschenberger_r_Page_150.jpg
3b0de3ac9ce8252aa35b469c9bb27858
6ed52805b77d8d9ef291e8802ac2725104e3716c
8848 F20110114_AABVPV rauschenberger_r_Page_091.QC.jpg
1df19d0571683efc2276a0a557225694
c53cfc39dfad5c14ffb37c9f22cef5a7f44429da
107906 F20110114_AABWMP rauschenberger_r_Page_213.jp2
33a0dbc6d042a1cae50a5cb69f9f4441
ec0771a8651d1bec423cf93c3954b22b61064fc0
73407 F20110114_AABVCL rauschenberger_r_Page_045.jpg
67c715046dcf422af8f83f76e9823ebc
b82df2e5243dcf5d4ea543eaf82aaf32e3027c4f
67625 F20110114_AABVPW rauschenberger_r_Page_040.jpg
a44a50a8381706206ced90d4b212c01b
8dc8ed3ac4f3e3bf7d47008ced48c5c9dfc29fea
111994 F20110114_AABWMQ rauschenberger_r_Page_217.jp2
f59640ecde8d884d8c05273ee65589e6
5facdf834912ee7bc0b206829fb0c169e803478d
86721 F20110114_AABVCM rauschenberger_r_Page_231.jpg
a37412f8cd337776cd4f85f6d5efe235
823b69e69e0ebc2bfa506ae37bf926d23bd2e606
70080 F20110114_AABVPX rauschenberger_r_Page_136.jpg
55a75a44ed481d14ae1bd94f220ad43c
fb27ec8785cb77343d7e65fdb3b31ee6c349bf47
111356 F20110114_AABWMR rauschenberger_r_Page_222.jp2
56099b53b4af6778b2eb29c33f8a9e01
fe5e7655d67d369877c948e10bbf2edf1ac2b991
64063 F20110114_AABVCN rauschenberger_r_Page_030.jpg
131706fdc7f7ef00e466af756053b64a
983bd2748413effd78f18879314565a56fe14164
F20110114_AABVPY rauschenberger_r_Page_228.tif
3e42b8c63e948e50865ebcd36116c314
96967d4ba32db1b766233ea49d651cae67892358
134978 F20110114_AABWMS rauschenberger_r_Page_225.jp2
228a37a0c038c543de29b49af4d0f357
abfe4b05dfa889692a5fd3ac3411b10d11625a45
F20110114_AABVCO rauschenberger_r_Page_087thm.jpg
4a3c527d635b2e83e2508cf40d09c313
16caf58807826f486b6c7ed308630ed1794686e9
6350 F20110114_AABVPZ rauschenberger_r_Page_168thm.jpg
a13e05aeb9df2af8c982b54d8749717b
646d5e78125ca6e9f1c7858a33255d115d5d9f56
F20110114_AABWMT rauschenberger_r_Page_008.tif
8539193d11e623a8a2313586d1af4d80
4eb3ec984481a552bfef519ba7e151e3da5618e9
F20110114_AABVCP rauschenberger_r_Page_164.tif
65de8731f44a5fd67a7a2db456523eb1
ff6369e79f9cea346e15f41c5b33b040bb8e2535
F20110114_AABWMU rauschenberger_r_Page_013.tif
c27795d3b2bb3d606e4503db27cab10f
35c7f80406e5186d42efc61b17f0c2d5b618c41e
113500 F20110114_AABUYA rauschenberger_r_Page_042.jp2
e841ad9c3ee62d1d3da50857aaaf1901
c2d154a2b3a2d15bf582f73863b126a43e811dec
107187 F20110114_AABVCQ rauschenberger_r_Page_166.jp2
a9e876ba1cf5a81ba3f4f092f7024608
32a38342af8483a1153fb2b4ea1318e125dfd0ca
F20110114_AABWMV rauschenberger_r_Page_014.tif
1690836bdd800535c9dab2bfebeb401a
aa3d318461355ec687dfc0be301aae5de3c81df9
44837 F20110114_AABUYB rauschenberger_r_Page_090.pro
e776f4ed087cc1466c7bbad4158d406b
561c00f26134436c336b18684f8d8aa292d6363e
2799 F20110114_AABVCR rauschenberger_r_Page_067thm.jpg
3b61e2633e07fd3e9ec0bf870a03f962
d1859c7134f5d4441ca8f342316e17ae96c74f64
F20110114_AABWMW rauschenberger_r_Page_017.tif
d06994647687a26cbf0ea3318e43ba05
bb82af3e420b8c58a02f1e4a7a0dca742a15d499
1912 F20110114_AABUYC rauschenberger_r_Page_168.txt
13172c626223e7c4280bd58ecda71b16
77359779f5bdc5ee363c46974266e1bb4efb7ff7
6188 F20110114_AABVCS rauschenberger_r_Page_170thm.jpg
4194729686e49c95bfc96847f4291c49
8efdf2462880a9f7f71a087cc702f052e428bd5f
F20110114_AABWMX rauschenberger_r_Page_021.tif
972b4ec14b1bb236e699b9b8d1d82da6
6ff26a492bc319a4f5852a754c6cfed172c64349
4135 F20110114_AABUYD rauschenberger_r_Page_038thm.jpg
7635c8d3cceab2448af5457151a3c172
16cfedd6a0a525e73a41854caf5c43f12256dad4
23568 F20110114_AABVCT rauschenberger_r_Page_222.QC.jpg
ec1b47038bd5765ca4863e9e4faecfb2
37435f01cd86697db17f01a480649df2ca8d389e
F20110114_AABWMY rauschenberger_r_Page_030.tif
20b113e08ce610186cb4b5c2d55a1ef3
dcf9990db4099b64f7bbc2e01fee8cb944ae1d1f
49570 F20110114_AABUYE rauschenberger_r_Page_104.pro
72ea87f0f42af4f029e6d88c06280f1f
b25b50af3cb392b1e7835f6451c8efa139ef81ba
F20110114_AABVCU rauschenberger_r_Page_140.tif
f4cda146fa96bc633f226e43f2c8331c
64985ce2408fce09d94dcbeffdba3df048038ac7
F20110114_AABWMZ rauschenberger_r_Page_035.tif
b9ab223dcf6a6a36b4a6667b388d2ae3
dfcc3eb4664358b3e7a0acc8f88a28fd10e25679
133963 F20110114_AABUYF rauschenberger_r_Page_231.jp2
862650c157bc339873e29eccd48a216a
4f67d9aa5904a1949965407d1d000d9407a605ea
F20110114_AABVCV rauschenberger_r_Page_055.tif
ee268389439afb1f6da6411f879abce4
373aa3b33af6a2151b5f692a27c55282fc0c1763
5403 F20110114_AABUYG rauschenberger_r_Page_056.pro
158c0f5929dcd8042465da2e983058d5
74153bd00c02a1d358ac5539a2c868d77f39c25e
21133 F20110114_AABVCW rauschenberger_r_Page_169.QC.jpg
694e40b0c7bd61b6d97464243388b7ee
2ecc734f7eb17f414ea148ea4255a9d81980be13
2858 F20110114_AABUYH rauschenberger_r_Page_153thm.jpg
3bf2be110e328ad9f93f7317c91c1b2f
24609af9e1ad44dc5f8f146c84c670b772a8802b
6704 F20110114_AABVVA rauschenberger_r_Page_163thm.jpg
ed797777ff891d885c9cee1eddfd1a3b
c39dfd1b6201e8faadbe1fea85d34191f82526d7
73568 F20110114_AABUYI rauschenberger_r_Page_102.jpg
a2649a1efd50e682ccc43df19eccd47e
b9a251e92466f94761f7fc02aabc91c3914da1a0
52712 F20110114_AABVCX rauschenberger_r_Page_019.pro
cba25d45168bacb744b20e2664a6b480
11ca80acd70d5b0ec4e47dafebeb15ff8acd6349
7665 F20110114_AABVVB rauschenberger_r_Page_196.pro
d3bf5857f0362691144709cd130cbbf2
9eef2b91d086a7fcae750fa95661ec19f59cdefb
F20110114_AABUYJ rauschenberger_r_Page_225.tif
ce99cacb51bee81fe47e09eb24f2bf28
fb8fa41176340e56aafd1ad41edb400a590da319
F20110114_AABVCY rauschenberger_r_Page_216.txt
093e25640cf8ecd758be67de4fda6e75
e09e6143364d032b4c7f3e16c5f1974610d9365f
79562 F20110114_AABVVC rauschenberger_r_Page_009.jpg
bd98fa655b498b4095efaeed32fabf5d
4dac255950c70249a4f8ae96cad0a56efec53306
24420 F20110114_AABUYK rauschenberger_r_Page_197.jp2
2914ed594061167d5992649f4b9d2983
24a902d1c4940391cea2c364a2f4b3b4dc536979
F20110114_AABVCZ rauschenberger_r_Page_015.tif
50ea0a4d314de3c9acc3b50c26426365
20ff73a772650812400eaafa20bbb37f7ad38397
11947 F20110114_AABVVD rauschenberger_r_Page_207.jpg
3b6bb9cfa6fda9b77015d97dcf73eb97
cb87a6ec14543efdb7e8eebbdea06169ae609823
F20110114_AABUYL rauschenberger_r_Page_053.txt
c6cf28d9c1b67d585147d0d2617e15f8
d760368af51d9c9f8fd2de09646ade2023a8d911
84476 F20110114_AABVVE rauschenberger_r_Page_141.jp2
78837f1160fc2fa4aef2a208afbd74f9
8cd9b85c7cc25d2acd84a7d101bc159dd310ed12
21823 F20110114_AABUYM rauschenberger_r_Page_011.pro
7b5b3375170add852169ba62ea339596
2b74814f3a3bd0e7c8b84ac7bb331258fff814c8
45180 F20110114_AABVVF rauschenberger_r_Page_059.pro
c460bf34b0e42efdd7813bea758c9adc
ec100c5dad926f797b10ad09fa8a78f1699d5ba8
5103 F20110114_AABUYN rauschenberger_r_Page_180thm.jpg
71aebf89c6b54fe1fb66f91b9c80da20
99bf6ab356145025eb8315db25382d20412e256d
F20110114_AABVVG rauschenberger_r_Page_028.txt
e643381a745134d8cb204b7411305ca1
7c83a27ab3f8456820834d2242917471336e980d
50610 F20110114_AABVVH rauschenberger_r_Page_176.pro
43e9604b7097466a8adf45ca5fbc9603
b868bf751c171c8c6a38e9331820066d7fbd5225
724 F20110114_AABWSA rauschenberger_r_Page_192.txt
23ea077068bbd758379fae1162e3d30f
0f09d7f379ecf4b1aada183d82a64f468de71da2
4898 F20110114_AABUYO rauschenberger_r_Page_233thm.jpg
3208e60fca1f10fd931db1b6e17d2a35
6b979e91e4fecaf884dc508db5426f4d5c8694e7
F20110114_AABVVI rauschenberger_r_Page_223.tif
7afce7fb5ee4355bb163316c5a5eb84c
08c029edbc384d9739db4423996b28efbe6c8388
788 F20110114_AABWSB rauschenberger_r_Page_197.txt
43057c05d49b3585da201cc58671775d
fcb1449155e722103494034cf24fb3d168e10386
97730 F20110114_AABUYP rauschenberger_r_Page_226.jpg
28692436503aae48d169edbcafcfee66
d1d94c2cd894d1b2904a6537dfefea6f17f62b15
54338 F20110114_AABVVJ rauschenberger_r_Page_083.pro
414efd3a2c5f1562487d5df8bd8368a1
4f3c5274d544303ee7342a6d1f59497cd2fa7256
2028 F20110114_AABWSC rauschenberger_r_Page_204.txt
758b6c965b4ea46966bdae9282c9b9c5
116668044e19e2cbc6a2f926875583de92d2afb0
2004 F20110114_AABUYQ rauschenberger_r_Page_172.txt
75b050b598e87ac709d08b22980522cd
b73c055c39fc17f6ec4ad55b005ac4387c4e85d1
2078 F20110114_AABWSD rauschenberger_r_Page_206.txt
21ce9f6afdb0af43d0927e2119d1604a
6b1aa4039bae52c1579b94bcbd108aba354239f7
965 F20110114_AABUYR rauschenberger_r_Page_064.txt
61ffb1ec4f9e7f845e3aa1b0891aa84a
3d42aaf50b31e7e8789a983461b1336b89426bbc
15097 F20110114_AABVVK rauschenberger_r_Page_209.pro
b9ac4f043e5f1458e0d7c7ca3211a845
b5e17c9120105d0ef417d31b9a9fa9f32c9ec2f7
837 F20110114_AABWSE rauschenberger_r_Page_209.txt
dd2f01ef5d6a3fcbee5ea55013ddea1f
9a212455f70144ee427594a724cb0cbc391d3fc1
72475 F20110114_AABVIA rauschenberger_r_Page_082.jpg
c430d462e6c523361b3d8f9c2029a373
66ada98949a29af82f9170f621deb10b040ad5b9
66255 F20110114_AABUYS rauschenberger_r_Page_004.jpg
32fdb3f206d57c9eb50e38ac9d1f25ae
d835ab13ba822a1015c93263af6f802261370224
105990 F20110114_AABVVL rauschenberger_r_Page_075.jp2
df5a2bcef45a04d8e8865ed8e9ca7d97
5c5d9fb0a5eebc808b832ed6bec5a3da19692f48
1695 F20110114_AABWSF rauschenberger_r_Page_210.txt
cbb865e9b981e478ce72fdd31210d3ed
3d094fface025c7539e4b611d104dfe135ec14b4
12340 F20110114_AABVIB rauschenberger_r_Page_146.QC.jpg
215460b2b3bb0f49247d7cf3347a4cc7
a1d8ef999b443fc2f38c1bf79815765c80cc6320
103398 F20110114_AABUYT rauschenberger_r_Page_181.jp2
9da6df6cd9029ce9acf0231adb6c2a13
083105566f3e52d5ded74bca64d6945f951c163c
112834 F20110114_AABVVM rauschenberger_r_Page_205.jp2
b56b1ed366597aa26ec807ef9ba29503
6547aaca4c9fe99905af4aa0a631ae81f0ac9620
1962 F20110114_AABVIC rauschenberger_r_Page_018.txt
b9069097f5880eaccfd9fa5a8cc97b0b
b9526d60f5133ce82380b7c437e9a9d6901aabf4
51360 F20110114_AABUYU rauschenberger_r_Page_054.pro
51aaeebcd1f429e4298f7a1632b7c736
65f2ea292eca2b5fb2866961a0975ad79aaea226
47964 F20110114_AABVVN rauschenberger_r_Page_035.pro
03ddd14b360c9fb5168cad27336f7dc7
f7888b6e004aeb31b367284420fe6ea772f4d05d
1993 F20110114_AABWSG rauschenberger_r_Page_219.txt
e7cfc5eb6e0271d6d6c7ca0de27c8916
6c65f95f36421a4fe376b966e6ed2706bf575c18
1098 F20110114_AABVID rauschenberger_r_Page_211.txt
2d749ab8e813a107f6e0c614e6faf392
d56c59bae0f6455b1d85379923f2db13aa0f2692
12680 F20110114_AABUYV rauschenberger_r_Page_140.QC.jpg
2d86f086310d4cda701cd83b4f783992
d9a51dd7568d44b62f70643c6accc5d379c94bcd
46931 F20110114_AABVVO rauschenberger_r_Page_028.pro
c748dbaad9ceaa1aba9542c816626923
c1d0e24db5233f65c657ba258f97cf49c86cb7cc
685 F20110114_AABWSH rauschenberger_r_Page_223.txt
37afc3407efa74233c26b92a49cdc9a5
e486f390b4c5155783714ab58e14dacc854d467a
25121 F20110114_AABVIE rauschenberger_r_Page_229.QC.jpg
714d6b9fbd19f073695a7ac3c1699615
a30e0048ed8fdf30981abdee00695159d57ab505
1909 F20110114_AABUYW rauschenberger_r_Page_136.txt
1f297d3fb5db46e8df5e36e5310c7236
1e5fd4a6a29930811f43afa621808bd20f9bbdbc
35601 F20110114_AABVVP rauschenberger_r_Page_089.jpg
3e1ece172fc2bd75518b27a56bd2070f
91e4589dbf2d0f81c80e944b4894b08b0e8e52c6
1937 F20110114_AABWSI rauschenberger_r_Page_224.txt
89a4190cc5d47c8b1b0dd627ce6c8e71
2fd60097e32adb723c148b7dccfb91936157360d
72300 F20110114_AABVIF rauschenberger_r_Page_084.jpg
4d4914979668cd6fd50f1840ee3f8f7c
5f6c279460c0b98eda8e013fd84f61eee153ee35
22716 F20110114_AABUYX rauschenberger_r_Page_097.QC.jpg
4f4e07435119ce45220cf14b1009dd1e
613afd4b766ac5280df23e0c228454c5a491985f
F20110114_AABVVQ rauschenberger_r_Page_067.tif
a5e6a9821eda50061c620c4498ab3f80
2b6f157e9cdd8fe5ba41502424814cbc6b2b53fd
2611 F20110114_AABWSJ rauschenberger_r_Page_225.txt
c1938fad06847497fe3db4dcde34ebec
a692b4c1c9f74a30eb644a78b23ce6b4343d0233
878 F20110114_AABVIG rauschenberger_r_Page_152.txt
d9d61dc66a65bff7613c55d2cb93a2c7
6717e19f1dbf01cd092dbcae16dfcd254806b5ee
F20110114_AABUYY rauschenberger_r_Page_188thm.jpg
4bd8e12b129290c2d7d50e6f9b05ceaf
d3ff3d2bbaee310256b8da3861bebeebe20dba4f
7150 F20110114_AABVVR rauschenberger_r_Page_194.QC.jpg
f1dd5a230f1d373fe0fc8663a73bea26
1d1303c8d5986ca5dd55b9499a76d00ebab69181
2827 F20110114_AABWSK rauschenberger_r_Page_226.txt
81e63cf84df45ecd7dd1a1eeee4397ff
596b01ba3a3505cd2d857ca1b83608922a3d1064
22891 F20110114_AABVIH rauschenberger_r_Page_108.QC.jpg
c1ea37d37d7a44a91a4fede33ea560a4
c097a2acdf9c416e8d833c6143d533e4adbc1673
F20110114_AABUYZ rauschenberger_r_Page_096.tif
e139ee45360906165256d59c1ddede6c
3aa2ef7e15a3ebee7d2753dafcb1b29eb57a5f43
1051979 F20110114_AABWFA rauschenberger_r_Page_008.jp2
38cd0dfd5743f7968796e57295db377b
4b5428bb69e053ac4cbccf29b7d3e7712b490688
70875 F20110114_AABVVS rauschenberger_r_Page_108.jpg
2059eab3decc735d141540c607de7b82
e328b8932d491b9145642ad8eef6ab3de6615872
10765 F20110114_AABWSL rauschenberger_r_Page_005.QC.jpg
a8b68e5c4275866c3d37cfdebd172a96
0a299802393f6547afe3b8115f2033a3e88c95cb
F20110114_AABVII rauschenberger_r_Page_048.tif
1435dc6be9210bc2993ce2eff628d4c7
0daf20b7a6d6d8c67b028345144b56ae31417a07
43204 F20110114_AABWFB rauschenberger_r_Page_070.pro
7b04c520155b6f649f69a4b8e17d3722
2f7725cb19c2f923c8ba93cb035f19767671194e
3884 F20110114_AABVVT rauschenberger_r_Page_138thm.jpg
7356a142798f44199399cd72191d1a9d
4b7edc47874a1481fcfbb0d2615c8c70dd064ad9
3338 F20110114_AABWSM rauschenberger_r_Page_005thm.jpg
d5e5b912302a9e4daf101b0f396d87e3
a379d7eddc2e91de59790ee9e957e719f2d20819
F20110114_AABVIJ rauschenberger_r_Page_230.tif
386328f0909579023bf897498e5738d7
1285dc84e85bc73723185384a99e8d45de8e6e4f
6311 F20110114_AABWFC rauschenberger_r_Page_134thm.jpg
0ae319b9e4ca99b2b6e0338a7f43e895
b082a718dce981d10fc279bfd07aed43423dd9a3
96587 F20110114_AABVVU rauschenberger_r_Page_070.jp2
1595d11a48d1fc4730a4bfd0a170520e
71b80527fd324431c10fbc34d03ab356860ea885
20861 F20110114_AABWSN rauschenberger_r_Page_006.QC.jpg
2045067b252894f6db8acfc9cf6e8de8
8b0c0c3e64a25cd41d05d6c227a2ed69cbccd215
38923 F20110114_AABVIK rauschenberger_r_Page_113.jp2
e89ef42d3d0e0c65f3c964383b344d47
ba78acaf1bc71464a4971e70b2ffba1d35a69393
F20110114_AABWFD rauschenberger_r_Page_051.tif
ee3b0a1925f6d78e9e6bb6909027c627
d03a87c1db831158f6358d321d01a94b8392e3a6
F20110114_AABVVV rauschenberger_r_Page_083.tif
a5b10e5d3a9d99e5b8223973fd6d13f1
0b3599c577fa471ebcaf7e968201e4dbf7051028
24523 F20110114_AABWSO rauschenberger_r_Page_019.QC.jpg
8ae3bb3959f057cede29454fbdf97dd4
9ac8b068a90c5291ba8bae64d80c03a5be501675
F20110114_AABVIL rauschenberger_r_Page_093thm.jpg
9d8e8fa00442ec7959f2a273560c1530
7d568b47e0c2d9256bd6b2b4c6f4e0a3c3fe1123
F20110114_AABWFE rauschenberger_r_Page_079.tif
0da190c9f0f95998c268411a63e6b865
2b06cbd76da3a82379e0774d824964f5c7ccbe13
75186 F20110114_AABVVW rauschenberger_r_Page_083.jpg
552a656a82aea748becf230eaf47bdcc
f37dc4f128ee8c46391271a9924b8a57593fcc68
6689 F20110114_AABWSP rauschenberger_r_Page_019thm.jpg
27ff59d3776e974b0eb3d451c6783fbd
ce5d27f0554c7f1c196c4825ab1e06e3709cded0
51203 F20110114_AABVIM rauschenberger_r_Page_214.pro
eb7191325d2b458805c37b9ae532e70d
bf5e38560be4a8b00efd66acd0af9eee7166fd39
20892 F20110114_AABWFF rauschenberger_r_Page_192.jpg
8d101da8e748c3b72544460111cc753f
eca7ad15ef1dfbb50049936f46dab4902d381822
105598 F20110114_AABVVX rauschenberger_r_Page_046.jp2
70a930c3ad9744778418e74b461ffbd1
a10f86c2aa6fe8b6ed6598d85895cf67dff52e40
22725 F20110114_AABWSQ rauschenberger_r_Page_022.QC.jpg
d349031752ed22503cc26733aae53b8a
3fb3a1d509a9fe159094417f85001697ecaf460d
6506 F20110114_AABVIN rauschenberger_r_Page_125thm.jpg
e0c82995facc9a79c06b0a6a6306e70c
946b5d5a6ffc848fd01962782c1408b2a5726402
73327 F20110114_AABWFG rauschenberger_r_Page_124.jpg
fa65b4bfdc22b5e13244c2a29bc85837
516ad8074b403b594605f6c16066e9d4a2aad095
F20110114_AABVVY rauschenberger_r_Page_118.QC.jpg
ea364c22db7a4325167858cc9d2418c6
ef17cffddca90cba7a1114a45eb9eb8aa269b4a4
22146 F20110114_AABWSR rauschenberger_r_Page_023.QC.jpg
72c92e43c3573a7bf2b89d57502a1201
9823008cfb8b28aafa06871d925b54a5799e834a
72558 F20110114_AABVIO rauschenberger_r_Page_177.jpg
90424fca9923336f358efa9e3b80a57c
a567817f890f3716133cc1d2df2cc7426f2d2e92
8822 F20110114_AABWFH rauschenberger_r_Page_183.QC.jpg
e705b7ed2393c997640a0c41e0e16e87
0f11e6acd74dfe1f9c6a3ab6465843313627df01
53683 F20110114_AABVVZ rauschenberger_r_Page_094.pro
d3d26ccb64929fac3ddb89906824f1b3
5a6a0f1b023a38b6f9c18d56d57c8c6cfcc37322
23833 F20110114_AABWSS rauschenberger_r_Page_024.QC.jpg
9a8457e26c260ab572f7fe4f5b03fb46
061efbe1a25565cac59512a3d73fbb926d6b59bc
F20110114_AABULV rauschenberger_r_Page_011.tif
94aee6ececcc314555e5762bdda94fbe
4cb39453485f2796ae23bcae21946db936808f73
234133 F20110114_AABVIP rauschenberger_r_Page_014.jp2
0c9ef01221104569083339851d5fd8b0
2a7dbc03b1802e66a7310be87247e1a991366c1e
6344 F20110114_AABWFI rauschenberger_r_Page_035thm.jpg
43b41e5d0e6654b0ad996d42f2932a6b
787c1678018217000b3fe6aa3ae9cbfcc463d994
F20110114_AABWST rauschenberger_r_Page_025.QC.jpg
cfea16c520cb2bdc00e1134bc41445c7
220dc12052879288eb25727b36c9d03a3ade6e79
6286 F20110114_AABULW rauschenberger_r_Page_043thm.jpg
3257c71069d1fb0930f209da5b7004f0
ebcf5622c3affc12e470e2ba2cb1ae613e503c1e
F20110114_AABVIQ rauschenberger_r_Page_098.tif
ff192f99e5dbe941c4ce74c1ddf143a4
4aca5b4b4d5218d58930e1ac966992d4d69756d1
109558 F20110114_AABWFJ rauschenberger_r_Page_074.jp2
7dfa83a12d81ab8dea73237ed969c6da
da9ebf050976cc3a4a93d3304a2f55fbc0229488
6001 F20110114_AABWSU rauschenberger_r_Page_030thm.jpg
3cc07b9d1b3fdf202f9658fc5cd1ff8e
7ba1d54c0398956541b754b2403f95d2a630f5fb
1724 F20110114_AABULX rauschenberger_r_Page_017.txt
2f1579730b49b991be9436b3d8bf9abc
e835373bd5facff7d410f222bfc77bbc30da63bf
106473 F20110114_AABVIR rauschenberger_r_Page_035.jp2
1a1e1615002eb059d730ba443d3d62ef
e3fb15b6e3ba433fd03b63f713896eeee3fb9503
F20110114_AABWFK rauschenberger_r_Page_036.tif
f69b5b19c4b1e26e510ea561bea287b3
8ae165212255838b04468ade532764bdad9202b3
23235 F20110114_AABWSV rauschenberger_r_Page_031.QC.jpg
82002e2166affc8b5d58fa7808c3a9f2
42880724a08e43c49587fcea3d48b547766a395f
95831 F20110114_AABULY rauschenberger_r_Page_198.jp2
3478ee31cc1317a7800825deaff4e470
dce8f946a83772aff7ec472e9e932967d178a520
F20110114_AABVIS rauschenberger_r_Page_193.txt
e4e646670cde4c8896c055c495b8de53
feb86130c0595d5adaeab86dde7365e9c55da8b0
257 F20110114_AABWFL rauschenberger_r_Page_056.txt
5174f26e95cc11ae4a89ec713cf384ae
59432622e0d58a71edf9423868c2e0fea7493476
F20110114_AABWSW rauschenberger_r_Page_031thm.jpg
a18dfa3f4c07930d994ce9fe537c5e05
4fe23a10a1cce1b3ba2ed29ce33c2906839cd572
6446 F20110114_AABULZ rauschenberger_r_Page_049thm.jpg
80a12e5cb5dee32b561d3ca07f25ecfc
b819f59d94b8cbf4b4ab0c5c2d6b9b6f12f2a97e
29590 F20110114_AABVIT rauschenberger_r_Page_063.jpg
571082131e8def5ef6ffe82dd8b84149
fb4052b9a85d8dee13ca4699da986b87decfab01
F20110114_AABWFM rauschenberger_r_Page_102.tif
1cd4e4347a7f13ce0b867cf74d1bb1df
99faf01c41503c65f243c795734f32dd28545222
6535 F20110114_AABWSX rauschenberger_r_Page_034thm.jpg
6d373fe2480522be0966ba905b2069b2
cebf59946be1999a5ad4c9ef2a9804175411b2c8
F20110114_AABVIU rauschenberger_r_Page_022.tif
fc95a8d1353a9052df3fcf2389d2a6d9
d36285a7ae6458a6b7db566080a118676fa42982
F20110114_AABWFN rauschenberger_r_Page_186.tif
2c25686da3e66dc006c2207bb621e6ff
87c0b2b6d1f54cfe4d31bbf9c90a2f9df7b4902b
7788 F20110114_AABWSY rauschenberger_r_Page_037.QC.jpg
6d472f290e171fec1e4d0eddba8b0bc2
0b70981680d4cf0d467f65f6b275640d6998e979
4279 F20110114_AABVIV rauschenberger_r_Page_114thm.jpg
9c5c96eafe932eaffce0fd812709e4c5
f45bfbf3a018c0d8f252a65aab2baefc4b95f006
24199 F20110114_AABWFO rauschenberger_r_Page_047.QC.jpg
85c0aba41ba62a4c2d256739636df464
87ae7bf4d210c272230d2c748edbded9c82d1a14
6321 F20110114_AABWSZ rauschenberger_r_Page_041thm.jpg
6fba97d9e099864be563aa1005cb79f1
d9b6e5268ca1dfb55710d1377f1eb0b7bd6cd1bd
8886 F20110114_AABVIW rauschenberger_r_Page_092.QC.jpg
c5ecd568c78a8f72d04bf77dcf8c2fa4
e06eb8ebcf7fba58baa847f62513fb5d93d49b53
98825 F20110114_AABWFP rauschenberger_r_Page_146.jp2
536bd2631a4ed7e7da67ba48f68178ed
2497dfe95a66f596121016a8d41624569f210c3e
21847 F20110114_AABVIX rauschenberger_r_Page_196.jpg
be48b9491bcad0160a9fbb715ed7675c
2a3c20eee0a12696893acb24da89d8dab7bde44a
F20110114_AABWFQ rauschenberger_r_Page_077.txt
af361484157ff3c2fe519c720084fb93
f408e3035354937ecc3dc8a0529e4de895105518
F20110114_AABVIY rauschenberger_r_Page_087.tif
6010d41ddfc6778eb7c3030fdd4bf039
67f496916f05ce366fb3ffa616b927de37b23638
F20110114_AABWFR rauschenberger_r_Page_001.tif
1523b9a13d6b2933eef704d6eff18655
ea8af16472e3e1519719175dd020ffd8b03a28bf
14540 F20110114_AABVIZ rauschenberger_r_Page_112.pro
a616be54ca3e18ae8f144c2244369a06
949bfe4e9a4e9fb11dae25a07f4cb6b5e2e70a95
13481 F20110114_AABWFS rauschenberger_r_Page_095.QC.jpg
4711ee94b7bac7b1c712a2b5ac95b2ce
d9163e2a87a62494c6433092157e61dd61fcb280
6224 F20110114_AABWFT rauschenberger_r_Page_171thm.jpg
2e3bdba6c7c0a615c838e6599ebd3eab
8c219a46e329dbad582ce8c13736ca957127364a
6364 F20110114_AABWFU rauschenberger_r_Page_216thm.jpg
65bd31f58d8fd4e327631459bf275612
315403a0848e7d418c4eebfd5ea2875da0a57824
109511 F20110114_AABURA rauschenberger_r_Page_177.jp2
4a990b61872853af6c8de6789cf478f9
e4b96480aaf64f84a29f455d433169fd490cc776
F20110114_AABWFV rauschenberger_r_Page_032.tif
0ad10e0280b6a0858c0c9316c031002d
d150aa9c8aa125d7052797f4375fc2b4f678cd8d
1436 F20110114_AABURB rauschenberger_r_Page_088.txt
89f86535154d3f4b1e13198ca12003bb
6c805984761b5d0f6efb35ecd08cd3f2de2869d3
10367 F20110114_AABWFW rauschenberger_r_Page_089.QC.jpg
16cc85ba610ae19ac8564a90d99f5222
1af78b8c93050d0382306a561a4a488039157112
F20110114_AABURC rauschenberger_r_Page_062.tif
96ce15ddc79e397b5b55aa1466496a98
9f5b2ddff1092e097e9e1803a6d84de32a6b471b
15156 F20110114_AABWFX rauschenberger_r_Page_158.pro
878fca05f3e0c70c7eded7ae213f730f
34102f14451b34d4621aa48c3f797bec66eb44f9
23700 F20110114_AABURD rauschenberger_r_Page_172.QC.jpg
c2b1723513746f9585f4184828ea1868
7fa7fea2fba7a177a31fe65810dcce5a82548a7c
1051938 F20110114_AABWFY rauschenberger_r_Page_009.jp2
a28594c0f001929972fcb4499ae68f0e
7383476c92153f81a30aca0094021f7444791dd7
23276 F20110114_AABURE rauschenberger_r_Page_150.QC.jpg
50de7618496faa5ee8e37801f7594b15
4adb82a4053aa0e6a4c2b1be1a2201712f16c37b
70446 F20110114_AABURF rauschenberger_r_Page_167.jpg
7100dc20181898a78e1a20da0217860c
26f8bc012324be1f5774c51da271d22e61ad3e10
22426 F20110114_AABWFZ rauschenberger_r_Page_053.QC.jpg
4d8b191b27289a48faa478b95237dd5d
627a07229798d74c558277460fe4c7aea5832fbc
182209 F20110114_AABURG rauschenberger_r_Page_196.jp2
33b648556e2f71e48e27b8415fd3156b
068f585faa8afa13c90ef22b2e0ecdee40fd84cf
23079 F20110114_AABVOA rauschenberger_r_Page_021.QC.jpg
10c91b02c6a27a1c41f8791cd7166d3b
afbceee49ef1042bf3374ceaec359677b0de771c
1862 F20110114_AABVOB rauschenberger_r_Page_115.txt
04673ed641d0a4db720dda929da1e081
80f85e8b1539856bb20f6b3ddbae5d297702201f
50755 F20110114_AABURH rauschenberger_r_Page_119.pro
30c542db5a360c0618f616a5997f3faf
a6a19980b1f97d7d4a49c49e09a65a58b94f6dbc
1422 F20110114_AABVOC rauschenberger_r_Page_148.txt
9d8e2a58321c6743e4a670eb758a39de
223530cb45e1bd42dd8b0f9f1e3947be9c07d91a
58375 F20110114_AABURI rauschenberger_r_Page_142.jpg
25df70bfcc5ad68c70cb4ce9dabd0bdb
ea1560760dc6e379e9ef19780ae1e2b61a5fd20b
72381 F20110114_AABURJ rauschenberger_r_Page_217.jpg
c5376d2378d8855b5aa5f003cb233621
24a027500183bd422798e9f2aa1c970062b6f049
643 F20110114_AABVOD rauschenberger_r_Page_069.txt
449d14b566e6364325e11bac09b6423c
69640cf9ed2b7ff4ec97c28f6e513fec7e383eeb
1948 F20110114_AABURK rauschenberger_r_Page_031.txt
3ba1df8ba0e448a650a06bd5afb70ef4
fa2893681a59cfb66b42cfe879cba6e6b72abd11
6279 F20110114_AABURL rauschenberger_r_Page_050thm.jpg
1807ac8dafc9a322bb1681fb4b8d3434
6542a960f3f058b496401d3e1c76c5df1602b5ad
F20110114_AABVOE rauschenberger_r_Page_135.tif
96f82c772414853e4624af664c42f806
9702999ac0f96e784acdbc81a1276867b92aec54
104673 F20110114_AABURM rauschenberger_r_Page_132.jp2
ff7e353960e26a5f1636c3f101cd744a
d62d6eccf74e1798a154f2d8a6e34fb01ca36c22
107501 F20110114_AABVOF rauschenberger_r_Page_136.jp2
a63e83fcc66f12254230eec475d2dce3
29290af5b77985f2bcbedded95dd33c47511018a
10977 F20110114_AABURN rauschenberger_r_Page_149.QC.jpg
a3c35764ba492f11c626d947a88793c7
e58c2d2422fb0ba6e347d58cee82ceb881078f50
2986 F20110114_AABVOG rauschenberger_r_Page_064thm.jpg
9c3bea47741bbe02637a15d1b74d3490
338b5faf17238bcbaaf687e9caec24d61237bcfa
66976 F20110114_AABWLA rauschenberger_r_Page_224.jpg
775c44da21bfeda9be793e3f6e490037
b3fd908c2c50846d8f0b71be3e31a119e2f9fdc1
F20110114_AABURO rauschenberger_r_Page_149.jpg
240b3e51a9f4d23b7c4a130e4d5b6681
b17f5f3d1cbb211482db4aeaa6c65d8dc4c37b8b
24298 F20110114_AABVOH rauschenberger_r_Page_052.QC.jpg
c841f93df70ef279a3ad615fd8b6f64a
3bdbbd52b617e25ece43800b0e984912ea362068
86754 F20110114_AABWLB rauschenberger_r_Page_230.jpg
fa2086ecf648d0805057f238f26836ae
4551098381be12ee726ed46d7d52cce9a8e4f0f2
2398 F20110114_AABURP rauschenberger_r_Page_062thm.jpg
c355d5be285524c81c15947238355e4c
58f92b96bcfe19d9561f3625fcc45afae782f5f4
1760 F20110114_AABVOI rauschenberger_r_Page_198.txt
151b852679770ba637dbdab48a1f197b
838287ab81c991acda4f6d9718dcf72f105d1e72
44835 F20110114_AABWLC rauschenberger_r_Page_005.jp2
6bbe496229a0f9b43ecc45f14fb516d6
648b0b834b7ba9a7fe40ebe726c130f675557194
108427 F20110114_AABURQ rauschenberger_r_Page_071.jp2
99b289391de928cdd15c48f47e2e602a
26715439d99c198776930ecd9bde8eee47710bba
6466 F20110114_AABVOJ rauschenberger_r_Page_055thm.jpg
c2ceac4da3a37bb2493d9fa2b434c3f1
0def8fd74dea15c9f1a0d56c7f85f554f1bccbde
102306 F20110114_AABWLD rauschenberger_r_Page_016.jp2
131b079570a927870d46e90ceceb2d92
f0b6cf37a46cef496b5eb055a91668221bee8f3e
F20110114_AABURR rauschenberger_r_Page_075.pro
07b0f503f3a4af988c9894f579a090bf
804dce5a49dfac91ec8aa1d5a3bf98edb4328a03
23628 F20110114_AABVOK rauschenberger_r_Page_104.QC.jpg
9a85b47d5258ad0bca257e272caac1cf
d9d4243c496edab2655ca41fa11093233dcc02f0
106536 F20110114_AABWLE rauschenberger_r_Page_021.jp2
f21c09dd27cae5b7ffaf9c8c2fde87f0
3e400ad1cfe245f23477757873dc9a253261316f
54143 F20110114_AABVBA rauschenberger_r_Page_185.jp2
92fea71c5ab845eb1c0d3050142cf7ac
485cc3760a0d837b0014c272a5a24adc9e466d12
6582 F20110114_AABURS rauschenberger_r_Page_022thm.jpg
bfc230ae38bc399291e5ed54ffec291c
e1195731ff4dca64c9843b68e18744e3a579c871
F20110114_AABVOL rauschenberger_r_Page_128.tif
d30778b95e94d18e0ed3e9259f2d36cd
d36e41a883f574e5b10ad137da22d8139cd07483
100280 F20110114_AABWLF rauschenberger_r_Page_026.jp2
088774bff9fe95a4066c69559170771f
8ba7ca2e5fe920875e81011c6cfa10eda082d55d
70629 F20110114_AABVBB rauschenberger_r_Page_077.jpg
87a5b2c21713ddbe90c6284d417f8016
a157773210cb0f5ef7ad0ffc89b9f82f8f71c993
F20110114_AABURT rauschenberger_r_Page_141.tif
085ff52a311164deb55186e050c74a1f
a5a8fb4e91d7d851a4373ac02bedf191de7e63e5
F20110114_AABVOM rauschenberger_r_Page_005.tif
cd436aa170a05a513080b204fa3702db
0c41d39823b91b737ad8d014cd661d48dd8154b1
108827 F20110114_AABWLG rauschenberger_r_Page_031.jp2
67710c96a0e03b1ba33d065ddc0c0ebb
91d6024935ac6b8ba7232d29351fbec214fd2f53
108928 F20110114_AABVBC rauschenberger_r_Page_077.jp2
008d46278a0933b4b92daac470f9b773
239326cce23dd6d3572b224876115c9d7af8dc6d
F20110114_AABURU rauschenberger_r_Page_116.txt
b639d159659e63987180cf441a69d52a
14226b4a48e7f9f48bf3498e62496fff60881842
50831 F20110114_AABVON rauschenberger_r_Page_100.pro
9911ef80328ec8ee4fe0101213fa1b4d
4e39dcaa2d8bfab460ddedae1563136650c806b3
103370 F20110114_AABWLH rauschenberger_r_Page_032.jp2
3bde3dbca421206459771ec51b05a379
24da161f545d8882d8d74586e3287ed9189fa32f
F20110114_AABVBD rauschenberger_r_Page_221.tif
edc5f58fcfe42a2a5b70ff6d79777303
578fef21c9d4b28faf2a205e8dc699fdd15ce38b
6979 F20110114_AABURV rauschenberger_r_Page_229thm.jpg
b2dd27af859819a43554f2bd0c3a902f
15a6469ff052a8af6c29b252fbaa3928a727e34f
49205 F20110114_AABVOO rauschenberger_r_Page_213.pro
3c4f82e64a1e943eeccb44d481e99739
1ed6a4799ce017e401705600806c49b1fdbf8492
111478 F20110114_AABWLI rauschenberger_r_Page_033.jp2
b9c7ee5a6824574aa2d1919ceb45b135
62174e38fc35fcb9bfa9f02fab07dbc71718ac07
F20110114_AABVBE rauschenberger_r_Page_192.tif
96644ef073bfa545d525c418875c0d81
a7a3a5eac5b385e3918e31dd08167e901974f68e
11127 F20110114_AABURW rauschenberger_r_Page_153.pro
6306122f85740caf7030c015bcf85277
a154f0521ddbf1d9739ca98470230053d0b40364
88662 F20110114_AABVOP rauschenberger_r_Page_015.jp2
d2348e43e944f318afd63e373c26258b
d36cb97be2b5d2764cc246773bfd8d637856f881
108898 F20110114_AABWLJ rauschenberger_r_Page_034.jp2
e2affebb9ee47b76de10be07c926fdc6
7ecee81cd37580db2e2b239f2b71de52b17f77da
F20110114_AABVBF rauschenberger_r_Page_164.txt
a73d9ebc3e4197184ff1f0aa5e91aa20
72f6ae977cae3ecaa1b80506160c508b2cb0a1ba
74299 F20110114_AABURX rauschenberger_r_Page_079.jpg
35da085030c4253ba7b9976dbd94128f
cdc741ece3ab304f2354481ae14c59afa82f4c8e
F20110114_AABVOQ rauschenberger_r_Page_111.tif
031681944e377ce9b542fd4f6e257a58
6c2ea12b08ee0299e5df8a52cbfcb9435cf27a27
105469 F20110114_AABWLK rauschenberger_r_Page_040.jp2
4c7d1cf5aefb017930875d34d79e5679
7c8ed6519077915262355d6d072561dbabd95ae6
1229 F20110114_AABVBG rauschenberger_r_Page_089.txt
9321fda4df6479451cea63f45a136e5c
0dddaf781d474d766b64352c0263299c9bb58364
F20110114_AABURY rauschenberger_r_Page_084.tif
676c1bb3bbcab8e6d1464aca54cfdd6f
f7821f5904a71cd73669ac86e6a1499dfec608cc
5120 F20110114_AABVOR rauschenberger_r_Page_088thm.jpg
9ff751f6e94a6a14aa6639e7785ecc6e
faa57dc1142fbe53895ccbca52554b9b85553b8b
105459 F20110114_AABWLL rauschenberger_r_Page_043.jp2
fa9c83a0a668798167cfca06efa5c1ae
d0dedce71da8b47d74af18cc38ae99a82db0bd64
1847 F20110114_AABVBH rauschenberger_r_Page_023.txt
e649170b79447a28beabb447263bc3d2
6294c2d3fcf04f714e66b8012867b536e37b3117
18542 F20110114_AABURZ rauschenberger_r_Page_003.jpg
e3947688052ddfa0f79342e637a9a876
76b404863ebc1ceaab93bf04132ccd3a34e86d29
49243 F20110114_AABVOS rauschenberger_r_Page_018.pro
a1abc3d95e4350a2a08b35a253a8d143
2f73c8dcd654243a82f74a99a59851653b3c957c
113553 F20110114_AABWLM rauschenberger_r_Page_054.jp2
fe3f50c834f8bf82b8f68d0af32813e1
71d0d26f951faf72b6558d67c338a9c4bd4488a3
F20110114_AABVBI rauschenberger_r_Page_217.tif
5ecb641e12b123e27fda459ec124e097
6f45ccb74fe004c90f3214b948ac2f67f58dacd3
95704 F20110114_AABVOT rauschenberger_r_Page_187.jp2
708f1e61e15c205726e36ff0ac1c1bb0
8d5ed7ba5e400f7a52e1216e40d8b2d1d9181371
111951 F20110114_AABWLN rauschenberger_r_Page_055.jp2
deb6c82c76bf45fef13a517da96122d9
bc5dd732b3d5b38c4add3fceb1f75c9979078709
F20110114_AABVBJ rauschenberger_r_Page_191.tif
28981c94b57228c9b0260a0941574adc
417105482eb9caf5a69e680641cf973c1a0bb3cb
12940 F20110114_AABVOU rauschenberger_r_Page_037.pro
f5e704eda41f0c138e62887c1149c876
fa6fa155c61c7fe61a67f7930ec3ffdb7b228c6b
28518 F20110114_AABWLO rauschenberger_r_Page_068.jp2
e2fab3f806533ecfa90ed5e80a7462cf
f88b278975ae82dc96cbb4e3fbba547df374748a
F20110114_AABVBK rauschenberger_r_Page_183.tif
bc484f9ea8202516993d0c764cbe9731
a1634eea3094c37753e0277a74c44f257bf02f4d
F20110114_AABVOV rauschenberger_r_Page_179.tif
fd2f0d9fce846d201419b73ce3dc2a94
83a3d395abc44b9f66eef7a129f97be58476315e
107892 F20110114_AABWLP rauschenberger_r_Page_076.jp2
e951929e229afde2a4238205ead43ade
06a4399f1af5970baf09e8ddaa84b4b6fbfe34b7
23881 F20110114_AABVBL rauschenberger_r_Page_211.pro
885094291147c24d1a2b56acf43822a7
e1cd22b655e51ced49508b9e0a7b8329ede54dbc
109082 F20110114_AABVOW rauschenberger_r_Page_168.jp2
cccf671c849daf5b64c3c2ed53d5ffc7
d544124fbaf21b9cd1fad499f9f1e3851ccebd05
107330 F20110114_AABWLQ rauschenberger_r_Page_078.jp2
01c2a90922ad155c438c1f26df1f3f2e
eb3a26a8b9e5780814cbf93ba22c00d92d8ea55f
11880 F20110114_AABVBM rauschenberger_r_Page_185.QC.jpg
aedbe64dc0203fb450f9aad9a1ee7882
b3c2cdee33c766d0215c342d13348863c2f0a5bb
24347 F20110114_AABVOX rauschenberger_r_Page_119.QC.jpg
ede9b2d704cac2fcc938076a1964ca38
72ae2221deef9f004026e6e608501f5c500151cf
111551 F20110114_AABWLR rauschenberger_r_Page_079.jp2
99d7eb9f9af98b0f9f88ae4a5041575a
f99093945aa1bd0cc9827ccc6bf3d90147eef514
F20110114_AABVBN rauschenberger_r_Page_104.tif
95c87da311373b5db948b024d1a7bf87
974ee0ab3f9017d430fb1cc0bfe6518f69364d25
1869 F20110114_AABVOY rauschenberger_r_Page_056thm.jpg
afd8e7faf6742f648ec5686cb7272a02
2b3ec1c7127834468a54e763d444bc028c7ca57a
115737 F20110114_AABWLS rauschenberger_r_Page_087.jp2
af93e59c1fb4eeebc590f1902c1f963a
b64af06f741c7b39aec68e83e707710b04654143
F20110114_AABVBO rauschenberger_r_Page_162.QC.jpg
18dc7be512af8f907f02dec3fe2e0976
f652a214ea97c91854c90d1886a9f2473262263e
49603 F20110114_AABVOZ rauschenberger_r_Page_121.pro
94a04ded7f7bf07c2644853f7293b44f
2ddc9f0049f3c87a41f1c1fb8cd133af12c8caa3
110556 F20110114_AABWLT rauschenberger_r_Page_094.jp2
05e82bd6d62fcb2b543af3c184fb9884
7a52c532795e8560dcdbef04a1466ad0fdbf1e5c
F20110114_AABVBP rauschenberger_r_Page_122.txt
1f820194aa97ce3f129653f1cabe9cd8
6dd3f1adb5aa7cfcd9d9c7478dadfbeb39f598a3
106526 F20110114_AABWLU rauschenberger_r_Page_101.jp2
f070eae8ad2baa40e27140de893053e2
f786ca8c92cfef43d6965add1daf4f5cd012a8df
51118 F20110114_AABUXA rauschenberger_r_Page_079.pro
f2e3fb85cc319bb1260ef599e4cd256e
54dd3164ee505fa13c1ab3533745c9a514ebbaa1
6544 F20110114_AABVBQ rauschenberger_r_Page_160thm.jpg
d1418e97505e77d6ab6f016aa52c013d
7c23d10b1ce79de3515eb2c7b17bb5467dcfc7a4
49624 F20110114_AABWLV rauschenberger_r_Page_110.jp2
f519201f76a38d72eb619b90d955a93a
56ad3a97e02f3416983ea2fa9131656243fd6cd7
13606 F20110114_AABUXB rauschenberger_r_Page_139.QC.jpg
5084dd9abe5ab4ecf7168adef802e714
2f4f16c691610d1aabe9741e938d43ac2f8b12c2
111198 F20110114_AABVBR rauschenberger_r_Page_082.jp2
fbf6f79d139167e8e6d051e30d90b68e
3d3e98787149b6cfe0d268e890b17efc6c6293af
88392 F20110114_AABWLW rauschenberger_r_Page_111.jp2
9162324665d34f1df14c827c3927184b
153326e7e8db333bc889c191a1ef6f4538bf4242
F20110114_AABUXC rauschenberger_r_Page_147thm.jpg
cc344d24bef1c28b5dc4dc93f758071f
2924ff4170639af3285c117df6bfb951235c6f96
47993 F20110114_AABVBS rauschenberger_r_Page_132.pro
75eae897257a02807c716af5a1ceba29
a48f5986916b50d03d0fd81a99af5fcea88b3243
761549 F20110114_AABWLX rauschenberger_r_Page_114.jp2
17eb715fe70938dfc482c3614ae765b6
0a92cc9e7bdcd57f8a33b868a86554c74aefcaef
48510 F20110114_AABUXD rauschenberger_r_Page_031.pro
26b5815476d82872b656e79362113b40
5325659722523f69f57ea095de7ad6c1060738f9
2013 F20110114_AABVBT rauschenberger_r_Page_161.txt
2997e4b75461e8cd95dfa586925fb47b
1eacfc49954b3d85991c6602af17287ca4c13967
101286 F20110114_AABWLY rauschenberger_r_Page_115.jp2
3b95bca0b12e814891d3b7dacb279f8d
8735e80a06594025fa69142034ef4ded8697ee0d
73933 F20110114_AABUXE rauschenberger_r_Page_201.jpg
02d50c1e8ff81c5a48524304e802cbfb
208ec03477465c04835e545ddcadc1aa6ce49cf5
16750 F20110114_AABVBU rauschenberger_r_Page_109.QC.jpg
eae769cf8eab36e3ab79834247ca0d6d
4f0af31e6678e036987119d76dbc8bf3ec0e355f
112811 F20110114_AABWLZ rauschenberger_r_Page_122.jp2
6a70365cafc793fe04190a6f12710cec
727879330dff8b7d374871f7f28738b52966c53d
50394 F20110114_AABUXF rauschenberger_r_Page_184.jpg
3183889168f5476a1003ae75d376fb45
39277fa829ba6367b7e63ee8bbc1c78018943940
1417 F20110114_AABVBV rauschenberger_r_Page_180.txt
33b052e0d51a740fd1145336975cbe4a
1320fdae45ca6d2667f14f7eed1428df76a58dde
107488 F20110114_AABUXG rauschenberger_r_Page_174.jp2
e1a758c52e0be66a8989569d87125991
fb4707b49726096952335874efac6485e5ad96d6
111468 F20110114_AABUXH rauschenberger_r_Page_121.jp2
06bb278b51894cd59c1f342dc041b9b1
cd9367441d6052295d8f370134cd878e8fc50a70
6608 F20110114_AABVBW rauschenberger_r_Page_103thm.jpg
1a9b2ce097c679d0daeb3e77374fac28
026fe588c2bc7077de97afe1158c64c8c63dedb9
24168 F20110114_AABVUA rauschenberger_r_Page_164.QC.jpg
f0179cf5b28b0eb4e36b61ad1b26054a
18aa1aad2b8443d8576c7de9892b6482d1e47804
22032 F20110114_AABUXI rauschenberger_r_Page_203.QC.jpg
d848a5d7e0e605f1e0e64e4d492e951e
d095450f1e268e8c9b4d8c1e5a60918dc89abe28
70171 F20110114_AABVBX rauschenberger_r_Page_085.jpg
ff4437e6355e616a0e1d810995d8e1a2
f4966148df4e191b2dea062d5850f8a446bb3daf
1837 F20110114_AABVUB rauschenberger_r_Page_212.txt
9194ef59c38cd92c93de1cab26c71f32
68adefe39bfe4e7672f09a3746df4a1f834bb198
161 F20110114_AABUXJ rauschenberger_r_Page_207.txt
a1f22c21e25df909cc67c1907a64ee67
2cc95e991e53b0b8a443d3963ba834dee483912d
71914 F20110114_AABVBY rauschenberger_r_Page_218.jpg
ec4afeddd129861320ab6d3738fc7e6e
2c7faa01615bf225b2d7fd411678c47f4e422f21
22818 F20110114_AABVUC rauschenberger_r_Page_040.QC.jpg
358bec4fef895333402155f5d6ee41d9
9b85eef5aeb4d164261931d96462a6c84dbe2e16
1963 F20110114_AABUXK rauschenberger_r_Page_218.txt
32c344a6a155621428c39abfdb7aea7c
e0055c33e1dda0c608cc616d1fb53cf86e42efe0
76182 F20110114_AABVBZ rauschenberger_r_Page_094.jpg
eb89be03600cad7448e5549d655c4414
d6a02a9f37ab5b3aa554f44ca4e560853bfd7cac
95903 F20110114_AABVUD rauschenberger_r_Page_169.jp2
035200beab3c68649a62345fd64acc47
ed5c8f608c6c75d184255b3872437716f1aa31c8
588 F20110114_AABUXL rauschenberger_r_Page_067.txt
1847aa0daa97a2c7d156378bbbf85ee5
996a44df40f3309e411f45d06c4e3904c9d23fe3
23576 F20110114_AABVUE rauschenberger_r_Page_204.QC.jpg
442943f7b333c09d1c316240955e7029
6eac1165ac0d05da01977fad41b5d94440de27ff
23023 F20110114_AABUXM rauschenberger_r_Page_008.QC.jpg
436c8f005a7a213be7f2f89388b72bbd
993dfa406bae2afe9f7e0737c958e713917036de
557 F20110114_AABVUF rauschenberger_r_Page_195.txt
6f01e67a3849e9790e694e202d6a480a
19ef00fec7d7a14e2cf5cefbd5131d6ba460fc46
F20110114_AABVUG rauschenberger_r_Page_078.txt
4640ed16eef8db37a1320469bf8d7434
bebb08962c3dfc8553c6eaec450d95cabf9de1aa
F20110114_AABWRA rauschenberger_r_Page_087.txt
ebfd37b92304ca52a9a4072debd3c0fb
04f9796ff4f20f4e8dc5082ae834e243551efd26
113545 F20110114_AABUXN rauschenberger_r_Page_129.jp2
2936640c5b01a7619bc7b95c7861f150
b8ab552beda1661ac5c939f863facb53b849ef54
50509 F20110114_AABVUH rauschenberger_r_Page_219.pro
6e521450043430484ea9fa8d76ad912b
80219f557799c2c35bbfbf7b5674e208f96765ea
880 F20110114_AABWRB rauschenberger_r_Page_095.txt
74712f9ba5892ddaf19ee68be1e4b18e
9c518d3d537935be54483c692c4c4d27eb4c80c4
59167 F20110114_AABUXO rauschenberger_r_Page_186.jpg
d8c7822615e81185ed30b8b97491369f
bc4d15939c247dde124ef8efa950ead2b2533ee3
F20110114_AABVUI rauschenberger_r_Page_014.pro
1f7d4a8562b99e39e76ca96d932ed960
ddf5794e99a83129f72e4b361248d6c0a3cb8d3e
1968 F20110114_AABWRC rauschenberger_r_Page_097.txt
0eab3d0f9fc00c3e93437300b1af7b43
cb4b054b535aa79c7b580a3f2b9f7dcb8a7a9480
26075 F20110114_AABUXP rauschenberger_r_Page_007.QC.jpg
7de3c4593f2d4179e57014192b1fd3c8
ea8b6fa8874b339f219fe901e2cbc642b6ffd01f
F20110114_AABWRD rauschenberger_r_Page_103.txt
6c40045ce2325d1a76649ca5c47c508d
f8fbc2b6284332b456b35c2e6d5915185f4f6e24
114411 F20110114_AABUXQ rauschenberger_r_Page_048.jp2
67c3e4aeadeb49e499e79ac7c93bb4ef
9fa9b8c51d9ba660a56d588d3e47576912356a9b
F20110114_AABVUJ rauschenberger_r_Page_172.tif
226ccc3d8103d5c95467dbd5d20dbae7
dbe56777b9f572d0160be8e7bb5b22c7f59e1b9b
F20110114_AABWRE rauschenberger_r_Page_104.txt
ae82342501912b5bb9ca68dcc750d4b4
dc04521d695fa32c788e264f0e13aab4b94736fa
46418 F20110114_AABUXR rauschenberger_r_Page_203.pro
ab975318b1de01004dfb7eab610ac685
918acc30fe700104571cda977fec688e973499e2
105095 F20110114_AABVUK rauschenberger_r_Page_023.jp2
e9a0d58c62a3d17efa86e6603052c763
a55e3102ebc6d2b2a2bdbb8d174eb82b4550ac86
45984 F20110114_AABVHA rauschenberger_r_Page_120.pro
d6bdae21e05a756bdb9ad9c681ab5be7
bf0657729e2e3f72e76e85ad0d32d550478932c1
F20110114_AABUXS rauschenberger_r_Page_089.tif
9e292fab4e6f4af56582b2deab9065aa
80a4bcee466c463c66e20f84e2ae80aa2a4b7a63
40705 F20110114_AABVUL rauschenberger_r_Page_185.jpg
c3ce7b12879c08464f2effbcf766967b
6a15bd516d526d6c8f1c6a4565d517dc7cc458a0
F20110114_AABWRF rauschenberger_r_Page_107.txt
9b61b156bd047b7cec7d5c5f1594cc9a
01757273237641687902589750befc9710fad9c0
65676 F20110114_AABVHB rauschenberger_r_Page_228.pro
621d1eada9fde972978352f3585f1bad
a0afee007962c5f041f846f5034dd98bae5083b1
18690 F20110114_AABUXT rauschenberger_r_Page_015.QC.jpg
8ae31ea90256468cc98277d5d767067f
dc2ef7657f2bc5a75bdaf5d5b96688346a5451b0
70928 F20110114_AABVUM rauschenberger_r_Page_073.jpg
db768f063b934c5c220e06c44f823774
21c47466634c61cab23ee925ebb131524003fe0f
17780 F20110114_AABVHC rauschenberger_r_Page_088.QC.jpg
aa5c68f793ffe2f993db11b09b681a7a
b8e9a75ca5cd19eabe62633e6939de213284b08c
F20110114_AABUXU rauschenberger_r_Page_009.tif
0759825a827424fb647d47493794948f
1ca5c8f563bfae165de60d579f950309717a2d62
107427 F20110114_AABVUN rauschenberger_r_Page_167.jp2
819639b8906ab13b896bef9801fc21fe
f4f8198ca215d6ed71c4a79a9c93f4373a66da06
631 F20110114_AABWRG rauschenberger_r_Page_112.txt
8578acce46a93022d65eade62d3b0276
50efdbeb90aac3d4bcc234426a52dc487b496e57
55553 F20110114_AABVHD rauschenberger_r_Page_186.pro
ac695b4ce7dc98f7c0a2cc28317739e2
3d94d7f49592bf2da5ac1f1f2e10f2238b38212e
7301 F20110114_AABUXV rauschenberger_r_Page_196.QC.jpg
44dcdcd96a5fa7afd414527afc269508
9c5e3b63db080702cbf19730e1b2b8106c577d5e
F20110114_AABVUO rauschenberger_r_Page_232.tif
da627e0b6815522a0a8721235695bf56
d21fde5af8d99c9a7aa5826ca26e12b9fb1f755a
2000 F20110114_AABWRH rauschenberger_r_Page_119.txt
263b5b01eee280c95776ca3de5afddb1
b74f77d362a95b64b649a832b25d03b96bbee6e6
29937 F20110114_AABVHE rauschenberger_r_Page_091.jpg
db0cf6d67234a9caab8bc3ca012a893a
bdc100c4e913afc3474fed97adfce52488b7c941
F20110114_AABUXW rauschenberger_r_Page_085.tif
d318363b08801bf279b7877a8b8269d5
6e66a33afb140229b39f9fc18dfc70199bcc1e66
106975 F20110114_AABVUP rauschenberger_r_Page_097.jp2
3ac27852d52b7a520e3415d80bf08747
cbc35426513e9593eb1e6abdc6cd73d9661d4672
2003 F20110114_AABWRI rauschenberger_r_Page_124.txt
09f6fc59c8eac2dd2dfc86afa2a3042f
44bf711102e8e4918e5d964f747f54039928f85a
F20110114_AABVHF rauschenberger_r_Page_204.tif
78ed2a1c8047a4a53452d20bd05354b3
ce09af060fc7981a34595b9b13b149baa1ff78b6
30742 F20110114_AABUXX rauschenberger_r_Page_143.jp2
fc17862245ba5a93dae372ba1d136e84
ca0bb1d5b8f9137949b1c41a8809db6eef74d92f
5811 F20110114_AABVUQ rauschenberger_r_Page_187thm.jpg
01036ba468b638454dde420d595ae89c
8e33375d4e1444f2245cc36679f56a0815c30aba
1958 F20110114_AABWRJ rauschenberger_r_Page_129.txt
5bad4de73bcb39215a632fb5dab1c478
b70056e5dee559d093a72ca40a0f19824658b362
12737 F20110114_AABVHG rauschenberger_r_Page_038.QC.jpg
49c5054d0693a7ff612a6714a3e5ece2
ba7eb825802491884019546fcd9601b9acd750d4
110748 F20110114_AABUXY rauschenberger_r_Page_161.jp2
8b6a9dd21457d40837f778d20da09c1d
808dc1f4d1d81659f33aaab477a2a3273d0768c9
F20110114_AABVUR rauschenberger_r_Page_081.tif
d787f95649a5b25a7672bad77bf48fc8
37b58509455b6ca899464da3cc6d7eafbc71b279
1941 F20110114_AABWRK rauschenberger_r_Page_133.txt
88cffe84fdf7b1854bd87229e92a9c6b
75acaaa2da0832ec9fd601f43c8a44923274a93b
1787 F20110114_AABVHH rauschenberger_r_Page_030.txt
6a951fffd817ab8f51ac9ec688c1d4cf
9e24bc68849d6cb1221283926a5ebbcc416b8dbd
7432 F20110114_AABUXZ rauschenberger_r_Page_062.QC.jpg
af978ee9c9200add7cb1a2c2c4570e1d
a4ac3bdbafa417daf9feba2b7875e4e9c74277a1
F20110114_AABWEA rauschenberger_r_Page_231.tif
28e6bba38c15e1eb1f1a4f017dfecf93
dbb95c2f9e1243dcdff4d9e20e3358869facd207
F20110114_AABVUS rauschenberger_r_Page_218.tif
89e68abcd8a7a25b1565039e16942335
40332e09007d8f42970888257249a42a67743196
1842 F20110114_AABWRL rauschenberger_r_Page_134.txt
817bc82e227553f57cbeeb14f7c2dab1
528ca55febf7b0fc1f586a83a67e00d138f874d0
109372 F20110114_AABVHI rauschenberger_r_Page_018.jp2
b6803b9a365c56cbddb9bc1be9d43147
46ccba9d7b150161af77dea7c7942b7abc69b4a7
23929 F20110114_AABWEB rauschenberger_r_Page_100.QC.jpg
71347cf65c8f7fb9f6b368b3d29fbe1a
d0d52ca90ff745dadf7227690563cd8d4e37e8ce
882 F20110114_AABVUT rauschenberger_r_Page_011.txt
3b9c27689ed39a41743bf6f6f37f2e74
7538823785fff24124c9a90fa79c5687a717d7ae
2040 F20110114_AABWRM rauschenberger_r_Page_140.txt
ee73601233c82d75e96486b73c9ccf58
13862eba59b183b0e88012c0b667a83311859f45
F20110114_AABVHJ rauschenberger_r_Page_160.tif
380b72d64a0f05f1af017456c9a7cc90
842d40e162e2d8d50b3510987cc48edbfe1e2adf
48795 F20110114_AABWEC rauschenberger_r_Page_128.pro
72c382778cfa87bf38ad09b906a775c9
f6614c8b64bd91f92a6897566016feafca197244
F20110114_AABVUU rauschenberger_r_Page_213.txt
e12a95db3bcfb1ab328e7ce6fd22cd26
4804cd5b173fb2f68be9a465b18017f1fb86203c
1836 F20110114_AABWRN rauschenberger_r_Page_141.txt
f420307d409626a12feae6de55aa7561
e0e088487f819b1c945856abb28bb79417862583
6686 F20110114_AABVHK rauschenberger_r_Page_085thm.jpg
d8e9cb1dc0507316311100f9b471ec68
63f5ec46502037ed1475ca488374ba73a619c046
24465 F20110114_AABWED rauschenberger_r_Page_220.QC.jpg
fab4176868b2ce3fbea008384d7f4a85
c3f465fa2efa693edf0333a4da50af409d7b8221
7043 F20110114_AABVUV rauschenberger_r_Page_013thm.jpg
3488fe0cbba9cfd505a1c7fdfdd1bdfe
7ed9ca518c5cf994035c6a5f4d9a174402c5912a
F20110114_AABWRO rauschenberger_r_Page_142.txt
208fa7357862d96ef9d14c89d69f14ac
f9ab55aefb3d21e55792beeeff36db1d5192f721
23029 F20110114_AABVHL rauschenberger_r_Page_101.QC.jpg
ede481bd99a458909b84e69adc000e9c
f6525c084569644b9af9b0c76314469fe4b16030
F20110114_AABWEE rauschenberger_r_Page_003thm.jpg
48f5288b6531327bf1b25b3095d41e0d
daec2c19809e01e827a059a5c8fdf55a78e272b5
F20110114_AABVUW rauschenberger_r_Page_053thm.jpg
7e55e2e0d46c13ccf9113a884f11c562
bdee3b1ecd9faba293c73562f038c3b263b7c1a5
783 F20110114_AABWRP rauschenberger_r_Page_150.txt
0c3071b1674a465327d43a7dd65c77bc
f2364d762279aa2b694e26d57e3a350b1b3a5740
6177 F20110114_AABVHM rauschenberger_r_Page_046thm.jpg
e12c188a2d342ce465ad3b1476ac9361
7aacae8e2322c133da088de708057a3c5ceefe7b
29220 F20110114_AABWEF rauschenberger_r_Page_064.jpg
4d01e09f7b1cffd45bf72309affbe3f9
8ff62cc8dddb3914cee24c40adcee5ebfd1bca5c
23325 F20110114_AABVUX rauschenberger_r_Page_216.QC.jpg
877491cceffb92460d2b3f5bc5bd6115
712362fd825eb12ef0e69541713dc2e62e5639a7
776 F20110114_AABWRQ rauschenberger_r_Page_154.txt
cba89738ed123ba5c5ccda4e9450d727
2478bd7db3385cd5080dd955d5cbbdda8b1fb0d0
F20110114_AABVHN rauschenberger_r_Page_102thm.jpg
416daf2312d9634ff7343cc65e6dfa30
a4aca08259212a73399e0be1f2eb105f23cda9be
115003 F20110114_AABWEG rauschenberger_r_Page_206.jp2
a489959602a590dc52a2a1b8b32eaada
291be6becee2538ac2dfbf0686092d0dd5a4e8e4
F20110114_AABVUY rauschenberger_r_Page_037.tif
112a2a5aa2e2f4da8fdcd475acbfcb59
760b9af520362741beda73ddf9c9087885754a91
639 F20110114_AABWRR rauschenberger_r_Page_155.txt
f449a3782d6f2683dae616d712dc26e6
e589969437c2fb7249a7046f12eca440789292b8
3470 F20110114_AABVHO rauschenberger_r_Page_002.QC.jpg
ce9792f40dc792014b3886e5570407f2
f07770a48693046863e998a4aff117055504a9e1
22392 F20110114_AABWEH rauschenberger_r_Page_028.QC.jpg
17fee13dc955c926380d283155094e2c
af93192571af27ff6461cd464c986976f082804a
2968 F20110114_AABVUZ rauschenberger_r_Page_207.pro
0991e551b6ca0f9ea25da66cf3cdbb45
e0eb67728da804aea94a7dd504c846832a5d23de
F20110114_AABWRS rauschenberger_r_Page_166.txt
1a6a3d3b8944a32e3af6b4734a914961
6e826b3dc5118b0ae5288792e0e3b075b391fbd8
F20110114_AABVHP rauschenberger_r_Page_120.tif
62adaf0fea1111705a69fdea86244239
9c1f8a961cda08ce21162d1f29a9f5e2df89e5f7
67093 F20110114_AABWEI rauschenberger_r_Page_026.jpg
153554248f69afb38864f3a1b9908622
b00f3d273d5c3eef702ae5758ea49648b1293c4d
1943 F20110114_AABWRT rauschenberger_r_Page_173.txt
e719a04aae487cfc82a768332049f8a4
1139faffc808d9b54aec80caabf1dbb29dee560f
50323 F20110114_AABVHQ rauschenberger_r_Page_160.pro
3e6f38c59d96c110876d54b3900d05ce
4de66e1db03bc214300a0115bab3fc50fbd60d13
23233 F20110114_AABWEJ rauschenberger_r_Page_167.QC.jpg
29a1dab774f7a5b53c4e066bf661925d
da374926bbaa2a88889a7231a53809682505258f
F20110114_AABWRU rauschenberger_r_Page_174.txt
c2e344fa333eb89473ed9e43160961f9
a9c08955af61b5f93654ddd42a14118def7be1d3
71106 F20110114_AABVHR rauschenberger_r_Page_029.jpg
48368d8a8603978e08ee2b2dd06170e1
d986f9d84b8dac6c7cbe1f8185e699ccf05cda99
24163 F20110114_AABWEK rauschenberger_r_Page_045.QC.jpg
b8fb4432545c10f4d74a916607d8df30
acb86bef64baf5deee5b928a24bc8cd309e0f7de
F20110114_AABWRV rauschenberger_r_Page_177.txt
429005ff096e88ff8f2e0834c347de35
f392a23aa70eb445c39d11c4542fb7dff2c3f3b3
9900 F20110114_AABVHS rauschenberger_r_Page_011.QC.jpg
8dc9ca7830758c5f611b8f974c10df4d
147c8c468b1941ed5106f08d6e651a7940dc7ad0
31463 F20110114_AABWEL rauschenberger_r_Page_183.jpg
ab78974b2bb7b668e752a9f8585faa76
4f4451297baace2ba3d166d971892263daca2f1a
2093 F20110114_AABWRW rauschenberger_r_Page_181.txt
8203fc2a5b9c363dc213e9d491928358
5802ac7a63f0789af32f1efb73bc1df097ce1ed5
15186 F20110114_AABVHT rauschenberger_r_Page_056.jp2
087f48011c17131684322ee309db6a94
aac71ba25d9c4155e575261b2ea8fd2ce323230e
23763 F20110114_AABWEM rauschenberger_r_Page_124.QC.jpg
8b6f1f14c5578fede275bf4458a91c22
4e78b97e66b36194a98420fef002e8bb02c38e7f
3654 F20110114_AABWRX rauschenberger_r_Page_184.txt
53ba93a059314d1bcac3dd2fe316d1c5
72a689218fc67f1e22b2ab5138fab19367e1ecec
63851 F20110114_AABVHU rauschenberger_r_Page_137.jpg
7a4decc1a608da06910262499fd5cebf
02f030b9fd40e36b4d68d8be5d09bbd884cea7b1
24831 F20110114_AABWEN rauschenberger_r_Page_195.jp2
7d0d97e69e6345030af25ed7a26ce8b3
b5b2ed38063f0d37364b645e3ce8cb42b45c0556
3267 F20110114_AABWRY rauschenberger_r_Page_186.txt
d39b3fd71cc38ab4ebc38eae7cc34014
ccf0c9067360c415469597bb76c14d229b0b6cae
5498 F20110114_AABVHV rauschenberger_r_Page_003.QC.jpg
7c1fea32e4c9d0bfeeea38e3331a0bc4
412a5cb1106f43af334a8c8b16567ae2fab85c16
2200 F20110114_AABWEO rauschenberger_r_Page_144.txt
b7f6f404a00d1e1670ddc7b71fcad15c
32b2363563a9dd71e4ac16f1e14fc39de8f914a2
2501 F20110114_AABWRZ rauschenberger_r_Page_187.txt
7c6c4bfbe5023845ab615657bdaecd1f
dab30a1ad865f90c8e4d9caf456881000d0fa7e0
1888 F20110114_AABVHW rauschenberger_r_Page_170.txt
ac58f34d836dd7b2dee65081f8f9f57c
72e0ae8ff207c28f22f950c79e5786184e691f66
21947 F20110114_AABWEP rauschenberger_r_Page_026.QC.jpg
5083cb107ac06fe0d7cc68f11257127b
2a6468abc4cf1c0c7af85f374c95f7c99922fc16
6951 F20110114_AABVHX rauschenberger_r_Page_231thm.jpg
57f6f0ae98fd76d98b5da380ca5e646b
b98c6004a8135898f5f3becbb2b2e4b30c94aa7c
70735 F20110114_AABWEQ rauschenberger_r_Page_031.jpg
a5c7c5d6cd8e7100541a17a94b8e4ed7
72e60f2856a7727fc6df4607f1d1ecd5bcf5689b
70914 F20110114_AABVHY rauschenberger_r_Page_101.jpg
4029f5097c5d7d733bbd0ad0a2b4b99b
e2e491896dce3fe25f81e24865547178d63ee084
3921 F20110114_AABWER rauschenberger_r_Page_144thm.jpg
ec964241cf89380ffb66420de2fe264e
8a5a46bd0a4cea0cdeca53182fb14f8c063cc0ad
1705 F20110114_AABVHZ rauschenberger_r_Page_137.txt
365d46a1043104b5d3242f206e6d22de
6586802108d3c052ab7e5b77455d6dddd353ddd1
6639 F20110114_AABWES rauschenberger_r_Page_167thm.jpg
df61330190034adbd484c2734726a4f3
dfe9e1d3bfbe5a94b4aed0d45faf9bedb0d75b53
F20110114_AABWET rauschenberger_r_Page_078.tif
596a1997a6cb0ec48d7452a82e9ce03e
6adfdd5f42d31bbd6e388ef0f4258d1862c68f14
4944 F20110114_AABWEU rauschenberger_r_Page_232thm.jpg
e9d0564270d8776f1cdadfe1151b16a4
9060d6ba39a5e72d9dd4c30c4d450e4045a1cf3d
3280 F20110114_AABUQA rauschenberger_r_Page_010.txt
ba477cefc8b4ee292f102f7783ae099e
413c4d98a345edcd910dc89cef682f788abcc4b9
110904 F20110114_AABWEV rauschenberger_r_Page_172.jp2
f4b77c368c83b51a5f9b990de1606a75
8fa58d63d2f9277f9b45fc61983ccdc2a3d98f77
1984 F20110114_AABUQB rauschenberger_r_Page_165.txt
c450f43a6b3d59d377d530d99094dd11
0b94d030d8cf40f3bed502fa57f6aecbf6c49d41
47477 F20110114_AABWEW rauschenberger_r_Page_073.pro
1f5066d5266473e87b5354713aa06ae2
764a2f1c530e229dfe4a972059afd0ed41b8bf3b
28525 F20110114_AABUQC rauschenberger_r_Page_157.jp2
d49bde17044be4b45ded89e0b552ff57
1b3c4948d62dcb9c35231820a9516497e7942dc4
41335 F20110114_AABWEX rauschenberger_r_Page_223.jp2
5fa27421ba398d81da80614a69019b4b
a348cf27e740b2e5efa43d6bd14779d1960123b7
3485 F20110114_AABUQD rauschenberger_r_Page_057thm.jpg
87596389bebbe52ab9bf94a799d7da93
4bbdfe01a35d597570e92071b5c465de90047935
1711 F20110114_AABUQE rauschenberger_r_Page_060.txt
917063b80ba9a9c63fb3146549ce5ec0
08f66c7dc0f6d65aea20049367a701e4da226294
35903 F20110114_AABWEY rauschenberger_r_Page_090.jpg
afd0e14ed1de38ff3bb4230a97669eeb
1dfefdd8b27f96e2f53e3287edc8b5596c3eded1
64487 F20110114_AABUQF rauschenberger_r_Page_169.jpg
7483259b8e90c6dda4770bd608de1618
529a1630aeb3b6e6bc124726eebf37b9ac594275
4072 F20110114_AABWEZ rauschenberger_r_Page_151thm.jpg
d87b0f70e57555a790f80dc2b2a8f04c
110f555fd52824ee0f6907a6d2b4fbb6ca877d25
116968 F20110114_AABVNA rauschenberger_r_Page_080.jp2
f764cfdb6adf192c8f4efe3ee9f8243a
61388d349ee7fbc5298ea056589072b9158dd01a
F20110114_AABUQG rauschenberger_r_Page_134.tif
8d2425c2042e6333ebd2c68e30e9ea7e
76c65f7fc9e5c297560917b0a2b68945a2d5f190
68690 F20110114_AABVNB rauschenberger_r_Page_115.jpg
93a40e2a676da48bd79d9cbed0719af1
df971c0eced1dbabbc768e500e5efd12f831bf00
F20110114_AABUQH rauschenberger_r_Page_202.tif
a445212a4bdaf84976ed9e80f3e1e7dd
cb60d7b76ef9772cc96591ae3073fb19477e051c
13239 F20110114_AABUQI rauschenberger_r_Page_144.QC.jpg
b3c0553cb200117ae98a5f851d843c00
95aa59748d8d88f947c268aacf9e2af0654efef4
7625 F20110114_AABVNC rauschenberger_r_Page_197.QC.jpg
44e53ec23387ab95ba9552882179b482
706f2992fad18825449a36279b729171f387f820
6371 F20110114_AABUQJ rauschenberger_r_Page_213thm.jpg
01cf37104bcb919f80bf0b17ede01137
e68adeabf6a7cde3bd50ab07162ed9f1bc13ee3c
114503 F20110114_AABVND rauschenberger_r_Page_221.jp2
c2b1c9e0570afd98af6e562724ce8190
23afe17e0a6df290aea4ee9f87a869451ed59bec
2474 F20110114_AABUQK rauschenberger_r_Page_001thm.jpg
bf53839461d58fabfe5df9dcc403bb7c
4e9dbb6c805559285d7d7a4d0762217ef187caf8
67232 F20110114_AABVNE rauschenberger_r_Page_051.jpg
8d5151dc4ea842551625e04b2c80601d
99f70254409c0afc3dc4ea3643efbbfa654ef899
23443 F20110114_AABUQL rauschenberger_r_Page_098.QC.jpg
6870ea39088934e5a4eb9ab3fb103366
65aed18e1246149bd882103912690047ec618cda
6584 F20110114_AABVNF rauschenberger_r_Page_117thm.jpg
0b5f5570ec3c9e4afe98d833684460a3
af85335539bceda65a57ba59c3d8e2160dd51b9b
6775 F20110114_AABUQM rauschenberger_r_Page_191.QC.jpg
b7a269533e2c3be692626b286668464a
1f63a4bd03edea122edbb66194836e7a2c9b984e
16676 F20110114_AABVNG rauschenberger_r_Page_064.pro
92ab5ad100a322e73fade99e34b8400d
1dca041354330edb9fa4c70c928c7dbcbd2f177d
2034 F20110114_AABUQN rauschenberger_r_Page_045.txt
6ec30763e76f6d3e2a28f72942e2530d
89cd4996160cc27bbd08f2590c4570b493620abe
2071 F20110114_AABVNH rauschenberger_r_Page_220.txt
3db9734bbb685d9a42c2245e24f5c9df
c702e9f06f6482884a0960f65f5dc6cb2f152d01
68612 F20110114_AABWKA rauschenberger_r_Page_125.jpg
854de212a4edd5b2c13bb7ba87cf4f26
615ddc0ff18b6a8ca47dc23e4b47b87e753b2a80
25529 F20110114_AABUQO rauschenberger_r_Page_225.QC.jpg
bffffd09be38d3cdbe048596460a6eb3
c244de069586f244075682c4dc036d0d8caa5e52
113526 F20110114_AABVNI rauschenberger_r_Page_200.jp2
a4693eb5475c64dfe2f50ade08115df0
7591d47206b80081e1d7e9eebec2b1c102ebe85c
76044 F20110114_AABWKB rauschenberger_r_Page_126.jpg
90713feac5aeb77efc0bee176bee1649
293ba6f2ce85e82048f8e1db0d9bf346f44f9697
49350 F20110114_AABUQP rauschenberger_r_Page_034.pro
03a3436670e1ac43ad9640cdfcbcde6f
8a0f618923bf27c7ff7495a01208609a54d7dbc4
56522 F20110114_AABVNJ rauschenberger_r_Page_139.pro
d78a1fb361eaa532bcec3c5d0d664126
a520e81f9a2dedc610ba4ca0bc8f7822b370340e
73678 F20110114_AABWKC rauschenberger_r_Page_130.jpg
d852aefdf536c70f3467d6bd222942b4
08f5c2cfec6c9e4c5ef31f6c6caf99b62f8795ab
69711 F20110114_AABUQQ rauschenberger_r_Page_171.jpg
3e4c2921ad0edfd837756877647ea4b9
791d1c66d1e86ba8b58e59a757124486990ab05c
71858 F20110114_AABVNK rauschenberger_r_Page_098.jpg
8448b63e93442f4d2c9fcf436a62c602
a71b88777a8d8346b8fd7d90e9aa49f10ad53d59
71326 F20110114_AABWKD rauschenberger_r_Page_132.jpg
cd555ec5ebcee40ec1b653968d58bb93
6ef4ea0e834306e61ae53a9a41261de7cd97a8ae
6412 F20110114_AABUQR rauschenberger_r_Page_205thm.jpg
d142e027187604f2daa81d5470ca34d4
6c3bf1d165e8307b676e41f9b8cce629551104c7
22202 F20110114_AABVNL rauschenberger_r_Page_039.QC.jpg
9ecf4838159f498968773d874e833d79
4cf990e3fd5919884c087811180c06a9120d84de
69421 F20110114_AABWKE rauschenberger_r_Page_134.jpg
38faa932b4c044eaecb48725cadda88c
157767c31fc73bb063cc3322e114398f60ebdb70
61486 F20110114_AABVAA rauschenberger_r_Page_190.jp2
a5abed49b1a43ad4598e00134077d795
e95cb1fa0be4007158433f5099ac81fd6c6bc40b
6416 F20110114_AABUQS rauschenberger_r_Page_135thm.jpg
c9c5ac94b732cd1fd969490969b5d1dc
4b930521fc8e9d7a32309eeb50960623712f0ed7
51487 F20110114_AABVNM rauschenberger_r_Page_042.pro
ae70c4239a4809ee92b980ce69bb8709
b512ffbe6217d9c437a85aca69546db3bdc36e21
71487 F20110114_AABWKF rauschenberger_r_Page_135.jpg
2440b7232f62bba8e2017e4cd7f9ebc6
1ee3c66b2de3f5fc4f3aeeea0689be4e98c75eb7
71291 F20110114_AABVAB rauschenberger_r_Page_175.jpg
3ab75ba94e1ee8651cd66eabcfc72a5c
23bc1cef1894cdd534f678aaac647fb6df1635c9
723 F20110114_AABUQT rauschenberger_r_Page_191.txt
f70a10f4ec2fa988565a9b37c5e5ec67
1d30b3ce06a844e829b87a904c9ba117be02abfc
1879 F20110114_AABVNN rauschenberger_r_Page_151.txt
b8cd4beae13ad986e756d97324458630
6b6c4e3c1e63c51191593c92f507278167546923
40745 F20110114_AABWKG rauschenberger_r_Page_141.jpg
59df67eb69dc3ef93cc161d9b4e59419
719b5b033189112ea0c54961a82455ebd9f79ba1
F20110114_AABVAC rauschenberger_r_Page_043.tif
ef65a1f53454c72429727115840bb914
f7857ca5006affc3af0299e9fc3c81fb5cbc94d1
110747 F20110114_AABUQU rauschenberger_r_Page_100.jp2
9f430c90cf29a8a3d86d95951885894d
736e7e1d9ce6305d38edd877b604e5504ee1d4c2
F20110114_AABVNO rauschenberger_r_Page_080.tif
94db70f60eba78c90a19956d6f20dc2b
4816563a547fa0be0cdd5346202b0c79b7bd96c8
43306 F20110114_AABWKH rauschenberger_r_Page_146.jpg
6f9c8dd5ffd1740fc52b9e05d2c341fa
1b722653b53df0d328f889f4554be2294f954681
50876 F20110114_AABVAD rauschenberger_r_Page_055.pro
fe8435edd72a30a0ac4b5c029f881285
a237c8f4740f660e9187636bd32298fe9cfe069d
6405 F20110114_AABUQV rauschenberger_r_Page_018thm.jpg
f795cbb1d53d7754bb72b919cc6e5a34
d6b7ed16aeb7cd93dcabc1029a7ab61f8b708475
17316 F20110114_AABVNP rauschenberger_r_Page_223.pro
532ecd34c3784c0da8364938ae19aa08
c84eb05a8bd000a45d3f26acddd8d806e7909292
39542 F20110114_AABWKI rauschenberger_r_Page_148.jpg
f24186c0b993163ceb85c7b935ef0f05
9889326c6245d4d890ac8e6b45c420df5438ae28
3915 F20110114_AABVAE rauschenberger_r_Page_139thm.jpg
ac6b5cb14b217ae855e97f6aa7720572
0c67321a9f2a7e6656ae4300ebda43c3d06a5f9e
50516 F20110114_AABUQW rauschenberger_r_Page_024.pro
92b639c3c5aa433b58c1d6ea60210746
a7da2f4bae435649df44cdc3bdc72b4811fe511a
25363 F20110114_AABWKJ rauschenberger_r_Page_152.jpg
fcc1ed54eef8eb0c7a7708065eddc433
1a71110cb24846aa2451503c31bf1b87730a8ec9
69289 F20110114_AABVAF rauschenberger_r_Page_050.jpg
4749eefd43384607022e99aa168b6bbc
22ef15321e79c1ce8c7086351f93e981e7f39e87
55089 F20110114_AABUQX rauschenberger_r_Page_109.jpg
948005f3321211fa22e3a81d3e9dd1a7
1620263bd31ce7f0d602e5bc0e1e6f76bbfe23e8
69466 F20110114_AABVNQ rauschenberger_r_Page_053.jpg
5679ab6c384b26d1edc509c5af099420
f08e4cb189f13b9d3902075a3b4b46727c46bd51
72244 F20110114_AABWKK rauschenberger_r_Page_161.jpg
585d459ea7cc0daa5bf53f945f535543
b1977d5a1b78ffeda06632e4f98f04c1488571bd
1870 F20110114_AABVAG rauschenberger_r_Page_112thm.jpg
3c6ddb0cfb78068208bada348c02f6c4
269044829055be3c27e466eec184ad8c8c40396f
51265 F20110114_AABUQY rauschenberger_r_Page_084.pro
1e9e43eed6d36b476d501147afea4765
a344d27682a63e08650849050507c5fe46f45fdc
2008 F20110114_AABVNR rauschenberger_r_Page_102.txt
e5be85fefcb4cab0e644b753abad881b
ece6a7ab1835d661885d6712092dfa92bf67031b



PAGE 1

DEVELOPMENTAL MORTALITY IN AMERICAN ALLIGATORS ( Alligator mississippiensis ) EXPOSED TO ORGANOCHLORINE PESTICIDES By RICHARD HEATH RAUSCHENBERGER A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2004

PAGE 2

Copyright 2004 by Richard Heath Rauschenberger

PAGE 3

To Jesus, my personal Lord and Savior. John 14:6. “Jesus said to him, I am the way, the truth, and the life: no man comes to th e Father, except by me.” Ephesians 2:8-9. “For by grace you have been sa ved through faith, and that not of yourselves; it is the gift of God, Not of works, le st anyone should boast.”

PAGE 4

iv ACKNOWLEDGMENTS I thank my wonderful wife, Alison; a nd my two sons, Heath and Ben. Their steadfast love, support, and s acrifices allowed me to succes sfully complete the arduous task of earning a Ph.D. I thank my pa rents, Richard E. and Mary Elizabeth Rauschenberger, for their ever-present love faith, and encouragement. I thank my mother-in-law, Sandra Pillow, for baby-sitting Heath and Ben while Alison and I were away at work and for her support and enc ouragement. I thank my parents-in-law, Tommy and Debbie Kirk, for their love and eve r-vigilant prayers. I thank my brother-inlaw, Matt Kirk; and sister-in-law, Kristin De ssert; for their support and encouragement. I thank my late grandfather, M. E. “Pappy” Walls, for showing me the outdoors; my high school biology teacher, Joe David White, for making me a better student; and my high school football coaches, Randy Tapley and Ji m Massarelli, for strengthening my work ethic and ability to deal with adversity. I am forever grateful to Tim Gross for taking me in as a student. I thank him and his wife Denise, for the kindness, generosity, and encouragement they’ve shown to my family and me. I thank my committee members (Marisol Seplveda; Bill Castleman; Richard Miles, Jr.; Franklin Percival; and Steve Roberts) for their support, friendship, and signi ficant contributions to my development as a research scientist. I also want to thank Kent Vliet for sh aring his vast literature and knowledge of alligator reproduction. I espe cially thank Jon Wiebe and Janet Buckland for their friendship and hard work. I am pr ivileged to have had the opportunity to work with the staff and students of our laborator y. I thank Wendy Mathis, Travis Smith, Jesse

PAGE 5

v Grosso, Eileen Monck, James Basto, Shane Ruessler, Carla Wieser, Alfred Harvey, Adriano Fazio, Nikki Kernaghen, Jennifer Mu ller, and Jessica Noggle for their help and friendship. I thank Ken Portier, Gary Steven s, Ramon Littell, Ron Marks, Jon Maul, and Linda Garzarella for providing statistical advice and assistance. I thank the National Institutes of Environmental Health Scienc es Superfund Basic Research Program (grant number P42ES-07375) and the Lake County Wa ter Authority for providing financial support for my education and research project. My name is listed al one as the author of this dissertation, but this work was the product of a team that I am honored to have been a part of and will always remember.

PAGE 6

vi TABLE OF CONTENTS Page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES.............................................................................................................ix LIST OF FIGURES..........................................................................................................xii CHAPTER 1 INTRODUCTION........................................................................................................1 Habitat Degradation in the Ocklawaha Basin...............................................................1 Alligators as Potential OCP Receptors.........................................................................2 Developmental Biology of the American Alligator......................................................3 Post-Ovipositional Development...........................................................................6 Ferguson’s Post-Ovipositional Staging Scheme...................................................8 Organochlorine Pesticide T oxicity in Vertebrates......................................................15 Classification, Mode of Action, and Pathology...................................................15 Exposure and Effects of OCPs in Crocodilians...................................................17 Reproductive Problems in Florida Alligators......................................................18 Specific Aims..............................................................................................................20 2 EGG AND EMBRYO QUALITY OF ALLI GATORS FROM REFERENCE AND ORGANOCHLORINE CONTAMINTED HABITATS............................................23 Materials and Methods...............................................................................................24 Egg Collections and Incubation...........................................................................24 Analysis of OCPs in Yolk...................................................................................26 GC/MS Analysis..................................................................................................27 Data Analysis.......................................................................................................28 Results........................................................................................................................ .30 Inter-Site Comparisons of Clutch Characteristics...............................................30 Organochlorine Pesticides Burden s and Clutch Characteristics.........................31 Clutch Survival and OCP Burdens in Egg Yolks................................................32 Average Egg Mass, Clutch Size and OCP Burdens............................................33 Discussion...................................................................................................................34 Inter-Site Comparisons of Clutch Characteristics...............................................34 Clutch Survival Parameters and OCP Burdens...................................................36 Egg and Clutch Size and OCP Burdens..............................................................38

PAGE 7

vii 3 MATERNAL TRANSFER OF OR GANOCHLORINE PESTICIDES.....................54 Materials and Methods...............................................................................................55 Site descriptions...................................................................................................55 Animal Collections..............................................................................................56 Analysis of OCPs in Maternal Tissues and Yolk................................................57 GC/MS Analysis..................................................................................................59 Data Analysis.......................................................................................................60 Results........................................................................................................................ .61 Female Morphological and Reproductive Characteristics..................................61 OCP concentrations in Yolk................................................................................62 OCP concentrations in maternal tissues..............................................................62 Relationships between Maternal Tissue and Yolk Burdens................................63 Relationships between Maternal Mass and OCP concentrations in Eggs and Tissues..............................................................................................................64 Discussion...................................................................................................................65 Evaluation of Predictive Models.........................................................................67 Relationships between Maternal Mass and OCP concentrations in Eggs and Tissues..............................................................................................................69 Maternal body burdens: Toxi cological Implications...........................................69 4 MATERNAL FACTORS ASSOCIA TED WITH DEVELOPMENTAL MORTALITY IN THE AMERICAN ALLIGATOR.................................................80 Materials and Methods...............................................................................................81 Site Descriptions..................................................................................................82 Animal Collections..............................................................................................82 Analysis of OCPs in Maternal Tissues and Yolk................................................83 GC/MS Analysis..................................................................................................86 Data Analysis.......................................................................................................87 Results........................................................................................................................ .88 Discussion...................................................................................................................89 5 MORPHOLOGY AND HISTOPATHOLOGY OF AMERICAN ALLIGATOR ( Alligator mississippiensis ) EMBRYOS FROM REFERENCE AND OCPCONTAMINATED HABITATS...............................................................................99 Materials and Methods.............................................................................................102 Site Descriptions................................................................................................102 Egg Collections.................................................................................................103 Embryo Sampling and Measurement................................................................103 Histopathology..................................................................................................105 Analysis of OCPs in Yolk.................................................................................106 GC/MS Analysis................................................................................................108 Results.......................................................................................................................1 09 Inter-Site Clutch Comparisons..........................................................................109 Intra-Site Live Embryo/Dead Embryo Morphological Comparisons...............110

PAGE 8

viii Inter-Site Comparisons of Morphology of Live Embryos................................111 Live Embryo Morphology and Embryo Survival Relationships.......................112 Live Embryo Morphology and Egg Yolk OCP Burdens...................................113 Embryo Morphological Age, Derived Mo rphometric Variables and Egg Yolk OCP Burdens.................................................................................................115 Histopathology of Live and Dead Embryos......................................................116 Discussion.................................................................................................................117 6 NUTRIENT AND CHLORINATED HYDRO CARBON CONCENTRATIONS IN AMERICAN ALLIGATOR EGGS AND ASSOCIATIONS WITH DECREASED CLUTCH VIABILITY.............................................................................................143 Materials and Methods.............................................................................................145 Egg Collections and Incubation.........................................................................145 Field studies................................................................................................146 Laboratory experiments..............................................................................147 Analysis of Chlorinated Hydrocarbons in Yolk................................................149 GC/MS Analysis................................................................................................150 Nutrient Analysis...............................................................................................151 Data Analysis.....................................................................................................152 Results.......................................................................................................................1 54 Field Study.........................................................................................................154 Case-control cohort study...........................................................................154 Expanded field study..................................................................................157 Laboratory Experiments....................................................................................160 Discussion.................................................................................................................162 7 REPRODUCTIVE EFFECTS OF ORGANOCHLORINE PESTICIDE EXPOSURE IN A CAPTIVE POPULATION OF AMERICAN ALLIGATORS ( Alligator mississippiensis ).......................................................................................................182 Materials and Methods.............................................................................................182 Results.......................................................................................................................1 85 Discussion.................................................................................................................186 8 CONCLUSIONS......................................................................................................196 Introduction...............................................................................................................196 Summary of Study’s Findings..................................................................................197 Future Considerations and Global Implications.......................................................204 LIST OF REFERENCES……………………………………………………………….208 BIOGRAPHICAL SKETCH...........................................................................................217

PAGE 9

ix LIST OF TABLES Table page 2-1. Reproductive, morphometric, and contam inant parameters measured on clutches of alligator eggs collected during summer 2000, 2001, and 2002................................41 2-2. Explanatory variables included in R DA with forward selection of four best variables...................................................................................................................42 2-3. Summary of clutch parameters and si te comparisons for clutches of American alligator eggs collected during 2000-2002...............................................................43 2-4. Organochlorine pesticide burdens and cl utch parameters and site comparisons for clutches of American alligator eggs collected during 2000-2002............................44 2-5. Results of RDA evaluating associations between clutch survival parameters and OCP variables...........................................................................................................47 2-6. Results of RDA evaluating associations between egg and clutch size parameters and OCP variables...........................................................................................................48 3-1. Morphological and reproductive characteris tics of adult female alligators collected during June 2001 and 2002 from Lakes Apopka Griffin, and Lochloosa in central Florida......................................................................................................................73 3-2. Pesticide concentrations (ng/g wet wt.) in tissues and yolks of adult female alligators collected during June 2001 and 2002 from Lakes Apopka, Griffin, and Lochloosa in central Florida......................................................................................................74 3-3. Regression equations for predicting orga nochlorine pesticide (O CP) concentrations in maternal tissues....................................................................................................78 4-1. Reproductive, morphometric, and contaminant parameters measured on adult female alligators collected during June 1999, 2000, 2001, and 2002..................................93 4-2. Explanatory variables included in R DA with forward selection of four best variables...................................................................................................................94 4-3. Reproductive, morphometric, and contaminant summary statisticsa of adult female alligators collected during June of 1999-2002.........................................................95

PAGE 10

x 4-4. Results of redundancy analysis with au tomatic selection of four best maternal factors associated with varia tion in reproductive efficiency....................................97 4-5. Results of redundancy analysis with au tomatic selection of four best maternal factors associated with variation in clutch size characteristics................................97 5-1. Summary statistics for parameters measured on American alligator clutches collected during June 2001 and 2002.....................................................................122 5-2. Comparisons of egg and embryo morpho metrics of live and dead embryos collected during June-August of 2001 and 2002...................................................................124 5-3. Morphometric comparisons of live embryos collected during June-August 2001 and 2002........................................................................................................................128 5-4. Explanatory variables included in partial redundancy an alysis evaluating relationship between organochlorine pest icide burdens in eggs and embryo morphometrics........................................................................................................131 5-6. Best five organochlorine pesticid e (OCP) variables that account for embryo morphological age and derived morphological parameters ..................................133 6-1. Classification matrix for clutches collected during 2002........................................165 6-2. Reproductive, morphometric, and contam inant parameters measured on clutches of alligator eggs collected during summer 2000, 2001, and 2002..............................165 6-3. Explanatory variables incl uded in RDA with forward selec tion of four best variables for case-control cohort and expanded field studies................................................166 6-4. Summary of clutch parameters on clutches collected during 2002 .......................168 6-5. Evaluation of the relationship between co ncentrations of nutrients, PAHs, and PCBs in eggs and clutch survival parameters via RDA analysis.....................................169 6-6. Evaluation of clutch size parameters a nd explanatory factors for clutches collected during 2002............................................................................................................169 6-7. Evaluation of the relationship between nutrient concentrati ons and explanatory variables for clutches collected during 2002..........................................................169 6-8. Summary and comparison of parameters measured on clutches collected during 2000-2002...............................................................................................................170 6-9. Evaluation of the relationships between clutch survival parameters and explanatory variables via RDA using age as the covariate........................................................171 6-10. Evaluation of the relationships between clutch size parameters and explanatory variables via RDA using age as the covariate........................................................171

PAGE 11

xi 6-11. Evaluation of the relationships between thiamine concentrations and explanatory variables via RDA using age as the covariate........................................................172 6-12. Site comparisons of parameters m easured on clutches collected during 2003......173 7-1. Summary statistics and comparisons of clutch parameters among treated and control groups for years 2002-2004....................................................................................192 7-2. Organochlorine concentrations and bl ood chemistry values of captive adult female alligators sacrificed during 2002............................................................................194 7-3. Explanatory parameters a nd clutch survival parameters with () indicating nature of association and value equal to concordance percentage........................................195

PAGE 12

xii LIST OF FIGURES Figure page 1-1. Map of Ocklawaha Basin..........................................................................................22 2-1. Biplot of clutch survival parameters (solid lines) and or ganochlorine pesticide variables (dashed lines) for clutches of alligator eggs collected from Lake Lochloosa during summer 2001-2002......................................................................49 2-2. Biplot of clutch survival parameters (solid lines) and or ganochlorine pesticide variables (dashed lines) for clutches of a lligator eggs collected from Lake Griffin during summer 2000-2002.......................................................................................50 2-3. Biplot of clutch survival parameters (solid lines) and or ganochlorine pesticide variables (dashed lines) for clutches of alligator eggs collected from Lake Apopka during summer 2000-2002.......................................................................................51 2-4. Biplot of clutch survival parameters (solid lines) and or ganochlorine pesticide variables (dashed lines) for clutches of alligator eggs collected from Emeralda Marsh during summer 2000-2002............................................................................52 2-5. Biplot of egg and clutch size parameters (solid lines) and organochlorine pesticide variables (dashed lines) for clutches of alligator eggs collected from Lake Lochloosa during summer 2001 and 2002...............................................................53 3-1. Linear regressi ons of total organochlorine pest icide (OCP) concentrations in maternal tissues against total OCP concentrations in egg yolks. ...........................79 4-1. Biplot of maternal factors (dashed lines) and clutch su rvival parameters (solid lines) of American alligators collected during June 1999-2002. .....................................98 5-1. Representative developmental stages of embryos that were collected from Lakes Lochloosa (reference site), Apopka, and Griffin, and Emeralda Marsh during 20012002. .....................................................................................................................134 5-2. Ordination biplot of embryo morphometric parameters (solid lines) and organochlorine pesticide (OCP) variables (dashed lines) for embryos collected at chronological age Day 14.......................................................................................135

PAGE 13

xiii 5-3. Ordination biplot of embryo morphometric parameters (solid lines) and organochlorine pesticide (OCP) variables (dashed lines) for embryos collected at chronological age Day 25 ......................................................................................136 5-4. Ordination biplot of embryo morphometric parameters (solid lines) and organochlorine pesticide (OCP) variables (dashed lines) for embryos collected at chronological age Day 33.......................................................................................137 5-5. Ordination biplot of embryo morphometric parameters (solid lines) and organochlorine pesticide (OCP) variables (dashed lines) for embryos collected at chronological age Day 43.......................................................................................138 5-6. Ordination biplot of derived embryo morphometric pa rameters (solid lines) and organochlorine pesticide (OCP) variables (dashed lines) for embryos collected at chronological age Day 14.......................................................................................139 5-7. Ordination biplot of derived embryo morphometric pa rameters (solid lines) and organochlorine pesticide (OCP) variables (dashed lines) for embryos collected at chronological age Day 25.......................................................................................140 5-8. Ordination biplot of derived embryo morphometric pa rameters (solid lines) and organochlorine pesticide (OCP) variables (dashed lines) for embryos collected at chronological age Day 33.......................................................................................141 5-9. Ordination biplot of derived embryo morphometric pa rameters (solid lines) and organochlorine pesticide (OCP) variable s (dashed lines) embryos collected at chronological age Day 43.......................................................................................142 6-1. Biplot of clutch surviv al parameters and explanatory factors for clutches collected during 2002............................................................................................................175 6-2. Biplot of clutch size parameters and explanatory vari ables for clutches collected during 2002............................................................................................................176 6-3. Biplot of nutrient concentrations in e ggs (solid arrows) and explanatory variables (dashed arrows)......................................................................................................177 6-4. Relationships between embryo age and thiamine phosphorylation in egg yolk for 29 clutches collected duri ng 2002 from Lakes Lochloosa, Griffin, Apopka, and Emeralda Marsh.....................................................................................................178 6-5. Biplot of clutch survival parameters and explanatory va riables for clutches collected during 2000-2002...................................................................................................179 6-6. Biplot of clutch size va riables (solid lines) and expl anatory variables (dashed lines) for clutches collected during 2000-2002................................................................180

PAGE 14

xiv 6-7. Biplot of thiamine egg yolk concentra tions (solid lines) and explanatory variables (dashed lines) measured on clut ches collected during 2000-2003.........................181

PAGE 15

xv Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy DEVELOPMENTAL MORTALITY IN AMERICAN ALLIGATORS ( Alligator mississippiensis ) EXPOSED TO ORGANOCHLORINE PESTICIDES By Richard Heath Rauschenberger December 2004 Chair: Timothy S. Gross Major Department: Veterinary Medicine—Physiological Sciences Since the early 1900s, the lake s of the Ocklawaha Basin in central Florida have experienced ecological degradation due to anthropogenic development. One species affected by degradation has been the American alligator ( Alligator mississippiensis ). Decreased clutch viability (pr oportion of eggs in a nest that yield a live hatchling) was observed in the years after a chemical spill in which large amounts of sulfuric acid and dicofol, an organochlorine pesticide (OCP), flowed into Lake Apopka. Lake Apopka and other lakes in the Ocklawaha basin have al so been contaminated by urban sewage and agricultural chemicals, with agricultural chem icals entering the lakes via rainfall run-off or back-pumping of water from agricultural la nds). Decreased hatch rates are a problem at Lake Apopka, as well as at other OCP-cont aminated sites in Florida. The purpose of my study was to determine the causes for decr eased clutch viabilit y, and to test the hypothesis that maternal exposure to OCPs is associated with embryonic mortality in alligators.

PAGE 16

xvi Field studies involved collec ting and artificially incuba ting eggs from reference sites (Lake Lochloosa) and from OCP-cont aminated sites (Lakes Apopka, Griffin, and Emeralda Marsh Restoration Area) to evaluate clutch viability as a function of egg and maternal OCP concentrations. Nutrient content of eggs and histopathology and morphometrics of embryos were also evaluate d to identify potential factors associated with embryo mortality. In addition, a novel laboratory experiment exposed a captive population of adult alligators to an OCP mixtur e, and compared OCP burdens in eggs and clutch viability with a captive control group. Results of field studies suggested that OCP concentrations (ng total OCP/g egg yolk, Mean SE) in reference site clutches (n = 19; 102 16) were significantly ( = 0.05) lower than those of Apopka (n = 23; 7,582 2,008), Griffin (n = 42; 1,169 423), and Emeralda Marsh (n = 31; 15,480 2,265). Clutches from reference sites also had significantly higher clutch viability (70 4 %) than those of Apopka (51 6%), Griffin (44 5%), and Emeralda Marsh (48 6 %). Furthermore, decreased thiamine concentrations in eggs may play a role in de creased clutch viability in wild clutches. Results of the captive study suggested that treated females produced eggs containing higher OCP concentrations (n = 7; 13,300 2,666) than controls (n = 9; 50 4). Eggs of treated females also exhibited decreased viability (9 6%) as compared to controls (44 11%). These field and laboratory studies s upport the hypothesis that maternal exposure to OCPs is associated with decreased clutch viability in American alligators, and that thiamine deficiency may also be a contribu ting factor in reduced clutch viability.

PAGE 17

1 CHAPTER 1 INTRODUCTION Habitat Degradation in the Ocklawaha Basin In central Florida, several lakes within the Ocklawaha River Basin (Fig. 1-1) have experienced severe degradation of habitat quality since the earl y 1900s, as agricultural and urban development progressed. Ind eed, Lake Apopka (headwaters of the Ocklawaha) was once renowned for its clear water and its excellent largemouth bass fishing. More recently, Lake Apopka has gain ed world-wide notorie ty as the “poster child” for polluted lakes, because of hi ghly publicized problem s associated with environmental contamination. Initial degradation of Lake Apopka and other lakes within the Ocklawaha Basin occurred as the result of the loss of thousands of hectares of marsh habitat through the agricu ltural practice known as muck farming (which involves installing levees around an area of marsh, so the marsh can be drained; allowing the fertile peat to be farmed). This farming practice began in the 1940s and continued into the 1980s (Benton et al., 1991). In addition to sewer discharge from the city of Winter Garden entering the Lake Apopka, organochlor ine pesticides (OCPs) were heavily and widely used to control crop-dest roying insect pests. Since the 1980s, use of most OCPs ha s been discontinued since they were determined to be persistent environmental c ontaminants that resist metabolic degradation and bioaccumulate in animal tissues, wh ere they are potenti ally carcinogenic, immunotoxic, endocrine disrupti ng, and developmentally toxic (Fairbrother et al., 1999;

PAGE 18

2 Ecobichon, 2001). Altered func tion of the reproductive a nd endocrine systems of wildlife and human populations have been sugge sted to occur after exposure to a variety of OCPs and OCP metabolites such as dichlorodiphenyltrichloroethane (DDT), dichlorodiphenyltrichloroethyl ene (DDE), methoxychlor, dico fol, chlordane, dieldrin, and toxaphene (Colborn et al., 1993 ; Longnecker et al., 2002). Further degradation and OCP contamina tion occurred in Lake Apopka in 1980. A chemical spill occurred when a highly acidi c wastewater pond at the Tower Chemical Company’s main facility overflowed into th e Gourd Neck area of Lake Apopka (Fig. 11). Because of the large volume and acidity (sulfuric acid), and the high levels of DDT, dicofol, and related OCP contaminants that en tered the relatively narrow area of the lake, aquatic vegetation and animals were severely affected. In 1983, the area was placed on the US Environmental Protection Agency’s (EPA ) National Priority Site List and became a part of the Superfund program; whic h was created by the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA), later amended by the Superfund Amendments and Reauthorizat ion Act (SARA). The CERCLA and SARA provide authority for the governme nt to respond to the release a nd/or threat of release of hazardous wastes, and allow cleanup and enfo rcement actions. Lake Apopka is still listed and groundwater toxicity testing is ongoing (EPA, 2004). Alligators as Potential OCP Receptors The American alligator is an important member of Florida wetlands and plays important roles in the ecology, es thetics, and economy of Flor ida. Therefore, identifying physiological and ecological charac teristics related to potential sus ceptibility to effects of contaminants, as well as potential exposure routes, is important in managing populations

PAGE 19

3 for optimal human use. Especially importa nt to consider, in regard to wildlife populations, are potential effect s of OCPs on reproduction. One of the first qualities that may be rela ted to an alligator’s susceptibility to reproductive effects of OCP contaminants is that alligators do not at tain sexual maturity until approximately 6-10 years of age, whic h allows exposure and bioaccumulation of OCPs to occur before reproductive maturity. Potential implications are that, as females begin to mobilize body stores during vitell ogenesis, the lipophilic OCPs that have accumulated in their fatty tissues during their li fespan would likely be deposited in what will later be the embryos’ sole source of nutrition (egg yolk) Secondly, adults exhibit a long reproductive period (over 30 years), and a long life span (over 50 years) (Ferguson, 1985), and are higher order predators (which allows for increased OCP exposure and bioaccumulation, possibly leading to altere d endocrine and reproductive function). Thirdly, alligators build nests th at can be identified from considerable distances (which aids in egg collections), lay a large number of eggs (approximately 40 eggs per clutch), and have a long developmental period of 65-72 days (Ferguson, 1985), allowing extended exposure at a potentially critic al stage of development. Thus, the propensity for OCPs to be bioaccumulated and biomagnified in biot a (combined with the alligator’s reproductive biology, longevity, ecological troph ic level, and relatively long in ovo developmental period) suggests the poten tial for OCPs to alter reproductive function. Developmental Biology of the American Alligator Understanding normal embryonic devel opment is an obvious necessity in determining the occurrence of abnormal embryonic development and identifying critical periods of development (e.g., organogenesis). Therefore, this brie f review summarizes pre-ovipositional and post-ovi positional development of the alligator embryo.

PAGE 20

4 Pre-ovipositional Development Overall, when compared to other verteb rate species such as the domestic chicken and domestic pig, there is a paucity of data re lated to crocodilian development. Despite the relatively low number of publications, th e quality of papers c overing early embryonic development is fairly high, considering that much of the research took place approximately a century ago. The most a ppropriate place to begin discussing embryonic development would be the point when fertiliz ation occurs. However, the precise timing and location of fertilization within a fe male alligator’s oviduct is unknown and inadequately studied. Pre-ovipositional development has been examined by sacrificing gravid females and collecting their eggs and embryos. Sacr ifice of gravid females was required since alligator embryos are at a more advanced stag e of development at the time of oviposition (Clarke, 1891). The earliest developmental stage examined in these pre-ovipositional studies were of Nile crocodile embryos ( Crocodylus niloticus ), in which all embryos exhibited body folds, a neural medullary gr oove, an embryonic shield, area opaca, early gut, and area pellucida (Voeltzkow, 1892). After the appearance of the neural folds, the amniotic head fold is formed from an anterior fold in the blastoderm. The head fo ld is crescent shaped, because it begins to develop with its free ends pointing toward th e posterior end of the embryo, and develops craniocaudally. The amniotic primordium deve lops in continuity with the head, and is derived from the somatopleure around the trunk. Craniocaudal separation of the embryo fr om the blastoderm occurs partly as a result of the development of the dorsal amniotic fold, but separation is not complete until

PAGE 21

5 post-ovipositional stage 3 (Day 3). The neural groove and blastopore become clearly demarcated as the ectoderm and endoderm of the blastoderm develop. The endoderm may form extensions that pe netrate the underlying yolk. Th e blastopore goes through the entire blastoderm, with the primitive str eak located posterior to the blastopore (Voeltzkow, 1892). As the body folds develop, the border between embryonic and extra-embryonic tissues becomes visible. At th is point, the beginning of the foregut is discernable, and the notochord stretches from the midline of the head fold to the anterior border of the blastopore. The primitive streak and primitive groove lie posterior to the blastopore, with the primitive groove being continuous at its posterior end. The primitive streak extends to a little less than halfwa y between the head fold and blastopore (Ferguson, 1985). Neural folds have two origins. The first is a secondary fold located anteriorly in the head region, and growing posteriorly along the median dorsal line to form a V-shaped process, with the apex pointing toward the bl astopore. The second is posterior folds that arise as ectodermal ridges extending forwar d from the blastopore, circumventing the neural groove. The apex of the V-shaped s econdary head fold later disappears, and each of the separate arms becomes continuous with the corresponding posterior neural fold. Thus, the secondary head fold forms the anterior part of the neural fo lds. Closure of the folds occurs first in the middle region of th e embryo closer to the anterior end of the neural groove in alligators (Ferguson, 1985) but closer to the posterior end in Nile crocodiles (Voeltzkow, 1892). After the closure of the neural canal, th e blastoporal or neurenteric canal is no longer visible. The neurenteric canal runs from its posterior cranioventral opening to

PAGE 22

6 where it opens into the neural groove at it s caudal limit. During this period, somites develop along the median axis, with the fi rst pair developing halfway between the anterior and posterior ends. The periphera l somatic cells are compactly arranged, and contain small myocoels within the center of the somites. The mesodermal layers cleave and form the somatic and splanchnic components as the foregut develops. The head fold of the embryo is positi oned ventrally into the underlying yolk, which is accentuated by the bending of the an terior neural folds, and by the cranial flexure that occurs later. At this pre-ovi positional stage of development, the embryo has not yet attached to the inner su rface of the eggshell membrane. Because embryos are at an advanced st age of development at the time of oviposition (and because an entire clutch ty pically hatches within a 2-day period, with most hatchlings being similar in size), it app ears that fertilization occurs over a short time period; and that embryos are actively developi ng during the next 2to 3-week period in which the ova receive albumin, eggshell me mbrane, and eggshell depositions (Ferguson, 1985). Presently, little information exists about gaseous exch ange and embryonic metabolism before oviposition, or about the processes that prev ent the embryo from attaching to the top of th e egg before oviposition. Post-Ovipositional Development Post-ovipositional development is better understood than pre-ovipositional development. Again, the amount of literat ure concerning crocodilian development is miniscule compared to the amount of literature deali ng with human and chicken embryology. One important area to address when disc ussing post-ovipositi onal development is the staging scheme. Establishing a staging scheme or a normal table of development for

PAGE 23

7 any species allows results of various studies to be compared (B illet et al., 1985). The currently accepted staging scheme for cr ocodilian embryology was proposed by Ferguson (1985). Before Ferguson’s, the only other staging systems related to crocodilians came from Voeltzkow (1892), Reese (1912), and We bb et al. (1983). These works were impressive, considering the conditions th ese pioneers faced; but many stages were missing, and incubation conditions were poorly controlled. Ferguson (1985) improved on their work by monitoring and controlling the temperature (30C) and the relative humi dity (95-100%) at which the eggs were incubated, allowing duplication of his e xperiment and standa rdization of the characteristics one should see in an embryo, given its stage. This accepted staging scheme is based on external morphological features, with limb and eye development being important diagnostic elements. With re spect to craniofacial development, a fair amount of data exists, because of Ferguson’ s focus on the structure and development of the palate in the alligator, and on how its de velopment relate s to stage (Ferguson, 1981). Although the relationship between craniofacial development and developmental stage has been studied, information relating stage a nd development in other organ systems is somewhat lacking. Alligator embryos are very sensitive to temperature. For example, 26-34C is the optimum incubation temperature; anything a bove or below for an extended period will result in increased mortality (Ferguson, 1985) Furthermore, 0.5-1 C changes can mean the difference between an entire clutch of embryos being 100% females or 100% males, since crocodilians exhibit temperature-depe ndent sex determinati on (Lang & Andrews, 1994).

PAGE 24

8 Ferguson’s Post-Oviposi tional Staging Scheme Because our study used Ferguson’s stagi ng scheme, a summary description of Ferguson’s (1985) staging scheme, it is su mmarized here. The normal table of development for crocodilians was based on examination of 1500 Alligator mississippiensis embryos, 300 Crocodylus porosus embryos, and 300 Crocodylus johnsoni embryos. One bias is that all of the alligator embryos used in developing this scheme originated from Rockefeller Wildlife Refuge, located in southern Louisiana. Alligator embryos from other geographic areas may develop at different rates, given the same incubation conditions. Alligators inhabiting Arkansas and North Carolina experience a shorter summer compared to popul ations inhabiting southern Louisiana or Florida. Shorter summers mean that optimal nest temperatures are maintained for a shorter period of time. Thus, embryos from more northerly latitudes may develop at an increased rate compared to embryos from sout herly latitudes (given identical incubation conditions), since the northern embryos must complete development within a shorter time frame. This hypothesis is supported by evidence that crocodilian species ( Crocodylus porosus and C johnsoni ) living along the equator have longer and more variable incubation periods and slower embryonic development than the (more northerly) Louisiana alligator (Deeming & Ferguson, 1990). Setting aside the potential bias descri bed above, developmental “stages” are determined by morphological characteristics alone, and are applicable to embryos regardless of incubation temperature. Howe ver, the developmental day(s) associated with each stage are only valid if the eggs ar e incubated at 30C with a relative humidity of 95-100%. Temperatures lower than 30 C slow the rate of development, and temperatures above 30C have been shown to increase the rate of development. Low

PAGE 25

9 humidity within the nest has been shown to dehydrate eggs, causing embryonic mortality and alterations in growth pa tterns (Deeming & Ferguson, 1990). Stage 1 covers the period from oviposition to the end of the first 24 hours, and is characterized by the embryo and blastoderm be ing not attached to the top of the inner eggshell membrane. The heart is a simple S-shaped tube. There are 16-18 pairs of somites along the trunk, and 3 pair s of somitomeres anterior to the otic vesicle. Although the brain has not yet regionalized, optic placode s and vesicles are present on the head. Body torsion has not begun. The notocord is ev ident, the gut is incomplete caudally and opens ventrally, and blood ve ssels are not present in th e extraembryonic membranes. Stage 2 (Day 2) embryos have 21-25 pair s of somites and a three-loop heart. However, one of the most notable characteristics is that the embryo attaches to the top of the egg, causing an opaque spot to form that is visible in an otherw ise translucent egg, when the egg is candled. Blood vessels are now visible, and the hindb rain is discernable as a clear transparent region. The lens placode and optic cup are defined, and no body torsion has occurred. Stage 3 (Day 3) embryos have 26-30 som ites, and are completely delineated from blastoderm. Forebrain, midbrain, and hindbrai n are now discernable, and the optic cup has an elongated horseshoe shape, extending below the lens vesicle to the primitive oronasal cavity. The head is positioned at a right angle to th e body, but no body torsion has occurred. Stage 4 (Day 4) embryos have 31-35 pairs of somites with the tail being distinct, straight, and unsegmented at the posterior e nd. Body torsion has started, with the cranial half rotated so that the right surface is contacting the shell membrane, while the left is

PAGE 26

10 parallel with underlying yolk. The caudal half of the embryo remains at a right angle to the yolk. The heart is displaced from midline to the left side of the embryo. Three cranial arches are present; and cranial nerves to the cranial arches are visible, using oblique or transmitted illumination. Stage 5 (Day 5) embryos have 36-40 pair s of somites, and the tail-tip bends ventrally at a right angle to th e body, with 3-5 somites visible at its base. Body torsion is complete except for the tail. The otic pit is dorsal to the junction of the 2nd and 3rd brachial arches, and its ex ternal opening is closed. Stage 6 (Day 6) embryos have visible nasa l placodes, and the hi ndlimbs are barely discernable on each side; with the right hind limb slightly advanced over the left. Forelimb buds are not yet present, and body tors ion is complete. The olfactory bulbs, forebrain, and midbrain are distinct. In the hindbrain, 4-6 neuromeres are discernible. Foregut and hindgut are formed, but midgut is incomplete ventrally. Major vitellogenic blood vessels emerge at the level of the 18th somite and smaller ones at the 6th and 11th somites. Embryos at Stage 7 (Day 7) have distin ct hind limb buds. In addition, forelimb buds are barely visible and extend over som ites 12-15. The midbrain bulge is evident, and the tail-tip is curled at 90 to the rest of the tail. Three brachial arches are present; and at the level of the heart, the cranial end is bent at 90 to the rest of body. Embryos at stage 8 (Day 8) have nasal pits external to the swellings of the olfactory bulbs, and distinct forelimb and hind limb buds that extend over somites 11-16 and 2732, respectively. An apical ectodermal ridge is developing on the hind limb bud, and the tail is coiled through 2 turns and has 12-18 somites.

PAGE 27

11 Stage 9 (Day 9) embryos have four brachia l arches, and a visible maxillary process extending to the midpoint of the eye. The optic cup is large and round but unpigmented. A distinct apical ectodermal ridge is present on the hind limb, and the hind limb bud extends beyond the forelimb. The tail is cu rled through three 90 turns. The heart exhibits distinct atria an d ventricles, and lung primord ia are visible through the pericardial sac. Midgut and body walls are open ventrally from the caudal limit of the pericardial sac to 2/3 of the way down the body, and the liver and mesonephros are barely visible. Stage 10 (Day 10-11) embryos have pigmen ted eyes (except for a central opaque lens) with the right eye developi ng pigmentation earlier and darker than the left eye. Five brachial arches are present, and medial and la teral processes are distinct elements on each side of the nasal pits. Maxillary processes delimit a distinct groove beneath the eye. The tail is coiled through four 90 turns, and th e liver and mesonephros are clearly visible through the body walls. Stage 11 (Day 12) embryos have a visible nasal pit slit forming between the medial and lateral processes. Forelimb and hi nd limb buds extend caudally from the body wall and exhibit distinct ap ical, ectodermal ridges. The fo relimb has a distinct constriction that separates the distal and proximal elemen ts, with constriction less obvious in the hind limb. A loop of midgut is visible at the um bilicus, the eye exhibits a distinct black pigment in the iris, and the chorioallantois extends 2/3 around the breadth of the shell membrane. Embryos at stage 12 (Day 13-14) have a di stinct notch in the midline of the face between the medial nasal processes. Fore limbs are starting to bend in the region of

PAGE 28

12 constriction, so that they are positioned cl oser to the pleuron of the embryo. The elongated hind limb shows little differentiati on into proximal and distal elements and, although there is a distinct ap ical ectodermal ridge, footplate formation is barely discernable. Stage 13 (Day 15) embryos have distinct na sal pit slits, and forelimbs are now bent toward the pericardium. The distal portion of the hind limb is flattened and enlarged into a footplate primordium. The chorioallant ois now extends as a ring around the inner circumference of the centr al eggshell membrane. Embryos at Stage 14 (Day 16-17) have nasa l pit slits that are closed due to the merging of the medial nasal, lateral nasal, a nd maxillary processes. Foot and hand plates are distinct, with the former more advanced than the latter. Lower jaw extends onequarter beneath the upper jaw, the upper ea rflap is overgrowing the external ear opening, and the embryonic face rests on the large bulge of the thorax. A large loop of gut herniates through the narrow umbilical stalk and touches the yolk, and the abdominal viscera are visible through body walls. The tail is coile d and kinked at the tip, and contralateral reflexes occur. Stage 15 (Day 18-20) embryos have lower jaws that extend one-third to one-half the length of the upper jaw. Th e anlage for the upper eyelid is an elevated rim of tissue above each eye. Distinct and proximal and dist al regions, as well as hand and foot plates are present on both the fore and hind limb. Th ere is a distinct hollow in the face beneath the anterior one-third of the eye. Stage 16 (Day 21) embryos exhibit faint di gital condensations in the footplate but not the hand plate. The lower jaw is now tw o-thirds the length of the upper jaw, with the

PAGE 29

13 upper jaw being hook-shaped around the perica rdial ridge. Caruncle development is observed, with two tiny widely spaced thickeni ngs that are just disc ernable on the tip of the snout. Embryos at stage 17 (Day 22-23) exhibit mesodermal condensations for the five forelimb digits and four hind limb digits, the head is extended off of the pericardial sac by neck elongation, and the exte rnal earflap is distinct. Stage 18 (Day 24-26) embryos have discernabl e, distinct cartilag inous digital rays on the hand and foot. The margins of upper eye lid anlage extend over the superior rim of the iris, forming a distinct groove between the eyelids and the eye. Dorsal scalation is now evident, and the pericardial sac is starting to submerge into the ventral thoracic wall. Stage 19 (Day 27-28) embryos have upper and lower eyelids, and the lower jaw lies behind the anterior margin of the upper jaw. Interdigital clefting has started, and slight marginal notches can be seen, particularly in the footplates. White flecks representing ossification are visible around the upper and lower jaws. Stage 20 (Day 29-30) embryos have nail an lages starting to develop, first on the most medial digit of the foot, then on adj acent digits; followed by the most medial digit on the hand, and finally on the adjacent hand digits. Interdigital clefting now extends one-quarter the length of the digits, and the lower jaw is in adult relationship with the upper jaw. The pericardial sac is one-quarter withdrawn into the body, and ossification is evident in the proximal and distal elements of limbs. Scale formation is evident dorsally, and scutes (osteoderms) are beginning to a ppear in the neck region near the skull. Stage 21 (Day 31-35) embryos have inte rdigital clefting no w extending threequarters down the digits, and phalanges can be distinguished. Scal es are now visible on

PAGE 30

14 the ventral body wall; and dorsally on the s nout, neck, body, and tail. Scutes on neck are clearly defined. The pericardial sac is one -half withdrawn into the body, and a white ring in the iris surrounds the outline of the lens of the eye. Both upper and lower eyelids overlap the eye. Stage 22 (Day 36-40) embryos have pigmented margins of the upper jaw, ventral flank, and proximal and distal elements of the limbs. Interdigital clef ting is at the adult level, and the eyelids are typica lly closed from this point fo rward. The pericardial sac is two-thirds withdrawn. Stage 23 (Day 41-45) embryos have more extensive pigmentation, with the embryos appearing light brown with dorsal st ripes. Scales are present on distal and proximal elements, and nails have a slight distal elevation. Th e sensory papillae are present on lateral jaw margins, and scales are evident on gular skin. The midbrain is visible as a white bulge at th e back of the cranium, and the pericardial sac is threequarters withdrawn. Stage 24 (Day 46-50) embryos are blacker Nails on hands have elevations at their tips, and the nails are starting to form curves. The midbrain is covered by pigmented skin. The pericardial sac is fully withdrawn and the mid line is closing. The volume of yolk outside the body cavity is large, and scales and scutes are evident all over embryo. Stage 25 (Day 51-60) embryos look identical to hatchlings, except smaller. The external yolk is beginning to be withdr awn, and few gross morphological changes are evident at this and later stages. Growth rela tionships (head length: total length ratio) and the amount of external yolk present are the major observable differences.

PAGE 31

15 Stage 26 is not present in alligators. This stage was esta blished using tooth eruption sequences and is useful only for saltwater crocodiles ( Crocodylus porosus ) and freshwater crocodiles ( Crocodylus johnsoni ). Stage 27 (Day 61-63) embryos have withdr awn the yolk sack into the body, ending with skin forming across the umbilical scar. The last stage before hatching (Stage 28, Day 64-70) ends with the umbilical scar being diminished in length and width. Overall, the first 35 days are a period of rapid orga nogenesis, and the second 35 days are characterized by embryo growth. Since organogenesis has been shown to be a sensitive period in regard to effects of developmental toxicants (Schmidt & Johnson, 1997), the first 35 days of incubation appear to be the most susceptible time points for toxicant-induced mortality. In summary, the establishe d staging scheme provides a way to estimate the age of the clutch at the time of co llection, and allows on e to later determine if a clutch is undergoing normal development. One can dete rmining if a clutch is undergoing normal development by examining embryos at preselected time points and comparing their morphological age to their calendar stage (i.e., does an embryo exhibit the normal morphological characteristics that it should exhibit, given its calendar age?). In addition, embryonic development may be compared among clutches and among populations, by collecting embryos at pre-determin ed stages of development. Organochlorine Pesticide T oxicity in Vertebrates Classification, Mode of Action, and Pathology Organochlorine pesticides (also known as chlorinated hydrocarbon insecticides) may be separated into five classes of com pounds. These classes are DDT and its analogs, cyclodienes and similar compounds, toxaphene (composed of several congeners), mirex

PAGE 32

16 and chlordecone (which have cage-like structur es), and benzene hexachloride (BHC). In rodent models, studies suggest that OCPs can adversely affect the function of neurons and cause cellular damage to the liver and kidneys (Smith, 1991). Organochlorine pesticides affect neural transmi ssion by altering enzyme activity (Ca2+-ATPase, phospokinase) and the electrophysical properties (K+, Na+ ion exchange) of nerve cell membranes. Different analytes may elicit si milar effects (neurona l hyperactivity), but by different mechanisms. For example, studies suggest DDT and its analogs affect the nerve axon by keeping Na+ channels open longer than normal. Cyclodienes, alternatively, may affect neural transmission at pres ynaptic terminals and may affect the -aminobutyric acid (GABA)-regulated chloride channel. Although they can cause severe neural dysfunction, little morphological ch anges are evident in neural ti ssue, even at lethal doses (Smith, 1991). Morphological changes are evident in the liver and include hepatocellular hypertrophy and focal necrosis. Hypertr ophy is due to enlargement of the smooth endoplasmic reticulum (SER) and formation of a lipid droplet in the center of the SER (caused by OCP-induced expression of mi crosomal enzymes within the SER). Functional alterations may also occur in hepatocytes, with disruption of intercellular communication (by hindering transfer of growth inhibitors) (Smith, 1991). Morphological changes have also been found in the liver and kidney of fish chronically exposed to organochlorine pestic ides. For example, chronic exposure to OCPs induce hepatic lesions, such as foci of vacuolated hepatocytes and spongiosis hepatic (lesions of hepatic parenchyma). Renal lesions induced by chronic OCP

PAGE 33

17 exposure include dilation of tubular lumina, and vacuol ization (degeneration) and necrosis of tubular epithelium (Metcalfe, 1998). In addition to morphological changes, or ganochlorine pesticides may adversely affect endocrine and reproductive function in la boratory models and wildlife populations. Mechanisms include direct toxicity on endoc rine glands (such as o,p’-DDD’s ability to permanently inactivate the adrenals), comp etitive binding of steroid hormone receptors, increased expression of steroid-metabolizing hepatic microsomal enzymes, and inhibition of hormone synthesis (such as DDE-induced in hibition of proglandin synthesis, leading to eggshell thinning in raptors) (Gross et al., 2003). Exposure and Effects of OCPs in Crocodilians Current knowledge on the effects of envi ronmental contaminants on crocodilian reproductive physiology is important in unders tanding the likelihood of developmental alterations occurring as a result of expos ure; and understanding which mechanisms may be involved. Campbell (2003) reviewed the effects of organic and inorganic contaminants on crocodilians. Campbell reported only 26 studi es related to the bioaccumulation of organic contaminants, with just 35% (8/23) of crocodilian sp ecies being represented. Of the 26 studies, 38% involve d American alligators ( Alligator mississippiensis ), 26% involved Nile crocodiles ( Crocodylus niloticus ), 13% involved American crocodiles ( Crocodylus acutus ), and 13% involved Morolet’s crocodile ( Crocodylus moreletii ). Slightly more studies were found that investig ated effects of organi c contaminants. With respect to these 39 studies, only 13% (3/23) of crocodilian species were represented, consisting of the American alligator (91% of studies), the Nile cr ocodile (5%), and the African dwarf crocodile ( Osteolaemus tetraspis 4%). Of these studies, American

PAGE 34

18 alligators are the only species in which an effort has been made to determine the relationship between OCPs and depressed hatch rates, with most of this work involving populations in central Florida. Reproductive Problems in Florida Alligators In the early to mid 1980s, studies showed that the population of juvenile alligators inhabiting the aquatic ecosys tem of Lake Apopka, Florida, declined by 90%. This decline was preceded by a 1980 chemical spil l and decades of OCP contamination via anthropogenic activities described earlier. Th e loss of juveniles was attributed primarily to a dramatic decrease in clutch viability (the proportion of eggs in a clutch that produce a live hatchling) (Woodward et al., 1993). Alterations in sexual diff erentiation, sex steroid ho rmone concentrations, and metabolism were also documented among La ke Apopka alligators. For example, testosterone was lower in male alligators from Lake Apopka as compared to those of control sites. Ovaries of female juven ile alligators from Lake Apopka showed abnormalities, suggesting that reproductive al terations were occurring in both sexes (Gross et al., 1994; Guillette et al., 1994; Gr oss, 1997). In addition, high concentrations of OCPs were measured in egg yolk, but concen trations were not clea rly associated with increased mortality (Heinz et al., 1991). Later studies suggested that the cause for the population decline was potentially more complex than previously suggested. First, poor egg viability for Lake Apopka alligators was more closely associated with muck farm reclamation (wetland restoration) sites than with tissue and egg concentrations of the predominant pe sticide residue (DDE) (Giroux, 1998). Second, altered endocrine f unction and decreased egg viability were documented among alligators at another si te, Lake Griffin, where tissue and egg

PAGE 35

19 concentrations of residues such as DDE are mo dest or intermediate compared with those of Lake Apopka. However, like Lake Apopka Lake Griffin is hi ghly eutrophic and has adjacent muck farms and muck farm reclama tion areas (Marburger et al., 1999). Third, poor reproductive success among Lake Apopka a lligators appeared to result from both decreased proportions of fertile eggs th at produce a live hatchling and decreased proportions of hatchlings that survive thr ough the first 20 days of life (which is the toxicant-sensitive organogenesis period); and decreased proportions of unbanded eggs (i.e., eggs that are nonviable on initial examination) (Masson, 1995; Wiebe et al., 2001). Unbanded eggs show no evidence of embr yo-eggshell attachment (as indicated by the presence of an opaque spot or band th at results from fusion of extraembryonic membranes to the dorsal portion of the inne r eggshell membrane). Unbanded eggs may result from very early embr yo mortality (fertilization has been confirmed in many cases by the presence of paternal DNA, via DNA micros atellite analysis); or may result from infertile eggs (Rotstein, 2000). The last similarity between alterations in alligator populations of Lake Griffin and Lake Apopka is increased mort ality among adult Lake Griffin alligators (Schoeb et al., 2002), which is similar to increased adult mo rtality on Lake Apopka in the early 1980s. These data indicate that alligato r populations are adversely aff ected at each of several life stages. Although anatomic and endoc rinologic effects of exposure to endocrine-disrupting OCPs could account for many of these effects, additional underlying mechanisms are almost certainly i nvolved. Overall, th ese data point to a complex process involving the introduction of OCPs into th ese aquatic ecosystems from

PAGE 36

20 chemical spillage or from muck farming a nd reclamation activities; possibly leading to developmental toxicity, in addi tion to endocrine disruption. Specific Aims The overall objective of our study was to determine the causes of decreased hatch rates among alligators in contaminated sites, and to determine if causal links could be established between specific adverse eff ects and exposure to individual OCPs or combinations of OCPs. The project consiste d of epidemiological field studies, which evaluated embryonic development and mort ality as a function of maternal and environmental exposure to OCPs and egg nutri ent composition; and controlled laboratory experiments to test hypothesized links be tween decreased hatch rates, altered egg composition, and exposure to selected OCPs. Specific aim 1 : Conduct field epidemiological studi es to determine the relative contributions of unbanded eggs, embryonic mo rtality in banded eggs, and decreased perinatal mortality to the overall decreased reproductive success in alligators at OCPcontaminated sites, to determine which OCPs or combinations of OCPs are most closely associated with adverse effect s at each life stage, and to ex amine the relationship between OCP burdens in maternal tissues and eggs For Specific Aim 1, the hypotheses were H1a : Adverse effects at early life stages are associ ated with muck farm environments, exposure to specific OCPs or OCP combinations, or both; H1b : Specific OCPs found in maternal tissues are highly correlate d to those present in eggs indicating maternal transfer of OCPs and that maternal size is correlated with OCP burdens and hatch rates; H1c : Eggs in which embryonic and perina tal mortality occur result from developmental abnormalities, altered stru cture or composition of the egg, or both. Specific aim 2 : Conduct controlled in ovo and in vivo experiments with alligators to confirm causal links between decreased ha tch rates and affected life stages as a

PAGE 37

21 function of exposure to selected OCPs or al tered egg qualities, or both. For Specific Aim 2, the hypotheses were H2a : Exposing a captive breeding populat ion of adult alligators to an environmentally relevant mixture of OCPs w ill elicit OCP concentrations in eggs and developmental effects similar to those obser ved in wild eggs from OCP-contaminated field sites; H2b : Exogenous in ovo alteration of egg nutrien ts based on data from field studies will alter embryonic development.

PAGE 38

22 Figure 1-1. Map of Ocklawaha Basin. Ocklawaha River Gourd Neck Area Lake Apopka Lake Griffin LakeLochloosa Orange Lake Lake George

PAGE 39

23 CHAPTER 2 EGG AND EMBRYO QUALITY OF ALLI GATORS FROM REFERENCE AND ORGANOCHLORINE CONTAMINTED HABITATS In the southeastern US, aquatic ecosystem s have experienced habitat degradation, alterations in water quality, and in some cases important declines in biodiversity due to increases in land development and associated anthropogenic im pacts. A case-in-point is the Ocklawaha River Basin in central Florida. Within this basin, American alligators ( Alligator mississippiensis ) from impacted lakes have e xhibited poor clutch viability (number eggs that yield a live hatchling / to tal number of eggs found in clutch) (Masson, 1995), abnormal reproductive hormone concen trations (Gross et al., 1994), and unexplained adult mortality (Schoeb et al., 2002). During the mid 1980s, clutches from alligators on Lake Apopka experienced severe declines in clutch viability (declined from 50% to 4%), and alligator clutches from other impacted lakes had only moderate viabilities (range of 40 to 60 %). These rates were below those observed in other less impacted Florida lakes (reference sites), including Lake Woodruff National Wildlife Refuge (79%), Orange Lake (82%), and th e Everglades Water Conservation Areas (6575%) (Woodward et al., 1993; Masson, 1995; Rice, 1996). Possible causal factors for reduced hatch ra tes in alligator popul ations within the impacted sites within the Ocklawaha Rive r Basin include pesticides, algal toxins, nutritional changes, density-related stress, and diseases. In one case, a chemical spill from a chemical manufacturing plant in 1980 near Lake Apopka (EPA, 2004) was temporally associated with the decline in reproductive success and consequent alligator

PAGE 40

24 population decline on Lake Apopka during th e early 1980s. However, decreases in clutch viability for Lake Apopka appeared to be more related to proximity to muck farm restoration areas as compared to yolk con centrations (Giroux, 1998), which is consistent with decreases in clutch viability on Lake Griffin and Emeralda Marsh, Griffin’s adjacent muck farm restoration area (S eplveda et al., 2001). Poor reproductive success threatens the long-term conservation of alligators, potentially altering the ecology of affected ecosystems, a nd substantially reducing the aesthetic and economic values of alligators in affected areas. Understanding and characterizing poor reproductive performance a nd determining associated factors is needed so that efficacious mitigation strategies may be developed. Thus, the overall objective of the present study was to determin e the relative contribu tions of losses during in ovo development in American alligators at imp acted sites in central Florida, and to evaluate whether organochlorine pesticid es (OCPs) are associated with adverse developmental effects and altered clutch characteristics. Materials and Methods Egg Collections and Incubation Lakes Apopka (N 28 35’, W 81 39’), Griffin (N 28 53’, W 81 46’), Emeralda Marsh Conservation Area ((N 28 55’, W 81 47’), and Lochloosa (N 29 30’, W 82 09’) in Florida were selected as collection sites because prior studies indicate vastly different levels of OCP exposure across th ese sites (Gross unpublis hed data, (Masson, 1995). Alligator nests were located via aerial (helicopter) and ground surveys (airboat), and clutches were subsequently collected by ground crews. The top of each egg was marked before eggs were removed from the nest to ensure prope r orientation; thus,

PAGE 41

25 preventing embryo mortality due to inversion. Embryo mortalit y due to inversion occurs if an embryo has attached to the top of the egg, inversion may either break embryonic attachment or cause the yolk mass to sett le on top of the embryo, crushing it. After marking each egg and placing about 5 cm of nest substrate in a uniquely numbered polypropylene pan (43 cm x 33 cm x 18 cm), all eggs found in each clutch were placed in the pan in five rows with six eggs per row. If a clutch contained more than 30 eggs, a second layer of nest substrate was added and the additi onal eggs were set. The top layer of eggs was covered with nest substrate so that there was no space left between the top of the pan and the top of the eggs (approximat ely 10 cm). Clutches were transported to the US Geological Survey’s Center for Aquatic Resources Studies, Gainesville, Florida (CARS). Upon arrival, clutches were evaluated for embryonic viability using a bright light candling procedure. Viable eggs (i.e. having a visible band) were nested in pans containing moist spha gnum moss and incubated at 30.5C and ~98% humidity, in an incubation building (7.3 m x 3.7 m). This intermediate incubation temperature will normally result in a 1:1 ma le/female sex ratio, since alligators have temperature dependent sex (or gender) diffe rentiation. One or two eggs were opened from each clutch to identify the embryonic st age of development at the time of collection, and to collect yolk samples for later measur ement of OCP burdens. From each clutch, information on the following parameters was collected: total number of eggs found per nest (fecundity); number of unbanded eggs, num ber of damaged eggs, number of dead banded eggs, number of live banded eggs, to tal clutch mass, and average egg mass of clutch.

PAGE 42

26 For years 2001 and 2002, some clutches were involved in an embryo development study. For these clutches, each clutch was even ly divided between two pans, with half of the clutch left relatively undi sturbed (except for weekly mo nitoring of embryo mortality) to determine clutch viability (the number of live hatchlings / fecundity), and the other half of the clutch used to study embryo de velopment and morphometry (Chapter 5). Analysis of OCPs in Yolk Analytical grade standards for the following compounds were purchased from the sources indicated: aldrin, al pha-benzene hexachloride ( -BHC), -BHC, lindane, -BHC, p,p’ -dichlorodiphenyldichloroethane ( p,p’ -DDD), p,p’ -dichlorodiphenyldichloroethylene ( p,p’ -DDE), dichlorodiphe nyltrichloroethane ( p,p’ -DDT), dieldrin, endosulfan, endosulfan II, endosulfan sulfate, endrin, e ndrin aldehyde, endrin ketone, heptachlor, heptachlor epoxide, hexachlorobenz ene, kepone, methoxychlor, mirex, cis -nonachlor, and trans -nonachlor from Ultra Scientific (Kingstown, RI, USA); cis -chlordane, trans chlordane, and the 525, 525.1 polychlorinated biphenyl (PCB) Mix from Supelco (Bellefonte, PA, USA); oxychlordane from Chem Service (West Chester, PA); o,p’DDD, o,p’DDE, o,p’DDT from Accustandard (New Haven, CT, USA); and toxaphene from Restek (Bellefonte, PA, USA). All reag ents were analytical grade unless otherwise indicated. Water was doubly distilled and deionized. Egg yolk samples were analyzed for OC P content using methods modified from Holstege et al. (1994) and Sc henck et al. (1994). For extraction, a 2 g tissue sample was homogenized with ~1 g of sodium sulfate a nd 8 mL of ethyl acetate. The supernatant was decanted and filtered t hough a Bchner funnel lined with Whatman #4 filter paper (Fisher Scientific, Hampton, NH, USA ) and filled to a depth of 1.25 cm with sodium sulfate. The homogenate was extracted twice with the filtrates collected together. The

PAGE 43

27 combined filtrate was concentrated to ~2 mL by rotary evaporation, and then further concentrated until solvent-free under a stre am of dry nitrogen. The residue was reconstituted in 2 mL of acetonitrile. Afte r vortexing (30 s), the supernatant was applied to a C18 solid phase extraction (SPE) car tridge (pre-conditio ned with 3 mL of acetonitrile; Agilent Technologies, Wilmingt on, DE, USA) and was allowed to pass under gravity. This procedure was repeated twice with the comb ined eluent collected in a culture tube. After the last addition, the car tridge was rinsed with 1 mL of acetonitrile which was also collected. The eluent was then applied to a 0.5 g NH2 SPE cartridge (Varian, Harbor City, CA, USA), was allowe d to pass under gravity, and collected in a graduated conical tube. The cartridge was rinsed with an additional 1 mL portion of acetonitrile which was also collected. The combined eluents were concentrated under a stream of dry nitrogen, to a volume of 300 L, and transferred to a gas chromatography (GC) vial for analysis. GC/MS Analysis Analysis of all samples was performed using a Hewlett Packard HP-6890 gas chromatograph (Wilmington, DE, USA) with a split/splitless inlet ope rated in splitless mode. The analytes were introduced in a 1 L injection and separa ted across the HP-5MS column (30 m x 0.25 mm; 0.25 m film thickne ss; J & W Scientific, Folsom, CA, USA) under a temperature program that began at 60 C, increased at 10 C/min to 270 C, was held for 5 min, then increased at 25 C/min to 300 C and was held for 5 min. Detection utilized an HP 5973 mass spectro meter in electron impact m ode. Identification for all analytes and quantitation for toxaphene was c onducted in full scan mode, where all ions are monitored. To improve sensitivity, se lected ion monitoring was used for the

PAGE 44

28 quantitation for all other analytes, except kepone. The above program was used as a screening tool for kepone which does not optim ally extract with mo st organochlorines. Samples found to contain kepone would be reex tracted and analyzed specifically for this compound. For quantitation, a five-point standard curve was prepared for each analyte ( r2 0.995). Fresh curves were analyzed with each se t of twenty samples. Each standard and sample was fortified to contain a deuterat ed internal standard, 5 L of US-108 (120 g/mL; Ultra Scientific), added just prior to analysis. All samples also contained a surrogate, 2 g/mL of tetrach loroxylene (Ultra Scientific) added after homogenization. Duplicate quality control samples were prepar ed and analyzed with every twenty samples (typically at a level of 1.00 or 2.50 g/mL of -BHC, heptachlor, aldr in, dieldrin, endrin, and p,p’ -DDT) with an acceptable recovery rangi ng from 70 – 130%. Limit of detection ranged from 0.1-1.5 ng/g for all OCP analyt es, except toxaphene (120-236 ng/g), and limit of quantitation was 1.5 ng/g for all anal ytes, except toxaphene (1500 ng/g). Repeated analyses were conducted as allo wed by matrix interferences and sample availability. Data Analysis Specific OCP analytes were removed from analysis if measurable concentrations were found in < 5% of all clutches. Numerical data were log-transf ormed [ln(x)], while proportional data were arcsine s quare root transformed to meet statistical assumptions. ANOVA (PROC GLM; SAS Institute Inc., 2002) was used for inter-site comparisons of adult female and clutch char acteristics, and the Tukey test was used for multiple comparisons among sites ( = 0.05). Because relationships between response variables and explanatory vari ables (Table 2-1) in ecologi cal studies are often complex

PAGE 45

29 with interactions occurring, an indirect gr adient multivariate analysis method, Detrended Correspondence Analysis (DCA) (ter Braak, 1986) was used to initially evaluate data structure. Two matrices were construc ted for DCA, with the first representing the response variables (clutch number x clutch parameters) and the second representing the explanatory variables (clutch number x OC P burdens) (Table 2-2). DCA results indicated that a direct gradient, multivariat e linear analysis, re dundancy analysis (RDA) (Rao, 1964), was appropriate since the gradient lengths of the DCA ordination axes were equal to or less than 2 standard deviations (ter Braak, 1995). RDA is the canonical form of principal compon ents analysis (PCA). In RDA, as in PCA, a straight line is fitted to each the re sponse variable (clutch su rvival parameters) in an attempt to explain the data of all response variables. Similar to PCA, the lower the residual sum of squares, the better the e nvironmental variable is at explaining the variation in response variables. RDA, unlike PCA, restricts the clutch scores (from the response variables measured on each clutch) to a linear combination of the environmental (explanatory variables). Because clutch scores are constrained to a linear combination of environmental variables, RDA explains slig htly less variance compared to PCA (ter Braak & Tongeren, 1995; ter Braak, 1994). Fo r RDA involving compositional data (i.e., clutch viability rates or percentages) and quantitative environmental variables, compositional data is log-transformed (ln (x + 1)) with correlation biplots being centered by the response variables (i.e., unbanded e gg percentage) and by the samples (i.e., clutches) (ter Braak, 1994). These corre lation biplots provide a way to examine relationships among a number of response variables and explanatory factors with response variable arrows forming a biplot of correlations with each other, environmental

PAGE 46

30 arrows forming a biplot among each othe r, and response variable arrows and environmental arrows forming a biplot of correlations with each other (ter Braak, 1995). For the RDA, separate matrices were cons tructed for response variables measured as a percentage (i.e., clutch viability) and re sponse variables measured as a number (i.e., clutch mass) because percentage data were ln(x+1) transformed and not standardized, while continuous data were ln (x) transformed and standardized(ter Braak & Smilauer, 2002). Automatic forward selection of the be st four explanatory variables was conducted for both sets of RDA analyses and tested for significance by Monte Carlo permutation tests. DCA and RDA were conducted us ing the program CA NOCO (ter Braak & Smilauer, 2002). Biplots of environmental variables and response variables were then constructed to interpret relationship between clutch parameters (response variables) and explanatory factors. Results Inter-Site Comparisons of Clutch Characteristics From 2000-2002, 168 clutches were collected from Lakes Lochloosa ( n = 44), Apopka ( n = 31), Griffin ( n = 47), and Emeralda Marsh ( n = 46). No significant differences were determined among sites with respect to clutch mass (overall mean standard error: 3.7 0.08 kg), egg mass (83 1.4 g), or percentage of unbanded eggs (15 1.7%) (Table 2-3). In contrast, significant diffe rences were determined am ong sites with respect to fecundity, clutch viability, pe rcentage of damaged eggs, percentage of early embryo mortality, and percentage of la te embryo mortality. Clutches from Lochloosa had lower fecundity and late embryo mortality rates co mpared to all other sites. In addition, Lochloosa clutches had greater clutch viability rates than all other sites and lower early

PAGE 47

31 embryo mortality rates than all other sites, except for Apopka. Clutches from Emeralda Marsh had greater incidence of damaged eggs than all other sites, except for those of Lake Griffin (Table 2-3). Organochlorine Pesticides Burdens and Clutch Characteristics From 2000-2002, clutch characteristics a nd OCP burdens were measured on 115 clutches collected from Lakes Lochloosa ( n = 19), Apopka ( n = 23), Griffin ( n = 42), and Emeralda Marsh ( n = 31). No significant differences were determined among sites with respect to clutch mass (overall mean standa rd error: 3.8 0.09), clutch viability (50 3.1), percentage of damaged eggs (4 1) unbanded eggs (13 1.6), early embryo mortality (21 2.3), and late embryo mort ality (11 1.7) (Table 2-4). However, significant differences were determined fo r fecundity and egg mass, with Lochloosa clutches having lower fecundity than all other sites, and greater average egg mass compared to those of all other sites, excep t for Lake Apopka. Furthermore, significant differences were detected among sites with re spect to individual OCP concentrations in egg yolks, total OCP concentrations in e gg yolks, and number of OCPs detected at measurable levels. For total OCP concentra tions and number of analytes detected at measurable levels, egg yolks of Lake Loch loosa clutches had si gnificantly lower total concentrations and a lower number of analytes de tected at measurable levels (Table 2-4). Individual OCP analyte concentrations in egg yolks of Lochloosa clutches were significantly less than those of the other sites, except for Lake Griffin with respect to aldrin and trans -nonachlor. Aldrin and trans -nonachlor egg yolk concentrations of Lochloosa clutches did not significantly diffe r from Lake Griffin, but egg burdens of these analytes of both sites were signifi cantly less than those of Lake Apopka and Emeralda Marsh (Table 2-4).

PAGE 48

32 Clutch Survival and OCP Burdens in Egg Yolks Because a number of site specific factor s may potentially affect clutch survival parameters and since OCP burdens varied greatly among sites, relationships between OCP egg yolk variables and clutch survival we re evaluated on a site-by-site basis. For Lake Lochloosa, redundancy analysis with Monte Carlo permutation tests for significance indicated that none of the four extracted OCP variables (Table 2-5) were found to be significantly correlate d with the clutch survival va riables. Number of OCPs detected (NOC) approached significance ( P = 0.07), was negatively associated with clutch viability, positively correlated with percentage unbanded eggs and late embryo mortality, and accounted for 11% of the variation in clutch surviv al parameters (Fig. 2-1). For Lake Griffin, redundancy analysis w ith Monte Carlo permutation tests for significance indicated th at three of the four extracted OCP variables were found to be significantly correlated w ith the clutch survival variable s and together accounted for 21% of the variance in clutch survival parame ters. The extracted OCP variables were concentration of p,p’ -DDE, toxaphene, and p,p’ -DDT, accounting for 8, 7, and 6%, respectively, of variation in clutch survival variables (Table 2-5). Clutch viability was positively associated with toxaphene and p,p’ -DDE egg yolk concentrations, but had little to no correlation with p,p’ -DDT yolk burdens. Early embryo mortality rates were negatively associated with p,p’ -DDE and toxaphene. Late embryo mortality rates were positively associated with toxaphene, and negatively associated with p,p’ -DDT, and p,p’ DDE. Unbanded egg rates were positively associated with p,p’ -DDT and p,p’ -DDE, but negatively associated with toxaphene (Fig. 2-2). For Lake Apopka, redundancy analysis w ith Monte Carlo permutation tests for significance also indicated that three of the four extracte d OCP variables were found to

PAGE 49

33 be significantly correlated with the clutch survival variables. These OCP variables were percentage dieldrin (lambda A = 17%), percen tage trans-chlordane (1 2%), and percentage aldrin (10%), and together accounted for 3% ( lambda A’s) of the variance in the clutch survival parameters (Table 2-5). Clutch vi ability was positively associated with aldrin, weakly associated with trans -chlordane, and negatively associ ated with dieldrin. Early embryo mortality and unbanded egg rates were positively associated with dieldrin and trans -chlordane and negatively associated with aldrin. Late embryo mortality rates were negatively with all three OC P variables (Fig. 2-3). For Emeralda Marsh, redundancy analysis with Monte Carlo permutation tests for significance also indicated that only percentage toxaphene was found to be significantly correlated with the clutch survival variables, and it accounted for 9% of the variance in the clutch survival parameters (Table 2-5). Percentage toxaphene was positively associated with clutch viability, weakly associated with late embryo mortality, and negatively associated with early embryo mo rtality and unbanded egg rates (Fig. 2-4). Percentage of heptachlor epoxide show ed a near signifi cant association ( P = 0.09) with clutch parameters, being negatively correla ted with clutch viability and positively correlated with early and late embryo mortality rates. Average Egg Mass, Clutch Size and OCP Burdens For Lochloosa clutches, three of four OC P variables were determined (via RDA analysis) to be significantly a ssociated with egg and clutch size parameters and accounted for 64% of the variation in egg and clut ch size parameters. These OCP variables included number of OCPs detected at m easurable levels (NOC) (lambda A = 31%), p,p’ DDT concentrations (20%), and trans -nonachlor concentrations (13%) (Table 2-6).

PAGE 50

34 NOC and trans -nonachlor concentrations were negatively associated with average egg mass but positively associated with f ecundity and clutch mass. In contrast, p,p’ -DDT concentrations were positively associated with egg mass, negativ ely associated with fecundity, and had little to no associ ation with clutch mass (Fig. 2-5). For Lake Griffin clutches, however, no signi ficant associations were found between OCP variables and egg and clutch size variables. In contrast, percentage o,p’ -DDT in Emeralda Marsh clutches was found to be pos itively associated with increasing egg and clutch mass but negatively associated with f ecundity. Lastly, Lake Apopka clutches were somewhat similar to Emeralda clutches in that one OCP variable ( p,p’ -DDD concentration) was found to be positively associated with egg and clutch mass and negatively associated with fecundity (Table 2-6). Discussion Inter-Site Comparisons of Clutch Characteristics The results of the present study suggested that the relative cont ributions 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. A lthough not significantly different among sites, infertility and/or embryo mo rtality before embryo attachment (unbanded eggs) also appears to be a major constituent of reduced cl utch viability among all sites. In order of importance, major constituents of reduced cl utch viability for all sites include early embryo mortality, unbanded eggs, late embryo mortality, and damaged eggs. In addition, clutches from OCP-contaminated sites had an average of 10 more eggs per clutch as compared to the reference site, but averag e clutch mass was not significantly different,

PAGE 51

35 making average egg mass of reference site clut ches greater than that of clutches of OCPcontaminated sites. The reduced clutch viability, increased rates of unbanded eggs and embryo mortality, and concurrent increa se in fecundity without propo rtional increase in clutch mass observed in clutches from OCP-contaminated sites, as compared to the reference site (Lochloosa), suggest that females and th eir embryos from contaminated sites may be responding to one or more environmental fact ors common to the three OCP-contaminated sites. Although measurement of all envir onmental factors is imp ractical, the large differences in OCP concentrations in al ligator eggs between reference and OCPcontaminated sites were found. Specifically, total OCP egg yolk burdens and number of OCPs detected at measurable levels in Lake Lochloosa were significantly less than those of Lake Griffin clutches, and OCP burdens in Lake Griffin clutches were, in turn, significantly less than those of Lake Apopka and Emeralda Marsh. Although Lake Apopka and Emeralda Ma rsh were not determined to be significantly different with respect to total OCP concentrations in egg yolks, significant differences were determined between these two high OCP exposure sites in regard to certain analytes, as well as the total number of OCPs detected at measurable levels. Clutches from Emeralda Marsh had a greater number of OCP analytes in their egg yolks and contained higher concentrations of cis-chlordane, p,p’-DDD, o,p-DDD, transchlordane, and toxaphene compared to thos e from Lake Apopka. Conversely, clutches from Lake Apopka had higher concentrations of aldrin, dieldrin, heptachlor epoxide, and oxychlordane compared to those of Emeralda Marsh.

PAGE 52

36 The differences in OCP exposure profiles am ong sites likely refl ect the differences in historic land-use and OCP applications, as opposed to differences in xenobiotic biotransformation among the different allig ator populations inha biting the respective sites. Importantly, although Emeralda Marsh is separate d from Lake Griffin by only a levee easily traversed by alligators, large differences in OCP egg burdens were noted between the two sites. Such differences in exposure suggest that the highly exposed adult females which oviposite within Emeralda Mars h likely have established territories and reside year round within Emeralda Marsh (a fo rmer agricultural property). Furthermore, the relatively high egg burdens in clutches of Emeralda Marsh likely occurred over a relatively short period since this 2,630 ha area was not flooded until the early 1990s (Marburger et al., 1999). In summary, the differences in OCP egg bur dens between the reference site and the contaminated sites support th e hypothesis that OCP contaminants may be associated with reduced clutch viability, given that OCPs have been causally linked to reduced reproductive success in other oviparous species (Donaldson & Fites, 1970; Fry, 1995). Clutch Survival Parameters and OCP Burdens Results of redundancy analyses more dire ctly addressed the question of whether OCPs are associated with reduced clutch viab ility by relationships on a site-by-site basis to control for potential site -associated confounding factors. For Lake Lochloosa, no significant correlations were determined alt hough significance might have been detected given a greater sample size. The positive bu t insignificant correlations between increases in unbanded egg and late embryo mortality perc entage and number of OCPs may suggest that increased OCP burdens in eggs play a role in clutch viability or it may simply indicate that older females ha ve increased levels of OCPs due to increased exposure time

PAGE 53

37 and that decreased clutch viability is due to decreased egg quality associated with senescence. For Emeralda Marsh, the weak associati ons between OCP variables and clutch survival variables suggests that other factor s may be involved in reduced embryo survival and increased rates of unbanded eggs. The weak associations for Emeralda Marsh are surprising given that relatively stronger associations were de termined for the other high exposure site (Lake Apopka; Table 2-5), as we ll as the intermediate exposure site (Lake Griffin, Table 2-5), with Emeralda Marsh be ing separated from La ke Griffin by only a non-fenced levee. Stronger associations were noted for La ke Apopka in contrast to the weak, associations noted for Emeralda Marsh. Th e positive association between early embryo mortality and unbanded egg rates and extracte d OCP variables for Lake Apopka clutches suggests that the percentages of dieldrin and trans -chlordane in eggs may play an important role in altered egg fertility and/or early embryo survival Interestingly, the percentage of aldrin, (dieldrin’s parent co mpound) had a negative association with late embryo mortality, a positive asso ciation with clutch viabilit y, and near-zero correlations with percentage unbanded eggs and early em bryo mortality. However, dieldrin (a metabolite formed from aldrin) had strong, positive correlations with percentage unbanded eggs and early embryo mortality, a nd a negative correla tion with clutch viability, suggesting this metabolite has gr eater efficacy than its parent compound in affecting embryo survival. Th e potential consequence exists that increasing a female alligator’s ability to biotransform aldrin to dieldrin may increase th e risk of early embryo mortality. Another important not e is that the level of diel drin in Apopka clutches was

PAGE 54

38 two-fold greater than those of Emeral da Marsh, suggesting that OCP mixture composition may be more important than sum OCP concentrations. For Lake Griffin, the negative to nea r-zero association between early embryo mortality rates and extracted OCP variables s uggests that OCP burdens in eggs may not play an important role in early embryo mort ality. However, the positive association between toxaphene burdens and late embryo mo rtality suggests that as toxaphene burdens increase, so does the risk for increased embryo death during the last 35 days of development. Furthermore, th e positive association between p,p’ -DDT concentrations and unbanded egg rates suggests that these an alytes may be involved in altered egg fertility and/or embryo survival (prior to eggshell membrane attachment) (Fig. 2-2). Egg and Clutch Size and OCP Burdens For Lochloosa, the strong associations between OCP burdens and egg and clutch size parameters suggest that, although a low OCP exposure site, certain patterns of OCP exposure are strongly associated with egg a nd clutch size characteristics. The positive associations p,p’ -DDT concentrations have with clut ch weight and average egg weight and p,p’ -DDT’s negative association with fec undity may be potentially related to senescent females, since older females have been reported to lay smaller clutches of larger eggs (Ferguson, 1985) and would likely have higher OCP burdens due to extended exposure period. In contrast, the positive associations that NOC and trans-nonachlor have with fecundity and clutch mass, and th e negative associations these OCP variables have with egg mass, suggests that increased OCP exposure may have altered clutch and egg size characteristics, as opposed to fema le age. Although these speculations are interesting from a low exposure effect sta ndpoint, they are irrelevant at the populationeffect level since clutch viability rates were unrelated.

PAGE 55

39 Since the low exposure site had stronger associations between OCP variables and egg and clutch size variables than intermed iate and high exposure sites, one might initially conclude that other factors are more important than OCP burdens in influencing egg and clutch size characteristics. While this may be the case, the fact that the intermediate and high exposure sites have significantly grea ter fecundity (averaging 10 more eggs per clutch compared to the low exposure site), signifi cantly less average egg mass, and similar clutch mass suggest that females attaining their maximum physiological response in regard to number of eggs ovulate d. These intermediate and highly exposed females appear to be produci ng more ova but are unable to sequester additional egg components (i.e., lighter eggs), in effect decreasi ng the amount of energy and structural supplies available to each embr yo and resulting in light er eggs and higher embryo mortality rates. In summary, our results suggest that, ove r all sampled clutches, clutch survival parameters and egg and clutch size paramete rs vary between the low OCP exposure site (Lochloosa) and the intermediate-high OCP e xposure sites. Furthermore, OCP burdens do not appear to be related to clutch survival for the low exposure site but are associated with clutch survival for the intermediate-hi gh OCP contaminated site s. In contrast, egg and clutch size parameters appear to be a se nsitive endpoint in OCP response in alligators due to the strong associations noted between OCP and clutch size variables for the low exposure site and the lack thereof for the intermediate-high expos ure sites, suggesting attainment of maximum response. In order to better determ ine the role of OCPs in the reduced reproductive efficiency of OCP-exposed alligator populations, suggested future studies should examine the relationship betw een maternal OCP burdens and respective

PAGE 56

40 egg burdens, presence of other environmental contaminants, maternal factors associated with clutch survival and OCP burdens, a nd how egg composition relates to clutch survival and OCP burdens.

PAGE 57

41 Table 2-1. Reproductive, morphometric, a nd contaminant parameters measured on clutches of alligator eggs collected during summer 2000, 2001, and 2002. Clutch Parameter Definition Measured as Response variables Fecundity Total No. of eggs in one clutch n Clutch mass Total mass of eggs in one clutch kg Ave. Egg Weight Clutch mass / Fecundity g Unbanded eggs% a No. of unbanded eggs / fecundity x 100 Percentage Early embryo mortality% No. of deaths < dev. Day 35 / fecundity x 100 Percentage Late embryo mortality% No. of deaths dev. Day 35 / fecundity x 100 Percentage Clutch Viability No. eggs yielding live hatchling / fecundity x 100 Percentage Explanatory variables [OCP analyte] in egg yolk ng OCP analyte / g egg yolk wet weight ppb % OCP analyte [OCP analyte] / [OCP] x 100 Percentage aAn egg with no evidence of embryonic attachment

PAGE 58

42 Table 2-2. Explanatory variab les included in RDA with forw ard selection of four best variables ( = 0.05). Variable Code Age Age No. OCPs at measurable levels NOC [OCP] TOC % Aldrin ALD% [Aldrin] [ALD] % cis -Chlordane CC% [ cis -Chlordane] [CC] % cis -Nonachlor CN% [ cis -Nonachlor] [CN] % Dieldrin DL% [Dieldrin] [DL] % Heptochlor epoxide HE% [Heptachlor epoxide] [HE] %Lipid content LPC% % Mirex MX% [Mirex] [MX] % o,p -DDT ODDT% [ o,p -DDT] [ODDT] % o,p -DDD ODDD% [ o,p -DDD] [ODDD] % Oxychlordane OX% [Oxychlordane] [OX] % p,p '-DDE PDDE% [ p,p '-DDE] [PDDE] % p,p '-DDD PDDD% [ p,p '-DDD] [PDDD] % p,p '-DDT PDDT% [ p,p '-DDT] [PDDT] % trans -Chlordane TC% trans -Chlordane [TC] % trans -Nonachlor TN% [ trans -Nonachlor] [TN] % Toxaphene TX% [Toxaphene] [TX]

PAGE 59

43Table 2-3. Summary of clutch parameters and site comparisons for clutches of American alligator eggs collected during 2000-200 2. Parametera Lochloosa Apopka Emeralda Griffin Summary No. Clutches 44 31 46 47 168 Fecundity ( n ) 36 1.2 B 46 1.3 A 46 1.1 A 45 1.2 A 43 0.7 (22–56) (28–56) (27–64) (19–58) (19–64) Clutch mass (kg) 3.4 0.15 4 0.13 3.8 0.21 3.6 0.13 3.7 0.08 (1.6–4.8) (2.4–5.1) (1.9–9.2) (1.5–5.2) (1.5–9.2) Egg mass (g) 87 2.2 86 2 83 4 80 1.6 83 1.4 (61–139) (62–120) (58–180) (46–113) (46–180) Clutch viability (%) 70 3.9 A 51 5.8 B 48 5.5 B 44 4.9 B 53 2.6 (0–100) (0–98) (0–97) (0–92) (0–100) Damaged eggs (%) 2 1.4 B 2 0.6 B 5 1.3 A 4 1.8 AB 3 0.7 (0–60) (0–16) (0–33) (0–63) (0–63) Unbanded eggs (%) 11 2.2 21 4.9 14 3.7 17 3.2 15 1.7 (0–84) (0–100) (0–100) (0–100) (0–100) Early Emb. Mort. (%) 12 2.7 B 15 4.2 AB 23 3.9 A 22 3.9 A 19 2 (0–69) (0–94) (0–95) (0–100) (0–100) Late Emb. Mort. (%) 6 1.7 B 12 3.5 A 10 2.4 A 13 3.1 A 11 1.4 (0–34) (0–77) (0–82) (0–89) (0–89) aValues indicate mean standard error of m ean with ranges in parentheses. Values w ith different letters (A-B) indicate signifi cant differences ( = 0.05); same letters indicate significant differences were not detected. Clutch viability = No. of eggs yielding a live hatchling / Fecundity x 100, Damaged eggs = No. damaged eggs / fecundity x 100, Unbanded eggs = No. of unbanded eggs / fecundity x 100, Early Emb. Mort. = No. of em bryonic deaths on or before developmenta l Day 35 / fecundity x 100, and Late Emb. Mort. = No. of embryonic deaths post dev. Day 35 / fecundity x 100).

PAGE 60

44Table 2-4. Organochlorine pesticide burdens and clutch parameters and site comparisons for clutches of American alligator eggs collected during 2000-2002. Parametera Loch. Apopka Emeralda Griffin Summary No. Clutches 19 23 31 42 115 Fecundity ( n ) 40 1.7 B 47 1.4 A 46 1.3 A 46 1.2 A 45 0.7 A (26–56) (31–56) (27–64) (24–58) (24–64) Clutch mass (kg) 3.6 0.17 4 0.16 3.8 0.25 3.7 0.13 3.8 0.09 (2.2–4.8) (2.5–5.1) (2.1–9.2) (1.5–5.2) (1.5–9.2) Egg mass (g) 90 2.9 A 86 2.5 AB 82 4.8 B 79 1.5 B 83 1.6 (78–139) (62–120) (58–180) (46–105) (46–180) Clutch viability (%) 65 5.5 52 6.4 50 6.9 43 5.1 50 3.1 (0–95) (0–98) (0–97) (0–92) (0–98) Damaged eggs (%) 4 3.1 2 0.8 6 1.6 5 2 4 1 (0–60) (0–16) (0–32) (0–63) (0–63) Unbanded eggs (%) 11 2 17 4.2 10 2.3 15 3.2 13 1.6 (0–33) (0–81) (0–58) (0–100) (0–100) Early Emb. Mort. (%) 13 3 15 4.2 26 5.1 24 4.3 21 2.3 (0–36) (0–90) (0–95) (0–100) (0–100) Late Emb. Mort. (%) 8 2.5 14 4.6 10 2.5 13 3.3 11 1.7 (0–34) (0–77) (0–61) (0–89) (0–89) Aldrin (ng/g) 0 0 C 4 0.3 A 2 0.3 B 0 0 C 3 0.3 (0–0) (2.9–5.2) (1.5–4.3) (0–0) (1.5–5.2) Methoxychlor (ng/g) 0 0 C 8 1 B 9 1 B 17 0.3 A 9 0.8 (0–0) (5.7–16.4) (5.8–18.4) (16.9–17.5) (5.7–18.4)

PAGE 61

45Table. 2-4. Continued. Parametera Orange/Loch Apopka Emer alda Griffin Summary Mirex (ng/g) 2 0.4 B 6 1.1 A 3 0.5 AB 3 0.2 AB 4 0.4 (1.2–2.7) (1.1–17.2) (0.1–10.3) (1.1–4.5) (0.1–17.2) Dieldrin (ng/g) 4 0.5 D 344 80.9 A 142 20.4 B 23 3.8 C 118 20.9 (1.3–8.2) (12.5–1783.2) (8.7–386.7) (2.9–124) (1.3–1783.2) Hep. Epoxide (ng/g) 3 0.8 C 17 5.6 A 7 1.4 B 7 1 B 8 1.4 (1.2–9.7) (1.2–135.5) (0.1–32.1) (1.1–29.6) (0.1–135.5) cis-Chlordane (ng/g) 2 0.2 D 43 7.6 B 90 13 A 11 0.9 C 37 5 (1.2–4.1) (6.6–179.2) (8.9–281) (4.3–31.8) (1.2–281) cis-Nonachlor (ng/g) 5 0.6 C 88 27.3 A 66 9.7 A 18 1.6 B 43 6.7 (2.4–12.5) (10.5–656.2) (11.6–232.2) (6.5–54.2) (2.4–656.2) Oxychlordane (ng/g) 4 1 D 51 14.6 A 23 3.8 B 10 1.3 C 21 3.4 (1.2–17.8) (3.9–353.8) (3.2–109.5) (1.1–41.9) (1.1–353.8) p,p' -DDE (ng/g) 74 11.7 C 5794 1794.7 A 8069 1402 A 271 31.3 B 3445 610.6 (28–231) (18.3–42653.4) (36.2–33554.8) (62.9–979.1) (18.3–42653.4) p,p'-DDD (ng/g) 2 0.2 D 42 8.5 B 1289 196.1 A 7 0.9 C 382 78.7 (1.2–3) (10.6–192.8) (10.3–2962.8) (2.7–28.9) (1.2–2962.8) p,p'-DDT (ng/g) 1 0 C 9 2.1 AB 12 1.2 A 5 0.8 B 10 1 (1.2–1.3) (1.2–45.6) (5.8–25.5) (1.1–7.2) (1.1–45.6) o,p'-DDD (ng/g) 0 0 C 5 0.7 B 37 5.1 A 1 0 B 29 4.5 (0–0) (3.1–9.2) (0.1–104) (1.3–1.3) (0.1–104) o,p'-DDT (ng/g) 1 0 C 11 1.9 A 170 161.6 A 4 0.3 B 48 42 (1.2–1.4) (1.2–38.5) (4.2–4372.8) (1.1–7.4) (1.1–4372.8)

PAGE 62

46Table. 2-4. Continued. Parametera Orange/Loch Apopka Emer alda Griffin Summary trans-Chlordane (ng/g) 3 0.7 C 8 1.5 B 25 3.3 A 2 0.2 C 11 1.5 (1.2–3.7) (1.3–27.4) (2.9–58.2) (1.1–8.7) (1.1–58.2) Toxaphene (ng/g) 0 0 C 2738 224.5 B 6865 552.4 A 3043 425.9 B 5456 483 (0–0) (1896.1–3809.1) (2300.6–12975.4) (1927.9–4533.2) (1896.1–12975.4) trans-Nonachlor (ng/g) 8 1.6 C 212 66.9 A 191 30.5 A 36 4.7 B 108 17.5 (2.5–24.6) (10.5–1569.2) (14.2–718.6) (8.6–155.2) (2.5–1569.2) OCPs (ng/g) 102 15.5 C 7582 2008.2 A 15480 2265.4 A 1169 422.8 B 6133 940.8 (42.7–289.4) (472.5–47333.8) (269.6–53559.7) (101.5–16795.4) (42.7–53559.7) No. OCPs 9 0.3 D 13 0.3 B 14 0.2 A 11 0.1 C 12 0.2 (7–11) (10–16) (13–17) (9–13) (7–17)

PAGE 63

47 Table 2-5. Results of RDA evaluating associat ions between clutch survival parameters and OCP variables. Site Variablea LambdaA P F Lochloosa NOC 0.11 0.074 2.25 [DL] 0.09 0.194 1.59 PDDT% 0.08 0.166 1.72 PDDE% 0.11 0.104 2.45 Apopka DL% 0.17 0.004 4.25 TC% 0.12 0.024 3.32 ALD% 0.10 0.042 3.16 LPC% 0.06 0.16 1.85 Emeralda Marsh TX% 0.09 0.044 2.99 HE% 0.06 0.09 2.27 ME% 0.06 0.15 1.85 [HE] 0.06 0.15 1.89 Griffin [PDDE] 0.08 0.024 3.67 [TX] 0.07 0.016 3.16 [PDDT] 0.06 0.04 2.71 [ODDD] 0.04 0.09 1.96 aSee Table 2-2 for definition of variable codes.

PAGE 64

48 Table 2-6. Results of RDA evaluating asso ciations between egg and clutch size parameters and OCP variables. Site Variable LambdaA P F Lochloosa NOC 0.31 0.004 10.15 [PDDT] 0.20 0.042 4.29 [TN] 0.13 0.006 6.77 OX% 0.08 0.088 2.8 Griffin PDDD% 0.05 0.134 2.32 [ODDT] 0.03 0.406 0.91 [PDDT] 0.02 0.236 0.95 [CC] 0.01 0.54 0.33 Emeralda [ODDT] 0.22 0.01 8.07 CC% 0.05 0.146 2 ODDT% 0.05 0.182 1.82 LPC% 0.04 0.21 1.7 Apopka [PDDD] 0.24 0.01 6.51 [ME] 0.08 0.112 2.54 [PDDT] 0.05 0.218 1.5 PDDE% 0.05 0.294 1.29

PAGE 65

49 -0.80.6-0.60.6 Clutch viability Unbanded egg% Early Emb. Mort. Late Emb. Mort. NOC [DL] PDDE% PDDT% A B Figure 2-1. Biplot of clutch survival parameters (solid lines) and organochlorine pesticide variables (dashed lines) for clutches of alligator eggs collected from Lake Lochloosa during summer 2001-2002. Arrows pointing in the same direction indicate a positiv e correlation (e.g., clutch viability and PDDE%), arrows that are approximately perpendicular indicate near-zero correlation (e.g., late emb. mort. and [DL]), and a rrows pointing in opposite directions indicate negative correlations (e.g., clut ch viability and [DL]. Arrow lengths indicate rank order of correlations. For example, late emb. mort. has higher positive correlation with NOC (A) compared to unbanded egg% (B). Cosine of angle formed between individual clutch variables and individual OCP variables (see Table 2-2 for code defin itions) equals correlation coefficient (r) (ter Braak, 1995). For example, arrows pointing in exactly opposite directions have an angle of 180, and since cos( 180) = -1.0, the arro ws are perfectly, negatively correlated (r ) (ter Braak, 1995).

PAGE 66

50 -1.01.0-0.80.6 Clutch Viability Unbanded egg% Early Emb. Mort. Late Emb. Mort. [ODDD] [PDDE] [PDDT] [TX] Figure 2-2. Biplot of clutch survival parameters (solid lines) and organochlorine pesticide variables (dashed lines) for clutches of alligator eggs collected from Lake Griffin during summer 2000-2002.

PAGE 67

51 -1.01.0-0.80.8 Clutch Viability Unbanded egg% Early Emb. Mort. Late Emb. Mort. LPC% ALD% DL% TC% Figure 2-3. Biplot of clutch survival parameters (solid lines) and organochlorine pesticide variables (dashed lines) for clutches of alligator eggs collected from Lake Apopka during summer 2000-2002.

PAGE 68

52 -0.80.6-0.60.6 Clutch Viability Unbanded egg% Early Emb. Mort. Late Emb. Mort. [HE] HE% ME% TX% Figure 2-4. Biplot of clutch survival parameters (solid lines) and organochlorine pesticide variables (dashed lines) for clutches of alligator eggs collected from Emeralda Marsh during summer 2000-2002.

PAGE 69

53 -1.01.0-0.60.4 Clutch Mass Egg Mass Fecundity NOC [PDDT] [TN] OX% Figure 2-5. Biplot of egg a nd clutch size parameters (sol id lines) and organochlorine pesticide variables (dashed lines) for clutches of alligator eggs collected from Lake Lochloosa during summer 2001 and 2002.

PAGE 70

54 CHAPTER 3 MATERNAL TRANSFER OF ORGANOCHLORINE PESTICIDES Studies have documented organochlorine pe sticide (OCP residues) in eggs and/or somatic tissues of several crocodilian species including the American alligator, Alligator mississippiensis (Heinz et al., 1991), Morelet’s crocodile, C moreletti (Wu et al., 2000a), the American crocodile, Crocodylus acutus (Hall et al., 1979; Wu et al., 2000b), and the Nile crocodile, C niloticus (Skaare et al., 1991). Indeed, alligator populations inhabiting Lake Apopka, where an OCP spill occurred in the 1980s, and other central Florida lakes contaminated with OCPs (through historic OCP use) produce eggs that contain concentrations of total OCPs that are over 100 times higher than concentrations found in eggs from reference lakes (Gross, unpublis hed data). In addition, the alligator populations inhabiting the OCP-contaminated lakes experience increased (and highly variable) rates of embryonic mortality, leading to reduced clutch success, and juvenile alligators appeared to have abnormal sex hormo ne concentrations as compared to those of reference sites (Masson, 1995; Rice, 1996; W oodward et al., 1993). However, a clear dose-response relationship has not been estab lished with respect to individual or total OCP concentrations in egg yolks and reduced clutch success (Heinz et al., 1991). The lack of a clear dose-response suggests othe r factors (e.g., diet, population dynamics, and specific OCP mixtures) might be involved and/or that developmental effects result from altered maternal physiology resulting from OCP exposure, as opposed to direct embryotoxicity.

PAGE 71

55 With respect to altered maternal physiol ogy, alterations in steroid hormone levels have also been shown in alligators inhabiti ng OCP-contaminated sites (Guillette et al., 1994). Furthermore, maternal exposure s uggests that OCPs may be maternally transferred from the adult female alligator to her offspring, as has been reported in other oviparous vertebrates (Russell et al., 1999). Assuming OCPs are maternally transferred, the possibility exists that yolks could be used as predictors of maternal exposure. A noninvasive method such as this would aid eco logical risk assessmen ts in understanding exposure levels for rare/endangered crocodilian species without having to capture and/or remove adults from the breeding population. Therefore, the objectives of the present study were to examine maternal transfer as a potential route for embryonic OCP exposure, and to evaluate the use of yolk bur dens for predicting OCP burdens in maternal tissues in alligators. Our hypothesis was that OCP burdens in maternal tissues and yolks would be strongly correlated, which would a llow yolk burdens to be used to predict maternal body burdens and suggest maternal tr ansfer of OCPs as the major route for embryonic OCP exposure. Materials and Methods Site descriptions Lakes Apopka (N 28 35’, W 81 39’), Griffin (N 28 53’, W 81 49’), and Lochloosa (N 29 30’, W 82 09’) in Florida were selected as co llection sites because prior studies by our laboratory indicate vastly different le vels of OCP exposure across these sites. All three lakes are part of th e Ocklawaha Basin. Lake Lochloosa (which is connected to Orange Lake) was selected as a low exposure (reference) site. Four years (1999-2002) of data indicate mean total OCP concentrations in egg yolks from the reference sites (Lakes Orange and Loch loosa) were 231 30 ppb (mean standard

PAGE 72

56 deviation [SD], n = 56 clutches) with a concurrent mean clutch viability rate (number of live hatchlings/total number of eggs in a ne st) of 71 21% (Gross, unpublished data). Lake Griffin was selected as an intermedia te exposure site since yolk concentrations averaged 4,414 617 ppb ( n = 47 clutches) and Lake A popka was selected as a high exposure site since yolk concen trations averaged 15,911 1,786 ppb ( n = 42) for the same time period (Gross, unpublished data). Furthermore, mean clutch viability rates during this time period for Lakes Apopka (51 31%, n = 42) and Griffin (44 33%, n = 47) have been below rates observed for the reference site. Animal Collections Adult female alligators and their corres ponding clutches of eggs were collected from Lakes Apopka ( n = 4), Griffin ( n = 8), and Lochloosa ( n = 3) over the course of two nesting seasons (June 2001 and June 2002). Nests were located by aerial survey (helicopter) and/or from the ground (airboat). Once nests were located, all eggs were collected, and the nest cavity was covered. A snare-trap was set perpendicular to the taildrag in order to capture the female as she cr ossed over the nest. After the traps were set, one member of the trapping crew subsequently transported the eggs to the Florida Fish and Wildlife Conservation Commission’s Wild life Research Unit (FWC; Gainesville, FL, USA) and placed the eggs in a temperaturecontrolled incubator. Snare-traps were checked later in the evening a nd early the next morning. Trapped females were secured and transported from each lake to the United States Geological Survey’s Florida In tegrated Science Center (USGS; Gainesville, FL, USA). Upon arrival, the animals were weighed, m easured, and blood samples were collected from the post-occipital sinus. Adult alligators were then euthanized by cervical dislocation followed by double pithing. A full necropsy was performed on each female.

PAGE 73

57 Bile, liver, adipose (composite of abdomina l fat and the abdominal fat pad), and tail muscle samples were collected for later dete rmination of OCP burdens. Liver, adipose tissue, and muscle were wrapped in aluminum foil, while bile and blood were placed in scintillation vials. All sa mples were grouped according to nest identification number (ID), placed in plastic bags labeled with the appropri ate ID, and stored in a –80 oC freezer. Each female’s corresponding clutch of eggs was then transferred from FWC to USGS where yolk samples were collected (t wo eggs/clutch) and stored with the corresponding maternal tissues. The remain ing eggs were set for incubation in a temperature/humidity-contr olled incubator (31-33 oC, 88-92% relative humidity) located at USGS. Analysis of OCPs in Ma ternal Tissues and Yolk Analytical grade standards for the following compounds were purchased from the sources indicated: aldrin, al pha-benzene hexachloride ( -BHC), -BHC, lindane, -BHC, p,p’ -dichlorodiphenyldichloroethane ( p,p’ -DDD), p,p’ -dichlorodiphenyldichloroethylene ( p,p’ -DDE), dichlorodiphe nyltrichloroethane ( p,p’ -DDT), dieldrin, endosulfan, endosulfan II, endosulfan sulfate, endrin, e ndrin aldehyde, endrin ketone, heptachlor, heptachlor epoxide, hexachlorobenz ene, kepone, methoxychlor, mirex, cis -nonachlor, and trans -nonachlor from Ultra Scientific (Kingstown, RI, USA); cis -chlordane, trans chlordane, and the 525, 525.1 polychlorinated biphenyl (PCB) Mix from Supelco (Bellefonte, PA, USA); oxychlordane from Chem Service (West Chester, PA); o,p’DDD, o,p’DDE, o,p’DDT from Accustandard (New Haven, CT, USA); and toxaphene from Restek (Bellefonte, PA, USA). All reag ents were analytical grade unless otherwise indicated. Water was doubly distilled and deionized.

PAGE 74

58 Adipose, liver, bile, and yolk samples were analyzed for OCP content using methods modified from Holste ge et al. (1994 and Schenck et al. (1994). For extraction, a 2 g tissue sample was homogenized with ~1 g of sodium sulfate and 8 mL of ethyl acetate. The supernatant was decanted and filtered though a Bchner funnel lined with Whatman #4 filter paper (Fisher Scientific, Ha mpton, NH, USA ) and filled to a depth of 1.25 cm with sodium sulfate. The homogenate was extracted twice with the filtrates collected together. The combined filtrat e was concentrated to ~2 mL by rotary evaporation, and then further concentrated until solvent-free unde r a stream of dry nitrogen. The residue was reconstituted in 2 mL of acetonitrile. After vortexing (30 s), the supernatant was applied to a C18 solid phase extrac tion (SPE) cartridge (preconditioned with 3 mL of acetonitrile; Agile nt Technologies, Wilmi ngton, DE, USA) and was allowed to pass under gravity. This proced ure was repeated twice with the combined eluent collected in a culture t ube. After the last addition, th e cartridge was rinsed with 1 mL of acetonitrile which was also collected. The eluent was then applied to a 0.5 g NH2 SPE cartridge (Varian, Harbor City, CA, US A), was allowed to pass under gravity, and collected in a graduated conical tube. The car tridge was rinsed with an additional 1 mL portion of acetonitrile whic h was also collected. The combined eluents were concentrated under a stream of dry nitrogen, to a volume of 300 L, and transferred to a gas chromotography (GC) vial for analysis. Whole blood was analyzed for OCP c ontent using methods modified from Guillette et al. (1999). A 10 mL aliquot was transferred from the homogenized bulk sample and extracted in 15 mL of acetone by vortex mixer. The mixture was centrifuged for 5 min at 3000 rpm, after which the supernatan t was transferred to a clean culture tube.

PAGE 75

59 This process was repeated with the supern atants collected and concentrated under a stream of dry nitrogen until solvent-free. Th e residue was re-extract ed in 11.5 mL of 1:1 methylene chloride-petroleum ether. After mixing, the sample was allowed to settle and the upper layer was tran sferred to a clean culture tube. This extraction was performed twice with the extracts collected together. Th e combined extracts we re then applied to a prepared florisil cartridge (5 mL Fisher Pr epSep, Fisher Scientific, Hampton, NH, USA). The cartridge had been prepared by filling the reservoir to a depth of 1.25 cm with anhydrous sodium sulfate and by prewashing the modified cartridge with 10 mL of 2:1:1 acetone: methylene chloride: petroleum ether. After the sample passed under gravity with the eluent collected in a 15-mL graduate d conical tube, the cart ridge was eluted with 4 mL of the 2:1:1 solv ent mixture which was also collected. The combined eluents were concentrated under a stream of dry nitrogen, to a volume of 300 L, and transferred to a GC vial for analysis. GC/MS Analysis Analysis of all samples was performed using a Hewlett Packard HP-6890 gas chromatograph (Wilmington, DE, USA) with a split/splitless inlet ope rated in splitless mode. The analytes were introduced in a 1 L injection and separa ted across the HP-5MS column (30 m x 0.25 mm; 0.25 m film thickne ss; J & W Scientific, Folsom, CA, USA) under a temperature program that began at 60 C, increased at 10 C/min to 270 C, was held for 5 min, then increased at 25 C/min to 300 C and was held for 5 min. Detection utilized an HP 5973 mass spectro meter in electron impact m ode. Identification for all analytes and quantitation for toxaphene was c onducted in full scan mode, where all ions are monitored. To improve sensitivity, se lected ion monitoring was used for the

PAGE 76

60 quantitation for all other analytes, except kepone. The above program was used as a screening tool for kepone which does not optim ally extract with mo st organochlorines. Samples found to contain kepone would be reex tracted and analyzed specifically for this compound. For quantitation, a five-point standard curve was prepared for each analyte ( r2 0.995). Fresh curves were analyzed with each se t of twenty samples. Each standard and sample was fortified to contain a deuterat ed internal standard, 5 L of US-108 (120 g/mL; Ultra Scientific), added just prior to analysis. All samples also contained a surrogate, 2 g/mL of tetrach loroxylene (Ultra Scientific) added after homogenization. Duplicate quality control samples were prepar ed and analyzed with every twenty samples (typically at a level of 1.00 or 2.50 g/mL of -BHC, heptachlor, aldr in, dieldrin, endrin, and p,p’ -DDT) with an acceptable recovery rangi ng from 70 – 130%. Limit of detection ranged from 0.1-1.5 ng/g for all OCP analyt es, except toxaphene (120-236 ng/g), and limit of quantitation was 1.5 ng/g for all anal ytes, except toxaphene (1500 ng/g). Repeated analyses were conducted as allo wed by matrix interferences and sample availability. Data Analysis OCP concentrations in maternal tissues and egg yolks were lipid-adjusted (wet weight concentration / proportion of lipid in tissue), and lipidadjusted tissue-to-egg yolk ratios (maternal tissue OCP concentrations /egg OCP concentrations) were examined. Predictive models were determined by linear regression analysis of OCP concentrations in yolk against those of maternal tissues (log -transformed wet weight concentrations). Each model’s ability to fit the data was evaluated by exam ining the p-value ( = 0.05), the r2 value and the residual plots (SAS Institute Inc., 2002). ANOVA was used for

PAGE 77

61 inter-site comparisons of adult female and clut ch characteristics, and the Tukey test was used for multiple comparisons among sites. The relationship between maternal mass (kg) and concentrations of OCPs in eggs and ma ternal tissues (log-transformed wet weight concentrations) were evaluated using linear regression to assess whether increasing mass was associated with increasing concentrations of OCPs in eggs and maternal tissues, which may suggest adult females continue to bioaccumulate OCPs as they grow throughout their life. Adult females were gr ouped by site since the extreme differences in OCP exposure among sites would likely confound results. Unless otherwise noted, values are reported as mean standard deviation. Results Female Morphological and Re productive Characteristics For all females, mass and snout-vent length (SVL) averaged 74 20 kg (range: 44-114) and 135 11 cm (119-156), respectively. Clutch mass (mass of all eggs from a single nest) and fecundity (number of eggs collected from a single nest) of these individuals were 3.65 0.86 kg ( 1.84-4.82) and 43 10 eggs/nest (19-56), respectively. No significant differences were detected across sites with respect to female mass (p = 0.14), total length (p = 0.90), SVL (p = 0.25), tail girth (p = 0.98), head length (p = 0.55), clutch mass (p = 0.23), or fecundity (p = 0.40, Table 1). With respect to lipid concentrations in egg yolk and muscle, no significant differences were detected across sites (p > 0.05) However, lipid concentration in liver of Lochloosa females was significantly higher (p < 0.05) than that of Apopka and Griffin females (which were not significantly different from one another). Furthermore, lipid concentration in abdominal adipose tissue of Apopka females was si gnificantly less (p < 0.05) than that of Lochloosa and Griffin females (Table 1).

PAGE 78

62 OCP concentrations in Yolk Egg yolks from Lake Apopka female s contained the highest total OCP concentration (15,108 13,704) and greatest number of individual OCPs detected above the limit of quantitation (n = 18) with p,p ’-DDE (66%) and toxaphene (32%) being main constituents. Lake Griffin females produced eggs with the next highest total OCP burdens (393 300 ng/g; n = 13) being main ly composed of p,p’-DDE (69%), transnonachlor (10%), and dieldrin (7%). Lake Lochloosa females produ ced egg yolks with the smallest total OCP burden (124 53 ng/g, n = 9), with main constituents being p,p’DDE (73%), trans-nonachlor (10%), and ci s-nonachlor (4%; Table 3-2). The OCP analytes with the highest average egg yolk concentrations were toxaphene (4,862 4,177 ng/g), which was detected above the limit of quantitation in 3 of 15 clutches, followed by p, p’-DDE (2,828 5,968 ng/g), dieldrin ( 191 474 ng/g), and trans-nonachlor (126 209 ng/g), which were above quantita tion limit in all 15 clutches. OCP concentrations in maternal tissues Adipose tissue (a composite of abdominal fat and fat pad) contained the highest concentration of total OCPs (12,805 31,678 ng/ g wet weight) of all tissues. p,p’-DDE (67%) composed the majority of the total burden, followed by dieldrin (5%), and transnonachlor (3%). Although toxaphene was only detected in 3 individuals from Lake Apopka, its average burden in adipose tis sue was 13,463 1,267 ng/g (Table 3-2). In liver, OCP analytes were detected above the quantitation limit in 9 of 15 individuals, and total OCP concentrations averaged 1,008 1,245 ng/g. Liver burdens were primarily composed of p,p’-DDE (76%) and dieldrin (6 %). Total OCP concentrations in muscle averaged 716 1,053 ng/g and were above qua ntitation limits in 10 of 15 individuals

PAGE 79

63 with most of the burden being composed of p,p’-DDE (83%), dieldrin (6%) and transnonachlor (6%). Total OCP burdens in bile (412 483 ng/g) were above quantitation limits in five individuals with p,p’-DDE (86 %) and dieldrin (6%) comprising the majority of the burden. Total OCP concentrations in blood (43 21 ng/g) we re above quantitation limits in 4 individuals with p,p’-DDE (64%) and dieldrin (14%) comprising most of the burden. Overall, Lake Apopka alligators ex hibited the highest OCP concentrations in maternal tissues and egg yolks, followed by Lakes Griffin and Lochloosa, respectively (Table 3-2). Relationships between Maternal Tissue and Yolk Burdens Examination of lipid-adjusted matern al tissue-to-egg yolk burdens showed differences among tissues. With respect to total OCPs, the adipose burden-yolk burden ratio was close to 1 (95% confidence interval (CI), 0.76 1.11). In contrast, the liver-yolk ratio was si gnificantly greater than 1 (95% CI, 1.49 9.19), and muscle ratios showed considerable variation (95% CI, -1.17 37.35). As would be expected, most individual OCPs follo wed the above trend. Howeve r, cis-chlordane was an exception as liver ratios (95% CI, 2.85 6.75) and muscle ratios (95% CI, 1.78 15.1) were greater than 1, while adipose ratios (95% CI, 0.59 0.84) were less than 1. With respect to total OCP concentrations significant linear relationships (predictive models) were found for adipose, liver, muscle, and bile (p 0.05, Fig. 1). With respect to individual OCP analytes, predictive models we re derived for 12 of 14 (78%) of the OCPs co-detected in adipose tissue and egg yolk, followed by liver (9/12, 75%), bile (8/11, 73%), and muscle (2/12, 17%; Table 3-3) Although nine OCP analytes were concurrently detected in blood of the females and their respective egg yolks, no significant linear correlations we re detected (p > 0.05).

PAGE 80

64 As for individual OCP analytes, p,p’-DDE c oncentrations in yolk was significantly correlated with those of liver, muscle, bile, and adipose tissue. Blood p,p’-DDE concentrations did not exhib it a significant linear relations hip (p > 0.05) with yolk p,p’DDE concentrations. Heptachlor epoxide, trans-chlordane, cis-chlordane, transnonachlor, cis-nonachlor, mirex, and dieldrin concentrations in yol k were significantly correlated to their respective concentrations in adipose, liver, and bile. With respect to oxychlordane, significant correl ations were only derived for liver and adipose tissue, and significant correlations for p,p’-DDD concentr ations were found only for adipose and bile. Toxaphene and o,p’-DDT concentrati ons in adipose tissue were significantly correlated with respective egg yol k concentrations (Table 3-3). Relationships between Maternal Mass and OC P concentrations in Eggs and Tissues For females collected from Lakes Apopka (n = 4) and Lochloosa (n = 3), no significant correlations (p > 0.05) were found when maternal mass (kg) was compared against either individual or total OCP con centrations (log-transformed wet weight) in maternal tissues and eggs. However, significan t correlations might have been difficult to detect because of the small sample size. In contrast, a larger nu mber of Lake Griffin females (n = 8) were collected, and analys es indicated significan t correlations between maternal mass and OCP concentrations in tissu es and eggs indicating that larger females have higher concentrations of OCPs in their tissues and eggs, which may suggest females continue to bioaccumulate OCPs as they gr ow (increase in mass). For Lake Griffin females, OCP burdens in eggs had the grea test number of significant correlations (p 0.05) with body mass (kg), which consisted of cis-nonachlor (r2 = 0.87), cis-chlordane (r2 = 0.75), trans-nonachlor (r2 = 0.73), dieldrin (r2 = 0.69), p,p’-DDE (r2 = 0.66), o,p’-DDT (r2 = 0.61), heptachlor epoxide (r2 = 0.59), oxychlordane (r2 = 0.58), trans-chlordane (r2 =

PAGE 81

65 0.57), and total OCPs (r2 = 0.71). Following egg concen trations, abdominal fat OCP burdens-to-body mass correlations consisted of cis-nonachlor (r2 = 0.67), cis-chlordane (r2 = 0.81), trans-nonachlor (r2 = 0.63), dieldrin (r2 = 0.62), p,p’-DDE (r2 = 0.58), heptachlor epoxide (r2 = 0.53), oxychlordane (r2 = 0.51), and total OCPs (r2= 0.64). Although egg burdens of o,p’-DDT and transchlordane were correlated with body mass, abdominal fat burdens were not. Lastl y, liver OCP burdens-to-body mass correlations included only trans-nonachlor (r2= 0.99) and p,p’-DDT (r2= 0.99). No significant correlations were found for ci s-chlordane, trans-chlordan e, oxychlordane, dieldrin, heptachlor epoxide, o,p’-DDT, and cis-nonachlor. Discussion The presence of OCPs in the eggs and tissu es of alligators is not novel; however, the value of our study was that OCP concen trations in maternal tissues and yolks appeared to be strongly correla ted with one another, allowing yolk burdens to be used as predictors of OCP burdens in tissues of adult reproductive alligators, which may be a useful noninvasive technique that would aid risk assessments involving endangered crocodilians. Furthermore, our results are cons istent with other stud ies that suggest OCPs are maternally transferred in wild alligators (Rausche nberger et al., 2004). Several OCP analytes were detected in both maternal tissues and yolk (Table 3-3) suggesting that mixture composition may be an important consideration in risk assessment. One reason for this is that different xenobiotic compounds may induce or inhibit certain biotransformation enzymes. Specifically, alligators from Louisiana express several different xenobi otic biotransformation enzymes (e.g., liver cytochrome P450 enzymes [CYP] such as CYP1A, CYP2B) in response to xenobiotic exposure (Ertl et al., 1999). Furthermore, genetic partitioning has been reported in spatially separated

PAGE 82

66 alligator populations (Ryberg et al., 2002). Therefore, the po ssibility exists that certain individuals or populations may lack the ge netic or epigenetic ability to produce a particular biotransformation enzyme, which may lead to increased risk of xenobioticinduced toxicity. For example, certai n populations of black -banded rainbowfish ( Melanotaenia nigrans ) were able to tolerate copper exposures (96-hr EC50) that were 8.3 fold greater than the toleran ce limits of other, spatially-sep arated populations of the same species. Genetic analyses sugge sted that allozyme frequencies of tolerant and susceptible populations were significantly di fferent at AAT-1 and GPI-1 loci, suggesting differences in allozymes of exposed fish may have a ssisted in the increas ed copper tolerance (Woosley, 1996). Examination of maternal tissue-to-egg con centration ratios (lipid-adjusted) showed differences among tissues. The adipose-toyolk concentration ratio was close to 1, suggesting that OCPs reach equilibrium within abdominal adipose tissue, and that lipids and OCPs are mobilized and subsequently in corporated into the developing yolks. In contrast, liver-to-yolk concentr ation ratios were significantly greater than 1, and muscleto-yolk concentration ratios showed consider able variation. One suggested explanation for the high liver-to-yolk ratios relates to one major function of the liver cells (hepatocytes), which is to accumulate and convert hydrophobic xenobiotics into hydrophilic metabolites to facilitate detoxicatio n, excretion, and elimination. In addition, the low lipid content of liver (relative to the lipid content of adipose tissue and yolk, Table 3-1) may have contribute d to the marked differences. With respect to the muscleto-yolk ratios, the reasons fo r the large degree of variabil ity are not as clear. One possible explanation is that muscle lipids are not mobilized during yolk formation and, as

PAGE 83

67 a result, OCP burdens may continually accumulate in muscle lipids. Another potential explanation relates to the low lipid content of muscle when compared to yolk (Table 3-1). Lastly, cis-chlordane’s exceptional liver, musc le, and adipose ratios underscore the fact that different OCP analytes may not al ways exhibit identi cal pharmacokinetics. When compared to other vertebrates, ad ipose tissue-to-egg ratios in alligators are similar to those reported in the freshwater catfish, Clarias batrachus in that adipose-toegg ratios are approximately equal to 1. Furthermore, C batrachus mobilizes lipids from its abdominal adipose tissue during vitelloge nesis (Lal & Singh, 1987) similar to what this study suggests occurs in the American alli gator. In contrast to adipose tissue OCP concentrations, muscle-to-egg OCP ratios in al ligators appear to be quite different from fish. Alligator muscle-to-egg ratios were highly variable and, for the most part, greater than 1, while fish ratios appear to be consiste ntly close to 1. With respect to more closely related species, muscle-to-egg OCP ratios ar e similar to those reported for the common snapping turtle ( Chelydra serpentina ) and several bird specie s with ratios exhibiting a great deal of variability and being greater than 1 (Russell et al., 1999). These differences suggest that fish differ from te rrestrial vertebrates in regard s to lipid content of muscle and/or lipid mobilization strategy (during vitell ogenesis), which could lead to differences in embryonal exposure given equivalent maternal exposure. Evaluation of Predictive Models Although significant linear models were found fo r most tissues with respect to total OCP concentrations, caution should be used in the application of these “total OCP” models since it is probable that the concentrations and ratios of individual OCP analytes may vary across different locations. The great est number of predictive linear models was derived for adipose tissue. This was not surp rising considering that (for most analytes)

PAGE 84

68 adipose-yolk lipid normalized ratios were close to 1. Next, with respect to the number of significant linear models, were liver and bile. The similarities between liver and bile should be expected since the liver produces bi le, which transports OCP analytes to the intestinal lumen, leading to their eventual elimination from the body. However, OCP analytes may be reabsorbed from the intestin e and redirected back to the liver via the portal vein through a process known as ente rohepatic circulati on, which may delay elimination of lipophilic xenobiotics, increa se hepatic exposure and bioaccumulation (Stenner et al., 1997). For OCP concentrations in muscle, regressi on analysis indicated that only two out of 12 mutually detected analytes could be predicted using OCP concentrations in eggs. Las tly, nine OCP analytes were c oncurrently detected in blood and egg yolk with none exhibiting significant re lationships. Possible explanations for the few significant linear re lationships include the low lipid co ntent of these tissues and thus the relatively low concentrations of OCP an alytes in these tissues, as well as the possibility that each of these tissue burden s exhibit a nonlinear relationship with yolk burdens. In addition, blood samples were co llected after the female had oviposited. Since blood was collected after eggs were ex creted from the body, it is likely that the overall maternal body burden decreased, which would in turn lower the steady-state OCP concentrations in blood. As for individual OCP analytes, predictiv e models for p,p’-DDE were derived for four of the five maternal tissues. One likely reason for this is that p,p’-DDE was detected in considerable concentrations in all eggs and in almost a ll tissues for all 15 females. Similarly, predictive models were derived for commonly detected analytes such as heptachlor epoxide, trans-chlo rdane, cis-chlordane, tran s-nonachlor, cis-nonachlor,

PAGE 85

69 mirex, and dieldrin for most tissues. Some what surprisingly, oxychlordane (a metabolite of cisand trans-chlordane) and p,p’-DDD (an intermediate metabolite of p,p’-DDT) showed significant linear models only with respect to liver an d adipose tissue. The fact that linear models for toxaphene and o,p’-DDT were derived only fo r adipose tissue was likely related to their low concentrations and in frequent detections in other tissues (Table 3-3). Relationships between Maternal Mass and OC P concentrations in Eggs and Tissues Although a portion of a female alligato r’s OCP body burden may be eliminated through egg deposition, adult female alligato rs from Lake Griffin had increased OCP concentrations in their tissues and eggs as they increased in mass, similar to size-related OCP bioaccumulation in smallmouth bass inha biting contaminated sites in Michigan (Henry et al., 1998). Corresponding increases in OCP burdens and mass indicate that larger and possibly older females accumulate OC Ps faster than they can excrete them. In addition, the relationship between OCP burdens in eggs and body mass was very similar to the relationship between abdomin al fat burdens and body mass. The correlation between OCP burdens in liver and body mass was significant for trans-nonachlor and p,p’-DDT; however the major metabolites of these compounds (oxychlordane and p,p’-DDE, respectively) we re not significantly correlated with body mass. These results contrast those of egg a nd abdominal fat burdens and suggest that that alligator liver may not sequester OCP metabolites to the same extent as abdominal fat or egg. Maternal body burdens: Toxicological Implications Although our study’s objective was to evaluate maternal transfer and prediction of the maternal OCP body burdens carried by the Am erican alligator, we would be remiss if

PAGE 86

70 we did not discuss whether these reported body burdens were capable of eliciting harmful effects. Although several stud ies report body and egg burdens in crocodilians, relatively few studies directly relate body and egg burdens to acute t oxicological effects (Campbell, 2003), so we will briefly discuss how p,p’-DDE burdens in maternal alligator liver compare to reported p,p’-DDE burdens in liver of birds (birds were not from the present study areas) that have been associated with mortality (Blus, 1996). In previous studies, mean DDE liver re sidues in birds which died due to DDT exposure ranged from 19,000–55,000 ng/g. When birds were exposed to DDE alone, liver residues of dead birds av eraged 3,883,000 ng/g (ra nge 460,000–11,725,000 ng/g) (Blus, 1996). When compared to the liver re sidues of the most contaminated alligators (Lake Apopka, upper 95% CI < 7,000 ng/g), it appears that death due to DDT/DDE exposure might be unlikely assuming bird and alligator susceptibilities are similar. However, since p,p’-DDE liver conc entrations in alligators are almost half of lethal liver concentrations in birds, there is reason for some concern. In addition, the assumption that bird and alligator susceptibil ities are similar might be ar gued as unfounded considering the variability in toxic responses between i ndividuals of the same species, different species, and different verteb rate classes (James et al., 2000). To account for these uncertainties the risk assessment process identifies the different sources of uncertainty and incorporates the uncertainty in attempti ng to determine a “safe” tissue concentration based on levels associated with no adverse effects (NOAEL) or lowest observed adverse effect levels (LOAEL). Typical ly, interspecies extrapolati on is assigned an uncertainty factor of 10, as are inter-indi vidual uncertainty, uncertainty related to comparing different study designs (e.g., acute doses rela ted to experimental bird studi es, in contrast to chronic

PAGE 87

71 exposure studies in wild alligators), and un certainty related to da tabase quality since DDE (p,p’-DDE + o,p-DDE) liver residues were reported, instead of p,p’-DDE. These four uncertainty factors cons titute an overall uncertainty factor of 10,000, which is an order of magnitude greater than commonly used uncertainty factors (range: 300-1000) (James et al,. 2000). Considering the high de gree of uncertainty, we suggest that more information is required before a “safe” leve l of p,p’-DDE exposure is determined for the American alligator based upon actual or predicted liver c oncentrations. Sublethal effects are anothe r possible consequence of OC P exposure. For example, exposure of the freshwater catfish, Clarias batrachus to an OCP analyte ( -BHC) at sublethal levels (2,000–8,000 ng/g) during vite llogenesis significantly decreased the biosynthesis and mobilization of phospholipids from liver to the developing follicles (Lal & Singh, 1987). Interestingly, alterations in fa tty acid profiles of alligator eggs have been associated with reduced clutch success. Specifically, fatty acid profiles from wild, alligator eggs (normal hatch rates) showed considerable differences when compared to those of eggs from captive alligators (reduc ed hatch rates). One suggested explanation for this association between altered fatty acid profiles and reduced clutch success in captive alligators was that certain fatty acids are critical for reproduc tive success and that captive diets were deficient in essential fatty acids (Noble et al., 1993). Thus, the possibility exists that exposure to OCPs may alter the liver’s ability to synthesize necessary fatty acids, leading to altered e gg quality and decreased clutch success in wild alligators that inhabit OCP-contaminated site s. Chronic exposure to low doses of OCPs prior to and during vitellogenesis has been s uggested as a cause for significant increases in OCP concentrations in egg yolk, as well as significantly decreased hatch rates in

PAGE 88

72 captive adult female alligators Importantly, the doses did not appear to induce acute toxicity in the adult female s (Rauschenberger et al., 2004). Presently, we are using a captive breeding population of adu lt alligators, as well as da ta from field studies, to further evaluate the relationships between OC P exposure, altered fatty acid biosynthesis, nutritional content of eggs, and embryonic mortality. In summary, the significant levels of OCP analytes observed across such a wide range of crocodilian species and geography s uggests the need for a greater understanding of xenobiotic metabolism and toxicologica l responses in crocodilians. Such understanding would aid in the conservation of this ancient group by determining what risks are posed by contaminants with respec t to species survival and how contaminantrelated risks compare to other risks, such as habitat destruction. The results of the present study provide some evidence suggesting that ma ternal transfer of OCP analytes is the major route for embryonic exposure. In a ddition, it provides several models for the prediction of OCP concentrations in maternal tissues of American alligators, which may be extrapolated to other crocodilians. H opefully, the present study will encourage new investigations into the phar macokinetics and pharmacodynamics of contaminants in other crocodilian species.

PAGE 89

73 Table 3-1. Morphological and reproductive characteristics of adult female alligators collected during June 2001 and 2002 from Lakes Apopka, Griffin, and Lochloosa in central Florida. Parameter a,b ApopkaGriffinLochloosa Number of females collected 483 Total Length (cm) 252 38258 17258 7 Snout-Vent Length (cm) 142 15134 9129 5 Mass (kg) 94 3070 1763 4 Clutch Mass (kg) 3.78 0.983.33 0.824.31 0.45 Fecundity (# eggs/clutch) 43 1040 1049 6 Lipid % Adipose 47.0 32.5 B78.1 8.0 A81.4 4.0 A Lipid % Liver 1.3 1.0 A0.8 0.2 A5.0 2.3 B Lipid % Muscle 0.8 0.91.3 0.90.2 0.02 Lipid % Yolk 19.9 1.118.1 1.718.2 1.6 a Values represent mean standard deviation. b Different letters i ndicate significant differences ( p < 0.05).

PAGE 90

74Table 3-2. Pesticide concentratio ns (ng/g wet wt.) in tissues and yolks of adult female alligators collected during June 2001 and 2002 from Lakes Apopka, Griffin, and Loch loosa in central Florida. Lake a Chemical b, c Bile Blood Adipose Liver Muscle Yolk Apopka Aldrin X X X X X 1 ( 4 ) -BHC X X X X X X -BHC X X 7.5 6.7 X X 2 1.4 cis -Nonachlor 10 3.2 2 0.4 521 602.7 31 6.7 23 18.3 123 81.9 cis -Chlordane 4 1.9 1 0.4 190 241.2 11 9.1 14.1 11.7 62 59.2 -BHC X X X X X X Dieldrin 38 10.2 5 0.4 2,376 3,770.9 105 80.2 68 48.3 663 803.0 Endosulfan I X X X X X X Endosulfan II X X X 21 X X Endosulfan Sulfate X X X X X X Endrin X X X X X X Endrin Aldehyde 3 0.4 X X X X X Endrin Ketone X X X X X X -BHC X X X X X X Heptachlor X X X X 8 11.7 1 0.04 Heptachlor Epoxide 3 2.1 0.3 0 67 81.5 6 2.2 4 3.6 26 15.0 Hexachlorobenzene 1 0 1 0 1 0 1 0.0 1 1 0.0 Kepone X X X X X X Methoxychlor X X X 5 X X Mirex 2 2.6 X 19 13.1 7 9.0 1 0.4 7 7.1 o,p’ -DDD X X 3 3.4 X X X o,p’ -DDE X X 52 55.8 X X 45 17.7 o,p’ -DDT 2 0.2 27 26.4 4 1.8 4 2.2 17 7.8 Oxychlordane 7 1.3 1 0.2 247 336.4 17 9.9 12 10.7 75 68.2 p,p’ -DDD 2 0.2 1 0.2 43 67.5 11 10.3 17 8.0 52 61.4 a p,p’ -DDE 806 341 42 5.7 29,840 34,366 1,846 918.1 1,392 1,0782 9,994 8,529 Toxaphene X X 13,436 12,670.2 X X 4,862 4,177 trans -Nonachlor 21 7.8 3 0.4 1,153 1,378.7 65 22.7 68 57.0 387 277.7 Total OCP 900 369.7 55 7 44,650 53,230 2,140 1,024 1,610 1,226 15,108 13,704

PAGE 91

75Table 3-2. (Continued) Lake Chemical Bile Blood Adipose Liver Muscle Yolk Griffin Aldrin X X X X X X ( 8 ) -BHC X X X X X X -BHC X X 2.1 1.1 X X X cis -Nonachlor 4 2.4 1 0.4 75 74.9 8 4.4 9 10.6 14 7.6 cis -Chlordane 2 0.5 1 0 30 10.8 2 0.7 3 3.4 11 3.7 -BHC X X X X X X Dieldrin 13 4.9 7 109 133.4 17 8.0 22 20.8 26 25.7 Endosulfan I X X X X X X Endosulfan II X X X X X X Endosulfan Sulfate X X X X X X Endrin X 5 X X X X Endrin Aldehyde X X X X X X Endrin Ketone X 2 X X X X -BHC X X 2 X X X Heptachlor X X X X 2 1.4 X Heptachlor Epoxide 3 3.3 1 34 45.7 5 3.6 10 12.0 8 8.8 Hexachlorobenzene 1 0 1 1 0 1 0 1 1 0.0 Kepone X X X X X X Methoxychlor X X X X X 2 Mirex 0.3 0 1 5 3.6 1 1 0.5 1 0.2 o,p’ -DDD X X X X X X o,p’ -DDE X X X X X 3 o,p’ -DDT 1 0.2 1 0.0 10 6.9 X 2 0.1 3 1.8 Oxychlordane 7 4.9 X 56 84 8 6.2 16 17.8 12 14.9 p,p’ -DDE 54 25.7 13 9.1 1,030 931.3 75 46.6 131 132.4 273 204.0 p,p’ -DDT 1 13 3 1.5 29 0.9 X 3 Toxaphene X X X X X X trans -Chlordane 1 0.3 1 0 3 1.7 1 0.3 1 2 1.0 trans -Nonachlor 9 5.9 1 0.09 171 213.5 18 13.2 28 36.0 40 38.3 Total OCP 87 46.4 31 28.4 1,533 1,439 153 78.1 208 227 393 299

PAGE 92

76Table 3-2. Continued. Lake Chemical Bile Blood Adipose Liver Muscle Yolk Lochloosa Aldrin X X X X X X ( 3 ) -BHC NA X X X X X -BHC NA X 1 X X X cis -Nonachlor NA X 17 1.8 1 X 5 1.9 cis -Chlordane NA X 8 1.4 X X 3 0.1 -BHC NA X X X X X Dieldrin NA X 14 4.7 2.6 1.4 4 2.8 Endosulfan I NA X X X 15.6 X Endosulfan II NA X X X X X Endosulfan Sulfate NA X X X X X Endrin NA X X X X X Endrin Aldehyde NA X X X X X Endrin Ketone NA X X X X X -BHC NA X X X X X Heptachlor NA X X X 18 9.1 X Heptachlor Epoxide NA X 11 9.6 X X 3 2.9 Hexachlorobenzene NA X X X X X Kepone NA X X X X X Methoxychlor NA X X X X X Mirex NA X 2.6 X X X o,p’ -DDD NA X X X X X o,p’ -DDE NA X X X X X o,p’ -DDT NA X 3 0.2 X 7.1 1 0.0 Oxychlordane NA X 17 11.0 1 X 5 4.3 p,p’ -DDD NA X 1 0.1 X 1 2 0.9 p,p’ -DDE NA X 297 90.1 20 20.9 11 6.7 91 32.5 p,p’ -DDT NA X 1.4 0.1 X 1.4 X Toxaphene NA X X X X X trans -Chlordane NA X 1 0.1 X 1.4 X trans -Nonachlor NA X 38 24.6 2.6 1.4 12 8.8 Total OCP NA X 407 143.6 28 32.6 33 33.9 124 53.3 a Number of females and clutches collected not ed in parentheses beneath name of lake. b Values represent mean standard deviation

PAGE 93

77[SD], values without SD indicate a single measurement. X indicat es values which were below lim it of detection (LOD) or below l imit of quantitation (LOQ) and NA indicates not analyzed. LOD range d from 0.1-1.5 ng/g for most OCP analytes (toxaphene LOD ranged from 120-236 ng/g), and LOQ ranged was 1.5 ng/g fo r all analytes except for toxaphene ( 1500 ng/g). Percent recovery ranged fro m 70-130%. The following chemicals were neithe r detected in females nor their eggs: -BHC, -BHC, endosulfan sulfate, and kepone. c BHC = Benzene hexachloride; DDD = Dichlorodiphenyldichloro ethane; DDE = Dichlorodiphenyldichloroethylene; DDT = Dichlorodiphenyltrichloroethane; Total OCP = organochlorine pesticide concentrations for all analytes.

PAGE 94

78 Table 3-3. Regression equations for pr edicting organochlorine pesticide (OCP) concentrations in maternal tissues, where LOG [Tissue-OCP] = bo + b1 LOG [Yolk-OCP]. Tissue Chemicala bo b1 n r2 p Adipose Dieldrin 0.6624 0.8785 150.87 < 0.0001 cis -Nonachlor 0.6737 0.9136 150.75 < 0.0001 cis -Chlordane 0.4037 0.9633 150.69 0.0001 Heptachlor Epoxide 0.6294 0.8134 140.62 0.0008 Mirex 0.8217 0.6030 6 0.89 0.0028 o,p -DDT 0.5840 0.6040 140.41 0.0141 Oxychlordane 0.6694 0.8544 150.80 <.0001 p,p’ -DDD 0.2375 0.7 597 140.50 0.0046 p,p’ -DDE 0.6968 0.9216 150.93 <.0001 Toxaphene 0.0880 1.0928 3 0.99 0.0486 trans -Chlordane 0.1733 0.9397 120.58 0.0041 trans -Nonachlor 0.6430 0.8960 150.84 < 0.0001 Bile Dieldrin -0.6196 0.9559 4 0.90 0.0494 cis -Nonachlor -0.3863 0.7646 5 0.97 0.0017 cis -Chlordane -0.4308 0.6314 5 0.83 0.0301 Heptachlor Epoxide -0.3207 0.6959 5 0.79 0.0435 p,p’ -DDD -1.1407 1.0748 4 0.95 0.0246 p,p’ -DDE -0.6385 0.9472 5 0.94 0.0057 trans -Nonachlor -0.2919 0.6867 5 0.96 0.0039 trans -Chlordane -0.2245 -0.4531 5 0.87 0.0220 Blood NSb Liver Dieldrin 0.0248 0.7162 7 0.98 <0.0001 cis -Nonachlor -0.2471 0.8448 8 0.92 0.0002 cis -Chlordane -0.5557 0.8876 7 0.97 <0.0001 Heptachlor Epoxide -0.3878 0.8323 6 0.85 0.0084 Mirex -0.0547 0.9557 5 0.89 0.0155 Oxychlordane -0.2855 0.8123 7 0.92 0.0005 p,p’ -DDE -0.7696 1.0156 100.93 <.0001 trans -Chlordane -0.0722 0.3300 7 0.94 0.0003 trans -Nonachlor -0.2854 0.8263 8 0.98 <.0001 Muscle p,p’ -DDE -0.3733 0.8153 100.54 0.0160 Mirex 0.1816 -0.2797 0.96 0.0040 a BHC = Benzene hexachloride; DDD = Dich lorodiphenyldichloroethane; DDE = Dichlorodiphenyldichloroethylene; DDT = Dichlorodiphenyltrichloroethane. b NS = no significant linear regressions were determined for the 9 chemicals which were detected both in blood and in yolk.

PAGE 95

79 Log Total OCPs in Yolk (ng/g) 100101102103104105 Log Total OCPs in Tail Muscle(ng/g) 100101102103104105106 100101102103104105 Log Total OCPs in Liver (ng/g) 100101102103104105106 100101102103104105 Log Total OCPs in Adipose (ng/g) 100101102103104105106 Apopka Griffin Lochloosa Log Total OCPs in Yolk (ng/g) 100101102103104105 Log Total OCPs in Bile (ng/g) 100101102103104105106 Figure 3-1. Linear regressions of total organochlorine pesticide (OCP) concentrations in maternal tissues against total OCP concentrations in egg yolks. A. Adipose tissue. B. Liver. C. Bile. D. Muscle. D. y = -0.0865 + 0.6688 x ( r2 = 0.55, p <0.05) A. y = -1338.60 + 3.318 x ( r2 = 0.95, p <0.05) B. y = -0.6330 + 0.9626 x ( r2 = 0.88, p <0.05) C. y = -0.4817 + 0.8342 x ( r2 = 0.89, p <0.05)

PAGE 96

80 CHAPTER 4 MATERNAL FACTORS ASSOCIATED WI TH DEVELOPMENTAL MORTALITY IN THE AMERICAN ALLIGATOR Recent data suggested maternal organoc hlorine pesticide (OCP) body burdens and OCP egg yolk concentrations are signif icantly correlated, an d that significant relationships between maternal size and maternal body burdens exist. Maternal age and size has also been shown to have a strong re lationship with clutch viability (number of live hatchings / total number of eggs) and cl utch size characteristics (i.e., fecundity, clutch mass). Specifically, females between 15 and 30 years old (~ 2.3–2.8 m in total length) produce larger clutches (35-40 eggs / clutch) with increased clutch viability compared to younger females, which themselves produce smaller clutches (15–25 eggs) with smaller eggs and have decreased clutch viability. Females older than 30 years tend to produce clutches similar to 15-30 year old females, with the only exception being smaller clutches (15–25 eggs) (Ferguson, 1985). Therefore, female size or age may be a confounding factor when examining the rela tionship between OCP burdens in yolk and reproductive performance. In addition, age (or size) and maternal OCP exposure could cause interactive effects. For example, females of optimum reproductive age may be more resistant to effects of OCPs; while, younger (or older) females may show increased susceptibility. Therefore, th e objective of the present study was to test the hypotheses that reproductive efficiency, clutch viabi lity, and mortality rate s are significantly correlated with maternal OCP body burdens, mate rnal size, or both; and (2) that clutch size characteristics are significantly correlated with maternal OCP body burdens, maternal size, or both.

PAGE 97

81 Materials and Methods The greatest difficulty in examining the relationship between maternal age and OCP exposure and effects is that determining th e age of an alligator requires either long term monitoring or counting the rings that form in the femur as a result of annual calcium deposition (Ferguson, 1985). However, this technique is not valid for reproductive females since femoral bone resorption provides calcium necessary for eggshell formation and egg yolk nutrition, and subsequently ca uses the removal of “bone rings” and underestimation of age (Elsey & Wink, 1985; Wink & Elsey, 1986). In addition, removing an alligator’s limb simply to age it is ethically unacceptable. Given these difficulties with assigning a chr onological age, female size will be used lieu of age. One potential limitation in using female size as an i ndicator of age class is that female growth rates between lakes may differ since dietar y composition has been suggested to differ among OCP-contaminated sites and referen ce sites (Rice, 2004). Therefore, the possibility exists that a fema le from a reference site may be smaller than one from a contaminated site, even though both are of the sa me age. This is important since age, in addition to size, has been shown to be an important determinant of sexual maturity in alligators. Indeed, alligator ranchers are able to accelerate growth so that a female may reach six feet in length in 3-4 years, howeve r, these females do not seem to be able to reproduce until they reach 8-10 years of ag e (Ferguson, 1985). To control for potential confounding due to differential growth rates, relationships between female size and OCP burdens and clutch viability will be evaluated using site and year as covariates. If the effects of covariates are determined statistically negligible, female data will be grouped together.

PAGE 98

82 Site Descriptions Lakes Apopka (N 28 35’, W 81 39’), Griffin (N 28 53’, W 81 49’), and Lochloosa (N 29 30’, W 82 09’) in Florida were selected as co llection sites because prior studies by our laboratory indicate vastly different le vels of OCP exposure across these sites. All three lakes are part of th e Ocklawaha Basin. Lake Lochloosa (which is connected to Orange Lake) was selected as a low exposure (reference) site. Three years (2000-2002) of data indicate mean total OCP concentrations in egg yolks from the reference sites (Lakes Orange and Loch loosa) were 102 16 ppb (mean standard deviation [SD], n = 19 clutches) with a concurre nt mean clutch viability rate (number of live hatchlings/total number of eggs in a ne st) of 70 4% (Gross, unpublished data). Lake Griffin was selected as an intermedia te exposure site since yolk concentrations averaged 1169 423 ppb (n = 42 clutches) a nd Lake Apopka was selected as a high exposure site since yolk concentrations av eraged 7,582 2,008 ppb (n = 23) for the same time period (Chapter 2). Furthermore, mean clutch viability rates during this time period for Lakes Apopka (52 6%, n = 23) and Griffin (43 5%, n = 42) have been below rates observed for the reference site. Animal Collections Adult female alligators and their corres ponding clutches of eggs were collected from Lakes Apopka ( n = 19), Griffin ( n = 18), and Lochloosa ( n = 3) over the course of four nesting seasons (June 1999 to June 2002) Nests were located by aerial survey (helicopter) and/or from the ground (airboat). Once nests were located, all eggs were collected, and the nest cavity was covered. A snare-trap was set perpendicular to the taildrag in order to capture the female as she cr ossed over the nest. After the traps were set, one member of the trapping crew subsequently transported the eggs to the Florida Fish

PAGE 99

83 and Wildlife Conservation Commission’s Wild life Research Unit (FWC; Gainesville, FL, USA) and placed the eggs in a temperaturecontrolled incubator. Snare-traps were checked later in the evening a nd early the next morning. In 1999 and 2000, trapped females were secu red and measurements (total length, snout-vent length, head length and tail girth) were collec ted along with a blood sample and a scute for OCP analysis. These females were then immediately released. In 2001 and 2002, females were captured and transporte d from each lake to the United States Geological Survey’s Florida In tegrated Science Center (USGS; Gainesville, FL, USA). Upon arrival, the animals were weighed, m easured, and blood samples were collected from the post-occipital sinus. Adult alligators were then euthanized by cervical dislocation followed by double pithing. A full necropsy was performed on each female. Bile, liver, adipose (composite of abdomina l fat and the abdominal fat pad), and tail muscle samples were collected for later dete rmination of OCP burdens. Liver, adipose tissue, and muscle were wrapped in aluminum foil, while bile and blood were placed in scintillation vials. All sa mples were grouped according to nest identification number (ID), placed in plastic bags labeled with the appropri ate ID, and stored in a –80 oC freezer. Each female’s corresponding clutch of eggs was then transferred from FWC to USGS where yolk samples were collected (t wo eggs/clutch) and stored with the corresponding maternal tissues. The remain ing eggs were set for incubation in a temperature/humidity-contr olled incubator (31-33 oC, 88-92% relative humidity) located at USGS. Analysis of OCPs in Ma ternal Tissues and Yolk Analytical grade standards for the following compounds were purchased from the sources indicated: aldrin, al pha-benzene hexachloride ( -BHC), -BHC, lindane, -BHC,

PAGE 100

84 p,p’ -dichlorodiphenyldichloroethane ( p,p’ -DDD), p,p’ -dichlorodiphenyldichloroethylene ( p,p’ -DDE), dichlorodiphe nyltrichloroethane ( p,p’ -DDT), dieldrin, endosulfan, endosulfan II, endosulfan sulfate, endrin, e ndrin aldehyde, endrin ketone, heptachlor, heptachlor epoxide, hexachlorobenz ene, kepone, methoxychlor, mirex, cis -nonachlor, and trans -nonachlor from Ultra Scientific (Kingstown, RI, USA); cis -chlordane, trans chlordane, and the 525, 525.1 polychlorinated biphenyl (PCB) Mix from Supelco (Bellefonte, PA, USA); oxychlordane from Chem Service (West Chester, PA); o,p’DDD, o,p’DDE, o,p’DDT from Accustandard (New Haven, CT, USA); and toxaphene from Restek (Bellefonte, PA, USA). All reag ents were analytical grade unless otherwise indicated. Water was doubly distilled and deionized. Adipose, liver, bile, and yolk samples were analyzed for OCP content using methods modified from Holste ge et al. (1994) an d Schenck et al. (1994). For extraction, a 2 g tissue sample was homogenized with ~1 g of sodium sulfate and 8 mL of ethyl acetate. The supernatant was decanted and filtered though a Bchner funnel lined with Whatman #4 filter paper (Fisher Scientific, Ha mpton, NH, USA) and filled to a depth of 1.25 cm with sodium sulfate. The homogenate was extracted twice with the filtrates collected together. The combined filtrat e was concentrated to ~2 mL by rotary evaporation, and then further concentrated until solvent-free unde r a stream of dry nitrogen. The residue was reconstituted in 2 mL of acetonitrile. After vortexing (30 s), the supernatant was applied to a C18 solid phase extrac tion (SPE) cartridge (preconditioned with 3 mL of acetonitrile; Agile nt Technologies, Wilmi ngton, DE, USA) and was allowed to pass under gravity. This proced ure was repeated twice with the combined eluent collected in a culture t ube. After the last addition, th e cartridge was rinsed with 1

PAGE 101

85 mL of acetonitrile which was also collected. The eluent was then applied to a 0.5 g NH2 SPE cartridge (Varian, Harbor City, CA, US A), was allowed to pass under gravity, and collected in a graduated conical tube. The car tridge was rinsed with an additional 1 mL portion of acetonitrile whic h was also collected. The combined eluents were concentrated under a stream of dry nitrogen, to a volume of 300 L, and transferred to a gas chromotography (GC) vial for analysis. Whole blood was analyzed for OCP c ontent using methods modified from Guillette et al. (1999). A 10 mL aliquot was transferred from the homogenized bulk sample and extracted in 15 mL of acetone by vortex mixer. The mixture was centrifuged for 5 min at 3000 rpm, after which the supernatan t was transferred to a clean culture tube. This process was repeated with the supern atants collected and concentrated under a stream of dry nitrogen until solvent-free. Th e residue was re-extract ed in 11.5 mL of 1:1 methylene chloride-petroleum ether. After mixing, the sample was allowed to settle and the upper layer was tran sferred to a clean culture tube. This extraction was performed twice with the extracts collected together. Th e combined extracts we re then applied to a prepared florisil cartridge (5 mL Fisher Pr epSep, Fisher Scientific, Hampton, NH, USA). The cartridge had been prepared by filling the reservoir to a depth of 1.25 cm with anhydrous sodium sulfate and by prewashing the modified cartridge with 10 mL of 2:1:1 acetone: methylene chloride: petroleum ether. After the sample passed under gravity with the eluent collected in a 15-mL graduate d conical tube, the cart ridge was eluted with 4 mL of the 2:1:1 solv ent mixture which was also collected. The combined eluents were concentrated under a stream of dry nitrogen, to a volume of 300 L, and transferred to a GC vial for analysis.

PAGE 102

86 GC/MS Analysis Analysis of all samples was performed using a Hewlett Packard HP-6890 gas chromatograph (Wilmington, DE, USA) with a split/splitless inlet ope rated in splitless mode. The analytes were introduced in a 1 L injection and separa ted across the HP-5MS column (30 m x 0.25 mm; 0.25 m film thickne ss; J & W Scientific, Folsom, CA, USA) under a temperature program that began at 60 C, increased at 10 C/min to 270 C, was held for 5 min, then increased at 25 C/min to 300 C and was held for 5 min. Detection utilized an HP 5973 mass spectro meter in electron impact m ode. Identification for all analytes and quantitation for toxaphene was c onducted in full scan mode, where all ions are monitored. To improve sensitivity, se lected ion monitoring was used for the quantitation for all other analytes, except kepone. The above program was used as a screening tool for kepone which does not optim ally extract with mo st organochlorines. Samples found to contain kepone would be reex tracted and analyzed specifically for this compound. For quantitation, a five-point standard curve was prepared for each analyte ( r2 0.995). Fresh curves were analyzed with each se t of twenty samples. Each standard and sample was fortified to contain a deuterat ed internal standard, 5 L of US-108 (120 g/mL; Ultra Scientific), added just prior to analysis. All samples also contained a surrogate, 2 g/mL of tetrach loroxylene (Ultra Scientific) added after homogenization. Duplicate quality control samples were prepar ed and analyzed with every twenty samples (typically at a level of 1.00 or 2.50 g/mL of -BHC, heptachlor, aldr in, dieldrin, endrin, and p,p’ -DDT) with an acceptable recovery rangi ng from 70 – 130%. Limit of detection (LOD) ranged from 0.1-1.5 ng/g for all OCP an alytes, except toxa phene (120-236 ng/g), and limit of quantitation (LOQ) was 1.5 ng/g for all analytes, except toxaphene (1500

PAGE 103

87 ng/g). Repeated analyses were conducted as allowed by matrix interferences and sample availability. Data Analysis Specific OCP analytes were removed from analysis if measurable concentrations were not found in at least 5% of the clutch es. Numerical data were log-transformed [ln(x)], while proportional da ta were arcsine square root transformed to conform to statistical assumptions. Maternal OCP bur dens for females collected during 1999 and 2000 were estimated using the females’ resp ective yolk burdens and predictive models described in Chapter 3. ANOVA (PROC GLM; SAS Institute Inc., 2002) was used for inter-site comparisons of adult female and clutch characteristics, and the Tukey test was used for multiple comparisons among sites ( = 0.05 since no interactions were tested). Because relationships between respons e variables and explanatory variables (Table 4-1) in ecological studies are often complex with inte ractions occurring, an indirect gradient multivariate analysis method, Detrended Co rrespondence Analysis (DCA) (ter Braak, 1986) was used to initially evaluate data stru cture. Two matrices were constructed for DCA, with the first representing the respons e variables (female-clutch pair number x clutch parameters) and the second representi ng the explanatory vari ables (female-clutch pair number x maternal size and OCP burdens) (Table 2). DCA results indicated that a direct gradient, multivariate linear anal ysis, Redundancy Analysis (RDA) (Rao, 1964), was appropriate since the lengths of the DCA ordination axes were equal to or less than 2 standard deviations (t er Braak, 1995). For the RDA, sim ilar matrices were constructed with the exception that response variables meas ured as a percentage (i.e., clutch viability) and response variables measured as a number (i .e., clutch mass) were split into separate

PAGE 104

88 matrices because percentage data were ln(x +1) transformed and not standardized, while continuous data were ln(x) transformed and st andardized (ter Braak & Smilauer, 2002). Automatic forward selection of the best f our explanatory variables was conducted for both sets of RDA analyses and tested for significance by Monte Carlo permutation test DCA and RDA were conducted using the pr ogram CANOCO (ter Braak & Smilauer, 2002). Biplots of environmental variables a nd response variables we re then constructed to facilitate interpretation. Results A total of 40 female alligators and their respective clutches (female-clutch pairs) were collected during the summers of 1999-2002 from Lake Apopka ( n = 19), Lake Griffin ( n = 18), and Lake Lochloosa ( n = 3). No significant di fferences between lakes were determined with respect to fecundity, av erage egg mass, clutch viability, percentage of unbanded eggs, percentage of early embryoni c mortality, percentage of late embryonic mortality, female head length, female snout-v ent length, female tail girth, female total length, and female body condition index (Table 4-3). Significant differences were detected with respect to total OCP concentration in female adipose tissue with Lake Apopka female burdens (22,737 5,767.6 ng/g) being greater than those of Lake Griffin (1,821 702.7 ng/g), and Lake Lochloosa female burdens (375 63.1 ng/g). No significant differences were determined betw een adipose burdens of Lake Griffin and Lake Lochloosa females (Table 4-3). Results of the forward selection RDA ev aluating reproductive efficiency (clutch viability, percentage unbanded eggs, early em bryo mortality and la te embryo mortality) indicated that the four explanat ory variables that best accounted for the variance of clutch survival parameters were interaction variable: % trans -chlordane (TC%) (lambda A =

PAGE 105

89 12%), percentage p p’ -DDE (DDE%) (lambda A = 6%), heptachlor epoxide ([HE]) (lambda A = 4%), and percentage oxychlordane ( A = 4%). These factors accounted for 28% of the total variation of reproductive efficiency. Two of the four variables, TC% and PDDE% were determined significant a nd together accounted for 18% of total variation (Table 4-4). Biplots of the extracted fact ors and reproductive variables (Fig. 4-1) suggested that clutch viability had strong ne gative correlations with TC% ( r = -0.5451) and %OX-[OX] ( r = -0.2885), but was weakly correlated with PDDE% ( r = 0.0548). Unbanded egg percentages, however, were positiv ely strongly correlated with DDE% ( r = 0.4012), weakly correlated with %OX-[OX] ( r = -0.1298) and TC%-[TC] ( r = 0.0228). Early embryonic mortality percentages were weakly correlated with TC%-[TC] ( r = 0.1860), %OX-[OX] ( r = 0.1556), and PDDE% ( r = 0.133). Late embryo mortality percentages were positively correlated with TC%-[TC] ( r = 0.3361) and %OX-[OX] ( r = 0.3144), but showed negative correl ations with DDE% ( r = -0.3230). For clutch size characteristics (fecundity, clutch mass, and average egg mass), RDA results indicated that the four explanatory variables that best explained clutch size variance were concentration of cis-chlordane ([CC]) (L ambdaA = 6%), (LambdaA = 6%), percentage dieldrin (DL%) (LambdaA = 6%), concentration of p,p ’-DDD ([PDDD]) (LambdaA = 4%), and concentration of toxa phene ([TOX]) (LambdaA = 4%). None of these variables were determined to be significan tly associated with clutch size parameters (Table 4-5). Discussion With respect to the first hypothesis, results of the present study suggest that certain OCPs in maternal adipose tissue were signifi cantly associated with decreased clutch

PAGE 106

90 survival parameters (clutch viability, per centage, unbanded eggs, ear ly and late embryo mortality), but that clutch survival parameters were neither significantly correlated with maternal morphometrics. Although maternal bur dens of certain OCPs were correlated with clutch survival parameters, extracted va riables only explained 18% of the variation. However, it is important to note that compos itional percentage of an OCP analyte appears to be an important factor with respect to clutch survival parame ters. Indeed both OCP variables found to be significantly associated with clutch survival parameters were compositional variables (TC% and pDE%) (T able 4-4). This suggests that the composition of the OCP mixture may be more im portant in altering clutch viability than the total OCP burden or total number of OCPs de tected in maternal tissues. Furthermore, biplots (Fig. 4-1) suggest that the rates of unbanded eggs, early embryo mortality, and late embryo mortality, which all contribute to reproductive efficien cy, have different relationships with the each of the extract ed OCP variables, suggesting that certain mixtures differentially affect certain aspects of reproducti ve function. Two reasons for the importance of mixture com position are that the effects and the toxicity (potency) of OCPs vary considerably from analyte to anal yte. For example, Japanese quail orally dosed with technical grade DDE (300 ppm) had decreased rates of fe rtility and increased rates of mortality; however, similar exposures to technical grade DDT (300 ppm) did not cause adverse effects (Robson et al., 1976). D ecreased fertility in quail exposed to DDE is consistent with the results of the presen t study in that as DDE% increased in female alligator adipose tissue, the percentage of unbanded eggs also increased. Although total OCP burdens in maternal ad ipose tissue were significantly higher in females from Lake Apopka, no significant diffe rences were detected between lakes with

PAGE 107

91 respect to clutch viability, percentages of unbanded eggs, early embryo mortality, or late embryo mortality,. The significant differences in total OCP burdens among sites and lack thereof for clutch parameters, again sugge sts that total OCP burdens may be less important than mixture composition. With respect to the second hypothesis, OCP burdens and maternal morphometrics were not found to be associated with clutch size characteristics. This may be due to similar sized females and clutches being collect ed with in and among sites (Table 4-3). The correlations between certain OCPs a nd clutch survival parameters suggest decreased reproductive efficiency may be re lated to increased maternal OCP burdens, however, correlations alone do not establis h causal relationships. Indeed, several viewpoints must be considered before cau sality is concluded, however, the only viewpoint, or “criterion”, that can rule out a cause-effect relationship is temporality (i.e., exposure must precede effect ) (Hill, 1965). The major cr iteria used in the current practice of causal inference is temporality, biological plau sibility, consistency of the association, strength of th e association, and biological gradient, (Weed et al., 2002; Gadbury & Schreuder, 2003). Since females were exposed to OCPs prior to vitellogenesis and oviposition, temporality is satisfied. The second criteri on, biological plausibilit y, is also satisfied since studies in other oviparous vertebrate s have shown that OC P exposure can cause adverse reproductive effects through a variety of mechanisms (Fry, 1995) The third criterion is consistency of asso ciation, which means that similar results have been found among other studies examini ng the same problem. Few studies have examined reproductive effects of maternal OC P exposure in alligators with one of these

PAGE 108

92 studies reporting no significan t correlations between DDE concentrations in maternal tissues and clutch anomalies for Lake A popka alligators (Giroux, 1998). The earlier study’s focus was on a single analyte while this study looked a several analytes concurrently, so the two studies differ some what. Given relatively small number of studies and the ambiguous interpretations, no clear conclusion can be reached as to whether the consistency criter ion has been satisfied. The fourth and fifth criteria, strength of association and biological gradient, are somewhat similar in nature. Strength of association refers to how strongly correlated the causal factor is to the respons e variable, and the biological gr adient refers to whether the response variable increases as the dose increases. With resp ect to strength of association, the present study’s results indicate weak-mode rate associations between maternal OCP burdens and clutch survival parameters (18% of variance explained). With respect to dose-response, biplots indicat ed that biological gradient s existed between certain maternal factors and reproductive responses in that as percentage p,p’-DDE and percentage trans-chlordane in creased, incidence of unbanded eggs increased and clutch viability decreased, respectively (Fig. 4-1). In summary, rarely does a single obser vational study estab lish clear causal relationships, and the presen t study is no exception. Howe ver, the present study does satisfy some of the criteria used for establ ishing causality. Importa ntly, results suggest that a moderate part of th e variation associated with reproductive function in the American alligator can be attributed to maternal OCP body burdens. Hopefully, the results of the present study will stimulat e future efforts aimed at increasing our understanding of the effects of enviro nmental contaminants of crocodilians.

PAGE 109

93 Table 4-1. Reproductive, mor phometric, and contaminant pa rameters measured on adult female alligators collected during June 1999, 2000, 2001, and 2002. Female Parameter Definition Measured as Response variables Fecundity Total No. of eggs in one clutch n Clutch mass Total mass of eggs in one clutch kg Ave. Egg Weight Clutch mass / Fecundity g % Unbanded eggsa No. of unbanded eggs / fecundity x 100 Percentage % Early embryo mortality No. of deaths < dev. Day 35 / fecundity x 100 Percentage % Late embryo mortality No. of deaths dev. Day 35 / fecundity x 100 Percentage Clutch Viability No. eggs yielding live hatchling / fecundity x 100 Percentage Explanatory variables Head Length Tip of snout to posterior base of skull (dorsal) cm Snout-Vent Length Tip of snout to posterior base of vent (dorsal) cm Tail Girth (cm) Circumferen ce of tail at vent cm Total Length (cm) Tip of snout to tip of tail (dorsal) cm Body condition index Snout-vent lengt h / Tail girth x 100 Percentage [OCP analyte] in adipose tissueb ng OCP analyte / g adipose tissue wet weight ppb % OCP analyte [OCP analyte] / [OCP] x 100 Percentage aAn egg with no evidence of embryonic attachment bSee text for list of measured OCP analytes. For 1999 and 2000 females, adipose OCP concentrations were estimated using pr edictive equations (see Chapter 3).

PAGE 110

94 Table 4-2. Explanatory variab les included in RDA with forw ard selection of four best variables ( = 0.05). Variable Code Head Length HL Snout-vent length SVL Total Length TL Tail Girth TG Body Index BI Age Age No. OCPs quantitated NOC [OCP] TOC % cis -Chlordane CC% [ cis -Chlordane] [CC] % cis -Nonachlor CN% [ cis -Nonachlor] [CN] % Dieldrin DL% [Dieldrin] [DL] % Heptochlor epoxide HE% [Heptachlor epoxide] [HE] % Mirex MX% [Mirex] [MX] % o,p -DDT ODDT% [ o,p -DDT] [ODDT] % Oxychlordane OX% [Oxychlordane] [OX] % p,p '-DDE PDDE% [ p,p '-DDE] PDDE] % p,p '-DDD PDDD% [ p,p '-DDD] [PDDD] % p,p '-DDT PDDT% [ p,p '-DDT] [PDDT] % trans -Chlordane TC% trans -Chlordane [TC] % trans -Nonachlor TN% [ trans -Nonachlor] [TN] % Toxaphene TX% [Toxaphene] [TX]

PAGE 111

95Table 4-3. Reproductive, morphometric and contaminant summary statisticsa of adult female alligators collected during June of 1999-2002. Parameter Apopka Griffin Lochloosa Summary Female-clutch pairs (n) 19 18 3 40 Fecundity (n) 45 2 44 1.9 49 3.5 44 1.3 (22–54) (19–56) (45–56) (19–56) Clutch mass (kg) 4.2 0.14 3.6 0.18 4.3 0.26 3.9 0.11 (2.4–5.1) (1.8–5.2) (4–4.8) (1.8–5.2) Egg Mass (g) 89 1.2 84 2.8 88 1.1 87 1.4 (77.6–100) (70.8–112.6) (86.1–90) (70.8–112.6) Clutch 'viability (%)b 61 6.8 42 8.5 60 8.3 52 5.2 (0–98) (0–92) (48–76) (0–98) Unbanded eggs (%)c 19 6.9 21 5.3 9 5.5 19 4 (0–100) (0–70) (3–20) (0–100) Early embryo mortality (%)d 12 3 14 3.4 25 3.2 14 2.1 (0–45) (0–52) (20–31) (0–52) Late embryo mortality (%)e 7 2 22 6.8 6 3 14 3.4 (0–25) (0–89) (0–10) (0–89) Head Length (cm) 37 1.3 36 0.7 35 0.4 36 0.7 (22–52) (28–41) (35–36) (22–52) Snout-Vent Length (cm) 140 3.6 135 1.9 129 2.8 137 2 (83–156) (120–148) (125–134) (83–156) Tail Girth (cm) 68 2.8 66 1.7 62 2 67 1.5 (36–92) (52–77) (59–66) (36–92) Total Length (cm) 263 7.9 260 4.5 258 4.2 262 4.2 (161–304) (220–298) (253–266) (161–304) Body indexf 2.09 0.059 2.05 0.04 2.07 0.023 2.07 0.033 (1.46–2.7) (1.79–2.38) (2.03–2.11) (1.46–2.7) TotalOCPs (ng/g)g 22,734 5767.6 A 1,821 702.7 B 375 63.1 B 11,648 3,200.9 (5,224.7–123,081.5) (355.6–12,938.7) (289.8–498.1) (289.8–123,081.5)

PAGE 112

96 96aValues represent mean standard error with ra nge in parentheses. Significant differences ( = 0.05) between sites indicated by letters (A-B) beside mean. Same letters = not significant b % of eggs in a clutch th at yield a live hatchling. c % of eggs with no eviden ce of embryonic attachment d % of embryos in a clutch that perish duri ng first half (35 days) of development. e % of embryos in a clutch that perish dur ing last half (35 days) of development. f snout-vent length / tail girth g [ ng OCP analyte / g adipos e tissue (wet weight)]

PAGE 113

97 Table 4-4. Results of redundancy analysis with automatic selection of four best maternal factors associated with varia tion in reproductive efficiency. Variable LambdaAa P F TC%* 0.12 0.004 5.18 pDE%* 0.06 0.04 2.78 [HE] 0.04 0.202 1.65 Lochloosa 0.04 0.146 1.80 aProportion of total variance explained by each variable (total variance = 1.0). P < 0.05 Table 4-5. Results of redundancy analysis with automatic selection of four best maternal factors associated with variation in clutch size characteristics. Variable LambdaA P F [CC] 0.06 0.174 2.21 [pDD] 0.04 0.190 1.94 [TOX] 0.04 0.162 1.49 [DL] 0.06 0.134 2.54

PAGE 114

98 -1.01.0-1.01.0 CLutch viability Unbanded egg% Early Emb. Mortality Late Emb. Mortality DDE% trans-Chlordane% [HE] Lochloosa Figure 4-1. Biplot of maternal factors (das hed lines) and clutch survival parameters (solid lines) of American alligators collected during June 1999-2002. Arrows pointing in the same direction indi cate a positive correlation (e.g., unbanded egg% and DDE%), arrows that are approxi mately perpendicular indicate nearzero correlation (e.g., clutch viability and DDE%), and arrows pointing in opposite directions indicate negative co rrelations (clutch viability and transchlordane%]. Arrow lengths indicate ra nk order of correlatio ns. For example, extending a perpendicular line from the early emb. mort. axis to tip of transchlordane% arrow indicates that early emb. mort. and trans-chlordane have a stronger positive correlation compared to early emb. mort. correlation and [HE]. The cosine of the angle formed at the origin between individual clutch variables and individual OC P variables is the correlation coefficient (r). For example, arrows pointing in exactly oppos ite directions have an angle of 180, and since cos(180) = -1.0, the arrows are perfectly negatively correlated (r) (ter Braak, 1995).

PAGE 115

CHAPTER 5 MORPHOLOGY AND HISTOPATHOLOGY OF AMERICAN ALLIGATOR ( ALLIGATOR MISSISSIPPIENSIS ) EMBRYOS FROM REFERENCE AND OCPCONTAMINATED HABITATS In central Florida, American alligators living in habitats contaminated with organochlorine pesticides (OCPs) have poor reproductive success in comparison to populations inhabiting reference sites (Wood ward et al., 1993) (Wiebe et al., 2001). Decreased reproductive efficiency has been largely attributed to increased rates of early embryo mortality (mortality occurring first 35 days of development) and, to a lesser extent, late embryo mortality (mortality afte r day 35), as well as increased incidence of unbanded eggs, with unbanded eggs likely being a product of in fertility, or preovipositional embryo mortality, or a combin ation of both (Masson, 1995; Wiebe et al., 2001; Rotstein et al., 2002). A clear dose-response relationship betw een embryo mortality and total OCP burdens in eggs has not been established (H einz et al., 1991), and recent studies, on Lake Apopka, suggested poor egg viab ility was more closely associated with muck farm reclamation (wetland restorati on) sites than with tissue a nd egg concentrations of the predominant pesticide residue (DDE) (Giroux, 19 98). Muck farming typically refers to a farming practice where a dike is built around a marshy area adjacent to a lake, then the water is pumped out of the marsh, and the fert ile peat (i.e., “muck”) is then used for crop production. In addition, altered endocrine f unction and decreased egg viability were documented among alligators at another si te, Lake Griffin, where tissue and egg concentrations of OCP residue s such as DDE are intermediate in comparison with Lake

PAGE 116

100 Apopka, but Lake Griffin is also highly eu trophic and has adjacent muck farms and muck farm reclamation areas. Although a clear dose-response relationship ha s not been established with respect to embryo mortality and total organochlorine pesticide burdens, great differences exist, nonetheless, between sites with respect to OCP egg burdens and OCP constituent composition, suggesting mixture composition may pl ay a role or even be more important than simple cumulative OCP burdens. OCPs are of concern because they are prevalent and persistent environmental contaminants that are lipid soluble, resistan t to metabolic degradation, bioaccumulate in animal tissues, and may cause altered function of the immune system, as well as neural toxicity (Blus, 1996). Furthermore, in vitro and in vivo experiments using laboratory organisms, as well as epidemiology studies involving OCP-exposed human and wildlife populations, suggest a variety of OC Ps and OCP metabolites, such as dichlorodiphenyltrichloroethane (DDT), dichlorodiphenyltrichloroethylene (DDE), methoxychlor, dicofol, chlordane, dieldri n, and toxaphene, may be associated with disrupted endocrine function and altered embryo development (Colborn et al., 1993; Gray et al.; 1997; Fairbrother et al., 1999; Longnecker et al., 2002; Rattner & Heath, 2003). Although increased early embryonic mortality and late embryo mortality have been documented, few histopathology studies have ex amined live, moribund, or dead alligator embryos to determine whether alterations in morphology and/or spec ific pathogenicities are associated with increased mortality rates or specific OCPs and OCP burdens in eggs. Such histopathology studies are arduous due to rapidity of tissue autolysis, confounded by the difficulty in determining whether an embryo is alive or dead. Determining

PAGE 117

101 whether an embryo is alive or dead, when it is still inside the egg, is difficult because bright light candling is currently the onl y practical method for determining embryo viability in large studies involving thousands of eggs. Using bright light candling, live embryos are differentiated from dead embryos based on the color of the illuminated egg, with bright red indicating a live embryo (or very recently dead) and orange-pink indicating a dead embryo (color changes may be related to breakdown of red bloods cells and general autolysis of egg and embryo membranes). Although difficult, examining morphologica l development and histopathology of live and dead embryos would aid in understand ing the causes and mechanisms associated with the embryo mortality. For example, histopathology may indicate occurrence of acute chemical toxicity since it is known that many types of pesticides, including OCP compounds, induce toxicopathic lesions in vita l organs, with liver being the predominant target organ (Metcalfe, 1998). Furthermor e, evaluating how changes in morphology and histopathology relate to clutch mortality ra tes and OCP egg burdens may provide insight as to whether OCPs play a role in the in creased incidence of em bryo mortality observed in OCP-contaminated lakes. Therefore, the objective of the presen t study was to evaluate embryo morphology as a function of embryo conditi on (live/dead), lake of origination, clutch quality, and OCP egg burden, and to evalua te the histopathology of em bryos from clutches with diverse OCP egg burdens and mortality rates. To accomplish this objective the following hypotheses will be tested. Firs t, morphological development of live alligator embryos is different from dead embryos of the same chronological age. Second, morphological development of live embryos of the same ch ronological age is different among reference

PAGE 118

102 and OCP-contaminated sites. Third, morphol ogy of live embryos from clutches with low mortality rates is different from those of cl utches with high mortality rates, and fourth, variation in morphological deve lopment of live embryos is associated with composition and/or concentration of OCPs in eggs. Lastly, histopathology of live embryos from clutches with low mortality rates and low OCP egg burdens is different from those of clutches with high mortality rates and high OCP egg burdens. Materials and Methods Site Descriptions Lakes Apopka (N 28 35’, W 81 39’), Griffin (N 28 53’, W 81 46’), Emeralda Marsh Conservation Area ((N 28 55’, W 81 47’), and Lochloosa (N 29 30’, W 82 09’) in Florida were selected as collection sites because prior studies by our laboratory indicate vastly different levels of OCP expos ure across these sites. All three lakes are part of the Ocklawaha Basin. Lake Lochloos a (which is connected to Orange Lake) was selected as a low exposure (reference) site. Four years (2000-2002) of data indicate mean total OCP concentrations in egg yolks from the reference sites (Lake Lochloosa) were 102 15 ppb (mean standard error [SE], n = 19 clutches) with a concurrent mean clutch viability rate (number of live hatchli ngs/total number of eggs in a nest) of 70 4% Lake Griffin was selected as an intermed iate exposure site sin ce yolk concentrations averaged 1,169 423 ppb ( n = 42 clutches) and Lake A popka was selected as a high exposure site since yolk concen trations averaged 7,582 2,008 ppb ( n = 23) for the same time period (Gross, unpublished data). Furthe rmore, mean clutch viability rates during this time period for Lakes Apopka (51 6%, n = 23) and Griffin (44 5%, n = 42) have been below rates observed for the reference site.

PAGE 119

103 Egg Collections In the field, clutches were located vi a aerial surveys (helicopter) and ground surveys (airboat). Each clutch was provide d with a unique identif ication number, and immediately transported in plastic pans (43 cm x 33 cm x 18 cm) c ontaining the original nest substrate material to the US Geologi cal Survey’s Center for Aquatic Resources Studies, Gainesville, Florida (CARS). Upon a rrival, complete clutches were evaluated for embryonic viability using a bright light ca ndling procedure. Viable eggs (i.e. having a visible band) were nested in pans cont aining moist sphagnum moss and incubated at 30.5C and ~98% humidity, in an incubation building (7.3 m x 3.7 m). This intermediate incubation temperature will normally result in a 1:1 male/female sex ratio. One or two eggs were sacrificed from each clutch to identify the embryonic stage of development at the time of collection, and to evaluate the concentrations of OCPs in yolk. From each clutch, information on the following paramete rs was collected: total number of eggs found per nest (fecundity); number of unbande d eggs, number of damaged eggs, number of dead banded eggs, number of live bande d eggs, total clutch mass and average egg mass of clutch. Then, each clutch was evenly divided between two pans, with half of the clutch left relatively undisturbed (except fo r weekly monitoring of embryo viability) to determine clutch viability (the number of live hatchlings / fe cundity), and the other half of the clutch used to study embr yo development and morphometry. Embryo Sampling and Measurement After initial determination of morphological ages (MA) for all clutches (Ferguson, 1985), 2-4 live embryos were collected from each clutch at each of four selected chronological ages. Morphol ogical age (MA) refers to the age of the embryo as determined by level of morphological developm ent and chronological ag e (CA) refers to

PAGE 120

104 the calendar age when an embryo was sampled. For example, two clutches are initially examined and it is determined that embryos of clutch A are of MA Day 12 and those of clutch B are of MA Day 10. W ith respect to the initial ag e determination, it is assumed that MA = CA. Furthermore, if embryos of both clutches are to be sampled at CA age14, then clutch A would be sampled two days after initial examination and Clutch B would be sampled 4 days after initial examina tion. MA can then be determined (based on morphological features), and can be compar ed to CA to see if actual morphological development (MA) differs from what would be expected, given the particular CA. The four chronological ages sampled were Day 14, Day 25, Day 33, Day 43. These ages were selected because each are clearly distingui shable from other ages, and provide a good representation of progression of organogenesi s and growth (Ferguson, 1985). These ages also correspond to periods of increased em bryo mortality, as determined by previous studies (Masson, 1995). The following parameters were measured on fresh embryos (live and dead): egg mass; embryo condition (live or dead), em bryo mass, embryo morphological age, eye length, head length, and total le ngth of embryo. Dead embryo s were differentiated from live embryos based on the lack of a visible ca rdiac contractions and signs of autolysis, such as atypical coloration and loss of tissue integrity. Other parame ters were derived in an attempt to determine more subtle differences in development. These derived parameters included the following ratios: eye leng th / head length (Eye L.: Head L.); head length / total length (Head L.: Total L.); a nd total length / embryo mass (Total L.: Emb. M.).

PAGE 121

105 Morphological age was determined using Ferguson’s (1985) crocodilian developmental staging scheme. Fresh em bryos were photographed with either a Olympus model DPII digital cam era mounted to a Zeiss model Stemi SV 6 dissecting scope (for embryos of age Day 9 or less) or with a Canon EOS D30 digital camera mounted to a photographer’s stand (for embryos of age Day 10 or greater) (Fig. 5-1). Embryos were measured from digitized photographs using an image analysis software program, SigmaScan Pro (Systat Inc., 1999). After being photographed, embryos were fixed in formalin and stored in labeled containers for histopathology. Histopathology Subsamples of live embryos from “best case” clutches (clutch viability > 71%, which is equal to overall mean clutch viabi lity + 1 standard deviation) and low total OCP egg burdens ( 350 ng/g) and live embryos from “wor st case” clutches (< 47%) and high total OCP egg burdens (i.e., 3,700) were selected and pr ocessed for histopathology. Comparing best case to worst case provided the best opportunity for determining if differences existed with respect to frequency of lesions and identifyi ng target organs and tissues. If large differences were found be tween embryos of best case and worst case clutches, subsequent examin ations could be conducted on embryos of intermediate quality clutches. Conversely, if no differences were found, it would be unlikely to detect differences in intermediate quality clutches ; therefore, subsequent examinations would not be warranted. Embryos were cross-sectioned, and then f our equidistance step-sections were taken from each of the following regions: the hea d, the thorax, and the abdomen. For each of the 7 m sections, distances between st ep-sections ranged from ~42-300 m, depending on the age of the embryo, with inter-sectiona l distances increasing with embryo size.

PAGE 122

106 Sections were mounted to slides and staine d with hematoxylin and eosin. Slides were screened for lesions, the type of lesion present, and the organ or tissue involved. Expected morphological changes due to chronic OCP exposure include hepatocellular hypertrophy and focal necrosis. Hypertrophy is due to enlargement of the smooth endoplasmic reticulum (SER) and formati on of a lipid droplet in the center of the SER (caused by OCP-induced expression of microsomal enzymes within the SER) (Smith, 1991). Other morphological changes found in the liver include foci of vacuolated hepatocytes and spongiosis hepatic (lesions of hepatic parenchyma). Renal lesions induced by chronic OCP exposure include dilati on of tubular lumina, and vacuolization (degeneration) and necrosis of tubul ar epithelium (Metcalfe, 1998). Other than hepatic and renal toxicopath ic lesions, OCPs may cause death by disrupting neural transmission to the point of cardiovascular failure. Neural morphology is rarely changed by OCP exposure, which causes difficulty in determining whether cardiovascular failure was caused by OCP exposure or some other factor. Analysis of OCPs in Yolk Analytical grade standards for the following compounds were purchased from the sources indicated: aldrin, al pha-benzene hexachloride ( -BHC), -BHC, lindane, -BHC, p,p’ -dichlorodiphenyldichloroethane ( p,p’ -DDD), p,p’ -dichlorodiphenyldichloroethylene ( p,p’ -DDE), dichlorodiphe nyltrichloroethane ( p,p’ -DDT), dieldrin, endosulfan, endosulfan II, endosulfan sulfate, endrin, e ndrin aldehyde, endrin ketone, heptachlor, heptachlor epoxide, hexachlorobenz ene, kepone, methoxychlor, mirex, cis -nonachlor, and trans -nonachlor from Ultra Scientific (Kingstown, RI, USA); cis -chlordane, trans chlordane, and the 525, 525.1 polychlorinated biphenyl (PCB) Mix from Supelco (Bellefonte, PA, USA); oxychlordane from Chem Service (West Chester, PA); o,p’-

PAGE 123

107 DDD, o,p’DDE, o,p’DDT from Accustandard (New Haven, CT, USA); and toxaphene from Restek (Bellefonte, PA, USA). All reag ents were analytical grade unless otherwise indicated. Water was doubly distilled and deionized. Egg yolk samples were analyzed for OC P content using methods modified from Holstege et al. (1994) and Sc henck et al. (1994). For extraction, a 2 g tissue sample was homogenized with ~1 g of sodium sulfate a nd 8 mL of ethyl acetate. The supernatant was decanted and filtered t hough a Bchner funnel lined with Whatman #4 filter paper (Fisher Scientific, Hampton, NH, USA ) and filled to a depth of 1.25 cm with sodium sulfate. The homogenate was extracted twice with the filtrates collected together. The combined filtrate was concentrated to ~2 mL by rotary evaporation, and then further concentrated until solvent-free under a stre am of dry nitrogen. The residue was reconstituted in 2 mL of acetonitrile. Afte r vortexing (30 s), the supernatant was applied to a C18 solid phase extraction (SPE) car tridge (pre-conditio ned with 3 mL of acetonitrile; Agilent Technologies, Wilmingt on, DE, USA) and was allowed to pass under gravity. This procedure was repeated twice with the comb ined eluent collected in a culture tube. After the last addition, the car tridge was rinsed with 1 mL of acetonitrile which was also collected. The eluent was then applied to a 0.5 g NH2 SPE cartridge (Varian, Harbor City, CA, USA), was allowe d to pass under gravity, and collected in a graduated conical tube. The cartridge was rinsed with an additional 1 mL portion of acetonitrile which was also collected. The combined eluents were concentrated under a stream of dry nitrogen, to a volume of 300 L, and transferred to a gas chromatography (GC) vial for analysis.

PAGE 124

108 GC/MS Analysis Analysis of all samples was performed using a Hewlett Packard HP-6890 gas chromatograph (Wilmington, DE, USA) with a split/splitless inlet ope rated in splitless mode. The analytes were introduced in a 1 L injection and separa ted across the HP-5MS column (30 m x 0.25 mm; 0.25 m film thickne ss; J & W Scientific, Folsom, CA, USA) under a temperature program that began at 60 C, increased at 10 C/min to 270 C, was held for 5 min, then increased at 25 C/min to 300 C and was held for 5 min. Detection utilized an HP 5973 mass spectro meter in electron impact m ode. Identification for all analytes and quantitation for toxaphene was c onducted in full scan mode, where all ions are monitored. To improve sensitivity, se lected ion monitoring was used for the quantitation for all other analytes, except kepone. The above program was used as a screening tool for kepone which does not optim ally extract with mo st organochlorines. Samples found to contain kepone would be reex tracted and analyzed specifically for this compound. For quantitation, a five-point standard curve was prepared for each analyte ( r2 0.995). Fresh curves were analyzed with each se t of twenty samples. Each standard and sample was fortified to contain a deuterat ed internal standard, 5 L of US-108 (120 g/mL; Ultra Scientific), added just prior to analysis. All samples also contained a surrogate, 2 g/mL of tetrach loroxylene (Ultra Scientific) added after homogenization. Duplicate quality control samples were prepar ed and analyzed with every twenty samples (typically at a level of 1.00 or 2.50 g/mL of -BHC, heptachlor, aldr in, dieldrin, endrin, and p,p’ -DDT) with an acceptable recovery rangi ng from 70 – 130%. Limit of detection ranged from 0.1-1.5 ng/g for all OCP analyt es, except toxaphene (120-236 ng/g), and limit of quantitation was 1.5 ng/g for all anal ytes, except toxaphene (1500 ng/g).

PAGE 125

109 Repeated analyses were conducted as allo wed by matrix interferences and sample availability. Results Inter-Site Clutch Comparisons A total of 58 clutches were coll ected during June 2001 and 2002 from Lakes Apopka, Griffin, Lochloosa and Emeralda Mars h Conservation Area. No differences ( = 0.05) were determined between sites with re spect to the following clutch parameters: fecundity (overall mean standard error: 43 1), clutch viability (56 3.9%), damaged eggs (4 1.7%), unbanded eggs (12 1.9%), early embryo mortality (i.e., mortality prior to Day 36; 14 2.7%), and late embryo mort ality (i.e., on or afte r Day 36; 14 2.5%). However, the average egg mass of clutches fr om Emeralda Marsh was greater than that of Lake Griffin (Table 5-1). Significant differences were noted between sites in recent studies (Chapter 2) which a had a larger total sample size (n = 168). The lack of significant differences was likely due to the large variance noted in Emeralda clutches (Table 5-3). Many differences were detected between sites with respect to egg yolk OCP concentrations. Alligator eggs from Emer alda Marsh and Lake Apopka were found to have a greater number of OCP analytes ( n = 12 and 11, respectively) as compared to Lake Griffin ( n = 10), which was greater than Lochloosa ( n = 8.5) (Table 1). Eggs from Emeralda Marsh yielded the highest tota l OCP concentrations (29,838 4,844 ng/g), which were over three-fold greater than thos e of Lake Apopka, 32-fold greater than those of Lake Griffin, and 290-fold greater than those of Lake Lochloosa. Furthermore, 45% of individual OCP analytes were at greater concentrations in Emeralda eggs as compared to those of Lake Apopka, with major differe nces in total OCP egg yolk concentrations

PAGE 126

110 related to amounts of toxaphene (thr ee-fold greater in Emeralda) and p,p’ -DDE (two-fold greater in Emeralda). Other OCP analyte c oncentrations were similar between Emeralda and Lake Apopka, except for a few DDT and ch lordane analytes. When individual OCP egg yolk concentrations from Apopka and Emeralda were compared against Lakes Griffin and Lochloosa, 82% of the individual OCP analyte con centrations were greater in eggs from Emeralda Marsh and Lake Apopka as compared to the other lakes (Table 5-1). Intra-Site Live Embryo/Dead Em bryo Morphological Comparisons Comparisons between live and dead embr yos sampled at chronological age (CA) Day 14 yielded the following results. For Lakes Lochloosa, Apopka, and Griffin clutches, live embryos sampled at CA Day 14 had an overall morphological age (MA) of 15 0.3 (mean standard error), which was greater than the MA (11 1) of dead embryos, and live embryos were of greater mass compared to dead embryos, suggesting that dead embryos may have been devel opmentally retarded. One other notable difference between live and dead embryos samp led at CA Day 14 was that eggs of dead embryos were greater in mass compared to live cohorts for Lakes Apopka and Griffin; however, for Lake Lochloosa, eggs of live embryos were greater in mass compared to dead cohorts (Table 5-2). Comparisons betw een eye length, head le ngth, and total length were not made because dead embryos coul d not be uniformLy positioned for photographs due to their size and fragility of tissues resulting from the early stage of development and limited autolysis. For Lake Lochloosa, live and dead embryos of CA Day 25 differed with respect to egg mass, embryo mass, and MA (for all endpoints: live > dead), and Total L.: Emb. M., with live Total L.: Emb. M. ratios being less than those of dead embryos. For Lake Apopka, live and dead embryos differed with respect to embryo mass, Eye L.: Head L.,

PAGE 127

111 morphological day (for all endpoin ts: live > dead), and Head L. : Total L. (live < dead). No significant differences we re detected between live and dead embryos from Emeralda clutches for the measured endpoints. For La ke Griffin, differences between live and dead embryos were determined for embryo mass, eye length, head lengt h, and MA (for all endpoints: live > dead) (Table 5-2). For Lake Lochloosa, live and dead embryos of CA Day 33 differed with respect to embryo mass and MA (for both endpoints: liv e > dead). For Lake Apopka, live embryos had greater mass, eye length, and MA than d ead embryos, but dead embryos had greater Total L.: Emb. M.. Live embryos from Emeralda Marsh clutches were of greater mass and Total L.: Emb. M. than dead cohorts. Li ve embryos from Lake Griffin clutches were also of greater mass and were of greater MA in comparison to dead cohorts (Table 5-2). For Lake Lochloosa, live and dead embryos sampled at CA Day 43 differed with respect to embryo mass, eye length, head length, Head L.:Total L., and MA (for all endpoints: live > dead). For Lake Apopka, live embryos were of greater mass, head length, total length, and morphological age compared to dead c ohorts. Live embryos of Emeralda Marsh were of greater morphological age than dead cohorts, however, power of detection was low since only one dead embryo was sampled. Lastly, live embryos of Lake Griffin were of greater mass and morphological age th an their dead cohorts (Table 5-2). Inter-Site Comparisons of Morphology of Live Embryos Egg mass of live embryos of CA Day 14 di ffered between sites, with respect to egg mass and MA. Eggs of CA Day 14 embryos from Lake Lochloosa clutches were of greater mass compared to those of Emeral da and Griffin, and MA of Lake Griffin embryos was greater than that of Emeralda Ma rsh (Table 3). For embryos sampled at CA

PAGE 128

112 Day 25, egg mass, embryo mass, eye lengt h, head length, total length, and TL: EM differed among sites. Eggs from Lake Loch loosa were of greater mass than all other sites, but embryo mass for Lochloosa clutches was less than that of Griffin. Embryos from Emeralda and Lake Griffin had the gr eater eye lengths in comparison to Lake Apopka with no significant differences dete cted for Lake Lochloosa embryos. With respect to head lengths and to tal lengths, embryos from La ke Apopka clutches were less than those of all other sites and embryos from Lake Griffin were greater than those of all other sites except for Emeralda. Lastly, Lochloosa embryos had higher Total L.: Emb. M. than all other sites except fo r Lake Apopka (Table 5-3). For CA Day 33 embryos, only egg mass and MA differed between sites. Similar to earlier sampling periods, Lake Lochloosa eggs were of greater mass than all other sites except for Apopka. Lake Griffin embryos were of greater MA than all other sites, and Lake Apopka embryos were of lesser mass than all other sites except for those of Emeralda. For CA Day 43 embryos, Lochloosa eggs were of greater mass than all other sites, and Lochloosa embryos were of lesser mass than all other sites except for Lake Apopka. Embryos of Lake Griffin were of greater ma ss than all other sites. Embryos of Lake Apopka and Lake Griffin also had greater eye lengths than those of Emeralda. For TL: EM ratios, Lake Griffin embryos had smaller ra tios compared to all other sites except for Emeralda. In addition, the MA of embryos of Lochloosa was less than all other sites except Emeralda (Table 5-3). Live Embryo Morphology and Em bryo Survival Relationships Redundancy analysis with forward select ion (Monte Carlo permutation tests for significance) was used to examine whethe r embryo morphometric parameters (eye

PAGE 129

113 length, head length, total lengt h, and embryo mass) were strongly associated with embryo survival parameters (clutch viability, early embryo mortality, and late embryo mortality percentages) for each of the chronological ages (CA) sampled (Day 14, Day 25, Day 33, Day 43). For CA Day 14 embryos, results of the RDA indicat ed only late embryo mortality percentage was significantly asso ciated with the observed variation embryo morphology, but accounted for only 7% of the morphological variation. For CA Day 25 and Day 33 embryos, no significant associa tions were found between morphological and embryo survival parameters. For CA Day 43, cl utch viability was determined significant but accounted for only 5% of the variation in morphology. Live Embryo Morphology and Eg g Yolk OCP Burdens In contrast to embryo survival paramete rs, OCP concentrations in egg yolks were significantly associated with variation in embryo morphology. For CA Day 14 embryos, partial-redundancy analysis usi ng site (i.e., Emeralda, Apopka Griffin, Lochloosa) as the covariate (since OCP burdens differed among site s), with forward selection of the best five OCP variables (Table 4), indicated that four of five sele cted variables were determined to be significant via Monte Carlo permutation tests. The four OCP variables were oxychlordane concentra tion ([OX]), heptachlor epoxid e percentage of total OCP burden (HE%), toxaphene percentage of total OCP burden (TX%), and trans -nonachlor percentage of total OCP burden (TN%). Th ese OCP variables accounted for 47% of the observed variation in embryo morphometric pa rameters. Individually, [OX] explained 20% of the variation in the morphometric parameters, followed by HE% (11%), TX% (10%), and TN% (6%) (Table 5). Embryo h ead length was negatively correlated with [OX] and HE%. Embryo eye length was ne gatively correlated with TN% and HE%.

PAGE 130

114 Embryo mass was positively correlated with [OX] and HE%, and embryo total length was negatively correlated with [OX] but pos itively correlated with TN%.(Fig. 5-2). For CA Day 25 embryos, four of five sele cted variables were determined to be significant, with HE%, cis-Nonachlor percen tage of total OCP burden (CN%), dieldrin percentage of total OCP burden (DL%), and dieldrin concentrati on ([DL]) accounting for 24% of variation of embryo morphological parameters. HE% accounted for 11% of embryo morphological variation, followed by CN% (6%), DL% (5%) and [DL] (2%) (Table 5). Embryo head length was positivel y correlated with DL%, CN%, HE%, and [DL]. Total embryo length was also positively correlated with HE%, DL%, and [DL], but showed little correlation with CN%. In contrast to head length and total length, embryo mass and eye length were negatively correlated with HE%, DL%, and [DL], but showed little correlation with CN% (Fig. 5-3). For CA Day 33 embryos, three of five sele cted variables were determined to be significant and consisted of CN%, DL%, cischlordane percentage of total OCP burden (CC%) and accounted for 24% of morphological variation (Table 5). Embryo head length and total length were positively correl ated with DL% and negatively correlated with CC% and CN%. In contrast, embryo mass and eye length were positively correlated with CC% and CN%, but showed little correlation with DL% (Fig. 5-4). For CA Day 43 embryos, three of five sele cted variables were determined to be significant and together accounted for 20% of the morphological variation. These variables consisted of p,p’ -DDT concentration ([pDDT]), cis -chlordane concentration ([CC]), and total number of individual OCP anal ytes detected in yolk (NOC) (Table 5). Embryo mass was positively correlated w ith NOC and [pDDT], but showed little

PAGE 131

115 correlation with [CC]. Embryo eye lengt h was positively correlated with NOC, negatively correlated with [CC], and showed no correlation with [pDDT]. Embryo head length was positively correlated with NOC and negatively correlated with [CC] and [pDDT]. Total embryo length was negatively correlated with NOC, not correlated with [pDDT], and positively correlated with [CC] (Fig. 5-4). Embryo Morphological Age, Derived Morphometric Variables and Egg Yolk OCP Burdens For embryos sampled at CA Day 14, four of five RDA-selected OCP variables were determined to be signifi cant and accounted for 44% of th e variation associated with morphological age (MA) and deri ved morphometric variables (DMV), which consisted of Eye L.: Head L., Head L.: Total L., and To tal L.: Emb. M.. The four extracted OCP variables were [OX], CN%, OX%, and HE%, and each respectively accounted for 21%, 12%, 7%, and 4% of variance associated w ith MA and DMV (Table 5-6). With the exception of Total L.: Emb. M., all DMV and MA were positively correlated with were [OX], OX%, and HE%. Total L.: Emb. M. was positively correlated with CN%, and CN% was negatively correla ted with MA and the other DMV (Fig. 5-6). For CA Day 25 embryos, three of five selected OCP variables were determined significant and accounted for 22% of MA and DMV variance. The three OCP variables consisted of HE%, NOC, and [pDD] and each respectively accounted for 12%, 7%, and 3% of the variation in MA and DMV (Table 6). HE% was positively correlated with Total L.: Emb. M. and negatively correlated with MA, Head L.: Total L., and Eye L.: Head L. NOC was negatively correlated with Total L.: Emb. M. and positively correlated with MA, Head L.: Total L., and Eye L. [p DD] was positively correlated with Head L.:

PAGE 132

116 Total L. and Eye L.: Head L., but showed little correlation with MA and Total L.: Emb. M (Fig. 5-7). For Day 33 embryos, three OCP variables (DL%, PDE%, and CN%) evenly accounted for 15% of variation in MA a nd DMV (Table 6). DL% was negatively correlated with Head L.: Total L. and Eye L. : Head L., but showed little correlation with MA and was positively correlated with Total L.: Emb. Mass. PDE%, and CN% were positively correlated with MA, Head L.: Tota l L. and Eye L.: Head L., but showed a negative correlation with Tota l L.: Emb. M. (Fig. 5-8). Lastly, for CA Day 43 embryos, four of five OCP variables selected via partial RDA were determined to be significant and accounted for 24% of variation in MA and DMV. These four OCP variables consisted of, with respect to amount of variation accounted for, PDT% (8%), [CC] (5%), NOC (5%), and [PDT] (4%) (Table 6). PDT%, NOC, and [PDT] were positively correlated with Head L.: Total L., Eye L.: Head L., and MA, but were negatively correlated with Total L.: Emb. M. [CC] was negatively correlated with Head L.: Total L., Eye L.: H ead L., and MA, but was positively correlated with Total L.: Emb. M. (Fig. 5-9). Histopathology of Live and Dead Embryos Results of histopathology of live embryos ( n = 34) from five reference clutches (clutch viability > 71% and OCP yolk burdens < 350 ng/g) and live embryos ( n = 26) from four OCP-contaminated clutches (clu tch viability < 47% and OCP yolk burdens > 3,700 ng/g) indicated that 16% of all embryos exhibited at least one type of hepatic lesion, followed by lesions of the skeletal mu scle (5%), and kidney (3%). Hepatic lesions included necrosis (characterized by pyknotic nuclei and vacuolated hepatocytes) and cholestasis. Lesions detect ed in skeletal muscle incl uded necrosis characterized by

PAGE 133

117 pyknotic nuclei and segmented sarcoplasm. Ki dney lesions included necrosis of tubules characterized by vacuolizati on and pyknotic nuclei. No significant differences were determined between reference embryos and OCP-contaminated embryos with respect to incidence of hepatic lesions ( 2 = 0.87, p = 0.49), renal lesions ( 2 = 1.58, p = 0.50), or muscular lesions ( 2 = 2.41, p = 0.25). Histopathology results of dead embryos ( n = 20) from OCP-contaminated sites indicated that generalized autolysis was the predominant finding, with fungi hyphae present in 2 embryos, and a si ngle case of menigoencephalitis that would be consistent with a bacterial infection. Advance autolysis, in some cases, likely impeded detection of cytoxic lesions. Discussion With respect to the first hypothesis, re sults suggest that certain morphological parameters of live alligator embryos differ from those of dead embryos of the same chronological age. Intra-site comparisons s uggested that among all sites and all sampled ages (CA) embryo MA and mass were greate r for live embryos as compared to dead embryos. Importantly, the concurrent decr eases in MA and mass of dead embryos suggests that embryos may have been deve loping normally up to a point at which development stalled and the embryo eventu ally perished, or embryos could have developed at a much slower overall rate until the point at which they perished. Either way it appears that the mass of dead embryos was appropriate for their MA. For example, live embryos of Lake Griffin samp led at CA Day 14 had an average MA of ~ Day 16 and an average mass of 0.41 g, which wa s similar to the MA (~ Day 15) and mass (0.41 g) of dead embryos sampled at CA Day 25 (Table 2). Other measured parameters and derived parameters showed variation in patterns among sites and ages, but one

PAGE 134

118 consistency was that when differences were detected, measured parameters were almost always greater in live embryos as compared to dead embryos. With the exception of Lake Apopka, few significant differences were found between live and dead embryos with respect to derived morphometric parameters (i.e., Eye L.: Head L.; Head L.: Total L.; and To tal L.: Emb. M.). Differences were found between derived morphometric parameters of live and dead embryos sampled at older ages (CA) from Lake Apopka, and suggest that morphology of dead embryos of Lake Apopka is disproportionate compared to liv e cohorts. The differences between the patterns of morphometric relationships of live and dead embryos from Lake Apopka as compared to other sites, may indicate the cause s or mechanisms associated with mortality of Lake Apopka embryos differ from other site s, since it has been s hown that the type of teratogenic effect may depend on the specifi c teratogenic agent or cause (Schmidt & Johnson, 1997). With respect to the second hypothesis, results suggested that morphology of live embryos was not consistently different among sites, except for live embryos of CA Day 25. For Day 25 live embryos, embryos of Lake Griffin and Emeralda Marsh were consistently larger, with respect to measurem ent parameters, than those of Lakes Apopka and Lochloosa. The only differences found, w ith respect to derived parameters, was for Total L.: Emb. M., with Lochloosa embryos app earing to be leaner embryos compared to those of Emeralda and Griffi n. Since Day 25 is during the middle of organogenesis, this stage of development may be more sensit ive to OCP exposure or variation in yolk nutrient content since it has been shown in other species the peri od of organogenesis is

PAGE 135

119 most susceptible to altera tions caused by teratogen expos ure or nutrient excess or deficiency (Schmidt & Johnson, 1997). With respect to the third hypothesis, re dundancy analysis results indicated that variation in morphometry of live embryos is not significantly related to variation in clutch mortality rates, suggesting that live embryos from clutches with high mortality rates develop similarly to those of low mort ality clutches. This finding may suggest a threshold-type response in which embryos expos ed to stressors below a certain threshold have the ability to overcome stressors through various cellu lar homeostatic mechanisms, but above a certain threshold, developmental retardation a nd lethality occur. Such threshold dose-response patterns have been acc epted as a major dose-response pattern in mammalian developmental toxico logy (Rogers & Kavlock, 2001). With respect to the fourth hypothesis, va riation in morphologi cal development of live embryos was significantly associated with variation in the composition and concentration of OCPs in eggs. However, th e strength of the relationships appeared to decrease with the age sampled (CA), with youngest embryos sampled (CA Day 14) showing the strongest rela tionships between OCP egg burden and morphometric parameters, followed by each subsequent CA, respectively (Table 5-5). Interestingly, the percentage of the total OCP burden (concentr ation) composed by an OCP analyte (i.e., HE%), appeared to be more important than OCP analyte concentrations alone. With respect to all sampled ages, except the elde st (CA Day 43), OCP percentage variables accounted from a minimum of 47% to a ma ximum of 100% of the total variation attributed to all OCP variables found to be significantly associated with variation in measured morphometric parameters (Table 55). For derived morphometric parameters

PAGE 136

120 and morphological age (MA), similar patterns were observed in that embryos sampled at younger CA showed stronger rela tionships with OCP burdens than older cohorts (Table 5-6). With respect to individual OCP analytes, the cyclodienes appear to be more important than the dichlorodi phenylethanes, in that cyclodienes accounted for an average of 70% of the morphometric variation that co uld be attributed to OCP variables for all sampled ages. This is surprising cons idering that dichlorodiphenylethanes ( p,p’ -DDT + p,p’ -DDD + p,p’ -DDE) make up an average of 66% of the total OCP burden among all sites (Table 5-1). Another important observation was that di fferent cyclodienes appeared to be associated with morphological variation of embryos of different ages (CA). Most important were the components of technical grade chlordane and its metabolites, which include cis and trans -chlordane, cis and trans -nonachlor, oxychlordane, and heptachlor epoxide. One or more of these components were found to be significantly associated with variation in embryo morphology for each CA sampled. These data suggest that the chlordane group may merit furthe r study in relation to developm ental effects in reptiles, especially considering other st udies have suggested that sexu al differentiation in turtles may be altered by low dose in ovo exposures of these compounds (Willingham, 2004). With respect to the final hypothesis, no si gnificant differences were found between the histopathology of live and dead embryos fr om best-case clutches (low mortality rates and low OCP egg burdens) compared to those of worst-case clutches (high mortality rates and high OCP egg burdens). Few signs of b acterial or fungal infections were found. These results may suggest that lesions were not a causal factor in death, and may not be

PAGE 137

121 associated with variation in OCP exposure or increased mortality rates. However, the death and autolysis of delicate embryonic tissu es may have obscured lesions associated with death and/or OCP exposure. In addi tion, OCPs may cause dysfunction in neural transmission, leading to cardiovascular fa ilure and mortality. Future studies may consider in ovo monitoring of neural transmissi on and cardiovascular function to determine if increased OCP exposure is associ ated with altered neural transmission and cardiovascular failure in alligator embryos. In conclusion, the present study found that embryo mortality occurring in alligator populations inhabiting refere nce and OCP-contaminated sites was characterized by developmental retardation without gross deformities or overt presence of lesions to vital organs. However, variation in embryo mo rphology appeared to be associated with variation in OCP burdens of eggs and the percentage composition composed by an OCP analyte was equally as important as concen tration, suggesting the importance of mixture composition. Younger embryos appeared more susceptible to OCP influence but OCP influence may not necessarily be the result of direct embryo effects. Similar types of embryo mortality has been documented in quail, with embryo mortality determined to be maternally mediated, where maternal liver function was altered, resulting in nutrient deficiencies in eggs that were severe enough to induce em bryo mortality (Donaldson & Fites, 1970). In summary, subsequent studi es should evaluate embryo mortality in alligators as a function of OCP exposure and egg nutrient content.

PAGE 138

122Table 5-1. Summary statistics for paramete rs measured on American alligator clut ches collected during June 2001 and 2002. Parameter Apopka Emeralda Griffin Lochloosa Summary No. Clutches (n) 15 7 18 18 58 Fecundity ( n ) 46 2 50 3 43 2 40 2 43 1 (28–56) (42–64) (19–58) (26–56) (19–64) Egg mass (g) 86 3.3 AB 107 18.1 A 79 3.2 B 89 3.2 AB 87 2.8 (67–120) (65–180) (46–113) (71–139) (46–180) Clutch viability (%) 53 8.6 63 13.8 46 6.7 64 5.7 56 3.9 (0–92) (0–96) (0–87) (0–95) (0–96) Damaged eggs (%) 1 0.4 1 0.7 7 4.2 4 3.3 4 1.7 (0–4) (0–4) (0–63) (0–60) (0–63) Unbanded eggs (%) 13 3.4 10 3.9 15 4.8 11 2.1 12 1.9 (0–40) (0–30) (0–65) (0–33) (0–65) Early Emb. Mort. (%) 14 6.2 24 12.7 13 4.4 13 3.1 14 2.7 (0–90) (0–95) (0–73) (0–36) (0–95) Late Emb. Mort. (%) 19 6.3 3 2.1 19 4.8 9 2.6 14 2.5 (0–77) (0–15) (0–58) (0–34) (0–77) Dieldrin (ng/g) 405.7 121.32 A 227.3 40.09 A 24.8 5.18 B 3.6 0.5 C 146.1 39.53 (23.5–1,859) (88–386.7) (4.4–76.9) (1.3–8.2) (1.3–1,859) Hep. Epoxide (ng/g) 14.4 3.01 A 5.3 1.38 AB 7.1 2.05 B 2.8 0.76 B 7.8 1.24 (1.2–46.8) (1.4–11.6) (1.1–29.6) (1.2–9.7) (1.1–46.8) cis -Chlordane (ng/g) 48.3 13.74 B 150.4 26.21 A 10.7 1.01 C 1.9 0.21 D 34.3 7.71 (6.6–179.2) (62.4–281) (4.3–16.9) (1.2–4.1) (1.2–281) cis -Nonachlor (ng/g) 70.4 16.41 A 89.2 15.7 A 17.1 2.7 B 4.6 0.63 C 35.7 6.29 (10.5–238.4) (55–166) (4.4–54.2) (2.4–12.5) (2.4–238.4) Oxychlordane (ng/g) 45.7 10.94 A 29.3 4.1 A 12.6 3.17 B 3.8 1.06 C 20.4 3.71 (3.9–176) (17.9–46.1) (1.1–45.9) (1.2–17.8) (1.1–176)

PAGE 139

123Table 5-1. Continued. Parameter Apopka Emeralda Griffin Lochloosa Summary Toxaphene (ng/g) 3,308 658.5 B 8,269 1,077.2 A 2,677 376.5 B nd C 4872 722.8 (1,896.1–9,678.3) (4,512.8–11,485.4) (1,927.9–3,110.8) (0–0) (1,896–11,485.4) p, p' -DDD (ng/g) 45.8 13.49 B 1,986 333.9 A 5.7 1.1 C 1.9 0.21 D 277.6 101.28 (10.6–192.8) (617.3–2962.8) (1.5–18.5) (1.2–2.9) (1.2–2962.8) p, p' -DDE (ng/g) 5,792 1,490.4 B 18,056.3 3,113.7 A 337 55.2 C 74.8 12.38 D 3,805 924 (18.3–22,421.9) (6,811.7–33,554.8) (94.6–979.1) (28–231) (18.3–33,554.8) p, p' -DDT (ng/g) 9.8 3.81 A 17.7 3.24 A 2.7 0.25 AB 1.3 0.02 B 9.4 2.17 (1.2–45.6) (5.8–25.3) (2.5–3) (1.2–1.3) (1.2–45.6) trans -Chlor. (ng/g) 7.4 2.38 B 44 4.61 A 1.6 0.19 C 2.6 0.73 BC 11.3 2.81 (1.3–27.4) (23.2–58.2) (0.8–3.4) (1.2–3.7) (0.8–58.2) trans -Nonachl. (ng/g) 202.5 54.77 A 278.2 56.93 A 42.4 9.31 B 8.4 1.67 C 101.7 20.46 (10.5–787.6) (148–554.9) (8.9–155.2) (2.5–24.6) (2.5–787.6) [OCP] (ng/g) 9,177 2,391.2 B 29,838 4,844.3 A 911 302.4 C 103 16.3 D 6,238.6 1,508.35 (555.2–35,587.8) (13,183.5–53,559.7) (128.7–4,487.7) (42.7–289.4) (42.7–53,559.7) No. OCPs ( n ) 11 0.28 A 12 0.22 A 10 0.18 B 8.6 0.28 C 10.1 0.2 (9–12) (11–13) (9–12) (6–10) (6–13) aValues represent mean standard error with ranges in parenthe ses. Letters beside values (A -D) indicate differences between si tes ( = 0.05). Clutch viability % = number of liv e hatchlings / fecundity x 100, damaged eggs % = number of damaged egg / fecundity x 100, unbanded eggs % = number of unbanded eggs / fecundity x 100, early embryo mortality % = number of embryos that died at age s Day 35 / fecundity x 100, late embryo mortality % = number of embr yos that died at ages >Day 35 / fecundity x 100, Hep. Epoxide = heptachlor epoxide, trans -Chlor. = trans -chlordane, trans -Nonachl. = trans -nonachlor, and No. OCPs = nu mber of OCPs detected at measurable levels.

PAGE 140

124Table 5-2. Comparisons of egg and embr yo morphometrics of live and dead embryos collected during June-August of 2001 and 2002. Agea Parameterb Apopka Emeralda Griffin Live Dead Live Dead Live Dead 14 n 7 9 12 23 8 Egg mass (g) 86.3 1.23 92.5 1.35* 78.6 1.35 81.6 1.92 86.7 2.6 Embryo mass (g) 0.38 0.12* 0.14 0.089 0.35 0.019 0.41 0.032 0.2 0 Eye L. (mm) 2.9 0 0 0 0 0 3.5 0.06 0 0 Head L. (mm) 8 0 0 0 0 0 9.1 0.28 0 0 Total L. (mm) 51.4 0 0 0 0 0 57.5 0.57 0 0 Eye L.: Head L. 0.36 0 0 0 0 0 0.39 0.012 0 0 Head L.: Total L. 0.16 0 0 0 0 0 0.16 0.006 0 0 Total L.: Emb. M. 171.33 0 0 0 0 0 111.5 15.353 0 0 Morph. Day 16 1.195 12.6 1.661 14 0 15.96 0.4* 12 3 25 n 20 13 24 6 30 37 Egg mass (g) 81.1 2.01 87.1 2.1 80 0.98 75.7 2.55 80.7 1.49 79.7 1.05 Embryo mass (g) 1.15 0.11* 0.56 0.131 1.45 0.116 1.57 0.27 1.51 0.088* 0.41 0.188 Eye L. (mm) 5 0.18 4.2 1.17 5.5 0.12 5.9 0 5.9 0.17* 2.5 0.98 Head L. (mm) 11.5 0.55 11.2 2.66 13.5 0.41 16.7 0 14.9 0.65* 7.7 3.54 Total L. (mm) 73.7 3.38 65.6 14.12 87.6 2.44 98.7 0 89.1 2.32 72.3 0 Eye L.: Head L. 0.44 0.01* 0.36 0.024 0.41 0.007 0.35 0 0.4 0.011 0.33 0.029 Head L.: Total L. 0.16 0.012 0.17 0.015 0.15 0.002 0.17 0 0.16 0.003 0.2 0* Total L.: Emb. M. 68.25 4.72 146.51 41.27* 58.89 2.47 47.02 0 52.24 3.526 0 0 Morph. Day 24.2 0.99* 17.63 1.475 26.42 0.58 28 0 26.13 0.619* 14.63 1.75 33 n 15 13 29 3 29 17 Egg mass (g) 82.4 2.08 81.5 3.4 80.8 1.15 75 3.09 77.7 1.42 81 1.4

PAGE 141

125Table 5-2. Continued. Agea Parameterb Apopka Emeralda Griffin Live Dead Live Dead Live Dead 33 Embryo mass (g) 3.14 0.09* 1.2 0.423 3.8 0.196* 1.2 0 4.04 0.203* 0.75 0.552 Eye L. (mm) 6.2 0.1* 3.3 0.52 6.4 0.12 6 0 6.5 0.1 6.6 0.27 Head L. (mm) 19.1 0.31* 10.7 3.21 20.1 0.49 19.1 0 20.6 0.35 19.9 0.34 Total L. (mm) 108.2 1.1* 67.8 13.25 114.4 2.54 108.8 0 117.8 2.19 111.9 5.02 Eye L.: Head L. 0.32 0.008 0.32 0.049 0.32 0.007 0.31 0 0.32 0.006 0.33 0.008 Head L.: Total L. 0.18 0.00* 0.15 0.017 0.18 0.002 0.18 0 0.18 0.001 0.18 0.005 Total L.: Emb. M. 34.77 0.81 291.4 254.57* 31.36 1.56 90.69 0* 31.41 1.467 30.53 0 Morph. Day 33.3 0.33* 19.83 3.323 35.69 0.57 33 0 39.28 0.854* 19 3.167 43 n 44 10 24 1 41 24 Egg mass (g) 81.2 1.45 85.4 2.56 79.8 1.19 73.5 0 78 1.32 80.6 1.34 Embryo mass (g) 9.91 0.27* 3.11 1.463 10.69 0.30 0 0 13.01 0.514* 6.77 2.706 Eye L. (mm) 7.3 0.14 7 0.4 6.5 0.11 0 0 7.5 0.2 6.8 0.75 Head L. (mm) 27.5 0.53* 21.6 5.57 26.1 0.41 0 0 27.4 0.9 24.6 4.08 Total L. (mm) 172.1 5.2* 130.1 36.33 170.1 2.14 0 0 184.9 7.05 167.8 29.4 Eye L.: Head L. 0.27 0.004 0.34 0.069* 0.25 0.006 0 0 0.35 0.08 0.29 0.041 Head L.: Total L. 0.16 0.003 0.17 0.004 0.15 0.002 0 0 0.19 0.042 0.15 0.003 Total L.: Emb. M. 15.89 1.00 238 188.667* 16.59 0.35 0 0 14.26 0.741 21.2 7.317 Morph. Day 47.8 0.68* 30.5 4.119 47.6 0.42* 38 0 48.5 0.482* 36 5.04

PAGE 142

126 Table 5-2. Continued. Agea Parameterb Lochloosa Live Dead 14 n 13 8 Egg mass (g) 90.4 1.64* 79.5 0.98 Embryo mass (g) 0.32 0.032 0 0 Eye L. (mm) 4.3 1.22 0 0 Head L. (mm) 12.2 3.18 0 0 Total L. (mm) 56.6 12.21 0 0 Eye L.: Head L. 0.35 0.025 0 0 Head L.: Total L. 0.18 0.03 0 0 Total L.: Emb. M. 151.48 52.437 0 0 Morph. Day 14.46 0.666* 8.33 1.202 25 n 32 22 Egg mass (g) 87.5 1.25* 81.7 1.36 Embryo mass (g) 1.03 0.051* 0.52 0.175 Eye L. (mm) 5.5 0.16 5 0.37 Head L. (mm) 13.4 0.51 13 1.89 Total L. (mm) 78.8 2.18 80.9 9.26 Eye L.: Head L. 0.42 0.007 0.39 0.025 Head L.: Total L. 0.16 0.003 0.16 0.005 Total L.: Emb. M. 74.64 12.461 186.08 78.758* Morph. Day 24.28 0.49* 14.5 2.045 33 n 27 11 Egg mass (g) 86.5 1.28 82.9 1.83 Embryo mass (g) 3.41 0.226* 3.13 1.788 Eye L. (mm) 6.4 0.18 7.4 0 Head L. (mm) 19.8 0.37 25.8 0* Total L. (mm) 115.1 2.63 144.8 0* Eye L.: Head L. 0.33 0.008 0.29 0 Head L.: Total L. 0.17 0.001 0.18 0 Total L.: Egg M. 31.93 1.262 0 0 Morph. Day 36.48 0.676* 16.75 4.304 43 n 40 14 Egg mass (g) 85.7 1 84.4 1.11 Embryo mass (g) 9.25 0.346* 5.74 3.25 Eye L. (mm) 7 0.14* 3 0 Head L. (mm) 26.2 0.63* 18.3 9.75 Total L. (mm) 165 4.15 212 0 Eye L.: Head L. 0.27 0.006 0.35 0 Head L.: Total L. 0.16 0.002* 0.13 0

PAGE 143

Table 5-2. Continued. Agea Parameterb Lochloosa Live Dead 43 Total L.: Emb. M. 14.29 1.212 139.47 139.47* Morph. Day 45.69 0.496* 20.74 4.456 aAge = chronological (calendar) age of embryo (days). bValues = mean standard error. L. = le ngth, Eye L.: Head length = eye length / head length, Head L.: Total L. = head length / to tal length, Total L.: Emb. M. = total length / embryo mass, and Morph. Day = age as dete rmined by morphological characteristics. *indicate significant differences ( = 0.05).

PAGE 144

128Table 5-3. Morphometric comparisons of live em bryos collected during June-August 2001 and 2002. Agea Parameterb Apopka Emeralda Griffin Lochloosa Summary 14 n 7 12 23 13 55 Egg mass (g) 86.3 1.23 AB 78.6 1.35 B 81.6 1.92 B 90.4 1.64 A 83.6 1.11 (82.8–90.9) (70.8–85.4) (61.8–100.1) (77–99.7) (61.8–100.1) Embryo mass (g) 0.38 0.115 A 0.35 0.02 AB 0.41 0.032 AB 0.32 0.03 B 2 0.37 0.022 (0–0.8) (0.2–0.4) (0.1–0.8) (0.2–0.5) (0–0.8) Eye length (mm) 2.9 0 0 0 3.5 0.06 4.3 1.22 3.8 0.53 (2.9–2.9) (0–0) (3.4–3.7) (2–6.6) (2–6.6) Head length (mm) 8 0 0 0 9.1 0.28 12.2 3.18 10.4 1.43 (8–8) (0–0) (8.7–9.9) (5.8–19.1) (5.8–19.1) Total length (mm) 51.4 0 0 0 57.5 0.57 56.6 12.21 56.4 4.07 (51.4–51.4) (0–0) (56.3–58.9) (44–81) (44–81) Eye L.: Head L. 0.36 0 0 0 0.39 0.012 0.35 0.025 0.37 0.013 (0.36–0.36) (0–0) (0.36–0.42) (0.29–0.41) (0.29–0.42) Head L.: Total L. 0.16 0 0 0 0.16 0.006 0.18 0.03 0.17 0.011 (0.16–0.16) (0–0) (0.15–0.18) (0.13–0.24) (0.13–0.24) Total L.: Emb. M. 171.33 0 0 0 111.49 15.353 151.48 52.437 135.91 23.698 (171.33–171.33) (0–0) (72.25–147.3) (0–223.86) (0–223.86) Morph. Day 16 1.195 AB 14 0 B 15.96 0.4 A 14.46 0.67 AB 15.18 0.291 (10–19) (14–14) (12–21) (9–18) (9–21) 25 n 20 24 30 32 106 Egg mass (g) 81.1 2.01 B 80 0.98 B 80.7 1.49 B 87.5 1.25 A 82.7 0.77 (62.4–96.4) (71.6–90.5) (59.4–100.2) (73.3–99) (59.4–100.2) Embryo mass (g) 1.15 0.111 AB 1.45 0.12 AB 1.51 0.088 A 1.03 0.051 B 1.29 0.049 (0.2–1.9) (0.2–2.7) (0.7–2.4) (0.3–1.6) (0.2–2.7) Eye length (mm) 5 0.18 B 5.5 0.12 A 5.9 0.17 A 5.5 0.16 AB 5.5 0.08 (4.5–7.2) (4.6–6.5) (4.9–8.6) (4.5–7.6) (4.5–8.6) Head length (mm) 11.5 0.55 B 13.5 0.41 A 14.9 0.65 A 13.4 0.51 A 13.5 0.29 (9.1–17.4) (10.4–17.7) (10.8–24.3) (10–18.5) (9.1–24.3)

PAGE 145

129Table 5-3. Continued. Agea Parameterb Apopka Emeralda Griffin Lochloosa Summary 25 Total length (mm) 73.7 3.38 C 87.6 2.44 AB 89.1 2.32 A 78.8 2.18 BC 82.9 1.43 (38.4–102.9) (71–105.6) (72.8–118.5) (63.4–102.5) (38.4–118.5) Eye L.: Head L. 0.44 0.01 0.41 0.007 0.4 0.011 0.42 0.007 0.42 0.005 (0.38–0.51) (0.36–0.48) (0.24–0.48) (0.34–0.46) (0.24–0.51) Head L.: Total L. 0.16 0.012 0.15 0.002 0.16 0.003 0.16 0.003 0.16 0.003 (0.13–0.32) (0.13–0.18) (0.14–0.21) (0.14–0.18) (0.13–0.32) Total L.: Emb. M. 68.25 4.717 AB 58.89 2.473 B52.24 3.526 B 74.64 12.461 A63.36 4.046 (46.28–102.39) (39.12–75.47) (0–79.12) (0–317) (0–317) Morph. Day 24.17 0.988 26.42 0.583 26.13 0.619 24.28 0.49 25.29 0.33 (12–33) (15–28) (17–30) (17–28) (12–33) 33 n 15 29 29 27 100 Egg mass (g) 82.4 2.08 AB 80.8 1.15 B 77.7 1.42 B 86.5 1.28 A 81.7 0.77 (65.9–92.6) (69.5–91.3) (62.4–90.7) (69.5–97.3) (62.4–97.3) Embryo mass (g) 3.14 0.09 3.81 0.196 4.04 0.203 3.41 0.226 3.67 0.107 (2.4–3.7) (1.7–6.1) (1.4–5.8) (1.1–8) (1.1–8) Eye length (mm) 6.2 0.1 6.4 0.12 6.5 0.1 6.4 0.18 6.4 0.06 (5.6–7) (4.9–7.7) (5.5–7.5) (4.3–7.5) (4.3–7.7) Head length (mm) 19.1 0.31 20.1 0.49 20.6 0.35 19.8 0.37 20 0.21 (16.7–21.4) (11.8–23.4) (14.5–23.2) (15.7–23.4) (11.8–23.4) Total length (mm) 108.2 1.11 114.4 2.54 117.8 2.19 115.1 2.63 114.6 1.22 (101–114.7) (76.6–135.9) (79.1–132.9) (91.2–153.6) (76.6–153.6) Eye L.: Head L. 0.32 0.008 0.32 0.007 0.32 0.006 0.33 0.008 0.32 0.004 (0.28–0.41) (0.26–0.42) (0.24–0.38) (0.26–0.38) (0.24–0.42) Head L.: Total L. 0.18 0.002 0.18 0.002 0.18 0.001 0.17 0.001 0.17 0.001 (0.16–0.19) (0.15–0.2) (0.17–0.19) (0.15–0.18) (0.15–0.2) Total L.: Emb. M. 34.77 0.812 31.36 1.562 31.41 1.467 31.93 1.262 32.07 0.726 (28.6–42.06) (22.28–52.18) (22.58–56.5) (19.2–49.51) (19.2–56.5) Morph. Day 33.33 0.333 C 35.7 0.57 BC 39.28 0.854 A 36.48 0.676 B 36.62 0.406 (33–38) (28–38) (28–48) (25–43) (25–48)

PAGE 146

130Table 5-3. Continued. Agea Parameterb Apopka Emeralda Griffin Lochloosa Summary 43 n 44 24 41 40 Egg mass (g) 81.2 1.45 B 79.8 1.19 B 78 1.32 B 85.7 1 A 81.4 0.68 (63.7–96) (67.5–91.5) (60.3–91.3) (69.6–97.9) (60.3–97.9) Embryo mass (g) 9.91 0.274 BC 10.69 0.297 B13.01 0.514 A 9.25 0.346 C 10.69 0.229 (7.3–14.41) (7.5–13.4) (7.71–23.53) (4.5–17.14) (4.5–23.53) Eye length (mm) 7.3 0.14 A 6.5 0.11 B 7.5 0.2 A 7 0.14 AB 7.2 0.08 (5.8–9.5) (5.5–7.1) (5.9–10.9) (4.3–8.6) (4.3–10.9) Head length (mm) 27.5 0.53 26.1 0.41 27.4 0.9 26.2 0.63 26.9 0.35 (15.8–36.9) (21.6–28.4) (3.2–35.8) (9.6–33.8) (3.2–36.9) Total length (mm) 172.1 5.19 170.1 2.14 184.9 7.05 165 4.15 173.6 2.87 (92.6–250.4) (147.5–181.3) (19.9–278.8) (122.5–251.3) (19.9–278.8) Eye L.: Head L. 0.27 0.004 0.25 0.006 0.35 0.08 0.27 0.006 0.29 0.02 (0.22–0.37) (0.21–0.3) (0.21–2.66) (0.23–0.45) (0.21–2.66) Head L.: Total L. 0.16 0.003 0.15 0.002 0.19 0.042 0.16 0.002 0.17 0.012 (0.13–0.22) (0.13–0.16) (0.01–1.48) (0.13–0.18) (0.01–1.48) Total L.: Emb.M. 15.89 1.004 A 16.6 0.35 AB 14.26 0.741 B 14.29 1.212 A 15.06 0.517 (0–23.01) (14.39–19.67) (0–24.48) (0–27.22) (0–27.22) Morph. Day 47.76 0.676 A 47.6 0.42 AB 48.54 0.482 A 45.69 0.496 B 47.35 0.289 (41.45–55) (38–48) (43–55) (37–48) (37–55) aAge = chronological (calendar) age of embryo (days). bValues represent mean standard error with ranges in parentheses. Letters beside values (A-D) indicate differences between si tes ( = 0.05). L. = length, Eye L.: Head length = eye length / head le ngth, Head L.: Total L. = head length / total length, Total L. : Emb. M. = total length / embryo mass, and Morph. Day = age as determined by morphological characteristics.

PAGE 147

131 Table 5-4. Explanatory vari ables included in partial re dundancy analysis evaluating relationship between organochlorine pest icide burdens in eggs and embryo morphometrics. Variablea Code Measured as cis -Chlordane [CC] ng/g yolk wet weight cis -Nonachlor [CN] ng/g yolk wet weight Dieldrin [DL] ng/g yolk wet weight Hep. Epoxide [HE] ng/g yolk wet weight o,p -DDD [ODD] ng/g yolk wet weight Oxychlordane [OX] ng/g yolk wet weight p,p’ -DDD [PDD] ng/g yolk wet weight p,p’ -DDE [PDE] ng/g yolk wet weight p,p’ -DDT [PDT] ng/g yolk wet weight Toxaphene [TX] ng/g yolk wet weight trans -Chlordane [TC] ng/g yolk wet weight trans -Nonachlor [TN] ng/g yolk wet weight All OCP burdens [TOC] ng/g yolk wet weight No. OCPs measured NOC n cis -Chlordane% CC% [CC] / [TOC] x 100 cis -Nonachlor% CN% [CN] / [TOC] x 100 Dieldrin% DL% [DL] / [TOC] x 100 Hep. Epoxide% HE% [HE] / [TOC] x 100 o,p -DDD% ODD% [ODD] / [TOC] x 100 o,p -DDT% ODT% [ODT] / [TOC] x 100 Oxychlordane% OX% [OX] / [TOC] x 100 p,p’ -DDD% PDD% [PDD] / [TOC] x 100 p,p’ -DDE% PDE% [PDE] / [TOC] x 100 p,p’ -DDT% PDT% [PDT] / [TOC] x 100 Toxaphene% TX% [TX] / [TOC] x 100 trans -Chlordane% TC% [TC] / [TOC] x 100 trans -Nonachlor% TN% [TN] / [TOC] x 100

PAGE 148

132 Table 5-5. Best five organochl orine pesticide (OCP ) variables accounting for variation in embryo morphology, selected using redundancy analysis with forward selection and Monte Carlo permutation tests for significance. Agea OCP variable LambdaAb P F 14 [OX] 0.20 0.012 12.31 HE% 0.11 0.02 7.03 TX% 0.1 0.028 8.48 ODT% 0.05 0.178 3.87 TN% 0.06 0.018 6.25 25 HE% 0.11 0.002 12.09 CN% 0.06 0.01 7.79 DL% 0.05 0.016 5.80 [DL] 0.02 0.04 3.49 PDD% 0.02 0.076 2.80 33 CC% 0.06 0.042 6.68 DL% 0.08 0.012 9.85 PDE% 0.03 0.082 4.00 CN% 0.1 0.006 14.51 PDD% 0.03 0.052 6.01 43 [CC] 0.07 0.002 11.11 [PDT] 0.08 0.002 12.58 NOC 0.05 0.002 9.60 [DL] 0.02 0.108 2.14 [HE] 0.01 0.064 2.84 aAge = chronological (calendar) age of embryo (days). bLambdaA = amount of morphometric varian ce accounted for by explanatory variable.

PAGE 149

133 Table 5-6. Best five organoc hlorine pesticide (OCP) variab les that account for embryo morphological age and derived morphologi cal parameters as determined by redundancy analysis with forward sel ection and Monte Carlo permutation tests for significance. Age Code LambdaA P F 14 [OX] 0.21 0.004 14.66 ODT% 0.08 0.064 5.41 CN% 0.12 0.006 11.56 OX% 0.07 0.026 6.67 [HE] 0.04 0.012 4.58 25 HE% 0.12 0.002 14.46 NOC 0.07 0.01 8.14 [PDD] 0.03 0.044 3.63 [CN] 0.01 0.154 2 [OX] 0.03 0.056 4.14 33 CC% 0.04 0.054 4.94 DL% 0.06 0.016 7 PDD% 0.03 0.082 4.07 PDE% 0.05 0.006 6.36 CN% 0.06 0.004 8.39 43 PDT% 0.08 0.008 11.2 [CC] 0.05 0.006 8.42 [PDT] 0.04 0.018 6.7 NOC 0.05 0.008 8.82 [DL] 0.02 0.068 3.02

PAGE 150

134 Figure 5-1. Representative developmental stag es of embryos that were collected from Lakes Lochloosa (reference site), Ap opka, and Griffin, and Emeralda Marsh during 2001-2002. A) Live embryo at Day 14 with red line indicating eye length. B) Saggital section of Day 14 embryo. C) Day 25 live embryo. D) Saggital section of Day 25 embryo. E) Day 33 live embryo with red line indicating head length. F) Saggital section of Day 33 embryo. G) Day 43 embryo with red line indicating total le ngth. H) Saggital section of Day 43 embryo (organogenesis nearly complete). 5 mm 5 mm 5 mm 5 mm E. F. G. H. 5mm B. 5 mm A. 5 mm 5 mm C. D.

PAGE 151

-0.80.8-0.60.8 Eye Length Head Length Total Length Embryo mass Heptachlor epoxide% o,p-DDT% Toxaphene% trans-Nonachlor% [Oxychlordane] Figure 5-2. Ordination biplot of embryo morphometric parameters (solid lines) and organochlorine pesticide (OCP) variable s (dashed lines) for embryos collected at chronological age Day 14. Arrows poin ting in the same direction indicate a positive correlation (e.g., embryo mass and [oxychlordane), arrows that are approximately perpendicular indicate near-zero correlation, and arrows pointing in opposite direc tions indicate negative corre lations (head length and [oxychlordane]. Arrow lengths indi cate rank order of correlations. For example, extending a perpendicular line (A) from the embryo mass axis to tip of [oxychlordane] arrow indicates that [oxychlordane] has a stronger positive correlation with embryo mass than hept achlor epoxide% (B). The cosine of the angle formed at the origin betw een individual clutch variables and individual OCP variables is the correla tion coefficient (r). For example, if arrows pointing in exactly opposite dire ctions have an angle of 180, and cos(180) = -1.0, then the arrows would be perfectly, negatively correlated (r) (ter Braak, 1995). A B

PAGE 152

136 -0.60.6-0.10.6 Eye Length Head Length Total Length Embryo mass cis-Nonachlor Dieldrin% Heptachlor expoxide p,p'-DDD% [Dieldrin] Figure 5-3. Ordination biplot of embryo morphometric parameters (solid lines) and organochlorine pesticide (OCP) variable s (dashed lines) for embryos collected at chronological age Day 25

PAGE 153

137 -0.60.6-0.30.4 Eye Length Head Length Total Length Embryo mass cis-Chlordane% cis-Nonachlor% Dieldrin% p,p'-DDD% p,p'-DDE% Figure 5-4. Ordination biplot of embryo morphometric parameters (solid lines) and organochlorine pesticide (OCP) variable s (dashed lines) for embryos collected at chronological age Day 33.

PAGE 154

138 -0.60.6-0.30.4 Eye Length Head Length Total Length Embryo mass NOC [cis-Chlordane] [Dieldrin] [Heptachlor epoxide [p,p'-DDT] Figure 5-5. Ordination biplot of embryo morphometric parameters (solid lines) and organochlorine pesticide (OCP) variable s (dashed lines) for embryos collected at chronological age Day 43.

PAGE 155

139 -0.80.8-0.40.2 Morphological day Eye L.: Head L. Head L.: Total L. Total L.: Emb. M. cis-Nonachlor% o,p-DDT% Oxychlordane% [Heptachlor epoxide] [Oxychlordane] Figure 5-6. Ordination bi plot of derived embryo morphometr ic parameters (solid lines) and organochlorine pesticide (OCP) va riables (dashed lines) for embryos collected at chronol ogical age Day 14.

PAGE 156

140 -0.600.60-0.150.15 Morphological day Eye L.: Head L. Head L.: Total L. Total L.: Emb. M. NOC Heptachlor epoxide% [cis-Nonachlor] [Oxychlordane] [p,p'-DDD] Figure 5-7. Ordination bi plot of derived embryo morphometr ic parameters (solid lines) and organochlorine pesticide (OCP) va riables (dashed lines) for embryos collected at chronol ogical age Day 25.

PAGE 157

141 -0.60.6-0.40.3 Morphological day Eye L.: Head L. Head L.: Total L. Total L.: Emb. M. cis-Chlordane% cis-Nonachlor% Dieldrin% p,p'-DDD% p,p'-DDE% Figure 5-8. Ordination bi plot of derived embryo morphometr ic parameters (solid lines) and organochlorine pesticide (OCP) va riables (dashed lines) for embryos collected at chronol ogical age Day 33.

PAGE 158

142 -0.60.6-0.20.8 Morphological day Eye L.: Head L. Head L.: Total L. Total L.: Emb. M. NOC p,p'-DDT% [cis-Chlordane] [Dieldrin] [p,p'-DDT] Figure 5-9. Ordination bi plot of derived embryo morphometr ic parameters (solid lines) and organochlorine pesticide (OCP) variables (dashed lines) embryos collected at chronol ogical age Day 43.

PAGE 159

143 CHAPTER 6 NUTRIENT AND CHLORINATED HYDRO CARBON CONCENTRATIONS IN AMERICAN ALLIGATOR EGGS AND ASSOCIATIONS WITH DECREASED CLUTCH VIABILITY In central Florida, American alligator ( Alligator mississippiensis ) populations inhabiting lakes contaminated with orga nochlorine pesticides (OCPs) have poor reproductive success, primarily due to increa sed embryo mortality ( Woodward et al., 1993; Woodward et al., 1989). During 2000-2002, cl utch viability (percentage of eggs that yield a live hatchling) was monitore d on 168 clutches from reference and OCPcontaminated sites, indicating that clutch es from a reference site, Lake LochloosaOrange, had higher clutch viability (mean clutch viability = 70%) as compared than the OCP-contaminated sites, Lake Apopka (51%), Emeralda Marsh Restoration Area (48%), and Lake Griffin (44%). Furthermore, 115 of these clutches were analyzed for OCPs, and results indicated that allig ators inhabiting Emeralda Rest oration Marsh (total average egg OCPs = 15,480 ng/g), Lake Apopka (7,582 ng/g), and Lake Griffin (1,169 ng/g) contained significantly higher OCP burdens in eggs compared to those of Lake Lochloosa-Orange (102 ng/g) (Chapter 2). Although total embryo mortality was highest in eggs from sites with high OCPs, the am ount of variation in embryo mortality rates explained by OCP egg burdens differs among OCP-contaminated sites (Chapter 2), suggesting the presence of additional factor(s). With respect to vertebrates, examples of non-OCP factors that ha ve been associated with increased embryo mortality include nut ritional deficiencies and excesses (Wilson, 1997; McEvoy et al., 2001), exposure to polyc hlorinated biphenyls (PCBs) (Summer et

PAGE 160

144 al., 1996), and exposure to polyaromatic hyd rocarbons (PAHs) (Hoffman, 1990). For example, early-life stage (embryo) morta lity has been associated with: thiamine deficiency in trout and salmon (Fitzsimons et al., 1999); PCB exposure in chickens (Summer et al., 1996); and PAH exposure in mallard eggs (Hoffman & Gay, 1981). More recent data suggested that thiami ne deficiency may be involved in the increased incidence of embryo mortality in American alligato rs inhabiting the aforementioned OCP-contaminated lakes in central Florida. Indeed, thiamine concentrations in egg yolks were positively co rrelated with clutch viability and accounted for 40% of variation in cl utch viability among Lakes Lochloosa, Griffin, Apopka, and Emeralda Marsh (Seplveda et al., 2004). Howe ver, further investigation into thiamine’s potential role in embryo mort ality is warranted before any conclusions are drawn. Reasons for further study are that only five clutches were sampled per site, sampling occurred during a single nes ting season (2000), and the potentia l role of other nutrients (i.e., vitamin E) and contaminants (i.e., PCBs) were not evaluated. With respect to other vitamins, vitamin E (tocopherol) has been sugge sted as having a potential role in the reduced clutch viability of captive alligators from Louisiana (Lance et al., 1983). Lastly, besides the embryotoxic eff ects of PCBs and PAHs, st udies indicate that these contaminants may reduce thiamine storage in laboratory animals (Yagi et al., 1979), and that the presence of high contaminant burde ns may affect thiamine’s role in the production of metabolic energy (d e Roode et al., 2002a). Toge ther these data suggest the need for a detailed examination of contaminan t burdens and nutrient content of eggs, and their association with clutch viability and embryo mortality in American alligators from OCP-contaminated sites in Florida.

PAGE 161

145 The present study’s spec ific aims were to conduct a case-control, cohort study to examine the relationship between multiple nutri ents and contaminants, to further examine hypotheses derived from the case-cohort study vi a an expanded field study, and to test hypotheses derived from the expanded field st udy using laboratory experiments. Materials and Methods Egg Collections and Incubation Alligator eggs were collected during 2001, 2002, and 2003 nesting seasons (JuneJuly) from the following OCP-contaminated s ites: Lakes Apopka (N 28 35’, W 81 39’), Griffin (N 28 53’, W 81 46’), and Emeral da Marsh Conservation Area ((N 28 55’, W 81 47’), and from a reference site, Lake Lochloosa (N 29 30’, W 82 09’) in central Florida. Alligator nests were located via ae rial (helicopter) and ground surveys (airboat), and clutches were subsequen tly collected by ground crews. The top of each egg was marked before eggs were removed from the nest to ensure prope r orientation; thus, preventing embryo mortality due to inversion. Embryo mortalit y due to inversion occurs because, once an embryo has attached to the top of the egg, inverting the egg’s orientation may either break embryonic attachme nt or cause the yolk mass to settle on top of the embryo, crushing it. After marking each egg and placing about 5 cm of nest substrate in a uniquely numbered plastic pan (43 cm x 33 cm x 18 cm), all eggs found in each clutch were placed in the pan in five rows with six eggs per row. If a clutch contained more than 30 eggs, a second layer of nest substrate was added and the remaining eggs were collected. The top layer of eggs was covered with nest substrat e so that there was no space left between the top of the pan and the top of the eggs (approx imately 10 cm). Clutches were transported to the US Geological Survey’s Center for Aqua tic Resources Studies in Gainesville, FL.

PAGE 162

146 Upon arrival, clutches were evaluated fo r embryonic viability using a bright light candling procedure. One or two eggs were opened from each clutch to identify the embryonic stage of development at the time of collection, and to collect yolk samples for later measurement of OCP, PAH, and PCB burde ns and selected nutrie nt content with all yolk and albumin samples being stored at -80 C. From each clutch, the following parameters were measured: total number of eggs per nest (fecundity); number of unbanded eggs, number of damaged eggs, numbe r of dead, banded eggs, number of live banded eggs, total clutch mass, and average e gg mass of clutch. Viable eggs (i.e. having a visible band) were nested in pans cont aining moist sphagnum moss and incubated at 30.5C and ~98% humidity in an incubation bu ilding (7.3 m x 3.7 m). This intermediate incubation temperature will normally result in a 1:1 male/female sex ratio, as alligators have temperature dependent sexual differen tiation (Ferguson, 1985). On a daily basis, temperature and humidity were monitored at several locations thr oughout the incubator, clutches were rotated within the incubator, and air was circulated to mitigate any thermal gradients. Eggs were monitored for viability via bright-light candling every 10 days during incubation. Clutches collected duri ng 2001 and 2002 were used for the field study and those collected during 2003 were used for th e laboratory experiment. Experimental Design Field studies A case-control cohort study was conducted th at involved the selection of clutches based upon their viability and their OCP egg burd ens. Clutches were assigned to one of nine possible categories based on clutch viabil ity and OCP egg burdens (Table 6-1). The purpose of the case-control cohort study was to determine if PAH, PCB, zinc, selenium, vitamins A, E, and B1 concentrations differed or show ed trends among clutch viability-

PAGE 163

147 OCP categories. Selenium (Spallholz & Hoffman, 2002), zinc, vitamins A, E, (Ashworth & Antipatis, 2001) and B1 (de Roode et al., 2002b)were examined because they are important for embryo development and survival, and their activity and/or levels may be affected by chlorinated hydrocarbons. This strategy aided in forming hypotheses related to the association between non-OCP factors and embryo mort ality and the relationship between non-OCP factors and OCP exposure. Fo r example, if increasing levels of a nonOCP factor showed a strong pos itive association with embr yo viability, regardless of OCP burden, and levels did not differ betw een OCP exposure groups, then it could be hypothesized that the potential effects were likely related to the non-OCP factor(s) and unrelated to OCP exposure(s). Conversely, if increasing leve ls of the non-OCP factor(s) showed a strong positive associ ation with embryo viability, bu t only with respect to low OCP exposure groups, then it could be hypothesize d that the potential effects were likely due to a combination of OCP exposure and the level of the non-OCP factor(s). Based on the case-control cohort study, hypot heses were derived that focused on the major non-OCP factors associated with em bryo mortality and OCP exposure. Lastly, results of this expanded field study were used to design an egg treatment experiment to examine the hypotheses in a more controlled setting. Laboratory experiments In 2003, laboratory experiments were c onducted using clutches collected from Lakes Dexter and Griffin, and from Emeral da Marsh. Based upon the case-control cohort study and the expanded field study (see results), the purpose of this experiment was to test the hypothesis that increas ing thiamine levels in eggs would result in decreased embryo mortality. This experiment consisted of increasing thiamine concentrations in eggs that were known to have low thiamine concentrations, moderate to high yolk OCP

PAGE 164

148 concentrations (Lakes Griffin and Emeral da Marsh), and high embryo mortality. Thiamine HCL was applied at high (60 mg thiamine/mL dimethyl sulfoxide, DMSO) and low concentrations (12 mg/mL DMSO) over th e surface of each egg (application volume of 50 l) using a micropipette. Controls receiv ed only vehicle treatment (DMSO). These doses were calculated to achie ve yolk thiamine concentrations similar to those measured from the reference site (Lakes Orange-Lochl oosa complex). Eggs from each site were labeled and randomLy distributed among each treat ment group, so that all clutches were equally represented in the study. There were two repli cates per treatment with a minimum of 26 eggs (maximum of 31) per re plicate. After being dosed, eggs were placed in the incubator, and candled weekly to determine effects on embryo and hatchling survival. For Emeralda Marsh clutches, thre e eggs from each replicate were sampled 7 days after treatment to determine the amount of thiamine present in albumin and yolk. Embryo mortality rates were recorded for each treatment group as the percentage of eggs failing to hatch over the number of eggs treated. The second experiment’s purpose was to test the hypothesis that, in the absence of high OCP exposure, decreased thiamine bioa ctivity (functional deficiency) would result in increased embryo mortality rates. This experiment involved inducing decreases in thiamine bioactivity in eggs known to have relatively high thiamine concentrations, low OCP burdens, and low embryo mo rtality. Since clutches fr om Lake Lochloosa-Orange were assigned to another study during 2003, eggs were collected from another reference site (Lake Dexter, N 29 98’, W 81 47’). To decrease thiamine bioactivity, oxythiamineHCL, a thiamine antagonist (Akerman et al., 1998), was topically applied at concentrations of 12 or 60 mg/mL using DMSO as the carrier. Cont rols received only

PAGE 165

149 DMSO. Eggs were labeled and randomLy distributed among each treatment group, so that all clutches were equally represented. There were two replicates per treatment with a minimum of 20 eggs (maximum of 21) per re plicate. After being dosed, eggs were placed in the incubator, and candled weekly to determine effects on embryo and hatchling survival. Hatch rates for each replicate were de termined as the percentage of eggs that produced a live hatchling. Analysis of Chlorinated Hydrocarbons in Yolk Analytical grade standards for the following compounds were purchased from the sources indicated: aldrin, al pha-benzene hexachloride ( -BHC), -BHC, lindane, -BHC, p,p’ -dichlorodiphenyldichloroethane ( p,p’ -DDD), p,p’ -dichlorodiphenyldichloroethylene ( p,p’ -DDE), dichlorodiphe nyltrichloroethane ( p,p’ -DDT), dieldrin, endosulfan, endosulfan II, endosulfan sulfate, endrin, e ndrin aldehyde, endrin ketone, heptachlor, heptachlor epoxide, hexachlorobenz ene, kepone, methoxychlor, mirex, cis -nonachlor, and trans -nonachlor from Ultra Scientific (Kingstown, RI, USA); cis -chlordane, trans chlordane, and the 525, 525.1 polychlorinated biphenyl (PCB) Mix from Supelco (Bellefonte, PA, USA); oxychlordane from Chem Service (West Chester, PA); o,p’DDD, o,p’DDE, o,p’DDT from Accustandard (New Haven, CT, USA); and toxaphene from Restek (Bellefonte, PA, USA). All reag ents were analytical grade unless otherwise indicated. Water was doubly distilled and deionized. Egg yolk samples were analyzed for chlorinated hydrocarbon content using methods modified from Holste ge et al. (1994) an d Schenck et al. (1994). For extraction, a 2 g tissue sample was homogenized with ~1 g of sodium sulfate and 8 mL of ethyl acetate. The supernatant was decanted and filtered though a Bchner funnel lined with Whatman #4 filter paper (Fisher Scientific, Ha mpton, NH, USA ) and filled to a depth of

PAGE 166

150 1.25 cm with sodium sulfate. The homogenate was extracted twice with the filtrates collected together. The combined filtrat e was concentrated to ~2 mL by rotary evaporation, and then further concentrated until solvent-free unde r a stream of dry nitrogen. The residue was reconstituted in 2 mL of acetonitrile. After vortexing (30 s), the supernatant was applied to a C18 solid phase extrac tion (SPE) cartridge (preconditioned with 3 mL of acetonitrile; Agile nt Technologies, Wilmi ngton, DE, USA) and was allowed to pass under gravity. This proced ure was repeated twice with the combined eluent collected in a culture t ube. After the last addition, th e cartridge was rinsed with 1 mL of acetonitrile which was also collected. The eluent was then applied to a 0.5 g NH2 SPE cartridge (Varian, Harbor City, CA, US A), was allowed to pass under gravity, and collected in a graduated conical tube. The car tridge was rinsed with an additional 1 mL portion of acetonitrile whic h was also collected. The combined eluents were concentrated under a stream of dry nitrogen, to a volume of 300 L, and transferred to a gas chromatography (GC) vial for analysis. GC/MS Analysis Analysis of all samples was performed using a Hewlett Packard HP-6890 gas chromatograph (Wilmington, DE, USA) with a split/splitless inlet ope rated in splitless mode. The analytes were introduced in a 1 L injection and separa ted across the HP-5MS column (30 m x 0.25 mm; 0.25 m film thickne ss; J & W Scientific, Folsom, CA, USA) under a temperature program that began at 60 C, increased at 10 C/min to 270 C, was held for 5 min, then increased at 25 C/min to 300 C and was held for 5 min. Detection utilized an HP 5973 mass spectro meter in electron impact m ode. Identification for all analytes and quantitation for toxaphene was c onducted in full scan mode, where all ions

PAGE 167

151 are monitored. To improve sensitivity, se lected ion monitoring was used for the quantitation for all other analytes, except kepone. The above program was used as a screening tool for kepone which does not optim ally extract with mo st organochlorines. Samples found to contain kepone would be reex tracted and analyzed specifically for this compound. For quantitation, a five-point standard curve was prepared for each analyte ( r2 0.995). Fresh curves were analyzed with each se t of twenty samples. Each standard and sample was fortified to contain a deuterat ed internal standard, 5 L of US-108 (120 g/mL; Ultra Scientific), added just prior to analysis. All samples also contained a surrogate, 2 g/mL of tetrach loroxylene (Ultra Scientific) added after homogenization. Duplicate quality control samples were prepar ed and analyzed with every twenty samples (typically at a level of 1.00 or 2.50 g/mL of -BHC, heptachlor, aldr in, dieldrin, endrin, and p,p’ -DDT) with an acceptable recovery rangi ng from 70 – 130%. Limit of detection ranged from 0.1-1.5 ng/g for all OCP analyt es, except toxaphene (120-236 ng/g), and limit of quantitation was 1.5 ng/g for all anal ytes, except toxaphene (1500 ng/g). Repeated analyses were conducted as allo wed by matrix interferences and sample availability. Nutrient Analysis Thiamine concentrations were measured in clutches coll ected during years 2001, 2002, and 2003. For analysis, samples were shipped overnight on dry ice (solid CO2) to the USGS Leetown Science Center, Appalach ian Research Laborator y in Wellsboro, PA. Thiamine concentrations were determined as described in (Brown et al., 1998). Briefly, a known amount of the frozen yolk sample was first placed in 2% trichloroacetic acid (TCA, Sigma, St. Louis, Missouri, USA) ho mogenization solution. The extract was then

PAGE 168

152 washed with ethyl acetate:he xane (3/2, vol/vol, Sigma) to remove excess TCA. An aliquot of the washed solution was reacted with potassium ferricyanide (Sigma) to produce thiochrome derivatives. The resulti ng derivatives were separated on a Hamilton PRP-1 column (Alltech, Deerfield, Illinois, USA) and detected with a spectrofluorometer set at 375 nm excitation wavelength a nd 433 nm emission (Shimadzu, Columbia, Maryland, USA). Authentic standards of thiamine pyrophosphate, thiamine monophosphate and thiamine-HCL (ICN Biomed icals, Montreal, Quebec, Canada) were used to quantify the amount of thiamine in each sample. In addition to thiamine analyses, samples from selected clutches collected during 2002 were sent to ABC Research Corp. in Ga inesville, FL, and analyzed for vitamin A (carotene, retinol, and activity) ( AOAC 960.45 and 941.15), vitamin E (tocopherol) (AOAC 948.26), zinc and sele nium (AOAC 990.8) using AOCAC methods (Horwitz, 2000). Data Analysis For the case-control cohort study, expande d field study, and laboratory experiments ANOVA (PROC GLM; SAS Institute Inc., 2002) was used for inter-site and inter-group comparisons of summary clutch characteristics, with the Tukey test for multiple comparisons among sites and groups ( = 0.05). Because relationships between response variables and explanatory vari ables (Table 6-2) in ecologi cal studies are often complex with interactions occurring, an indirect gr adient multivariate analysis method, Detrended Correspondence Analysis (DCA) (ter Braak, 1986) was used to initially evaluate data structure for the case-control cohort study, as well as the expanded field study. Two matrices were constructed for DCA, with th e first representing th e response variables clutch ID number x clutch parameters) a nd the second represen ting the explanatory

PAGE 169

153 variables (clutch ID number x OCP burdens) (Table 6-3). DCA results indicated that a direct gradient, multivariate linear analys is, redundancy analysis (RDA) (Rao, 1964), was appropriate for the case-control cohort st udy and the expanded field study since the gradient lengths of the DCA ordination axes were never more than (approximately 2 standard deviations (ter Braak, 1995). For the RDA, similar matrices were cons tructed with the exception that response variables measured as a percentage (i.e ., clutch viability) and response variables measured as a number (i.e., clutch mass) were divided into separate matrices because percentage data were ln(x+1) transformed and not standardized, while continuous data were ln(x) transformed and standard ized(ter Braak & Smilauer, 2002). Automatic forward selection of the best f our explanatory variables was for all RDA analyses and Monte Carlo permutation test s were used to determine significance ( = 0.05). DCA and RDA were conducted usi ng the program CANOC O (ter Braak & Smilauer, 2002), and CANODRAW (ter Braak & Smilauer, 2002) was used to construct biplots of environmental vari ables and response variables to interpret relationships between clutch parameters (response va riables) and explanatory factors. Specific OCP analytes were removed from analysis if measurable concentrations were found in less than 5% of all clutches. Numerical data, such as fecundity, were logtransformed [ln(x)], while proportional data (c lutch viability) were arcsine square root transformed to meet statistical assumptions and [ln (x+1)] transformed for RDA analysis

PAGE 170

154 Results Field Study Case-control cohort study In 2002, 32 clutches were collected from Emeralda Marsh, and Lakes Apopka, Griffin, and Lochloosa. Of the 32 clutches 20 were selected and each of the 20 was assigned to one of nine categ ories based on clutch viabilit y and total OCP burdens in eggs (Table 6-1). Only seven categories were filled with six cate gories being represented by three clutches. The remaining category, “good viability-high OCP burden”, was represented by two. Although the number of cl utches within each ca tegory was not large, clutches assigned to good and intermediate vi ability categories had significantly greater viability rates compared to poor category cl utches, which supports the assignment of these clutches to their resp ective categories. Similarly, clutches assigned to high, intermediate, and low OCP categories were sign ificantly different from one another with respect to total OCP burdens, further supporting the statistical and bi ological validity of assigned categories (Table 6-4) Differences among OCP analytes were not determined. In addition to the somewhat expected diffe rences in clutch viability rates and OCP burdens among categories, significant differenc es were found with respect to total PCB burdens, total PAH burdens, thiamine m onophosphate (TP), and thiamine pyrophosphate (TPP) in eggs (Table 6-4). Although total PCB and PAH burdens differed among categories, levels were below those known to elicit adverse effect s on avian development (Summer et al., 1996). Vitamin A was not detect ed in any of the eggs with the lack of detection likely due to the relatively higher limit of detec tion (0.3 ppm) compared to the other nutrients (e.g., 0.1 ppm for vitamin E) Since vitamin A was not detected or

PAGE 171

155 quantified in any eggs, no conclusions can be reached regarding its potential role in embryo mortality in alligators. No other significant differences were not ed for non-OCP variables likely due in part to the relatively small sample sizes and considerable variation in values of clutch parameters. Since the purpose of the study was to develop hypothe sis; some important non-significant differences shoul d be pointed out. For exam ple, mean values of total thiamine and free thiamine concentrations of good viability-low OCP clutches and poor viability-high OCP clutches were nearly fou r-fold those of intermediate viability-low OCP clutches (Table 6-4). This four-fold di fference may suggest that reduced viability in clutches with low OCP burdens may be associat ed with reduced thiamine levels, and that poor viability in clutches with high OCP bur dens may not be associated with reduced thiamine levels. Redundancy analysis (RDA) with forward selection of best four explanatory variables (Table 6-3) provided a way to ev aluate the relationships between the non-OCP variables and clutch variables, and allowed each clutch’s site to be included in the analysis. Including site in th e analysis aided in identifying whether site differences, as opposed to other factors, were related to clutch survival a nd related parameters. For the 20 clutches included in the RDA, thiami ne monophosphate, TP, (lambda A = 26%) and thiamine pyrophosphate, TPP, (12%) were signifi cantly correlated with clutch survival parameters, accounting for 38% of th e variation in clutch survival parameters (Table 6-5). Indeed, TP had a strong positive associat ion with clutch viability and a strong negative association with early embryo mort ality, while TPP had a strong negative association with late embr yo mortality (Fig. 6-1).

PAGE 172

156 These results are biologically plausible because TP and TPP are the bioactive forms of thiamine needed for the production of me tabolic energy and deficiencies have been associated with intrauterine growth retarda tion in laboratory models (Roecklein et al., 1985). In contrast, the positive relationshi p between unbanded egg% and TPP (Fig. 6-1) has little biological implications because an embryo must be present for TPP to be produced. Important to note is that PAH and PCB burdens did not appear to be significantly associated with em bryo survival parameters. In contrast to clutch su rvival parameters, clutch si ze parameters (e.g., fecundity) appeared to be associated with the site, as three of the four extracted explanatory variables were the nominal site variables. Of these extracted variables, only Lochloosa was determined to be significantly associated with clutch size pa rameters (Table 6-6), accounting for 27% of the variation. Furtherm ore, Lochloosa clutches appear to have higher average egg masses and lower fecundity compared to other sites (Fig. 6-2). Lastly, the relationship between nutrien ts and chlorinated hydrocarbons were examined via RDA. Interestingly, all four extracted explanatory variables, heptachlor epoxide concentration (lambda A = 18%), dieldrin% (17%), trans-chlordane concentration (15%), Lochloosa (site eff ect, 9%) were found to be significantly associated with nutrient levels in eggs, accounting for 59% of the variation in egg nutrient content (Table 6-7). Heptachlor e poxide concentrations had a strong negative correlation with thiamine pyrophosphate, but weak positive correlations with the other thiamine forms and nutrients. Dieldrin% had strong negative asso ciations with free thiamine and total thiamine, but strong positive relationships with vitamin E, zinc, and selenium. Trans-chlordane concentrations ha d strong negative correlations with vitamin

PAGE 173

157 E, zinc, and selenium and little to near-zero correl ation with thiamine concentrations. Lochloosa clutches appeared to be associat ed with increasing thiamine concentrations and decreasing zinc, selenium, and vitamin E concentrations (Fig. 6-3). In summary, results of the case-contro l cohort study suggest the main factors associated with reduced clutch viability a nd increased embryo mortality are decreasing thiamine concentrations (Table 6-5, Fig. 6-1), and that reductions in thiamine concentrations may be associated with orga nochlorine pesticides (T able 6-7, Fig. 6-3). Therefore, the expanded field study was de signed in order to examine how clutch survival parameters vary as a function of OCP burdens and thiamine concentrations in eggs. Expanded field study The purpose of the expanded field study was to examine the relationships between thiamine and OCP concentrations in eggs and clutch viability and cl utch size parameters. Since consistent methods were used for OCP and thiamine analysis, as well as for egg collections and incubation, data from year 2000 (Seplveda et al., 2004), was combined with data from years 2001 and 2002. Using a larger number of clutches (n = 72) over multiple nesting seasons increased ecological validity of conclusions, as well as power in testing the hypothesis that th iamine deficiency and OCP ex posure are associated with altered clutch survival parameters and altered clutch size. Clutches from the Lochloosa-Orange co mplex (n = 18), Emeralda Marsh (n = 19), and Lakes Apopka (n = 14) and Griffin (n=21) No significant di fferences were noted among sites with respect to clutch survival pa rameters, clutch size pa rameters, or the four thiamine parameters. However, biologi cal significance should be noted in that Lochloosa-Orange complex clutches had m ean clutch viability rates that were

PAGE 174

158 consistently greater than all other sites by an average of 18%. Furthermore, LochloosaOrange clutches had lower embryo mortality rates that were less than all other sites, by an average of 8%. The paucity of statistically significant differences is likely due to the high variance of clutch survival in OCP-contamin ated sites. Significant differences were found among sites with respect to many OCP anal yte burdens in eggs. Indeed, mean total OCP burdens and number of OCP analytes dete cted at quantifiable levels significantly differed among all sites (Table 6-8). RDA was used to evaluate the relationships between the many OCP and thiamine parameters (explanatory variables) and th e clutch survival parameters (response variables) (Table 6-3). Initial RDA showed that embryo age at the time of collection was an important factor, but not a specific factor of interest. Further examination of age effects indicated that for all sites, phosphorylation of free thiamine increased with age (Fig. 6-4). Therefore, another RDA was conducted usi ng age as a covariate. The best four explanatory variables determined via this RDA accounted for 30% of the variation in clutch survival parameters and consisted of total thiamine concentration (lambda A = 16%), thiamine pyrophosphate (7%), thiamine monophosphate (4%), and methoxychlor% (3%), with all explanatory variables dete rmined to be significant (Table 6-9). Total thiamine (TT) and thiamine monophosphate (TP) were strongly and positively correlated with clutch viability, and negatively correlated with unbanded egg% and early embryo mortality but showed near-zero correlation with late embryo mortality. Thiamine pyrophosphate (TPP) was strongly and negatively correlated with late embryo mortality and had weak to near-zero corre lations with remaining clutch survival

PAGE 175

159 parameters. Methoxychlor% (ME%) had posit ive correlations with unbanded egg% and early embryo mortality and near -zero correlations with other clutch survival parameters (Fig. 6-5). In addition to clutch survival parameters redundancy analysis was used to examine relationships between clutch size variables a nd explanatory variable s. Results of the RDA indicated that two of four extracted variables were found to be significant and explained 15% of the variation in clutch size parameters. Extracted variables found to be significantly associated with clutch size va riables included free thiamine (lambda A = 9%) and thiamine pyrophosphate (6 %). Site effect may be im portant regarding variation in clutch size parameters, as the nominal variable “GR” (Lake Griffin) approached significance (Table 6-10). Interestingly, all thiamine forms were pos itively associated with egg weight and negatively associated with fecundity. Thia min pyrophosphate was negatively correlated with Griffin (meaning clutches from Lake Gr iffin had reduced levels of TPP), and had near-zero correlations with clutch mass. Total thiamine and free thiamine had strong, negative correlations with clutch mass and n ear-zero correlations with GR (Fig. 6-6). Lastly, redundancy analysis was used to examine the relationship between the various thiamine forms (response variables) and explanatory va riables to see if thiamine deficiency was associated with OCP variables or other clutch variables. Results indicated that four extracted variable were significan tly correlated with thiamine concentrations and accounted for 31% of the variation in thiami ne levels. Interestin gly, lipid content of eggs (%) accounted for 16% of thiamine variation, followed by mirex concentrations (5%), trans-chlordane concentr ations (6%), and oxychlordane concentrations (4%) (Table

PAGE 176

160 6-11). Lipid content had strong negative co rrelations with free thiamine and total thiamine and positive correlations with thiamine monoand pyrophosphate. Transchlordane concentrations were positively correlated with thiamine pyrophosphate and negatively correlated with the remaining th iamine forms. Oxychlordane and mirex concentrations in eggs were positively correlated with thiamine monophosphate, had near-zero correlations with total and free th iamine, and weak negative correlations with thiamine pyrophosphate (Fig. 6-7). In summary, results of the expande d field study suggested that thiamine concentrations and certain OCP variables accounted for a signif icant amount of the variation in clutch survival and size char acteristics, supporting the hypothesis that thiamine deficiency and OCP exposure contri butes to decreased clutch viability and altered clutch size characteristics (Figs. 66; 6-5). Furthermore, decreasing thiamine levels were associated with increasing lip id content in egg yolks, suggesting that alterations in yolk composition are occurring and may be indicative of altered maternal liver function, possibly due to a number of reasons including OCP exposure and female age. In addition, alterations may be related to dietary fact ors. Indeed, altered liver function, leading to altered yolk composition ha s been documented in laboratory studies in catfish exposed to similar pesticides (Lal & Singh, 1987), and diets and body condition of alligators have been suggested to differ am ong two of the lakes included in the present study (Lakes Apopka and Griffin) in central Florida (Rice, 2004). Laboratory Experiments Because the case-control cohort and expanded field studies supported the hypothesis that thiamine deficiency and OCPs ar e associated with altered clutch survival and clutch size parameters, two laboratory ex periments were conducted to more directly

PAGE 177

161 test this hypothesis. The purpose of the firs t experiment was to te st the hypothesis that increasing in ovo thiamine concentrations would in crease embryo survival (thiamine topical exposure experiment ) in clutches with high OCP burdens, and the second experiment tested the hypothesis that decrea sing thiamine concentrations (via thiamine activity inhibitor) would decr ease embryo survival in clut ches with low OCP burdens. A total of 14 clutches were used in the two experime nts, with clutches having relatively high embryo mortality, low thiamine levels in eggs, and intermediate (Lake Griffin, n = 5) to high OCP burdens in eggs (E meralda Marsh, n = 5) used in the thiamine topical exposure study. Convers ely, clutches (Lake Dexter, n = 4) having relatively low embryo mortality, high thiamine levels in eggs, and low OCPs were used in the oxythiamine (thiamine-antagonist) topical expos ure study. Clutch characteristics differed significantly among sites with respect to f ecundity, clutch mass, egg mass, many OCP analytes, total OCP burdens, and number of OC Ps detected at quantifiable levels (Table 6-12). Seven days after topical treatment, three eggs from three different clutches (same clutches sampled for all replicates) were anal yzed to determine the amount of thiamine that was transferred into the egg. For Emeralda clutches, results indicated that total thiamine concentrations in egg albumin of the high and low thiamine treatment groups were significantly greater than controls. Ind eed, total thiamine concentrations in albumin of the high thiamine and low thiamine treatment groups were over 40-fold and over 30fold greater, respectively, than those of c ontrols, confirming a significant increase in thiamine levels in these eggs. Thiamine con centrations in egg yolk of Emeralda clutches were also greater in high and low thiamine treatment groups in a dose-dependent manner,

PAGE 178

162 but the difference was not significant, with thiamine concentrations in high treatment groups being only 1.1-fold greater than controls (Table 6-13). Similar results were noted for Lake Griffin clutches, with thiamine tr eatments showing a dose-dependent, but nonsignificant increase in thiamine concentrati ons. High treatment clutches had thiamine concentrations that were 1.2-fold those of controls (Table 6-13). Thiamine concentrations in egg yolk of control groups for both Emeralda and Gr iffin clutches were high than means reported in the expanded fiel d study but still within respective ranges. Changes in embryo mortality rates were the primary interest and analysis indicated no significant differe nces were noted between th iamine treatment groups and controls. However, Emeralda clutches fr om both thiamine treatment groups had embryo mortality rates which averaged 10% less than those of controls. However, for Lake Griffin clutches, thiamine treatments were associated with a 5-7% increase in embryo mortality (Table 6-13). For the oxythiamine (thiamine antagonist) study using Lake De xter clutches, no significant differences were noted between oxythiamine treatment groups and controls. Surprisingly, embryo survival of controls (mean standard error: 81 9%) was slightly less than those of the low exposure groups (98 2%), and high exposure groups (88 8%). Oxythiamine concentrations were not measured because oxythiamine has physicochemical properties very similar to thiamine ; therefore, transf er rates across the eggshell were assumed to be similar. Discussion The present study examined associations between egg nutrients, OCP egg burdens, and clutch survival and size characteristics us ing a three-tiered approach that identified potentially important associations, and th en more rigorously examined hypothesized

PAGE 179

163 associations using large field studies and laboratory experiments. The first tier of the present study, case-control cohor t study, suggested that PAH and PCB concentrations as well as non-thiamine nutrients, were not likely to be the cause of decreased clutch viability as their levels did not show larg e differences across sites, nor were they significantly associated with alter clutch su rvival parameters. In addition, the casecontrol study indicated that th iamine concentrations were significantly associated with clutch survival parameters and that the associ ation suggested decreased thiamine levels in eggs were associated with decreased clutch viability, which is consistent with similar studies involving fish ((Fitzsim ons et al., 1999). Lastly, as dieldrin% increased (i.e., the proportion of total OCP burden composed by dieldrin) thiamine levels decreased, suggesting that OCPs may be indirectly i nvolved in decreased clutch viability via thiamine reduction, as OCP exposure has been suggested to decrease thiamine concentrations in laboratory models (Yagi et al., 1979). Results of the expanded field study provi ded more support for the hypothesis that thiamine deficiency may be involved in decr eased clutch viability and that OCP burdens and lipid content were significantly associated with variation of thia mine concentrations. However, the laboratory experiments, ove rall, did not support the hypothesis that thiamine is related to embryo viability in al ligators as thiamine amelioration or inhibition did not altered embryo mortality rates. One poten tial reason for the lack of effects is that thiamine levels were already sufficient fo r adequate embryo survival and therefore increasing concentrations were biologically irrelevant. With the thiamine antagonist experiment, two potential reasons for the la ck of effects are that oxythiamine may not have transferred into the yolk compartment and/or the concentra tion was not high enough

PAGE 180

164 to inhibit thiamine activity to the point that effects were elicited. Although thiamine and oxythiamine treatment experiments were ineff ective in the present study, similar studies involving in ovo treatments of fish eggs have been effective in demonstrating the effects of induced thiamine deficiency and thiami ne amelioration on embryo and fry survival (Fitzsimons et al., 2001). In conclusion, decreasing thiamine levels in eggs may be associated with decreased clutch success and lipid content, and OCP burde ns may be associated with variation in thiamine concentrations. However, it should be noted that thiamine levels in eggs only explained 38% of the variation in clutch su rvival parameters in the case-control cohort study and 27% of the clutch survival varia tion in the expanded fi eld study, which suggest that other factors are likely invo lved as well. Because of th e lack of effects observed in the experimental studies, future studies should try to induce th iamine deficiency in eggs through maternal dietary restriction, especially since embryos are at a relatively advanced stage of development by the time oviposition oc curs (Clarke, 1891). A concurrent study involving a captive adult alligator breeding popula tion will be able to control for diet and examine relationships between maternal OC P exposure and thiamine levels in eggs.

PAGE 181

165 Table 6-1. Classification matrix for clutches coll ected during 2002. Total OCP Burdena Clutch Viability >3700 3700 x > 350 350 (10071%) Good viab./High OCP Good via b./ Inter. OCP Good viab./Low OCP (70-48%) Inter. viab./High OCP Inter. viab./ Inter OCP Inter viab./Low OCP (47-0%) Poor viab./High OCP Poor via b./ Inter. OCP Poor viab./Low OCP ang/g yolk wet weight. Good and High = grea ter than mean + 1 standard deviation, Intermediate = mean 1 standard deviati on, Low and Poor = less than mean–1 standard deviation. Table 6-2. Reproductive, morphometric, a nd contaminant parameters measured on clutches of alligator eggs collected during summer 2000, 2001, and 2002. Parameter Definition Measured as Response variables Fecundity Total No. of eggs in one clutch n Clutch mass Total mass of eggs in one clutch kg Ave. Egg Weight Clutch mass / Fecundity g Unbanded eggs% a No. of unbanded eggs / fecundity x 100 Percentage Early embryo mort.% No. of deaths < dev. Day 35 / fecundity x 100 Percentage Late embryo mort.% No. of deaths dev. Day 35 / fecundity x 100 Percentage Clutch Viability No. eggs yielding live hatchling / fecundity x 100 Percentage Explanatory variables [OCP analyte] in ng OCP analyte / g egg yolk wet weight ppb OCP analyte% [OCP analyte] / [OCP] x 100 Percentage [PCBs] in egg yolk ng PCBs / g egg yolk wet weight ppb [PAHs] in egg yolk ng PAHs analyt e / g egg yolk wet weight ppb Thiamine in egg yolkb Pmoles / g egg yolk wet weight pmol/g Zn, Se, Vit. A, Ec ng analyte / g egg yolk wet weight ppb aAn egg with no evidence of embryonic attachment. bThiamine was measured in pmoles because various bioactive forms of were measured. cThese analytes were only measured in clutches collected during year 2002.

PAGE 182

166 Table 6-3. Explanatory variab les included in RDA with forw ard selection of four best variables for case-control cohor t and expanded field studies. Variablea Code Embryo age at time of collection Age Lake Griffin GR Lake Apopka AP Lake Lochloosa-Orange LO Emeralda Marsh EM No. OCP analytes at measurable levels NOC [OCP] TOC % Aldrin ALD% [Aldrin] [ALD] % cis -Chlordane CC% [ cis -Chlordane] [CC] % cis -Nonachlor CN% [ cis -Nonachlor] [CN] % Dieldrin DL% [Dieldrin] [DL] % Heptochlor epoxide HE% [Heptachlor epoxide] [HE] %Lipid content LPC% % Mirex MX% [Mirex] [MX] % o,p -DDT ODDT% [ o,p -DDT] [ODDT] [Methoxychlor] [ME] % Methoxychlor ME% % o,p -DDD ODDD% [ o,p -DDD] [ODDD] % Oxychlordane OX% [Oxychlordane] [OX] % p,p '-DDE PDDE% [ p,p '-DDE] [PDDE] % p,p '-DDD PDDD% [ p,p '-DDD] [PDDD] % p,p '-DDT PDDT% [ p,p '-DDT] [PDDT] % trans -Chlordane TC% trans -Chlordane [TC] % trans -Nonachlor TN% [ trans -Nonachlor] [TN] % Toxaphene TX% [Toxaphene] [TX] PCBs [PCB] PAHs [PAHs] Free Thiamine FT

PAGE 183

167 Table 6-3. Continued. Variablea Code Thiamine monophosphate TP Thiamine pyrophosphate TPP Vitamin E Vit.E Zinc Zn Selenium Se aFor the case-control cohort study, no OCP va riables were included in RDA involving clutch survival or size parameters, since clutches were selected a priori based on total OCP egg burdens. OCP variables were incl uded in the RDA evaluating the relationship between egg nutrients and chlorinated hydro carbons. For the expanded field study, only thiamine variables and OCP variables were in cluded after it was determined they were the more important explanatory factors (see results).

PAGE 184

168Table 6-4. Summary of cl utch parameters on clut ches collected during 2002. Parametera Good-High Good-Int. Good-Low Int.-Low Poor-High Poor-Int. Poor-Low No. Clutches 2 3 3 3 3 3 3 Fecundity ( n ) 51 5 49 1.9 41 2 49 5.2 55 5.8 46 4.5 47 0.9 Clutch mass 4 0.5 3 0.7 4 0.2 4 0.4 4 0.4 4 0.6 4 0.1 Egg mass (g) 86 0.8 69 12.5 88 6 86 1.5 76 6.4 78 6.2 79 0.3 Clutch viability 92 3.4 A 80 3.8 A 79 4.3 A 60 5 A 11 9.4 B 15 10.9 B 25 13 B Damaged eggs 0 0 1 1.3 0 0 0 0 2 1.1 1 1.1 0 0 Unbanded eggs 6 5.7 6 1.5 10 4.9 15 4.2 25 10.5 5 2.7 21 6.8 Early emb. mort. 2 2.3 10 5.1 12 8.1 7 3.3 54 21.6 36 26.9 30 3.9 Late emb. mort. 0 0 3 1.4 0 0 18 4 8 4.3 42 21.8 24 16.9 Dieldrin 248 20.2 72 47.8 6 1.1 9 5 157 66 264 134.8 16 5.4 Hep.Epoxide 3 0.8 11 4.2 4 2.3 2 0.5 6 2.6 4 1.8 5 1.7 cis-Chlordane 161 14.9 15 1.1 4 1.3 7 3.6 145 69.7 25 9.2 13 0.8 cis-Nonachlor 82 11.2 24 4.1 7 1.3 9 4.2 89 38.4 21 6.3 11 0.8 Oxychlordane 23 4.1 25 13.4 8 5 4 1.4 31 6.8 14 5.2 7 2.8 Toxaphene 10289 313.6 0 0 0 0 0 0 4670 1268.9 1928 0 0 0 p,p'DDD 2614 348.7 10 4.9 3 0.6 4 0.7 897 578.1 18 4.8 4 0.8 p,p'-DDE 19136 3277.6 1167 830.3 139 30.9 117 45.6 9149 3668.7 1019 795 153 28.6 p,p'-DDT 24 0 0 0 0 0 0 0 10 2.1 0 0 0 0 trans-Chlordane 51 1.5 1 0 1 0 1 0 34 15.3 1 0 2 0.5 trans-Nonachlor 251 46 55 13.9 16 4.7 15 5.8 273 141.4 41 15.8 22 2.8 [OCPs] 32959 3891.1 A 1391 923 B 188 41.9 C 168 67.9 C 15508 5426.2 A 2057 770.2 B 234 42.5 C NOC 14 0 11 0 10 0.7 10 0.9 14 0.3 11 0.7 11 0 [PAHs] 21 1.3 BC 33 2.8 AB 34 7.6 AB 31 2.9 AB 18 2.5 C 27 3.5 ABC 43 7.3 A [PCBs] 39 0 B 168 28.9 A 83 14.6 A 111 47 A 40 2.5 A 55 10.7 A 92 16 A Selenium 1000 100 1233 176.4 1067 66.7 1133 166.7 1000 57.7 933 120.2 833 145.3 TP 23 5.7 AB 13 1.6 AB 41 8.4 A 24 1.7 AB 2 1.8 C 12 6.7 BC 13 6.4 AB TPP 14 3.6 A 0 0.3 C 21 3.1 A 16 2.1 A 18 8.1 AB 4 3.8 BC 7 5.3 AB FT 463 92.6 719 427.9 868 231.1 238 40.6 891 261.2 553 482.3 326 115.4 Thiamine 500 83.3 733 427.7 931 220.0 278 38.9 912 267.4 569 486.5 345 104.3 Vit. E 16287 2389.7 26397 2978.6 19118 7352.9 12255 122.5 21054 1889.2 20025 5831 15221 1784.5 Zinc 15900 300 15433 809 24900 10570.9 15300 750.6 15267 437.2 12367 800.7 12967 788.1 aCodes for parameters are listed in Table 6-3.

PAGE 185

169 Table 6-5. Evaluation of the relationship betw een concentrations of nutrients, PAHs, and PCBs in eggs and clutch survival parameters via RDA analysis ( = 0.05). Variable Lambda A P F Thiamine monophosphate 0.26 0.004 6.28 Thiamine pyrophosphate 0.12 0.014 3.95 Free thiamine 0.08 0.102 2.1 Thiamine forms 0.08 0.142 2.08 Table 6-6. Evaluation of clut ch size parameters and explan atory factors for clutches collected during 2002. Variable Lambda A P F Lochloosa 0.27 0.018 6.77 Apopka 0.06 0.198 1.54 PCB concentrations 0.05 0.272 1.25 Emeralda Marsh 0.07 0.164 1.81 Table 6-7. Evaluation of the relationshi p between nutrient co ncentrations and explanatory variables for cl utches collected during 2002. Variable Lambda A P F Heptachlor epoxide conc. 0.18 0.006 4.07 trans-Chlordane conc. 0.15 0.022 3.67 Dieldrin% 0.17 0.002 5.43 Lochloosa 0.09 0.048 3.13

PAGE 186

170 Table 6-8. Summary and comparison of para meters measured on clutches collected during 2000-2002. Parametera Loch.-Orange Griffin Apopka Emeralda No. clutches 18 21 14 19 Fecundity 40 1.8 45 1.8 47 2 46 1.8 (26–56) (24–58) (31–56) (34–64) Clutch mass 3.6 0.18 3.5 0.19 4 0.2 4 0.38 (2.2–4.8) (1.8–4.8) (2.6–4.9) (2.3–9.2) Egg mass 90 3.1 78 2.2 86 3.5 87 7.4 (78–139) (46–89) (67–120) (60–180) Clutch viability 63 5.6 40 6.7 49 8.6 46 9.1 (0–95) (0–87) (0–80) (0–97) Damaged% 4 3.3 5 2.5 2 0.9 5 1.8 (0–60) (0–46) (0–12) (0–27) Unbanded% 11 2 12 2.3 13 3.6 12 3.5 (0–33) (0–32) (0–40) (0–58) Early Emb. Mort. 13 3 26 6.3 17 6.4 25 6.7 (0–36) (0–93) (0–90) (0–95) Late Emb. Mort. 8 2.6 18 4.9 19 6.8 11 3.8 (0–34) (0–58) (0–77) (0–61) TP 20 3.7 19 3.8 20 3.9 19 5.2 (0–52) (0–72) (0–60) (0–67) TPP 12 2.6 7 2.7 8 3.6 11 3.8 (0–31) (0–53) (0–46) (0–54) FT 747 102.8 536 93.1 573 110.8 657 125.9 (77–1324) (109–1570) (50–1412) (57–2171) TT 780 102.8 562 94.2 601 111.1 688 128.5 (77–1364) (152–1583) (62–1431) (57–2212) ALD 0 0 0 0 3 0.1 3 0.3 (0–0) (0–0) (3–3) (3–4) oDD 0 0 C 1 0 B 5 2.3 B 47 5.7 A (0–0) (1–1) (1–9) (8–104) oDT 1 0 B 3 0.4 B 10 2.1 A 301 290.9 A (1–1) (1–6) (1–29) (4–4373) ME 0 0 17 0.3 8 2.6 10 1.9 (0–0) (17–17) (6–16) (6–18) MI 2 0.4 2 0.4 4 1.4 3 0.9 (1–3) (1–4) (1–17) (0–10) DL 4 0.5 D 20 3.4 C 323 66.8 A 186 25.4 A (1–8) (6–70) (24–957) (30–387) HE 3 0.8 C 6 1.4 B 12 2.2 A 6 1.6 B

PAGE 187

171 Table 6-8. Continued. Parametera Loch.-Orange Griffin Apopka Emeralda (1–10) (1–30) (1–30) (0–29) CC 2 0.2 D 11 0.7 C 46 12.2 B 109 15.6 A (1–4) (6–17) (7–179) (15–281) CN 5 0.6 C 18 2.4 B 61 12.4 A 71 8.9 A (2–13) (8–54) (10–171) (17–166) OX 4 1.1 C 11 2.1 B 38 6.1 A 24 3.4 A (1–18) (1–42) (4–72) (3–57) TX 0 0 C 2678 376.5 B 2738 224.5 B 7558 703.6 A (0–0) (1928–3111) (1896–3809) (3216–12975) pDD 2 0.2 D 7 1 C 49 13.3 B 1711 225.1 A (1–3) (3–18) (11–193) (10–2963) pDE 76 12.3 D 283 47.4 C 4576 948.3 B 11304 1872.1 A (28–231) (70–979) (18–13294) (36–33555) pDT 1 0 C 2 0.7 BC 9 3.8 B 15 1.7 A (1–1) (1–2) (1–46) (6–25) TC 3 0.7 BC 2 0.2 C 7 2.2 B 31 4.1 A (1–4) (1–3) (1–27) (3–58) TN 8 1.7 C 37 7.2 B 157 36.8 A 208 30.8 A (3–25) (10–155) (10–532) (14–555) TOC 104 16.2 D 783 264.9 C 6855 1267.2 B 20417 2969.9 A (43–289) (127–4488) (555–18471) (672–53560) NOC 9 0.3 D 11 0.2 C 13 0.4 B 14 0.2 A (7–11) (10–13) (10–15) (13–16) aSee Table 6-3 for parameter codes. Values = mean standard error with range in parentheses. Table 6-9. Evaluation of the relationships between clutch survival parameters and explanatory variables via RDA using age as the covariate. Explanatory Variable LambdaA P F Total Thiamine 0.16 0.002 13.96 Thiamine Pyrophosphate 0.07 0.002 6.14 Thiamine Monophosphate 0.04 0.01 3.56 Methoxychlor% 0.03 0.036 2.71 Table 6-10. Evaluation of the relationships between clutch size parameters and explanatory variables via RDA using age as the covariate. Explanatory Variable LambdaA P F Free Thiamine 0.09 0.012 6.52 Thiamine Pyrophosphate 0.04 0.01 4.82 GR 0.03 0.054 3.36 Total Thiamine 0.04 0.06 3.9

PAGE 188

172 Table 6-11. Evaluation of the relationships between thiamine concentrations and explanatory variables via RDA using age as the covariate. Variable LambdaAP F Lipid content % 0.16 0.002 13.4 Trans-chlordane concentrations 0.06 0.002 6.5 Mirex concentrations 0.05 0.024 4.23 Oxychlordane concentrations 0.04 0.048 3.62

PAGE 189

173 Table 6-12. Site comparisons of parameters measured on clutches collected during 2003. Parameter Dexter Griffin Emeralda No. Clutches 4 5 5 Fecundity 37 3.8 B 46 1.2 AB 46 1.9 A Clutch mass 3 0.5 B 4 0.1 A 4 0.1 AB Egg mass 82 4.1 B 93 2.7 A 80 2.6 B Unbanded eggs 3 2 6 3 5 3.4 Damaged eggs 0 0 1 0.5 4 3.1 Dieldrin 5 1.4 29 9.3 188 78.2 Hep. Epoxide 2 0.4 8 3.1 4 1.5 cis-Chlordane 1 0 B 1 0 B 8 2.6 A cis-Nonachlor 6 1.8 B 22 7 AB 60 19.8 A Oxychlordane 4 1 B 14 5 AB 26 10.7 A Toxaphene 0 0 B 0 0 B 6765 2240.4 A o,p'-DDD 1 0 B 0 0 B 13 0 A o,p'-DDT 1 0 3 0.8 3 0.4 p,p'-DDD 1 0 3 0.4 981 407.8 p,p'-DDE 117 28.1 B 399 114.7 B 13166 5918.5 A p,p'-DDT 1 0 B 2 0.4 B 16 5 A trans-chlordane 1 0 1 0 3 0.6 Endrin ketone 0 0 B 0 0 B 3 0 A Mirex 4 1.3 2 0.3 2 0.5 trans-Nonachlor 10 3.7 B 54 19.9 AB 168 60.3 A OCP burdens 171 38 B 556 159.2 B 21410 8499.4 A No. OCP analytes 12 0.5 B 12 0.4 AB 14 0.5 A

PAGE 190

174 Table 6-13. Comparisons of parameters meas ured on the three thiamine treatment groups during 2003. Treatment Group Site Component Parametera control low high Emeralda Albumin FT (g/ng) 68 9.2 B 2414 464.3 A 3120 56.8 A (58–77) (1950–2878) (3063–3176) TMP 2 0.3 B 7 1.2 A 6 1 A (2–3) (6–8) (5–7) TPP 4 2.5 9 2.5 7 1.2 (1–6) (6–11) (6–8) TT 76 5.6 B 2436 459.2 A 3138 53.7 A (70–82) (1976–2895) (3084–3191) Yolk FT 1047 54.5 1122 80.7 1176 4.3 (992–1101) (1041–1203) (1172–1181) TMP 41 5.5 31 4.1 36 3.1 (35–46) (27–35) (33–39) TPP 21 0.2 18 2.8 24 0.3 (20–21) (15–21) (24–24) TT 1125 60.5 1185 71.8 1254 8.4 (1064–1185) (1113–1257) (1246–1263) Embryo Mort. 25 1.5 12 4.3 18 9.5 (23–26) (8–17) (8–27) Griffin Yolk FT 1041 115.9 1215 29.9 1233 18.7 (925–1157) (1185–1245) (1214–1252) TMP 6 0.6 9 0.4 11 4 (6–7) (9–9) (7–15) TPP 2 0.1 2 0.6 4 0.9 (2–2) (2–3) (3–5) TT 1052 116.7 1229 28.6 1252 24.7 (935–1168) (1200–1257) (1227–1277) Embryo Mort. 36 2.3 43 10.1 41 3.4 (33–38) (33–54) (38–45)

PAGE 191

175 -1.01.0-0.60.6 Clutch viability Unbanded eggs% Early Emb. Mort.% Late Emb. Mort.% TPP TP FT TT Figure 6-1. Biplot of clutch survival parameters and expl anatory factors for clutches collected during 2002.

PAGE 192

176 -1.01.0-0.80.8 Clutch mass Egg mass Fecundity [PCBs] Apopka Emeralda Marsh Griffin Lochloosa Figure 6-2. Biplot of clutch size parameters and explanatory variables for clutches collected during 2002.

PAGE 193

177 -1.01.0-1.01.0 Se Vit. E Zn TPP TP FT TT [HE] [TC] DL% LO Figure 6-3. Biplot of nutrien t concentrations in eggs (s olid arrows) and explanatory variables (dashed arrows).

PAGE 194

178 0% 20% 40% 60% 80% 100% 14253343 Figure 6-4. Relationships between embryo ag e and thiamine phosphorylation in egg yolk for 29 clutches collected during 2002 from Lakes Lochloosa (n = 6), Griffin (n = 10, Apopka (6), and Emeralda Marsh (n = 7). Embr y o a g e ( da y s ) FT TP TPP

PAGE 195

179 -1.00.6-0.61.0 Clutch viability Unbanded egg% Early Emb. Mort. Late Emb. Mort. TPP TP TT ME% Figure 6-5. Biplot of clutch survival parameters and explan atory variables for clutches collected during 2000-2002. See text and Table 6-3 for definition of explanatory variable codes.

PAGE 196

180 -1.01.0-1.00.4 Clutch mass Egg mass Fecundity TPP FT TT GR Figure 6-6. Biplot of clutch size variables (solid lines) a nd explanatory va riables (dashed lines) for clutches co llected during 2000-2002.

PAGE 197

181 -1.00.6-0.40.8 TPP TP FT TT LPC [MI] [OX] [TC] Figure 6-7. Biplot of thiamine egg yolk c oncentrations (solid lines) and explanatory variables (dashed lines) measured on clutches collected during 2000-2003.

PAGE 198

182 CHAPTER 7 REPRODUCTIVE EFFECTS OF ORGANOCHLORINE PESTICIDE EXPOSURE IN A CAPTIVE POPULATION OF AM ERICAN ALLIGATORS (ALLIGATOR MISSISSIPPIENSIS) In central Florida, American alligator ( Alligator mississippiensis ) eggs collected from organochlorine pesticide (OCP) contam inated sites (Lakes Apopka, Griffin, and Emeralda Marsh) contain total concentrati ons of OCPs that range from 4,000-30,000 ng/g yolk wet weight. This is several orders of magnitude greater than the reference sites (Lakes Orange and Lochloosa) (Chapter 2). In addition, alligato r populations inhabiting OCP-contaminated sites have experienced increased embryonic mortality resulting in reduced clutch success (Masson, 1995; Rotste in et al., 2002; Chapter 2). One possible explanation for these increased rates of embryonic mortality is embryonic exposure to OCPs, as similar effects have been reported in birds (Summer et al., 1996). The present study utilized a population of cap tive adult alligators to test the hypotheses that maternal exposure to OCPs would increase OCP burde ns in egg yolks, leading to increased embryonic mortality and decreased hatch rates. Materials and Methods Alligators were obtained from JungleL and Zoo (Kissimmee, FL) and Gatorland Zoo (Orlando, FL). Thirteen male and 14 female adult alligators were randomLy assigned to one of 13 pens (a pproximately 30 m x 30 m) at a ratio of 1 male: 1 female, except for one large pen which housed two females and one male. Seven pens were designated as treatment pens and six as cont rol. Prior to random group assignment (i.e.,

PAGE 199

183 control vs. OCP group), head length, total leng th, tail girth, and estimated mass [calculated using total lengt h and tail girth (Woodward et al., 1992)] were determined, with by-sex comparisons (T-test) indicati ng no significant differ ences among endpoints ( P > 0.3617 for all comparisons). Male and fe male head lengths averaged 35 3 cm (mean standard deviation) and 30 2 cm, respectively. Total lengths for males and females were 2.53 0.15 m and 2.23 0.18 m, respectively, and estimated mass was 69.5 13.1 kg for males and 46 13.8 kg for females. In addition to the breeding pairs, two extra treated females were housed separa tely to monitor bioaccumulation of OCPs and health status via monthly blood assessments of hematocrit, glucose, and total protein (Mader, 1996). Selection of specific OCP analytes and dose calculations were based on OCP concentrations in alligator yol ks collected from contaminated sites in Florida and avian maternal transfer rates (Fairbrother et al ., 1999). The dosing regime was designed to coincide with oocyte development and yol k formation (vitellogenesis), which for alligators begins in early fall and continues through late spri ng (Lance, 1986; Guillette, et al., 1997). Dosing began on 16 and 17 October, 2001. Animals were randomized with OCP-treated individuals receiving one intram uscular (IM) and one intraperitoneal (IP) injection consisting of a mixture of p,p ’-DDE (36.5 mg/kg), t oxaphene (2.6 mg/kg), chlordane (2.5 mg/kg), and diel drin (8.4 mg/kg) solubilized in reagent grade olive oil (cumulative injection volume of 40 mL). C ontrol animals received the same volume of olive oil. Animals did not receive oral doses of OCPs until they resumed feeding the following spring. On 16 April, 2002 oral dos ing began and continued to 20 October, 2002 when animals went began winter fast. Th is pattern of animals receiving oral doses

PAGE 200

184 from April to October continued through 2003 and 2004, so animals were exposed for a period of three years. Treat ed animals received oral dos es of p,p’-DDE (0.18 mg/kg), toxaphene (0.13 mg/kg), chlordane (0.014 mg /kg), and dieldrin (0.018 mg/kg). The chemicals were mixed with reagent grade oliv e oil (total mixture volume per weekly dose = 8 mL). Control animals received the sa me feed ration minus the OCP mixture. When females in breeding groups began nesting (24 June–10 July 2002), the two extra OCP-treated females housed in separate enclosures for monthly health status monitoring, and two females which did not produce clutches were sacrificed (via decapitation/cervical dislocation with double p ithing) to determine bioaccumulation rates of OCPs. Tissue samples (adipose, liver, a nd blood) were collected for analytical chemistry, along with one or two egg yolks from each of the females that oviposited, with eggs being collected and incubated using methods described in Chapter 2, except no helicopter or airboat was necessary. Ti ssues and yolks were screened for 30 OCP analytes by GC-MS according to procedures de scribed in Chapter 3. Lipid content (%) was determined gravimetrically for liver, and adipose tissue, wh ile GC-MS techniques were used for blood (Chapter 3). For 2002 and 2003 clutches, a subset of yolks from five control clutches and four treated clutches were analyzed for thiamine content to determine if thiamine levels were related to OCP exposure and/or clutch viability. Thiamine analysis was conducted using methods described in Chapter 6. For treated versus control comparisons, T-tests (PROC TTEST; SAS Institute Inc., 2002) and Wilcoxon two-sample tests (PROC NPAR1WAY WILCOXON) were used for parametric and nonparametric clutch paramete rs, respectively. Nume rical data were logtransformed [ln(x)], while propor tional data were arcsine squa re root transformed to aid

PAGE 201

185 in meeting statistical assumptions for parame tric tests. Logistic regression (PROC LOGISTIC; SAS Institute Inc., 2002) was used to evaluate associations between clutch survival parameters and potential explanat ory factors (i.e., tota l OCP concentrations, thiamine concentrations, and clutch size parameters). Results Nine clutches were collected from the control group and seven from the treated group over a period of three years. Clutch pa rameters that signif icantly differed among the groups included clutch vi ability, incidence of unbanded eggs, lipid content, egg concentrations of seven of eight OCP analyt es, and total OCP concentrations in eggs (Table 7-1). Specifically, clutch viability of the control group wa s 30% higher than the treated group, and the inciden ce of unbanded eggs was 40% lo wer in the control group as compared to the treated group. In addition, eggs of the treated group had significantly higher lipid content and total OCP concentra tions over those of controls. Importantly, OCP burdens in yolks from the control group (5 0 3.6 ng/g) were less than those of the reference site (102 15.5 ng/g), and the treated group yielded yolk burdens (13,300 2,666 ng/g) that fell within the range of the mean OCP concentrations (1,169-15,480 ng/g) observed in contaminated sites (Chapt er 2). No significant differences were detected with respect to number of clut ches produced by each group, fecundity, clutch mass, egg mass, oxychlordane c oncentrations, or thiamine concentrations, with thiamine being analyzed on five control and four treated clutches during year 2002-2003. Monthly health status assessments on two “ex tra” females, which were housed apart from breeding females, indicated that blood chemis try values appeared to be within normal limits (Table 7-2). After 10 months of dos ing and concurrent with nesting of other captive females (June 2002), the two extra females and two non-reproductive females

PAGE 202

186 were sacrificed and tissues were analyzed for OCP content. Individual chemicals exhibited differing concentrations among ti ssues, with the differing levels possibly related to varying lipid content of the tissue and the level of the administered dose. Results of logistic regression indicated that OCP variables an d clutch-egg size variables appeared to be associated with clutch survival parameters. Based on the differences between treated and control groups it was not surprising to find that total OCP concentrations (TOC) in egg yolk was ne gatively associated w ith clutch viability and positively associated with incidence of unbanded eggs. Fecundity, egg mass, and clutch mass, which were not correlated with (TOC), but were positively correlated with clutch viability, early embryo mortality, and late embryo mortality, and negatively correlated with unbanded egg incidence. Lipi d content in eggs wa s positively correlated with early and late embryo mortality, and as shown in group comparisons, lipid content was positively correlated with TOC (i.e., si gnificantly higher in the treated group). Thiamine concentrations in eggs were determin ed to be significantly correlated with one another. In addition, thiamine monophosphate (TP) was found to be positively associated with clutch viability, clutch mass, and f ecundity, and negatively associated with unbanded egg incidence. Thiamine pyrophosphate (TPP) was not associated with any of the clutch survival parameters. In contra st, free thiamine and total thiamine were negatively associated with unbanded egg incide nce and positively associated with early and late embryo mortality (Table 7-3). Discussion The results of this study support th e hypothesis that OCPs are maternally transferred to the developing e gg, and that maternal exposure is associated with reduced clutch success and increased embryonic mortalit y. In addition, this is the first study to

PAGE 203

187 develop a method for exposing alligator embryos to endogenous concentrations of OCPs, in contrast to prior studies that have exogenously applied OCPs to eggs to elicit embryonic exposure (Matter, et al., 1998). Im portantly, the dosing regime did not induce adult mortality, or alterations in monthl y blood chemistry assessments, food intake, weight gain, and behavior (e.g., females fi ercely defended their nests). However, subclinical, cytotoxic effects on the liver, gona ds, or kidneys may have been undetected. In addition, OCPs may cause f unctional defects in neural tr ansmission, leading to subtle increases in stress due to subl ethal neuronal hyperactivity. The decreased clutch viability noted in the OCP treated group was due to increased incidence of unbanded eggs. Sin ce unbanded eggs may be the result of embryo mortality occurring prior to embryo attachme nt (Rotstein et al., 2002) or possibly the result of infertility (or both), and since bot h parents received sim ilar doses of OCPs and since effects of OCPs vary from species to species and analyte to analyte (Rattner & Heath, 2003), the potential mechanisms by which OCP exposure might possibly induce increased incidence of unbanded eggs are many and may include altered egg quality, direct embryo toxicity, or decreased reproductive function in males. Other factors besides OCP exposure and e gg concentrations that were associated with variation in clutch survival parameters (and that weren’t concurrently associated with OCP exposure) included: fecundity, clut ch mass (fecundity and clutch mass were collinear), egg mass, lipid c ontent, thiamine monophosphate (TP), free thiamine (FT), and total thiamine (TT) concentrations (all thiamine forms were collinear with one another).

PAGE 204

188 In order to accurately interpret associa tions, consideration should be given to biological relevance and plausibility, as well as the experimental design and results. Given that the major contribution to decrea sed clutch viability for both control and treated groups was the increased incidence of unbanded eggs, fact ors associated with unbanded eggs are due careful consideration. In this respect, concentrations of OCPs in eggs were positively associated with unbande d egg incidence and negatively associated with clutch viability, as e xpected given the experimental design and results of group comparisons. In contrast to OCPs, clutch fecundity, clutch mass, egg mass, and TP had negative associations with unbanded egg incide nce and positive associations with clutch viability. However, none of these explan atory factors were correlated with OCPs, suggesting that they may independently ac count for a portion of the incidence of unbanded eggs in OCP exposed group and, mo re importantly, the control group. The biological implications are that in a control situ ation the incidence of unbanded eggs are related to smaller clutches and lighter e ggs, and that the rate of unbanded eggs is basically doubled when captive animals are exposed to OCPs. Factors that were not correlated with clutch viability were FT, TT, and lipid content. FT and TT were negatively co rrelated with unbanded egg incidence and positively correlated with early and late embryo mortality. These associations may be a result of the significant correlation FT and TT ha ve with TP, and might not be a result of a direct link with unbanded egg incidence. Positive associations between FT, TT, and early and late embryo mortality, suggest pos sible thiamine hypervitaminosis. Although unlikely, thiamine concentrations of experime ntal clutches (both gr oups) were three-fold to five-fold greater than thos e of wild clutches, and thiamine hypervitaminosis has been

PAGE 205

189 shown to induce neurotoxicosis in laborator y models (Snodgrass, 1992), which suggests that thiamine toxicity can’t be completely discounted. Also correlated with increased early and late embryo mortality were fecundity, clutch mass, and egg mass. Although a biologically relevant reason for this association may be pres ent, the association between embryo mortality and fecundity, clutch and egg ma ss may be attributed to the fact that as unbanded egg incidence decreases, the more embryos are pres ent and allows the potential for embryo mortality to occur. In contrast to thiamine concentrations, lipid content was found to be significantly associated only with early and late embryo mortality, and OCP concentrations, which is to be expected as treated groups had signifi cantly higher levels comp ared to controls. One might conclude that the reasons for the positive association between OCPs and lipid content is simply because OCPs are hydr ophobic and lipophilic; however, because lipid content was different between treatment gr oups, it may be that OCP exposure altered liver and/or follicle function in producing and sequestering yolk components. These results may suggest maternally-mediated alterations in egg quality and resulting decreased clutch viability. Ma ternally-mediated reductions in clutch viability is a likely scenario as liver is a known target organ for OCP-induced toxicity (Metcalfe, 1998), and exposure to similar organochlorine compounds has been shown to alter liver function (phospholipid production and transfer), vitello genesis, egg component profiles of other oviparous vertebrates ((Lal & Singh, 1987), a nd up-regulates biotransformation enzymes that are involved in xenobiotic biotransforma tion and lipid metabolism (Ertl et al., 1998). With respect to lipid contents associ ation with embryo mortality, an important finding was that OCP concentrations were not associated with early or late embryo

PAGE 206

190 mortality, suggesting lipid associ ation is not simply due to multicollinearity with OCPs. These results may indicate that maternal exposur e to OCPs alters lipid content, leading to increased embryo mortality. This association may be biologically re levant and plausible since differences in egg yolk fatty acid prof iles are suggested to be related to reduced clutch viability in captive alligators (Noble et al., 1993; Millstein, 1995). In summary, results of the present stu dy supports the hypothesis that parental OCP exposure may decrease clutch viability by increasing the incidence of unbanded eggs. These results differ from observations in wild clutches from OCP-contaminated sites in which reduced clutch viability is primarily due to increases in early and late embryo mortality (Chapter 2). However, unbanded eggs may be products of very early embryo mortality (Rotstein et al., 2002), with very early embr yo mortality in the captive population being likely related to OCP effects that have been exacerbated by the stress of captivity. Also important to consider is that alligators (less than 50 years old) from OCPcontaminated sites have likely been exposed to OCPs since con ception, and therefore may have been reproductively altered duri ng development {Gross et al., 1994} and may respond differently to OCP exposure as compared to previously unexposed adults. This study confirms, as somewhat expected, th at OCPs are maternally transferred in the alligator and that this is likely the major ro ute for embryonic exposure. This study is also the first induce, via maternal OCP exposur e, endogenous OCP exposure in developing alligator embryos. Importantl y, this ecological relevant e xperiment demonstrates that parental exposure to OCPs results in decreases in clutch viability sim ilar to what has been observed in wild alligator populations inhabiti ng OCP-contaminated sites. Lastly, this study provides experimental evidence linking pa rental OCP exposure to decreased clutch

PAGE 207

191 viability in the American alligator, and suggests a maternally-mediated mechanism may be involved.

PAGE 208

192 Table 7-1. Summary statistics and comparisons of clutch parameters among treated and control groups for years 2002-2004. Parameter Control Treated Summary No. Clutches 9 7 16 Fecundity ( n ) 32 2.4 30 2.5 31 1.7 (19–40) (20–37) (19–40) Clutch mass (kg) 2.31 0.227 2.22 0.203 2.27 0.151 (1.33–3.3) (1.51–2.9) (1.33–3.3) Egg mass (g) 73 3 73 1.2 73 1.7 (53–83) (70–78) (53–83) Clutch viability (%) 44 11* 9 6 29 7.9 (0–95) (0–35) (0–95) Unbanded eggs (%) 39 12.4* 81 12.3 58 10.1 (3–100) (22–100) (3–100) Damaged eggs (%) 3 3 0 0 2 1.7 (0–27) (0–0) (0–27) Early emb. mort. (%) 8 4 4 2.8 6 2.5 (0–36) (0–16) (0–36) Late emb. mort. (%) 6 2.7 5 5 6 2.6 (0–20) (0–35) (0–35) Lipid content (%) 19 0.7* 22 0.7 20 0.7 (15–21) (20–25) (15–25) TP (pmoles/g) 24 6.1 16 8 20 4.8 (4–39) (3–39) (3–39) TPP (pmoles/g) 21 6 15 3.7 18 3.6 (11–44) (6–22) (6–44) Thiamine (pmoles/g) 3088 182.8 3035 343.4 3065 170.4 (2623–3694) (2576–4045) (2576–4045) Thiamine (pmoles/g) 3133 182.6 3066 352.8 3103 173.6 (2640–3731) (2585–4105) (2585–4105) CC (ng/g) 1 0* 24 9.4 14 6.1 (1–1) (3–64) (1–64) CN (ng/g) 1 0* 10 2.6 7 2 (1–2) (1–18) (1–18) Dield. (ng/g) 6 1.2* 773 122.1 335 116.4 (3–11) (475–1143) (3–1143) Oxychl. (ng/g) 1 0.1 2 0.4 2 0.2 (1–2) (1–3) (1–3) p,p'-DDE (ng/g) 19 2.6* 11729 2200.4 5038 1838.8 (6–30) (5801–18448) (6–18448)

PAGE 209

193 Table 7-1. Continued. Parameter Control Treated Summary Oxychl. (ng/g) 1 0.1 2 0.4 2 0.2 (1–2) (1–3) (1–3) p,p'-DDE (ng/g) 19 2.6* 11729 2200.4 5038 1838.8 (6–30) (5801–18448) (6–18448) TC (ng/g) 1 0* 25 9.9 17 7.5 (1–1) (3–66) (1–66) TN (ng/g) 2 0.4* 36 8 17 5.6 (1–4) (11–56) (1–56) Toxa. (ng/g) 0 0* 2035 720 2035 720 (0–0) (1315–2755) (1315–2755) [OCPs] (ng/g) 50 3.6* 13300 2666.1 5728 2116.4 (29–60) (6393–21991) (29–21991) No. OCPs ( n ) 5 0.7 7 1.2 6 0.7 (0–7) (0–9) (0–9)

PAGE 210

194 Table 7-2. Organochlorine concentrations and blood chemistry values of captive adult female alligators sacrificed during 2 002 (Rauschenberger et al., 2004). Mean standard deviation (sample size). Parameter Control Treated Adipose Tissue (ng/ga) p,p’-DDE No datab 68,315 35,275 (4) Toxaphene No datab 8,385 1486 (4) Chlordane No datab 708 200 (4) Dieldrin No datab 4,372 1,237 (4) Lipid Content (%) No datab 82 6 (4) Liver Tissue (ng/ga) p,p’-DDE No datab 8,168 3,750 (4) Toxaphene No datab Not detectedc Chlordane No datab 23 13 (4) Dieldrin No datab 143 92 (4) Lipid Content (%) No datab 4 2 (4) Whole Blood (ng/ga) p,p’-DDE No datab 179 184 (4) Toxaphene No datab Not detectedc Chlordane No datab Not detectedd Dieldrin No datab 15 5 (4) Lipid Content (%) No datab 0.10 0.02 (4) Hematocrit ( %) e 20-30 20 4 (2) Glucose (mg/dl) e 74 63 17 (2) Tot.Plasma Protein mg/dl) e5.1 6 1 (2) ang chemical/g yolk wet weight (not lipid normalized). bNo control females were sacrificed. cLimit of detection for toxaphene = 230 ng/g. dLimit of detection for chlordane = 0.2 ng/g. eFor controls, blood chemistry valu es reported by Mader (1996). Values for treated group reflect mean of 10 samples collected evenly over 10 months.

PAGE 211

195 Table 7-3. Explanatory parame ters and clutch survival parameters with () indicating nature of association and value equal to concordance percentage. Parametera Unbanded egg% Clutch Viability Early Emb. Mort. Late Emb. Mort. TOCb (+) 64 (–) 65 ns ns Fecundityc (–) 71 (+) 65 (+) 54 (+) 65 clutch massc (–) 77 (+) 72 (+) 67 (+) 76 egg mass (–) 63 (+) 58 (+) 55 (+) 66 lipid %b ns ns (+) 58 (+) 68 TPcd (–) 59 (+) 59 ns ns TPPd ns ns ns ns FTd (–) 68 ns (+) 74 (+) 72 TTd (–) 69 ns (+) 74 (+) 72 aTOC = total OCP concentrations in egg yolk; TP = thiamine monophosphate; TPP = thiamine pyrophosphate; FT = free thiamine; TT = total ( ) thiamine concentrations in yolk. b-d Parameters sharing same superscript letters are significantly correlated with each other, those not sharing letters are not correlated. NS = not significant ( P > 0.05)

PAGE 212

196 CHAPTER 8 CONCLUSIONS Introduction The American alligator has a significant role in the ecology, esthetics, and economy of Florida. Thus, maintaining viable popul ations of alligators is desirable for many reasons. As reproduction is critical for ma intaining a species p opulation, any relatively sudden or sustained decrease in repro duction may be cause for concern. Over the last quarter century, alligat or populations in organochlorine (OCP) contaminated lakes in central Florida have garnered intense study and much attention due to their decreased reproductive performan ce (Woodward et al., 1989; Woodward et al., 1993; Wiebe et al., 2001). Decreased reproduct ive performance has been attributed to decreased clutch viability due to increased embryo mortality, with mortality typically occurring during the first 20 days of devel opment (Masson, 1995). Furthermore, prior study suggests OCP exposure may be a potential contributing factor to increased embryo mortality since increased mortality had been reported only in sites heavily contaminated with OCPs and since alligator eggs from thes e sites contained increased levels of OCPs, but a clear relationship was not ev ident (Heinz et al., 1991). Understanding biological and environmental characteristics related to embryo development, egg quality, and clutch viability in alligators is necessary in evaluating whether OCPs and/or some other factor(s) may be causally linked to decreased clutch viability. Identifying an d understanding factors associated with decreased clutch viability may benefit management of alligator populations and ensure sustainable human use. On

PAGE 213

197 a larger scale, understandi ng the relationship between OCP exposure and developmental mortality in alligators ma y provide some insight into the potential impact of organochlorine pesticides on the ecol ogical health of Florida’s wetlands. Evaluating the relationships between cl utch viability, OCP exposure, embryo development, and other potentially important biological and environmental factors has been the theme of this disse rtation. These evaluations have been accomplished through field studies designed to identify importan t factors and associations and to test hypothesized associations with laboratory experiments. Summary of Study’s Findings The first study (Chapter 2) examined clutch viability on OCP-contaminated and reference sites from 2000-2002. Results indicate d that clutch viability is significantly lower in contaminated sites, with these site s having higher rates of early and late embryo mortality, and that unbanded eggs also appear to be an important constituent of reduced clutch viability for reference and contaminat ed sites. In order of importance, major constituents of reduced clutch viability for all sites include early embryo mortality, unbanded eggs, late embryo mortality, and dama ged eggs. In addition, clutches from OCP-contaminated sites had an average of 10 more eggs per clutch as compared to the reference site, but average clutch mass wa s not significantly different, making average egg mass of reference site clutches greater than that of clutches of OCP-contaminated sites. In addition to differences in clutch viability and size, la rge differences in OCP concentrations in alligator eggs between reference and OCP-contaminated sites were found. Although not surprising given the histor y of the sites, these results support the continued problems with alligator repr oduction in OCP-contaminated sites.

PAGE 214

198 Results of redundancy analyses indicate that for Lake Lochloosa, no significant correlations were determined although significance might have been detected given a greater sample size. For Emeralda Marsh, th e weak associations between OCP variables and clutch survival variables suggests that other factors may be involved in reduced embryo survival and increased rates of unbanded eggs. The weak associations for Emeralda Marsh are surprising given that relatively stronger associations were determined for the other high exposure site (Lake Apopka; Table 2-5), as well as the intermediate exposure site (Lake Griffin, Table 2-5), with Emeralda Marsh being separated from Lake Griffin by easily traver sable, non-fenced levee. The positive association between early em bryo mortality and unbanded eg g rates and extracted OCP variables for Lake Apopka clutches suggest s that the percentage s of dieldrin and trans chlordane in eggs may play an important role in altered egg fertility and/or early embryo survival. For Lake Griffin, the negative to near-zero association between early embryo mortality rates and extracted OCP variables s uggests that OCP burdens in eggs may not play an important role in early embryo mort ality. However, the positive association between toxaphene burdens and late embryo mo rtality suggests that as toxaphene burdens increase, so does the risk for increased embryo death during the last 35 days of development. Furthermore, th e positive association between p,p’ -DDT concentrations and unbanded egg rates suggests that these an alytes may be involved in altered egg fertility and/or embryo survival (prior to eggshell membrane attachment) (Fig. 2-2). In summary, the first study suggested that, over all sampled clutches, clutch survival parameters and egg and clutch size parameters vary between the low OCP exposure site (Lochloosa) and the intermedia te-high OCP exposure sites. Furthermore,

PAGE 215

199 OCP burdens do not appear to be related to cl utch survival for the low exposure site but are associated with clutch survival for the intermediately OCP contaminated site and one of the highly OCP contaminated sites. The next study (Chapter 3) evaluated the relationship between OCP burdens in eggs and in maternal tissues in order to exam ine the extent of maternal transfer of OCPs in the alligator and to determine if eggs coul d be used as predicto rs of maternal tissue burdens. Major finding of this study were that adipose tissue and yolk burdens were similar when adjusted for lipid content a nd that yolk was an ex cellent predictor of adipose tissue burdens. Convers ely, blood and yolk burdens were not linearly related. Importantly, liver had higher burdens than yolk after adjustment for lipid content suggesting liver may sequester OCPs, supporti ng the possibility that liver function may be altered due to chronic OCP exposure. Altered liver function due to OCP exposure may affect lipid metabolism, vitellogene sis, and egg quality (Lal & Singh, 1987), potentially resulting in maternally-med iated embryo mortality. However, non-OCP related maternal factor(s), such as size and ag e, could also affect clutch viability rates. The following study (Chapter 4) addressed the potential influence of maternal factors on clutch viability. Re sults indicated maternal body si ze was not associated with variation in clutch survival parameters, but moderate associations existed between maternal OCP burdens and clutch survival parameters (18% of variance explained, P < 0.05). Specifically, as p,p’-DDE proportions increased in relation to total OCP egg burdens, the incidence of unbanded eggs incr eased, and as trans-chlordane proportions increased in relation to total OCP burdens in eggs, clutch viability decreased and early embryo mortality increased.

PAGE 216

200 Since increased rates of embryo mortality were the reason for decreased clutch viability, the fourth study (Cha pter 5) sought to evaluate the histopathology, growth, and development of embryos, and their associati ons with OCP exposure (egg burdens). Intrasite comparisons suggested that among all sites and all sampled ages (calendar age = CA), that morphological age (MA) of embr yos and embryo mass were greater for live embryos as compared to dead embryos, whic h suggested that embryos may have been developing normally up to a point at wh ich development stalled and the embryo eventually died, or embryos could have devel oped at a much slower overall rate until the point at which they perished. Either way it appears that the mass of dead embryos was appropriate for their MA. Morphometry of live embryos did not appear to be significantly related to variati on in clutch mortality rates, suggesting that live embryos from clutches with high mortality rates de velop similarly to those of low mortality clutches. This finding may suggest a threshol d-type response in which embryos exposed to stressors below a certain threshold have the ability to overcome stressors through various cellular homeostatic mechanisms, but above a certain threshold, developmental retardation and lethality occu r. Such threshold dose-response patterns have been accepted as a major dose-response pattern in mammalian developmental toxicology (Rogers & Kavlock, 2001). Furthermore, variation in morphologi cal development of live embryos was significantly associated with variation in th e composition and concentration of OCPs in eggs. The strength of the relationships appear ed to decrease with the age sampled (CA), with youngest embryos sampled (CA Day 14) showing the strongest relationships between OCP egg burden and morphometric parameters, followed by each subsequent

PAGE 217

201 CA, respectively (Table 5-5). Interestingly, the percenta ge of the total OCP burden (concentration) composed by an OCP analyte (i .e., HE%), appeared to be more important than OCP analyte concentrations alone. W ith respect to all sampled ages, except the eldest (CA Day 43), relative proportions of th e OCP analyte(s) appeared to be more important than concentrations alone (Table 5-5). For derived mor phometric parameters and morphological age (MA), similar patterns were observed in that embryos sampled at younger CA showed stronger rela tionships with OCP burdens than older cohorts (Table 5-6). Another important observation was that di fferent cyclodienes appeared to be associated with morphological variation of embryos of different ages (CA). Most important were the components of technical grade chlordane and its metabolites, which include cis and trans -chlordane, cis and trans -nonachlor, oxychlordane, and heptachlor epoxide. One or more of these components were found to be significantly associated with variation in embryo morphology for each CA sampled. These data suggest that the chlordane group may merit furthe r study in relation to developm ental effects in reptiles, especially considering other st udies have suggested that sexu al differentiation in turtles may be altered by low dose in ovo exposures of these compounds (Willingham, 2004). In conclusion, the embryo morphology a nd histopathology study found that embryo mortality occurring in alligator populations inhabiting reference and OCP-contaminated sites was characterized by developmental reta rdation without gross deformities, or overt presence of lesions to vital organs. Howe ver, variation in embryo morphology appeared to be associated with variation in OCP bur dens of eggs and the percentage composition composed by an OCP analyte was equally as important as concen tration, suggesting the

PAGE 218

202 importance of mixture composition. Younger embryos appeared more susceptible to OCP influence but OCP influence may not nece ssarily be the result of direct embryo effects. Similar types of embryo mortality, characterized by developmental retardation) has been documented in quail, with embryo mortality determined to be maternally mediated, where maternal liver function was al tered, resulting in nutrient deficiencies in eggs that were severe enough to induce em bryo mortality (Donaldson & Fites, 1970). Because nutrition and non-OCP contaminants have been associated with developmental retardation in salmonids (Fitzsimons et al ., 1999) and birds (Wilson, 1997; Gilbertson et al., 1991)., the next study (Chapter 6) ev aluated embryo mortality in alligators of reference and OCP-contaminated sites as a function of exposure to OCPs, polychlorinated biphenyls (PCBs), and pol yaromatic hydrocarbons (PAHs), as well as egg nutrient content. Results of this study suggested that decreasing thiamine levels in eggs may be associated with decreased clutch success and lipid content, and OCP burdens may be associated with variation in thiamine concentrations In addition, PCBs, PAHs, and non-thiamine nutrients were not found to be significan tly associated with clutch viability. Thiamine levels in eggs explained 38% of the variation in clutch survival parameters in the case-control c ohort study and 27% of the clutch survival variation in the expanded fiel d study, which suggest that factors in addition to thiamine are likely involved. A lack of effects on clutch viability observed in experiments involving thiamine amelioration and inhibition via topical egg treatments may suggest a number of potential conclusions including th at either thiamine has no effect on the embryo or that the embryo was not exposed to enough thiamine or th iamine inhibitor to elicit effects.

PAGE 219

203 The last experiment of this dissertati on (Chapter 7) attempted to examine the relationship between OCP exposur e and decreased clutch viabili ty in alligators in a more direct way which involved orally dosing a cap tive breeding population of adult alligators with OCPs to test the hypothesi s that adult alligators exposed to OCPs yield clutches with decreased clutch viability as compared to controls. The results of this study found that clutch viability was decreased in the OCP tr eated group, but that the decrease was due to increased incidence of unbanded eggs and not mortality in banded eggs (i.e., after embryo attachment). However, very early embryo (conceptus) mortality has been documented in unbanded eggs via determination of paternal DNA {Rotstein et al., 2002), so it is likely that a portion of the unbanded eggs were products of early embryo mortality, especially considering captives had not been raised in a contaminated habitat and may have responded more severely to OCP exposure in comparison to their wild cohorts. In addition to OCP factors, the captive study s uggested other factors, not concurrently associated with OCP exposure, were associat ed with clutch viability. These factors included fecundity, clutch mass (fecundity a nd clutch mass were correlated with each other), egg mass, lipid content, thiamine monophosphate (TP), free thiamine (FT), and total thiamine (TT) concentrations (all thiamine forms were correlated with one another). In summary, results of the captive parental dosing study support the hypothesis that OCP exposure may decrease clutch viab ility by increasing the incidence of unbanded eggs and/or early embryo mortality. The bi ological implications ar e that in a control situation incidence of unbanded eggs are related to smaller cl utches and lighter eggs, and that the rate of unbanded eggs is basically doubled when captive animals are exposed to

PAGE 220

204 OCPs. The study confirms that OCPs are matern ally transferred in th e alligator and that maternal OCP exposure alters lipid content of eggs and reduces clutch viability. Overall, our studies (Chapt ers 2-7) suggest that OCPs may indeed be contributing to the decreased clutch viab ility in alligator populations inhabiting OCP-contaminated sites. In addition, thiamine leve ls and clutch size parameters appear to be associated with embryo mortality and decreased clutch viab ility. These data combined with dead embryos being developmentally retarded s uggest that alterations in growth and metabolism are the probable mechanism by whic h mortality results, as opposed to acute toxicity to organs or specific deformities since these were not gr eatly observed. Lastly, the captive exposure study provided experiment al evidence that parental OCP exposure can reduce clutch viability. Continuing studi es beyond this dissertation are investigating the relationship between fatty acid prof iles, OCPs, and clutch viability. Future Considerations and Global Implications Although difficult and expensive, conducti ng an expanded captive dosing study is likely the only way to separate which OCPs are actually causing the decreased clutch viability from those that are just collinear. A large number of alligators would have to be involved so that hypothesized OCP analytes could be given individually to determine what component of the OCP mixture was resp onsible or if decreased clutch viability resulted from some type of mixture effect. A challenge in trying to re late the present findings to other OCP exposure studies involving birds, mammals, and fish is that the basic metabolic function of an adult alligator is vastly different from most models. For example, blood flow of a 70 kg alligator (0.26 L/min) is less than 8% of th at of a 70 kg human, and 0.3% of that of a 70 kg shrew (Coulson & Hernandez, 1983). Thes e differences mean that xenobiotics and

PAGE 221

205 endogenous compounds circulate throughout the al ligator at a decreased rate which can affect excretion and elimination, as well as the amount of time target organs are exposed (i.e., decreased blood flow through liver may mean increased OCP e xposure to liver). Another factor that may affect OCP toxicity is the temperature of the alligator, as low temperatures have been associated with incr eased DDT toxicity in exposed fish (Rattner & Heath, 2003). Although it is often believed that being cold-blooded causes low blood flow in alligators, low blood flow is actually related to their relati vely small hearts and low blood hemoglobin, and not temperature (Coulson & Hernandez, 1983). Speculatively, low blood flow, seasonally lo wer body temperatures, and seasonal fasting (possibly resulting in mobilization of lipids and hydrophobic contaminants) may contribute to this species su sceptibility to reproductive modulation via OCP exposure. Another aspect concerning the biochemistry of the alligator is that they are true predators in that they cannot di gest complex sugars or starches or plant proteins. Indeed, their sources of glucose are mainly glucone ogenesis, in which the liver uses carbon skeletons of catabolized amino acids to synt hesize glucose, and u tilization of glucose stores in the carcasses of pr ey. The implications are the importance of normal liver function in producing energy. Furthermore, liver functions in amino acid storage, in that it has been shown that alligator s can store excess amino acids in liver that can be later mobilized for protein production. The ecologi cal trophic level of th e alligator obviously contributes to increased orga nochlorine pesticide exposure vi a biomagnification, but their apparent susceptibility to maternally mediated development mortality may be exacerbated by the physiological requirements of being a predator. This speculation is supported by studies indicating p,p’-DDE causes eggshell thinning in predatory birds but

PAGE 222

206 not domestic fowl, suggesting predatory sp ecies may be more susceptible to the reproductive effects of OCP exposure (Fairbroth er et al., 1999). Lastly, alligators are a poikilothermic species that fast for up to si x months during a time when vitellogenesis is underway. The concurrent fasting and vite llogenesis means that OCPs are mobilized along with lipid stores to meet the meta bolic demands of homeostasis and follicle development, likely increasing risk of OCP-a ssociated alterations in liver function. Identifying other species that may be susceptible to similar organochlorineassociated embryo mortality is important for both helping to maintain biodiversity and for better understanding of the mechanisms of organochlorine-associated developmental mortality. Key ecological and physiological ch aracteristics to look for in a potential model are that the species be a season ally fasting, oviparous, highly fecund, poikilothermic predator. Given these attribut es, species which may be potential models for examining OCP-induced reproductive toxicity include predatory turtles, such as the common snapper ( Chelydra serpentina ), the softshell ( Apalone muticus ), the alligator snapper ( Macrochelys temminckii) water snakes ( Nerodia spp .), and predatory fish, such as largemouth bass ( Micropterus salmoides ), and bowfin ( Amia calva ). Indeed, alterations in endocrine function and increase d developmental mortality have been noted in largemouth bass inhabiting Emeralda Mars h (Seplveda et al ., 2004). In addition, turtle eggs from Lake Apopka were found to have abnormalities and poor hatch rates around the time reproductive problems with alligat ors began to be investigated (Franklin Percival, pers. comm..). The global implications of this dissertati on’s results and postula tions suggest that predatory reptiles and fish inhabiting areas of the world that receive(d) high inputs of

PAGE 223

207 organochlorine pesticides may be at risk of increased rates of embryo mortality or decreased reproductive performance. Many of these areas are in tropical, third-world countries that continue to buy DDT from U. S. manufacturers because it is an economical way to control malarial mosquitoes and cropdestroying pests (Brema n et al., 2004). The combination of concern for human health and ecological inte grity underscore the exigency for better understanding of the eff ects associated with OCPs and similar persistent organic compounds, so that best management practices may be developed in order to protect human health and ecological integrity.

PAGE 224

208 LIST OF REFERENCES Akerman, G., Tjarnlund, U., Noaksson, E., & Balk, L. (1998). Studies with oxythiamine to mimic reproduction disorders among fish early life stages. Marine Environ Res 46: 493-497. Ashworth, C. J. & Antipatis, C. (2001) Micronutrient programming of development throughout gestation. Reproduction 122: 527-35. Benton, J., Douglas, D., & Prevatt, L. (1991) Completion report as required by federal aid in fish restoration: Wallop-Breaux project F-30-18, Ocklawaha Basin Fisheries Investigations Study XII. State of Flor ida Game and Freshwater Fish Commission. Tallahassee, FL. Billet, F., Gans, C., & Maderson, P. F. A. (1985). Why study reptilian development? In Biology of the Repti lia. 14. Development A : 3-25. Gans, C., Billet, F. & Maderson, P. F. A. (Eds.). New York, NY: John Wiley and Sons. Blus, L. J. (1996). DDT, DDD, and DDE in Birds. In Environmental contaminants in wildlife : Beyer, W. (Ed.). Boca Raton: Lewis Publishers. Breman, J. G., Alilio, M. S., & Mills, A. ( 2004). Conquering the intolerable burden of malaria: what’s new, what’s needed: a summary. Am J Trop Med Hyg 71: 1-15. Brown, S., Honeyfield, D. C., & Vandenbyllaardt, D. L. (1998). Thiamine analysis in fish tissues. American Fisheries Society Sym posium 21. 21, 73-81. American Fisheries Society. Campbell, K. R. (2003). Ecotoxicology of crocodilians. Applied Herpetology 1: 45-163. Clarke, S. F. (1891). The habits and embryology of the American alligator. J Morph 5: 181-205. Colborn, T., Von Saal, F. S., & Soto, A. M. (1993). Developmental effects of endocrinedisrupting chemicals in wildlife and humans. Environ Health Perspect 101: 378384. Coulson, R. A. and Hernandez, T. (1983). Alligator metabolism: studies on chemical reactions in vivo. Oxford, UK, Pergamon.

PAGE 225

209 Deeming, D. C. & Ferguson, M. W. J. ( 1990). Morphometric analysis of embryonic development in Alligator mississippiensis Crocodylus johnstoni and Crocodylus porosus J Zool 221: 419-439. de Roode, D. F., Gustavsson, M. B., Rantalaine n, A. L., Klomp, A. V., Koeman, J. H., & Bosveldt, A. T. (2002a). Embryotoxic potenti al of persistent organic pollutants extracted from tissues of guillemots (Uria aalge) from the Baltic Sea and the Atlantic Ocean. Environ Toxicol Chem 21: 2401-11. de Roode, D. F., Vuorinen, P. J., & Bosveld, A. T. (2002b). Effects of furazolidone, PCB77, PCB126, Aroclor 1248, paraquat and p, p'-DDE on transketolase activity in embryonal chicken brain. Toxicology 173: 203-10. Donaldson, W. E. & Fites, B. L. (1970) Embryo mortality in quail induced by cyclopropene fatty acids: Re duction by maternal diets high in unsaturated fatty acids. J Nutr 100: 605-10. Ecobichon, D. J. (2001). Toxic effects of pesticides. In Caasarett and Doull's toxicology: the basic science of poisons : 763-810. Klaasen, C. D. (Ed.). New York, NY: McGraw-Hill. Elsey, R. M. & Wink, C. S. (1985). Femoral bone as a possible source of calcium for eggshell deposition in Alligator mississippiensis Anat Rec 211: A57. EPA. (2004). http://www.epa.gov/region4/wa ste/npl/nplfls/towercfl.htm, accessed October 8. US Environmental Pr otection Agency, Washington DC. Ertl, R. P., Bandiera, S. M., Buhler, D. R ., Stegeman, J. J., & Winston, G. W. (1999). Immunochemical analysis of liver microsomal cytochromes P450 of the American alligator, Alligator mississippiensis Toxicol Appl Pharmacol 157: 157-165. Ertl, R. P., Stegeman, J. J., & Winston, G. W. (1998). Induction time course of cytochromes P450 by phenobarbital and 3-met hylcholanthrene pretreatment in liver microsomes of Alligator mississippiensis Biochem Pharmacol 55: 1513-1521. Fairbrother, A., Ankley, G. T ., Birnbaum, L. S., Bradbury, S. P., Francis, B., Gray, E., Hinton, D., Johnson, L. L., Peterson, R. E., & Van Der Kraak, G. (1999). Reproductive and developmental toxicology of contaminants in oviparous animals. In Reproductive and developmental effect s of contaminants in oviparous vertebrates : 283-361. Di Giulio, R. T. & Tillit, D. E. (Ed.). Pensacola, FL : SETAC press. Ferguson, M. W. J. (1981). The valu e of the American alligator ( Alligator mississippiensis ) as a model for research in craniofacial development. J Craniofac Genet Dev Biol 1: 123-144.

PAGE 226

210 Ferguson, M. W. J. (1985). The reproductiv e biology and embryology of crocodilians. In Biology of the reptilia: Vol. 14, Development A : 329-491. Gans, C. (Ed.). New York, NY: John Wiley and Sons. Fitzsimons, J. D., Brown, S. B., Honeyfield, D. C., & Hnath, J. G. (1999). A Review of Early Mortality Syndrome (Ems) in Great Lakes Salmonids: Relationship With Thiamine Deficiency. Ambio 28: 9-15. Fitzsimons, J. D., Vandenbyllaardt, L., & Brow n, S. B. (2001). The use of thiamine and thiamine antagonists to inves tigate the etiology of early mortality syndrome in lake trout (Salvelinus namaycush). Aquat Toxicol 52: 229-39. Fry, D. M. (1995). Reproductive effects in bi rds exposed to pestic ides and industrial chemicals. Environ Health Perspect 103 Suppl 7: 165-71. Gadbury, G. L. & Schreuder, H. T. (2003). Ca use-effect relationshi ps in analytical surveys: an illustration of statistical issues. Environ Monit Assess 83: 205-227. Gilbertson, M., Kubiak, T., Ludwig, J., & F ox, G. (1991). Great La kes embryo mortality, edema, and deformities syndrome (GLEMEDS) in colonial fish-eating birds: similarity to chick-edema disease. J Toxicol Environ Health 33: 455-520. Giroux, D. (1998). Lake Apopka revisited: a co rrelational analysis of nesting anomalies and DDT contaminants. Master of Science Thesis. Gainesville, Florida. University of Florida. Gray, L. E. Jr., Kelce, W. R., Wiese, T., Tyl, R., Gaido, K., Cook, J., Klinefelder, G., Desaulniers, D., Wilson, E., Zacharewski, T ., Waller, C., Foster, P., Lasky, J., Reel, J. G. J., Laws, S., McLachlan, J., Bres lin, W., Cooper, R., DiGiulio, R., Johnson, R., Purdy, R., Mihaich, E., Safe, S., S onnenschein, C., Weshons, W., Miller, R., McMaster, S., & Colborn. T. (1997). Endocrine Screening Methods. Workshop report: detection of estrogenic and an drogenic hormonal and antihormonal activity for chemicals that act via receptor or steroidogenic enzyme mechanisms. Reprod Toxicol 11: 719-750. Gross, D. A. (1997). Thymus, spleen and bone marrow hypoplasia and decreased antibody responses in hatchli ng lake Apopka alligators. Ma ster of Science Thesis. Gainesville, Florida. University of Florida. Gross, T. S., Arnold, B. S., Sepulveda, M. S., & McDonald, K. (2003). Endocrine disrupting chemicals and e ndocrine active agents. In Handbook of Ecotoxicology : 1033–1098. Hoffman, D. J., Rattner, B. A., Burt on, G. A. Jr. & Cairns, J. Jr. (Ed.). Boca Raton, FL USA: Lewis Publishers. Gross, T. S., Guillette, L. J., Percival, H. F., Masson, G. R., Matter, J. M., & Woodward, A. R. (1994). Contaminant-induced reproducti ve anomalies in Florida alligators. J Comp Pathol 26: 1-8.

PAGE 227

211 Guillette, L. J., Brock, J. W., Rooney, A. A., & Woodward, A. R. (1999). Serum Concentrations of Various Environmental Contaminants and Their Relationship to Sex Steroid Concentrations and Phallus Size in Juvenile American Alligators. Arch Environ Contam Toxicol 36: 447-455. Guillette, L. J. Jr, Gross, T. S., Masson, G. R., Matter, J. M., Percival, H. F., & Woodward, A. R. (1994). Developmental a bnormalities of the gonad and abnormal sex hormone concentrations in juvenile al ligators from contaminated and control lakes in Florida. Environ Health Perspect 102: 680-8. Guillette, L. J. Jr, Woodward, A. R., Crain, D. A., Masson, G. R., Palmer, B. D., Cox, M. C., You-Xiang, Q., & Orlando, E. F. (1997) The reproductive cycle of the female American alligator (Alligator mississippiensis). Gen Comp Endocrinol 108: 87101. Hall, R. J., Kaiser, T. E., Robertson, W. B. Jr, & Patty, P. C. (1979). Organochlorine residues in eggs in the endangered Amer ican crocodile (Crocodylus acutus). Bull Environ Contam Toxicol 23: 87-90. Heinz, G. H., Percival, H. F., & Jennings, M. L. (1991). Contaminants in American Alligator Eggs From Lake Apopka, Lake Griffin, and Lake Okeechobee, Florida. Environ Monit Assess 16: 277-285. Henry, K. S., Kannan, K., Nagy, B. W., Kever n, N. R., Zabik, M. J., & Giesy, J. P. (1998). Concentrations and hazard assessmen t of organochlorine contaminants and mercury in smallmouth bass from a remote lake in the upper peninsula of Michigan. Arch Environ Contam Toxicol 34: 81-86. Hill, A. B. (1965). The environment a nd disease: association or causation? Proc R Soc Med 58: 295-300. Hoffman, D. J. (1990). Embryotoxicity and tera togenicity of environmental contaminants to bird eggs. Rev Environ Contam Toxicol 115: 40-89. Hoffman, D. J. & Gay, M. L. (1981). Embryot oxic effects of benzo[ a]pyrene, chrysene, and 7,12-dimethylbenz[a]anthracene in petr oleum hydrocarbon mixtures in mallard ducks. J Toxicol Environ Health 7: 775-787. Holstege, D. M., Scharberg, D. L., Tor, E. R ., Hart, L. C., & Galey, F. D. (1994). A rapid multiresidue screen for organophosphorus, organochlorine, and N-methyl carbamate insecticides in plant and animal tissues. J AOAC Int 77: 1263-74. Horwitz, W. (2000). Official methods of analys is of AOAC Interna tional. Gaithersburg, MD, AOAC International. James, R. C. Warren, D. A. Halmes, N. C., & Roberts, S. M. (2000). Risk assessment. In Principles of Toxicology : 437-478. Williams, P. L. James, R. C. & Roberts, S. M. (Ed.). New York: John Wiley & Sons.

PAGE 228

212 Lal, B. & Singh, T. P. (1987). Impact of pesticides on lipid metabolism in the freshwater catfish, Clarias batrachus during the vitellogenic phase of its annual reproductive cycle. Ecotoxicol Environ Saf 13: 13-23. Lance, V. (1986). Hormonal control of reproduction in crocodilians. In Wildlife Management: Crocodiles and Alligators : 409-415. Webb, G. J. W., Manolis, S. C. & Whitehead, P. J. (Ed.). Chipping No rton, NSW: Surrey Beatty and Sons. Lance, V., Joanene, T., & McNease, L. (1983) Selenium, vitamin E, and trace elements in the plasma of wild and farm reared alligators during the reproductive cycle. Can J Zool 61: 1744-1751. Lang, J. W. & Andrews, H. V. (1994). Temp erature-dependent sex determination in crocodilians. J Exp Zool 270: 28-44. Longnecker, M. P., Klebanoff, M. A., Brock, J. W., Zhou, H., Gray, K. A., Needham, L. L., & Wilcox, A. J. (2002). Maternal serum level of 1,1-dichloro-2,2-bis(pchlorophenyl)ethylene and ri sk of cryptorchidism, hypos padias, and polythelia among male offspring. Am J Epidemiol 155: 313-22. Mader, D. R. (1996). Reptile Medicine and Surgery Philadelphia, PA: W. B. Saunders. Marburger, J. E., Johnson, W. E., Douglas, D. R., & Gross, T. S. (1999). Pesticide contamination of fish and sediments in the Emeralda Marsh Conservation Area: relevance to fisheries establishmen t in flooded muck farms. Technical Memorandum No. 31 to the St. John’s Wate r Management District. Palatka, FL. Masson, G. R. (1995). Environmental influe nces on reproductive potential, clutch viability and embryonic mortality of the American alligator in Florida. PhD dissertation. Gainesville, FL, University of Florida. Matter, J. M., McMurry, C. S., Anthony, A. B., & Dickerson, R. L. (1998). Development and implementation of endocrine biomarkers of exposure and effects in American alligators (Alligator mississippiensis). Chemosphere 37: 1905-1914. McEvoy, T. G., Robinson, C. J., Ashworth, J. A., Rooke, J. A., & Sinclair, K. D. (2001). Feed and forage toxicants affecting em bryo survival and fetal development. Theriogenology 55: 113-129. Metcalfe, C. D. (1998). Toxicopathic responses to organic compounds. In Fish diseases and disorders : 133-161. Leatherland, J. F. & Woo, P. T. K. (Ed.). Cambridge, MA: CABI Publishing. Millstein, S. R. (1995). Nutrient therapy in the American allig ator and its association with egg yolk long-chain fatty acids and egg fertility. Master of Science Thesis Gainesville, FL. University of Florida.

PAGE 229

213 Noble, R. C., Mccartney, R., & Ferguson, M. W. J. (1993). Lipid and fatty-acid compositional differences between eggs of wild and captive-breeding alligators ( Alligatormississippiensis )–an association with reduced hatchability. J Zool 230: 639-649. Rao, C. R. (1964). The use and interpretation of principal component analysis in applied research. Sankhya A 26: 329-358. Rattner, B. A. & Heath, A. G. (2003). Envi ronmental factors affecting contaminant toxicity in aquatic and te rrestrial vertebrates. In Handbook of Ecotoxicology : 679699. Hoffman, D. J., Rattner, B. A., Burton, G. A. Jr. & Cairns, J. Jr. (Ed.). Boca Raton, FL USA: Lewis Publishers. Rauschenberger, R. H., Wiebe, J. J., Buckland, J. E., Smith, J. T., Seplveda, M. S., & Gross, T. S. (2004). Achieving environmen tally relevant organochlorine pesticide concentrations in eggs through maternal exposure in Alligator mississippiensis Marine Environ Res 58: 851-856. Reese, A. M. (1912). The embryol ogy of the Florida alligator ( A. mississippiensis ) Proc 7th Int Zool Congr 1907: 535-537. Rice, A. K. (2004). Diet a nd condition of alligators ( Alligator mississippiensis ) in three central Florida lakes. Master of Science Thesis. Gainesville, FL. University of Florida. Rice, K. G. (1996). Dynamics of exploitati on on the American alligator: environmental contaminants and harvest. PhD Dissertation. Ga inesville, FL. University of Florida. Robson, W. A., Arscott, G. H., & Tinsley, I. J. (1976). Effect of DDE, DDT and calcium on the performance of adult Japanese quail ( Coturnix coturnix japonica ). Poult Sci 55: 2222-7. Roecklein, B., Levin, S. W., ComLy, M., & Mukh erjee, A. B. (1985). Intrauterine growth retardation induced by thiamine deficiency and pyrithiamine during pregnancy in the rat. Am J Obstet Gynecol 151: 455-60. Rogers, J. M. & Kavlock, R. J. (2001). Developmental toxicology. In Casarett and Doull's Toxicology: The Basic Science of Poisons : 351-386. Klaasen, C. D. (Ed.). New York: McGraw-Hill. Rotstein, D. S. (2000). An evaluation of deve lopmental mortality and fertilization failure in alligators using microsatelli te analyses. Master of Science Thesis. Gainesville, FL. University of Florida. Rotstein, D. S., Schoeb, T. R., Davis, L. M., Glenn, T. C., Arnold, B. S., & Gross, T. S. (2002). Detection by microsatellite analys is of early embryonic mortality in an alligator population in Florida. J Wildl Dis 38: 160-165.

PAGE 230

214 Russell R. W, Gobas, F. P. C., & Haffner, G. D. (1999). Maternal transfer and in ovo exposure of organochlorines in ovipa rous organisms: A model and field verification. Environ Sci Technol 33: 416-420. Ryberg, W. A., Fitzgerald, L. A., Honeycutt, R. L., & Cathey, J. C. (2002). Genetic relationships of American alligator p opulations distributed across different ecological and geographic scales. J Exp Zool 294: 325-333. SAS Institute Inc. (2002). SAS version 9.0. Cary, NC. Schenck, F. J., Wagner, R., Hennessy, M. K., & Okrasinski, J. L. Jr. (1994). Screening procedure for organochlorine and orga nophosphorus pesticide residues in eggs using a solid-phase extraction cleanup and gas chromatographic detection. J AOAC Int 77: 1036-40. Schmidt, R. R. & Johnson, E. M. (1997). Principles of teratology. In Handbook of Developmental Toxicology : 3-12. Hood, R. D. (Ed.). Boca Raton, Florida: CRC Press. Schoeb, T. R., Heaton-Jones, T. G., Clemm ons, R. M., Carbonnea u, D. A., Woodward, A. R., Shelton, D., & Poppenga, R. H. (2002). Clinical and necropsy findings associated with increased mortality am ong American alligators of Lake Griffin, Florida. J Wildl Dis 38: 320-337. Seplveda, M. S., Wiebe, J. J., Harvey, A ., Basto, J., Ruessler, D. S., Roldan, E., & Gross, T. S. (2001). An evaluation of environmental contaminants and developmental toxicity for the Ameri can alligator in central Florida. 21st Society of Toxicology Annual Meeting Abstract B ook. March 15-19. San Francisco, CA. pp. 151. Seplveda, M. S., Wiebe, J. J., Honeyfield, D. C., Rauschenberger, R. H., Hinterkopf, J. P., Johnson, W. E., & Gross, T. S. (2004). Organochlorine pesticides and thiamine in eggs of largemouth bass and American alligators and their relationship with early life-stage mortality. J Wildl Dis In press Skaare, J. U., Ingebrigsten, K., Aulie, A., & Kanui, T. I. (1991). Organochlorines in crocodile ( Crocodylus niloticus) Bull Environ Contam Toxicol 47: 126-130. Smith, A. G. (1991). Chlorinate d hydrocarbon insecticides. In Handbook of pesticide toxicology: Vol. 2: 731–915. Hayes, W. J., Jr. & Laws, E. R., Jr. (Ed). San Diego, CA: Academic Press. Snodgrass, S. R. (1992). Vitamin neurotoxicity. Mol Neurobiol 6: 41-73. Spallholz, J. E. & Hoffman, D. J. (2002). Sele nium toxicity: cause a nd effects in aquatic birds. Aquat Toxicol 57: 27-37.

PAGE 231

215 Stenner, R. D., Merdink, J. L., Stevens, D. K., Springer, D. L., & Bull, R. J. (1997). Enterohepatic recirculation of trichloroeth anol glucuronide as a significant source of trichloroacetic acid. Metabolit es of trichloroethylene. Drug Metab Dispos 25 : 529-535. Summer, C. L., Giesy, J. P., Bursian, S. J., Re nder, J. A., Kubiak, T. J., Jones, P. D., Verbrugge, D. A., & Aulerich, R. J. (1996). Effects induced by feeding organochlorine-contaminated carp from Saginaw Bay, Lake Huron, to laying White Leghorn hens. II. Embryotoxic and teratogenic effects. J Toxicol Environ Health 49: 409-38. Systat, Inc. (1999). SigmaScan Pro. Point Richmond, CA. ter Braak, C. J. F. (1986). Canonical co rrespondence analysis: a new eigenvector technique for multivariate direct gradient analysis. Ecology 67: 1167-1179. ter Braak, C. J. F. (1994). Canonical commun ity ordination. Part I: Basic theory and linear methods. Ecoscience 1: 127-140. ter Braak, C. J. F. (1995). Ordination. In Data analysis in community and landscape ecology : 91-173. Jongman, R. H. G., ter Braak, C. J. F. & Van Tongeren, O. F. R. (Ed.). Cambridge, UK: Cambridge University Press. ter Braak, C. J. F. & Smilauer, P. (2002) CANOCO reference manual and Canodraw for Windows Users' guide: software for canonical community ordination (version 4.52). Ithaca, NY, USA, Microcomputer Power. Voeltzkow, A. (1892). On the oviposition and embryonic development of the crocodile. Ann Mag Nat Hist (Ser 6) 9: 66-72. Webb, G. J. W., Buckworkth, R., Sack, G. C., & Manolis, S. C. (1983). An interim method for estimating the age of Crocodylus porosus embryos. Aust Wildl Res 10: 563-570. Weed, D. L. (2002). Environmental epidemio logy: basics and proof of cause-effect. Toxicology 181: 399-403. Wiebe, J. J., Rotstein, D., Percival, H. F., Woodward, A., Schoeb, T., & Gross, T. S. (2001). Evidence of developmen tal toxicity in th e American alligator from central Florida lakes. 20th Annual meeting of Society for Environmental Toxicology and Chemistry Abstract Book. November 15-19. San Francisco, CA. pp. 231. Wiebe, J. J., Sepulveda, M., Woodward, A., Pe rcival, H. F., Abecrombie, A., Wilkinson, P., Harvey, A., Basto, J., Ruessler, D. S ., Roldan, E., & Gross, T. S. (2001). An evaluation of environmental contaminants and decreased egg viability in the American Alligator ( Alligator mississippiensis ). 2001 Society of Toxicology Annual Meeting, Nov. 13-17. Baltimore, MD. pp. 160.

PAGE 232

216 Willingham, E. (2004). Endocrine-disrupti ng compounds and mixtures: unexpected doseresponse. Arch Environ Contam Toxicol 46: 265-9. Wilson, H. R. (1997). Effects of ma ternal nutrition on hatchability. Poult Sci 76: 134143. Wink, C. S. & Elsey, R. M. (1986). Changes in femoral morphology during egg-laying in Alligator mississippiensis J Morphol 189: 183-188. Woodward, A. R., Jennings, M. L., & Percival H. F. (1989). Egg collecting and hatch rates of American alligat or eggs in Florida. Wildl Soc Bull 17: 124-130. Woodward, A. R., Moore, C. T., & Delany, M. F. (1992). Experimental alligator harvest. Florida Fish and Wildlife Conservati on Commission. Gainesville, FL. pp. 170. Woodward, A. R., Percival, H. F., Jennings, M. L., & Moore, C. T. (1993). Low clutch viability of American alligators on Lake Apopka. Florida-Scientist 56 (1) 52-63. Woosley, R. L. (1996). Cardiac Actions of Antihistamines. Annu Rev PharmacolToxicol 36: 233-252. Wu, T. H., Rainwater, T. R., Platt, S. G ., McMurry, S. T., & Anderson, T. A. (2000a). Organochlorine contaminants in Morelet's crocodile (Crocodylus moreletii) eggs from Belize. Chemosphere 40: 671-8. Wu, T. H., Rainwater, T. R., Platt, S. G ., McMurry, S. T., & Anderson, T. A. (2000b). DDE in Eggs of Two Crocodile Species From Belize. J A g ric Food Chem 48: 6416-6420. Yagi, M., Kamohara, K., & Itowaka, Y. (1979). Thiamine deficiency induced by polychlorinated biphenyls (PCB) and di chlorodiphenyltrichloroethane (DDT) administration to rats. J Environ Pathol Toxicol 2: 1119-1125.

PAGE 233

217 BIOGRAPHICAL SKETCH Richard Heath Rauschenberger was born in North Little Rock, Arkansas in 1970, and is the son of Richard Edward and Mary Elizabeth Rauschenberger. Heath graduated from Greenbrier High School in Greenbrier, Arkansas in 1988; and received a BS in wildlife management in 1993, from Arkansas St ate University in Jonesboro, Arkansas. After gaining professional work experience as a pest contro l technician and later as a private lands wildlife biologist Heath returned to Arkansas State in 1999. He entered graduate school and received his MS in biology in 2001. After ear ning his MS degree, Heath immediately entered the University of Florida College of Ve terinary Medicine’s doctoral program (under the mentorship of Dr. Timothy S. Gross) and majored in physiological sciences, with a concentration in interdisciplinary toxicology. Heath has held diverse positions such as lifeguard, vete rinary technician, grocery store clerk, pest control technician, wildlife bi ologist, and currently, research graduate assistant, which have aided in rounding out his professional experience. Heat h is married, has two sons, and enjoys spending time with them and the re st of his family. He also enjoys outdoor activities and is an active Christian a nd member of a local church.


Permanent Link: http://ufdc.ufl.edu/UFE0008223/00001

Material Information

Title: Developmental Mortality in American Alligators (Alligator mississippiensis) Exposed to Organochlorine Pesticides
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: UFE0008223:00001

Permanent Link: http://ufdc.ufl.edu/UFE0008223/00001

Material Information

Title: Developmental Mortality in American Alligators (Alligator mississippiensis) Exposed to Organochlorine Pesticides
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: UFE0008223:00001


This item has the following downloads:


Full Text










DEVELOPMENTAL MORTALITY IN AMERICAN ALLIGATORS (Alligator
mississippiensis) EXPOSED TO ORGANOCHLORINE PESTICIDES














By

RICHARD HEATH RAUSCHENBERGER


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2004

































Copyright 2004

by

Richard Heath Rauschenberger

































To Jesus, my personal Lord and Savior. John 14:6. "Jesus said to him, I am the
way, the truth, and the life: no man comes to the Father, except by me." Ephesians 2:8-9.
"For by grace you have been saved through faith, and that not of yourselves; it is the gift
of God, Not of works, lest anyone should boast."















ACKNOWLEDGMENTS

I thank my wonderful wife, Alison; and my two sons, Heath and Ben. Their

steadfast love, support, and sacrifices allowed me to successfully complete the arduous

task of earning a Ph.D. I thank my parents, Richard E. and Mary Elizabeth

Rauschenberger, for their ever-present love, faith, and encouragement. I thank my

mother-in-law, Sandra Pillow, for baby-sitting Heath and Ben while Alison and I were

away at work and for her support and encouragement. I thank my parents-in-law,

Tommy and Debbie Kirk, for their love and ever-vigilant prayers. I thank my brother-in-

law, Matt Kirk; and sister-in-law, Kristin Dessert; for their support and encouragement. I

thank my late grandfather, M. E. "Pappy" Walls, for showing me the outdoors; my high

school biology teacher, Joe David White, for making me a better student; and my high

school football coaches, Randy Tapley and Jim Massarelli, for strengthening my work

ethic and ability to deal with adversity. I am forever grateful to Tim Gross for taking me

in as a student. I thank him and his wife, Denise, for the kindness, generosity, and

encouragement they've shown to my family and me. I thank my committee members

(Marisol Sepulveda; Bill Castleman; Richard Miles, Jr.; Franklin Percival; and Steve

Roberts) for their support, friendship, and significant contributions to my development as

a research scientist. I also want to thank Kent Vliet for sharing his vast literature and

knowledge of alligator reproduction. I especially thank Jon Wiebe and Janet Buckland

for their friendship and hard work. I am privileged to have had the opportunity to work

with the staff and students of our laboratory. I thank Wendy Mathis, Travis Smith, Jesse









Grosso, Eileen Monck, James Basto, Shane Ruessler, Carla Wieser, Alfred Harvey,

Adriano Fazio, Nikki Kernaghen, Jennifer Muller, and Jessica Noggle for their help and

friendship. I thank Ken Portier, Gary Stevens, Ramon Littell, Ron Marks, Jon Maul, and

Linda Garzarella for providing statistical advice and assistance. I thank the National

Institutes of Environmental Health Sciences Superfund Basic Research Program (grant

number P42ES-07375) and the Lake County Water Authority for providing financial

support for my education and research project. My name is listed alone as the author of

this dissertation, but this work was the product of a team that I am honored to have been a

part of and will always remember.














TABLE OF CONTENTS
Page
A C K N O W L E D G M E N T S ................................................................................................. iv

LIST OF TABLES ..................................................... ix

LIST OF FIGURES ............................. ............ .................................... xii

CHAPTER

1 IN T R O D U C T IO N .................................................................. .. ... .... ............... 1

H habitat D egradation in the Ocklaw aha Basin.......................................... ...............1...
A lligators as Potential O CP R eceptors.................................................... ...............2...
Developmental Biology of the American Alligator................................................3...
Post-O vipositional D evelopm ent...................................................... ...............6...
Ferguson's Post-Ovipositional Staging Scheme .............................................8...
Organochlorine Pesticide Toxicity in Vertebrates ................................................ 15
Classification, Mode of Action, and Pathology............................................. 15
Exposure and Effects of OCPs in Crocodilians............................................. 17
Reproductive Problems in Florida Alligators ................................................ 18
S p ecific A im s.............................................................................................................. 2 0

2 EGG AND EMBRYO QUALITY OF ALLIGATORS FROM REFERENCE AND
ORGANOCHLORINE CONTAMINTED HABITATS ................. ..................... 23

M materials and M ethods ............................................ ........................... ................ 24
Egg C collections and Incubation...................................................... ................ 24
A analysis of O CPs in Y olk ........................................................ 26
G C/M S A analysis ... .. ................................. ......................................... 27
D ata A n aly sis....................................................................................................... 2 8
R e su lts.......................................................................................................... ........ 3 0
Inter-Site Comparisons of Clutch Characteristics ..........................................30
Organochlorine Pesticides Burdens and Clutch Characteristics ......................31
Clutch Survival and OCP Burdens in Egg Yolks...........................................32
Average Egg Mass, Clutch Size and OCP Burdens ......................................33
D isc u ssio n .. ............... ........ ... .... ........................................................... 3 4
Inter-Site Comparisons of Clutch Characteristics .........................................34
Clutch Survival Parameters and OCP Burdens ..............................................36
Egg and Clutch Size and OCP Burdens ......................................... ................ 38









3 MATERNAL TRANSFER OF ORGANOCHLORINE PESTICIDES.................. 54

M materials an d M eth o d s ...............................................................................................5 5
Site descriptions ..................................................................... ............... 55
A nim al C collections ....................................................... .... ......... ..... .. ........ ..... 56
Analysis of OCPs in Maternal Tissues and Yolk...........................................57
G C/M S A analysis ... .. ................................. ......................................... 59
D ata A n aly sis....................................................................................................... 6 0
R e su lts........................................... ... ............................................................. ........ 6 1
Female Morphological and Reproductive Characteristics ................................61
O C P concentrations in Y olk ........................................................... ................ 62
O CP concentrations in m aternal tissues ......................................... ................ 62
Relationships between Maternal Tissue and Yolk Burdens...............................63
Relationships between Maternal Mass and OCP concentrations in Eggs and
T issue e s......................................................................................................... . 6 4
D isc u ssio n ................................................................................................................... 6 5
Evaluation of Predictive M odels .................................................... ................ 67
Relationships between Maternal Mass and OCP concentrations in Eggs and
Tissues.................. ............ . .... .. ............... 69
Maternal body burdens: Toxicological Implications.....................................69

4 MATERNAL FACTORS ASSOCIATED WITH DEVELOPMENTAL
MORTALITY IN THE AMERICAN ALLIGATOR............................................80

M materials and M ethods .. ..................................................................... ................ 81
Site D escriptions......................................................................................... 82
A nim al C collections ....................................................... .... ......... ..... .. ........ ..... 82
Analysis of OCPs in Maternal Tissues and Yolk...........................................83
G C/M S A analysis ... .. ................................. ............................. ............ 86
D ata A n aly sis....................................................................................................... 8 7
R e su lts....................................................................................................... ....... .. 8 8
D isc u ssio n ............................................................................................................... ... 8 9

5 MORPHOLOGY AND HISTOPATHOLOGY OF AMERICAN ALLIGATOR
(Alligator mississippiensis) EMBRYOS FROM REFERENCE AND OCP-
CON TAM IN A TED H ABITA TS .......................................................... ................ 99

M materials and M ethods ................... .............................................................. 102
Site D escriptions... ................................................................... ........... 102
E gg C collections ........................................................................................... 103
Embryo Sam pling and M easurem ent ....... .......... ....................................... 103
H istopathology ..................................................................................... 105
A analysis of O CPs in Y olk ........................................................ 106
G C /M S A naly sis ... ................................................................................. 108
R esults.................................... .................................... ................... 109
Inter-Site C lutch C om prisons ..................................................... ................ 109
Intra-Site Live Embryo/Dead Embryo Morphological Comparisons .............110









Inter-Site Comparisons of Morphology of Live Embryos .............. ................111
Live Embryo Morphology and Embryo Survival Relationships.................... 112
Live Embryo Morphology and Egg Yolk OCP Burdens................................ 113
Embryo Morphological Age, Derived Morphometric Variables and Egg Yolk
O C P B u rd e n s .............. ..... ...... ...................................................................... 1 1 5
Histopathology of Live and Dead Embryos ......................... .................. 116
D isc u ssio n ............................................................................................................. .. 1 1 7

6 NUTRIENT AND CHLORINATED HYDROCARBON CONCENTRATIONS IN
AMERICAN ALLIGATOR EGGS AND ASSOCIATIONS WITH DECREASED
C L U T C H V IA B IL IT Y ............................................................................................. 143

M materials and M ethods ........................................ .......................... ............... 145
Egg Collections and Incubation...... ........ ......................145
F ield stu dies ..................................................................... ............ 14 6
Laboratory experiments............................................ 147
Analysis of Chlorinated Hydrocarbons in Yolk..................... .................. 149
G C /M S A analysis ... .................................................................. ............... 150
Nutrient Analysis ........................................................................ 151
D ata A n aly sis..................................................................................................... 152
R e su lts..................................................................................................... ......... 15 4
F ield S tu d y ..................................... .................................................................... 1 5 4
Case-control cohort study...... ........... ........ ..................... 154
E expanded fi eld study ....................................................... ............... 157
Laboratory Experim ents ........................................................ 160
D isc u ssio n ............................................................................................................... .. 1 6 2

7 REPRODUCTIVE EFFECTS OF ORGANOCHLORINE PESTICIDE EXPOSURE
IN A CAPTIVE POPULATION OF AMERICAN ALLIGATORS (Alligator
m ississip p ien sis) ....................................................................................................... 1 8 2

M materials and M ethods ................... .............................................................. 182
R e su lts..................................................................................................... ......... 18 5
D isc u ssio n ............................................................................................................. .. 1 8 6

8 CONCLUSIONS ..................................................... ...... .... ............... 196

In tro d u ctio n .............................................................................................................. 19 6
Sum m ary of Study's Findings ............................................................. 197
Future Considerations and Global Implications .............................................204

LIST OF REFERENCES .......................................................... ............ 208

BIOGRAPH ICAL SKETCH .................. .............................................................. 217















LIST OF TABLES


Table page

2-1. Reproductive, morphometric, and contaminant parameters measured on clutches of
alligator eggs collected during summer 2000, 2001, and 2002...............................41

2-2. Explanatory variables included in RDA with forward selection of four best
v a riab le s ............................................................................................................. .. 4 2

2-3. Summary of clutch parameters and site comparisons for clutches of American
alligator eggs collected during 2000-2002. ............... .................................... 43

2-4. Organochlorine pesticide burdens and clutch parameters and site comparisons for
clutches of American alligator eggs collected during 2000-2002.........................44

2-5. Results of RDA evaluating associations between clutch survival parameters and
O C P v ariab les........................................................................................................... 4 7

2-6. Results of RDA evaluating associations between egg and clutch size parameters and
O C P v ariab les........................................................................................................... 4 8

3-1. Morphological and reproductive characteristics of adult female alligators collected
during June 2001 and 2002 from Lakes Apopka, Griffin, and Lochloosa in central
F lo rid a .................................................................. ............................................... ... 7 3

3-2. Pesticide concentrations (ng/g wet wt.) in tissues and yolks of adult female alligators
collected during June 2001 and 2002 from Lakes Apopka, Griffin, and Lochloosa
in cen trial F lo rid a ..................................................................................................... 7 4

3-3. Regression equations for predicting organochlorine pesticide (OCP) concentrations
in m atern al tissue es .................................................................................................... 7 8

4-1. Reproductive, morphometric, and contaminant parameters measured on adult female
alligators collected during June 1999, 2000, 2001, and 2002..............................93

4-2. Explanatory variables included in RDA with forward selection of four best
v a riab le s ............................................................................................................. .. 9 4

4-3. Reproductive, morphometric, and contaminant summary statistics of adult female
alligators collected during June of 1999-2002. ................................... ................ 95









4-4. Results of redundancy analysis with automatic selection of four best maternal
factors associated with variation in reproductive efficiency...............................97

4-5. Results of redundancy analysis with automatic selection of four best maternal
factors associated with variation in clutch size characteristics ..............................97

5-1. Summary statistics for parameters measured on American alligator clutches
collected during June 2001 and 2002. ...... ... ........................................... 122

5-2. Comparisons of egg and embryo morphometrics of live and dead embryos collected
during June-August of 2001 and 2002. .......... ... ......................................... 124

5-3. Morphometric comparisons of live embryos collected during June-August 2001 and
2 0 0 2 ...................................................................................................... .......... 12 8

5-4. Explanatory variables included in partial redundancy analysis evaluating
relationship between organochlorine pesticide burdens in eggs and embryo
m orp h om etrics........................................................................................................ 13 1

5-6. Best five organochlorine pesticide (OCP) variables that account for embryo
morphological age and derived morphological parameters .............................133

6-1. Classification matrix for clutches collected during 2002 ................................165

6-2. Reproductive, morphometric, and contaminant parameters measured on clutches of
alligator eggs collected during summer 2000, 2001, and 2002........................... 165

6-3. Explanatory variables included in RDA with forward selection of four best variables
for case-control cohort and expanded field studies........................................166

6-4. Summary of clutch parameters on clutches collected during 2002 .....................168

6-5. Evaluation of the relationship between concentrations of nutrients, PAHs, and PCBs
in eggs and clutch survival parameters via RDA analysis. ...............................169

6-6. Evaluation of clutch size parameters and explanatory factors for clutches collected
d u rin g 2 0 0 2 ........................................................................................................... 16 9

6-7. Evaluation of the relationship between nutrient concentrations and explanatory
variables for clutches collected during 2002....... ... ..................................... 169

6-8. Summary and comparison of parameters measured on clutches collected during
2 0 0 0 -2 0 0 2 .............................................................................................................. 17 0

6-9. Evaluation of the relationships between clutch survival parameters and explanatory
variables via RDA using age as the covariate. ...... ... ................................... 171

6-10. Evaluation of the relationships between clutch size parameters and explanatory
variables via RDA using age as the covariate. ...... ... ................................... 171









6-11. Evaluation of the relationships between thiamine concentrations and explanatory
variables via RDA using age as the covariate. ...... ... ................ ................... 172

6-12. Site comparisons of parameters measured on clutches collected during 2003...... 173

7-1. Summary statistics and comparisons of clutch parameters among treated and control
groups for years 2002-2004................................... ...................... ............... 192

7-2. Organochlorine concentrations and blood chemistry values of captive adult female
alligators sacrificed during 2002. ...... ....... ........ ...................... 194

7-3. Explanatory parameters and clutch survival parameters with () indicating nature of
association and value equal to concordance percentage. .................................195















LIST OF FIGURES


Figure page

1-1. M ap of O cklaw aha B asin. ................. ............................................................. 22

2-1. Biplot of clutch survival parameters (solid lines) and organochlorine pesticide
variables (dashed lines) for clutches of alligator eggs collected from Lake
Lochloosa during sum m er 2001-2002................................................. ................ 49

2-2. Biplot of clutch survival parameters (solid lines) and organochlorine pesticide
variables (dashed lines) for clutches of alligator eggs collected from Lake Griffin
during sum m er 2000-2002 ........................................ ....................... ................ 50

2-3. Biplot of clutch survival parameters (solid lines) and organochlorine pesticide
variables (dashed lines) for clutches of alligator eggs collected from Lake Apopka
during sum m er 2000-2002 ........................................ ....................... ................ 5 1

2-4. Biplot of clutch survival parameters (solid lines) and organochlorine pesticide
variables (dashed lines) for clutches of alligator eggs collected from Emeralda
M arsh during sum m er 2000-2002 ...................................................... ................ 52

2-5. Biplot of egg and clutch size parameters (solid lines) and organochlorine pesticide
variables (dashed lines) for clutches of alligator eggs collected from Lake
Lochloosa during sum m er 2001 and 2002 ......................................... ................ 53

3-1. Linear regressions of total organochlorine pesticide (OCP) concentrations in
maternal tissues against total OCP concentrations in egg yolks. ..........................79

4-1. Biplot of maternal factors (dashed lines) and clutch survival parameters (solid lines)
of American alligators collected during June 1999-2002. ...............................98

5-1. Representative developmental stages of embryos that were collected from Lakes
Lochloosa (reference site), Apopka, and Griffin, and Emeralda Marsh during 2001-
2 0 0 2 .................................................................................................................... 1 3 4

5-2. Ordination biplot of embryo morphometric parameters (solid lines) and
organochlorine pesticide (OCP) variables (dashed lines) for embryos collected at
chronological age D ay 14..................................... ....................... ............... 135









5-3. Ordination biplot of embryo morphometric parameters (solid lines) and
organochlorine pesticide (OCP) variables (dashed lines) for embryos collected at
chronological age D ay 25 ........ ............................... ..................... 136

5-4. Ordination biplot of embryo morphometric parameters (solid lines) and
organochlorine pesticide (OCP) variables (dashed lines) for embryos collected at
chronological age D ay 33 .................. ......................................................... 137

5-5. Ordination biplot of embryo morphometric parameters (solid lines) and
organochlorine pesticide (OCP) variables (dashed lines) for embryos collected at
chronological age D ay 43 .................. ......................................................... 138

5-6. Ordination biplot of derived embryo morphometric parameters (solid lines) and
organochlorine pesticide (OCP) variables (dashed lines) for embryos collected at
chronological age D ay 14 .................................... ....................... ............... 139

5-7. Ordination biplot of derived embryo morphometric parameters (solid lines) and
organochlorine pesticide (OCP) variables (dashed lines) for embryos collected at
chronological age D ay 25 .................................... ....................... ............... 140

5-8. Ordination biplot of derived embryo morphometric parameters (solid lines) and
organochlorine pesticide (OCP) variables (dashed lines) for embryos collected at
chronological age D ay 33 .................. ......................................................... 141

5-9. Ordination biplot of derived embryo morphometric parameters (solid lines) and
organochlorine pesticide (OCP) variables (dashed lines) embryos collected at
chronological age D ay 43 .................. ......................................................... 142

6-1. Biplot of clutch survival parameters and explanatory factors for clutches collected
d u rin g 2 0 0 2 ........................................................................................................... 17 5

6-2. Biplot of clutch size parameters and explanatory variables for clutches collected
d u rin g 2 0 0 2 ........................................................................................................... 17 6

6-3. Biplot of nutrient concentrations in eggs (solid arrows) and explanatory variables
(d ash ed arrow s). ..................................................................................................... 17 7

6-4. Relationships between embryo age and thiamine phosphorylation in egg yolk for 29
clutches collected during 2002 from Lakes Lochloosa, Griffin, Apopka, and
Em eralda M arsh. .......... .. .... ............................. .............. ............... 178

6-5. Biplot of clutch survival parameters and explanatory variables for clutches collected
during 2000-2002.. ............... .......... .. ..... ............................... 179

6-6. Biplot of clutch size variables (solid lines) and explanatory variables (dashed lines)
for clutches collected during 2000-2002 ....... ... ....................................... 180









6-7. Biplot of thiamine egg yolk concentrations (solid lines) and explanatory variables
(dashed lines) measured on clutches collected during 2000-2003 .......................181















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

DEVELOPMENTAL MORTALITY IN AMERICAN ALLIGATORS (Alligator
mississippiensis) EXPOSED TO ORGANOCHLORINE PESTICIDES

By

Richard Heath Rauschenberger

December 2004

Chair: Timothy S. Gross
Major Department: Veterinary Medicine-Physiological Sciences

Since the early 1900s, the lakes of the Ocklawaha Basin in central Florida have

experienced ecological degradation due to anthropogenic development. One species

affected by degradation has been the American alligator (Alligator mississippiensis).

Decreased clutch viability (proportion of eggs in a nest that yield a live hatchling) was

observed in the years after a chemical spill in which large amounts of sulfuric acid and

dicofol, an organochlorine pesticide (OCP), flowed into Lake Apopka. Lake Apopka and

other lakes in the Ocklawaha basin have also been contaminated by urban sewage and

agricultural chemicals, with agricultural chemicals entering the lakes via rainfall run-off

or back-pumping of water from agricultural lands). Decreased hatch rates are a problem

at Lake Apopka, as well as at other OCP-contaminated sites in Florida. The purpose of

my study was to determine the causes for decreased clutch viability, and to test the

hypothesis that maternal exposure to OCPs is associated with embryonic mortality in

alligators.









Field studies involved collecting and artificially incubating eggs from reference

sites (Lake Lochloosa) and from OCP-contaminated sites (Lakes Apopka, Griffin, and

Emeralda Marsh Restoration Area) to evaluate clutch viability as a function of egg and

maternal OCP concentrations. Nutrient content of eggs and histopathology and

morphometrics of embryos were also evaluated to identify potential factors associated

with embryo mortality. In addition, a novel laboratory experiment exposed a captive

population of adult alligators to an OCP mixture, and compared OCP burdens in eggs and

clutch viability with a captive control group.

Results of field studies suggested that OCP concentrations (ng total OCP/g egg

yolk, Mean SE) in reference site clutches (n = 19; 102 16) were significantly (a =

0.05) lower than those of Apopka (n = 23; 7,582 2,008), Griffin (n = 42; 1,169 423),

and Emeralda Marsh (n = 31; 15,480 + 2,265). Clutches from reference sites also had

significantly higher clutch viability (70 4%) than those of Apopka (51 6%), Griffin

(44 5%), and Emeralda Marsh (48 6%). Furthermore, decreased thiamine

concentrations in eggs may play a role in decreased clutch viability in wild clutches.

Results of the captive study suggested that treated females produced eggs containing

higher OCP concentrations (n = 7; 13,300 2,666) than controls (n = 9; 50 4). Eggs of

treated females also exhibited decreased viability (9 6%) as compared to controls (44 +

11%). These field and laboratory studies support the hypothesis that maternal exposure

to OCPs is associated with decreased clutch viability in American alligators, and that

thiamine deficiency may also be a contributing factor in reduced clutch viability.















CHAPTER 1
INTRODUCTION

Habitat Degradation in the Ocklawaha Basin

In central Florida, several lakes within the Ocklawaha River Basin (Fig. 1-1) have

experienced severe degradation of habitat quality since the early 1900s, as agricultural

and urban development progressed. Indeed, Lake Apopka (headwaters of the

Ocklawaha) was once renowned for its clear water and its excellent largemouth bass

fishing. More recently, Lake Apopka has gained world-wide notoriety as the "poster

child" for polluted lakes, because of highly publicized problems associated with

environmental contamination. Initial degradation of Lake Apopka and other lakes within

the Ocklawaha Basin occurred as the result of the loss of thousands of hectares of marsh

habitat through the agricultural practice known as muck farming (which involves

installing levees around an area of marsh, so the marsh can be drained; allowing the

fertile peat to be farmed). This farming practice began in the 1940s and continued into

the 1980s (Benton et al., 1991). In addition to sewer discharge from the city of Winter

Garden entering the Lake Apopka, organochlorine pesticides (OCPs) were heavily and

widely used to control crop-destroying insect pests.

Since the 1980s, use of most OCPs has been discontinued since they were

determined to be persistent environmental contaminants that resist metabolic degradation

and bioaccumulate in animal tissues, where they are potentially carcinogenic,

immunotoxic, endocrine disrupting, and developmentally toxic (Fairbrother et al., 1999;









Ecobichon, 2001). Altered function of the reproductive and endocrine systems of

wildlife and human populations have been suggested to occur after exposure to a variety

of OCPs and OCP metabolites such as dichlorodiphenyltrichloroethane (DDT),

dichlorodiphenyltrichloroethylene (DDE), methoxychlor, dicofol, chlordane, dieldrin,

and toxaphene (Colborn et al., 1993; Longnecker et al., 2002).

Further degradation and OCP contamination occurred in Lake Apopka in 1980. A

chemical spill occurred when a highly acidic wastewater pond at the Tower Chemical

Company's main facility overflowed into the Gourd Neck area of Lake Apopka (Fig. 1-

1). Because of the large volume and acidity (sulfuric acid), and the high levels of DDT,

dicofol, and related OCP contaminants that entered the relatively narrow area of the lake,

aquatic vegetation and animals were severely affected. In 1983, the area was placed on

the US Environmental Protection Agency's (EPA) National Priority Site List and became

a part of the Superfund program; which was created by the Comprehensive

Environmental Response, Compensation, and Liability Act (CERCLA), later amended by

the Superfund Amendments and Reauthorization Act (SARA). The CERCLA and SARA

provide authority for the government to respond to the release and/or threat of release of

hazardous wastes, and allow cleanup and enforcement actions. Lake Apopka is still

listed and groundwater toxicity testing is ongoing (EPA, 2004).

Alligators as Potential OCP Receptors

The American alligator is an important member of Florida wetlands and plays

important roles in the ecology, esthetics, and economy of Florida. Therefore, identifying

physiological and ecological characteristics related to potential susceptibility to effects of

contaminants, as well as potential exposure routes, is important in managing populations









for optimal human use. Especially important to consider, in regard to wildlife

populations, are potential effects of OCPs on reproduction.

One of the first qualities that may be related to an alligator's susceptibility to

reproductive effects of OCP contaminants is that alligators do not attain sexual maturity

until approximately 6-10 years of age, which allows exposure and bioaccumulation of

OCPs to occur before reproductive maturity. Potential implications are that, as females

begin to mobilize body stores during vitellogenesis, the lipophilic OCPs that have

accumulated in their fatty tissues during their lifespan would likely be deposited in what

will later be the embryos' sole source of nutrition (egg yolk). Secondly, adults exhibit a

long reproductive period (over 30 years), and a long life span (over 50 years) (Ferguson,

1985), and are higher order predators (which allows for increased OCP exposure and

bioaccumulation, possibly leading to altered endocrine and reproductive function).

Thirdly, alligators build nests that can be identified from considerable distances (which

aids in egg collections), lay a large number of eggs (approximately 40 eggs per clutch),

and have a long developmental period of 65-72 days (Ferguson, 1985), allowing extended

exposure at a potentially critical stage of development. Thus, the propensity for OCPs to

be bioaccumulated and biomagnified in biota (combined with the alligator's reproductive

biology, longevity, ecological trophic level, and relatively long in ovo developmental

period) suggests the potential for OCPs to alter reproductive function.

Developmental Biology of the American Alligator

Understanding normal embryonic development is an obvious necessity in

determining the occurrence of abnormal embryonic development and identifying critical

periods of development (e.g., organogenesis). Therefore, this brief review summarizes

pre-ovipositional and post-ovipositional development of the alligator embryo.









Pre-ovipositional Development

Overall, when compared to other vertebrate species such as the domestic chicken

and domestic pig, there is a paucity of data related to crocodilian development. Despite

the relatively low number of publications, the quality of papers covering early embryonic

development is fairly high, considering that much of the research took place

approximately a century ago. The most appropriate place to begin discussing embryonic

development would be the point when fertilization occurs. However, the precise timing

and location of fertilization within a female alligator's oviduct is unknown and

inadequately studied.

Pre-ovipositional development has been examined by sacrificing gravid females

and collecting their eggs and embryos. Sacrifice of gravid females was required since

alligator embryos are at a more advanced stage of development at the time of oviposition

(Clarke, 1891). The earliest developmental stage examined in these pre-ovipositional

studies were of Nile crocodile embryos (Crocodylus niloticus), in which all embryos

exhibited body folds, a neural medullary groove, an embryonic shield, area opaca, early

gut, and area pellucida (Voeltzkow, 1892).

After the appearance of the neural folds, the amniotic head fold is formed from an

anterior fold in the blastoderm. The head fold is crescent shaped, because it begins to

develop with its free ends pointing toward the posterior end of the embryo, and develops

craniocaudally. The amniotic primordium develops in continuity with the head, and is

derived from the somatopleure around the trunk.

Craniocaudal separation of the embryo from the blastoderm occurs partly as a

result of the development of the dorsal amniotic fold, but separation is not complete until









post-ovipositional stage 3 (Day 3). The neural groove and blastopore become clearly

demarcated as the ectoderm and endoderm of the blastoderm develop. The endoderm

may form extensions that penetrate the underlying yolk. The blastopore goes through the

entire blastoderm, with the primitive streak located posterior to the blastopore

(Voeltzkow, 1892).

As the body folds develop, the border between embryonic and extra-embryonic

tissues becomes visible. At this point, the beginning of the foregut is discernable, and the

notochord stretches from the midline of the head fold to the anterior border of the

blastopore. The primitive streak and primitive groove lie posterior to the blastopore, with

the primitive groove being continuous at its posterior end. The primitive streak extends

to a little less than halfway between the head fold and blastopore (Ferguson, 1985).

Neural folds have two origins. The first is a secondary fold located anteriorly in

the head region, and growing posteriorly along the median dorsal line to form a V-shaped

process, with the apex pointing toward the blastopore. The second is posterior folds that

arise as ectodermal ridges extending forward from the blastopore, circumventing the

neural groove. The apex of the V-shaped secondary head fold later disappears, and each

of the separate arms becomes continuous with the corresponding posterior neural fold.

Thus, the secondary head fold forms the anterior part of the neural folds. Closure of the

folds occurs first in the middle region of the embryo closer to the anterior end of the

neural groove in alligators (Ferguson, 1985) but closer to the posterior end in Nile

crocodiles (Voeltzkow, 1892).

After the closure of the neural canal, the blastoporal or neurenteric canal is no

longer visible. The neurenteric canal runs from its posterior cranioventral opening to









where it opens into the neural groove at its caudal limit. During this period, somites

develop along the median axis, with the first pair developing halfway between the

anterior and posterior ends. The peripheral somatic cells are compactly arranged, and

contain small myocoels within the center of the somites. The mesodermal layers cleave

and form the somatic and splanchnic components as the foregut develops.

The head fold of the embryo is positioned ventrally into the underlying yolk,

which is accentuated by the bending of the anterior neural folds, and by the cranial

flexure that occurs later. At this pre-ovipositional stage of development, the embryo has

not yet attached to the inner surface of the eggshell membrane.

Because embryos are at an advanced stage of development at the time of

oviposition (and because an entire clutch typically hatches within a 2-day period, with

most hatchlings being similar in size), it appears that fertilization occurs over a short time

period; and that embryos are actively developing during the next 2- to 3-week period in

which the ova receive albumin, eggshell membrane, and eggshell depositions (Ferguson,

1985). Presently, little information exists about gaseous exchange and embryonic

metabolism before oviposition, or about the processes that prevent the embryo from

attaching to the top of the egg before oviposition.

Post-Ovipositional Development

Post-ovipositional development is better understood than pre-ovipositional

development. Again, the amount of literature concerning crocodilian development is

miniscule compared to the amount of literature dealing with human and chicken

embryology.

One important area to address when discussing post-ovipositional development is

the staging scheme. Establishing a staging scheme or a normal table of development for









any species allows results of various studies to be compared (Billet et al., 1985). The

currently accepted staging scheme for crocodilian embryology was proposed by Ferguson

(1985). Before Ferguson's, the only other staging systems related to crocodilians came

from Voeltzkow (1892), Reese (1912), and Webb et al. (1983). These works were

impressive, considering the conditions these pioneers faced; but many stages were

missing, and incubation conditions were poorly controlled.

Ferguson (1985) improved on their work by monitoring and controlling the

temperature (300C) and the relative humidity (95-100%) at which the eggs were

incubated, allowing duplication of his experiment and standardization of the

characteristics one should see in an embryo, given its stage. This accepted staging

scheme is based on external morphological features, with limb and eye development

being important diagnostic elements. With respect to craniofacial development, a fair

amount of data exists, because of Ferguson's focus on the structure and development of

the palate in the alligator, and on how its development relates to stage (Ferguson, 1981).

Although the relationship between craniofacial development and developmental stage has

been studied, information relating stage and development in other organ systems is

somewhat lacking.

Alligator embryos are very sensitive to temperature. For example, 26-340C is the

optimum incubation temperature; anything above or below for an extended period will

result in increased mortality (Ferguson, 1985). Furthermore, 0.5-1 C changes can mean

the difference between an entire clutch of embryos being 100% females or 100% males,

since crocodilians exhibit temperature-dependent sex determination (Lang & Andrews,

1994).









Ferguson's Post-Ovipositional Staging Scheme

Because our study used Ferguson's staging scheme, a summary description of

Ferguson's (1985) staging scheme, it is summarized here. The normal table of

development for crocodilians was based on examination of 1500 Alligator

mississippiensis embryos, 300 Crocodylusporosus embryos, and 300 Crocodylus

johnsoni embryos. One bias is that all of the alligator embryos used in developing this

scheme originated from Rockefeller Wildlife Refuge, located in southern Louisiana.

Alligator embryos from other geographic areas may develop at different rates, given the

same incubation conditions. Alligators inhabiting Arkansas and North Carolina

experience a shorter summer compared to populations inhabiting southern Louisiana or

Florida. Shorter summers mean that optimal nest temperatures are maintained for a

shorter period of time. Thus, embryos from more northerly latitudes may develop at an

increased rate compared to embryos from southerly latitudes (given identical incubation

conditions), since the northern embryos must complete development within a shorter time

frame. This hypothesis is supported by evidence that crocodilian species (Crocodylus

porosus and C. johnsoni) living along the equator have longer and more variable

incubation periods and slower embryonic development than the (more northerly)

Louisiana alligator (Deeming & Ferguson, 1990).

Setting aside the potential bias described above, developmental "stages" are

determined by morphological characteristics alone, and are applicable to embryos

regardless of incubation temperature. However, the developmental day(s) associated

with each stage are only valid if the eggs are incubated at 300C with a relative humidity

of 95-100%. Temperatures lower than 300C slow the rate of development, and

temperatures above 300C have been shown to increase the rate of development. Low









humidity within the nest has been shown to dehydrate eggs, causing embryonic mortality

and alterations in growth patterns (Deeming & Ferguson, 1990).

Stage 1 covers the period from oviposition to the end of the first 24 hours, and is

characterized by the embryo and blastoderm being not attached to the top of the inner

eggshell membrane. The heart is a simple S-shaped tube. There are 16-18 pairs of

somites along the trunk, and 3 pairs of somitomeres anterior to the otic vesicle. Although

the brain has not yet regionalized, optic placodes and vesicles are present on the head.

Body torsion has not begun. The notocord is evident, the gut is incomplete caudally and

opens ventrally, and blood vessels are not present in the extraembryonic membranes.

Stage 2 (Day 2) embryos have 21-25 pairs of somites and a three-loop heart.

However, one of the most notable characteristics is that the embryo attaches to the top of

the egg, causing an opaque spot to form that is visible in an otherwise translucent egg,

when the egg is candled. Blood vessels are now visible, and the hindbrain is discernable

as a clear transparent region. The lens placode and optic cup are defined, and no body

torsion has occurred.

Stage 3 (Day 3) embryos have 26-30 somites, and are completely delineated from

blastoderm. Forebrain, midbrain, and hindbrain are now discernable, and the optic cup

has an elongated horseshoe shape, extending below the lens vesicle to the primitive

oronasal cavity. The head is positioned at a right angle to the body, but no body torsion

has occurred.

Stage 4 (Day 4) embryos have 31-35 pairs of somites with the tail being distinct,

straight, and unsegmented at the posterior end. Body torsion has started, with the cranial

half rotated so that the right surface is contacting the shell membrane, while the left is









parallel with underlying yolk. The caudal half of the embryo remains at a right angle to

the yolk. The heart is displaced from midline to the left side of the embryo. Three

cranial arches are present; and cranial nerves to the cranial arches are visible, using

oblique or transmitted illumination.

Stage 5 (Day 5) embryos have 36-40 pairs of somites, and the tail-tip bends

ventrally at a right angle to the body, with 3-5 somites visible at its base. Body torsion is

complete except for the tail. The otic pit is dorsal to the junction of the 2nd and 3rd

brachial arches, and its external opening is closed.

Stage 6 (Day 6) embryos have visible nasal placodes, and the hindlimbs are barely

discernable on each side; with the right hind limb slightly advanced over the left.

Forelimb buds are not yet present, and body torsion is complete. The olfactory bulbs,

forebrain, and midbrain are distinct. In the hindbrain, 4-6 neuromeres are discernible.

Foregut and hindgut are formed, but midgut is incomplete ventrally. Major vitellogenic

blood vessels emerge at the level of the 18th somite and smaller ones at the 6th and 11th

somites.

Embryos at Stage 7 (Day 7) have distinct hind limb buds. In addition, forelimb

buds are barely visible and extend over somites 12-15. The midbrain bulge is evident,

and the tail-tip is curled at 900 to the rest of the tail. Three brachial arches are present;

and at the level of the heart, the cranial end is bent at 900 to the rest of body.

Embryos at stage 8 (Day 8) have nasal pits external to the swellings of the olfactory

bulbs, and distinct forelimb and hind limb buds that extend over somites 11-16 and 27-

32, respectively. An apical ectodermal ridge is developing on the hind limb bud, and the

tail is coiled through 2 turns and has 12-18 somites.









Stage 9 (Day 9) embryos have four brachial arches, and a visible maxillary process

extending to the midpoint of the eye. The optic cup is large and round but unpigmented.

A distinct apical ectodermal ridge is present on the hind limb, and the hind limb bud

extends beyond the forelimb. The tail is curled through three 900 turns. The heart

exhibits distinct atria and ventricles, and lung primordia are visible through the

pericardial sac. Midgut and body walls are open ventrally from the caudal limit of the

pericardial sac to 2/3 of the way down the body, and the liver and mesonephros are barely

visible.

Stage 10 (Day 10-11) embryos have pigmented eyes (except for a central opaque

lens) with the right eye developing pigmentation earlier and darker than the left eye. Five

brachial arches are present, and medial and lateral processes are distinct elements on each

side of the nasal pits. Maxillary processes delimit a distinct groove beneath the eye. The

tail is coiled through four 900 turns, and the liver and mesonephros are clearly visible

through the body walls.

Stage 11 (Day 12) embryos have a visible nasal pit slit forming between the medial

and lateral processes. Forelimb and hind limb buds extend caudally from the body wall

and exhibit distinct apical, ectodermal ridges. The forelimb has a distinct constriction

that separates the distal and proximal elements, with constriction less obvious in the hind

limb. A loop of midgut is visible at the umbilicus, the eye exhibits a distinct black

pigment in the iris, and the chorioallantois extends 2/3 around the breadth of the shell

membrane.

Embryos at stage 12 (Day 13-14) have a distinct notch in the midline of the face

between the medial nasal processes. Forelimbs are starting to bend in the region of









constriction, so that they are positioned closer to the pleuron of the embryo. The

elongated hind limb shows little differentiation into proximal and distal elements and,

although there is a distinct apical ectodermal ridge, footplate formation is barely

discernable.

Stage 13 (Day 15) embryos have distinct nasal pit slits, and forelimbs are now bent

toward the pericardium. The distal portion of the hind limb is flattened and enlarged into

a footplate primordium. The chorioallantois now extends as a ring around the inner

circumference of the central eggshell membrane.

Embryos at Stage 14 (Day 16-17) have nasal pit slits that are closed due to the

merging of the medial nasal, lateral nasal, and maxillary processes. Foot and hand plates

are distinct, with the former more advanced than the latter. Lower jaw extends one-

quarter beneath the upper jaw, the upper earflap is overgrowing the external ear opening,

and the embryonic face rests on the large bulge of the thorax. A large loop of gut

herniates through the narrow umbilical stalk and touches the yolk, and the abdominal

viscera are visible through body walls. The tail is coiled and kinked at the tip, and

contralateral reflexes occur.

Stage 15 (Day 18-20) embryos have lower jaws that extend one-third to one-half

the length of the upper jaw. The anlage for the upper eyelid is an elevated rim of tissue

above each eye. Distinct and proximal and distal regions, as well as hand and foot plates

are present on both the fore and hind limb. There is a distinct hollow in the face beneath

the anterior one-third of the eye.

Stage 16 (Day 21) embryos exhibit faint digital condensations in the footplate but

not the hand plate. The lower jaw is now two-thirds the length of the upper jaw, with the









upper jaw being hook-shaped around the pericardial ridge. Caruncle development is

observed, with two tiny widely spaced thickenings that are just discernable on the tip of

the snout.

Embryos at stage 17 (Day 22-23) exhibit mesodermal condensations for the five

forelimb digits and four hind limb digits, the head is extended off of the pericardial sac

by neck elongation, and the external earflap is distinct.

Stage 18 (Day 24-26) embryos have discernable, distinct cartilaginous digital rays

on the hand and foot. The margins of upper eyelid anlage extend over the superior rim of

the iris, forming a distinct groove between the eyelids and the eye. Dorsal scalation is

now evident, and the pericardial sac is starting to submerge into the ventral thoracic wall.

Stage 19 (Day 27-28) embryos have upper and lower eyelids, and the lower jaw lies

behind the anterior margin of the upper jaw. Interdigital clefting has started, and slight

marginal notches can be seen, particularly in the footplates. White flecks representing

ossification are visible around the upper and lower jaws.

Stage 20 (Day 29-30) embryos have nail anlages starting to develop, first on the

most medial digit of the foot, then on adjacent digits; followed by the most medial digit

on the hand, and finally on the adjacent hand digits. Interdigital clefting now extends

one-quarter the length of the digits, and the lower jaw is in adult relationship with the

upper jaw. The pericardial sac is one-quarter withdrawn into the body, and ossification is

evident in the proximal and distal elements of limbs. Scale formation is evident dorsally,

and scutes (osteoderms) are beginning to appear in the neck region near the skull.

Stage 21 (Day 31-35) embryos have interdigital clefting now extending three-

quarters down the digits, and phalanges can be distinguished. Scales are now visible on









the ventral body wall; and dorsally on the snout, neck, body, and tail. Scutes on neck are

clearly defined. The pericardial sac is one-half withdrawn into the body, and a white ring

in the iris surrounds the outline of the lens of the eye. Both upper and lower eyelids

overlap the eye.

Stage 22 (Day 36-40) embryos have pigmented margins of the upper jaw, ventral

flank, and proximal and distal elements of the limbs. Interdigital clefting is at the adult

level, and the eyelids are typically closed from this point forward. The pericardial sac is

two-thirds withdrawn.

Stage 23 (Day 41-45) embryos have more extensive pigmentation, with the

embryos appearing light brown with dorsal stripes. Scales are present on distal and

proximal elements, and nails have a slight distal elevation. The sensory papillae are

present on lateral jaw margins, and scales are evident on gular skin. The midbrain is

visible as a white bulge at the back of the cranium, and the pericardial sac is three-

quarters withdrawn.

Stage 24 (Day 46-50) embryos are blacker. Nails on hands have elevations at

their tips, and the nails are starting to form curves. The midbrain is covered by

pigmented skin. The pericardial sac is fully withdrawn and the midline is closing. The

volume of yolk outside the body cavity is large, and scales and scutes are evident all over

embryo.

Stage 25 (Day 51-60) embryos look identical to hatchlings, except smaller. The

external yolk is beginning to be withdrawn, and few gross morphological changes are

evident at this and later stages. Growth relationships (head length: total length ratio) and

the amount of external yolk present are the major observable differences.









Stage 26 is not present in alligators. This stage was established using tooth

eruption sequences and is useful only for saltwater crocodiles (Crocodylusporosus) and

freshwater crocodiles (Crocodylusjohnsoni).

Stage 27 (Day 61-63) embryos have withdrawn the yolk sack into the body, ending

with skin forming across the umbilical scar. The last stage before hatching (Stage 28,

Day 64-70) ends with the umbilical scar being diminished in length and width.

Overall, the first 35 days are a period of rapid organogenesis, and the second 35

days are characterized by embryo growth. Since organogenesis has been shown to be a

sensitive period in regard to effects of developmental toxicants (Schmidt & Johnson,

1997), the first 35 days of incubation appear to be the most susceptible time points for

toxicant-induced mortality.

In summary, the established staging scheme provides a way to estimate the age of

the clutch at the time of collection, and allows one to later determine if a clutch is

undergoing normal development. One can determining if a clutch is undergoing normal

development by examining embryos at pre-selected time points and comparing their

morphological age to their calendar stage (i.e., does an embryo exhibit the normal

morphological characteristics that it should exhibit, given its calendar age?). In addition,

embryonic development may be compared among clutches and among populations, by

collecting embryos at pre-determined stages of development.

Organochlorine Pesticide Toxicity in Vertebrates

Classification, Mode of Action, and Pathology

Organochlorine pesticides (also known as chlorinated hydrocarbon insecticides)

may be separated into five classes of compounds. These classes are DDT and its analogs,

cyclodienes and similar compounds, toxaphene (composed of several congeners), mirex









and chlordecone (which have cage-like structures), and benzene hexachloride (BHC). In

rodent models, studies suggest that OCPs can adversely affect the function of neurons

and cause cellular damage to the liver and kidneys (Smith, 1991). Organochlorine

pesticides affect neural transmission by altering enzyme activity (Ca2+-ATPase,

phospokinase) and the electrophysical properties (K+, Na+ ion exchange) of nerve cell

membranes. Different analytes may elicit similar effects (neuronal hyperactivity), but by

different mechanisms. For example, studies suggest DDT and its analogs affect the nerve

axon by keeping Na+ channels open longer than normal. Cyclodienes, alternatively, may

affect neural transmission at presynaptic terminals and may affect the y-aminobutyric

acid (GABA)-regulated chloride channel. Although they can cause severe neural

dysfunction, little morphological changes are evident in neural tissue, even at lethal doses

(Smith, 1991).

Morphological changes are evident in the liver and include hepatocellular

hypertrophy and focal necrosis. Hypertrophy is due to enlargement of the smooth

endoplasmic reticulum (SER) and formation of a lipid droplet in the center of the SER

(caused by OCP-induced expression of microsomal enzymes within the SER).

Functional alterations may also occur in hepatocytes, with disruption of intercellular

communication (by hindering transfer of growth inhibitors) (Smith, 1991).

Morphological changes have also been found in the liver and kidney of fish

chronically exposed to organochlorine pesticides. For example, chronic exposure to

OCPs induce hepatic lesions, such as foci of vacuolated hepatocytes and spongiosis

hepatic (lesions of hepatic parenchyma). Renal lesions induced by chronic OCP









exposure include dilation of tubular lumina, and vacuolization (degeneration) and

necrosis of tubular epithelium (Metcalfe, 1998).

In addition to morphological changes, organochlorine pesticides may adversely

affect endocrine and reproductive function in laboratory models and wildlife populations.

Mechanisms include direct toxicity on endocrine glands (such as o,p'-DDD's ability to

permanently inactivate the adrenals), competitive binding of steroid hormone receptors,

increased expression of steroid-metabolizing hepatic microsomal enzymes, and inhibition

of hormone synthesis (such as DDE-induced inhibition of proglandin synthesis, leading

to eggshell thinning in raptors) (Gross et al., 2003).

Exposure and Effects of OCPs in Crocodilians

Current knowledge on the effects of environmental contaminants on crocodilian

reproductive physiology is important in understanding the likelihood of developmental

alterations occurring as a result of exposure; and understanding which mechanisms may

be involved.

Campbell (2003) reviewed the effects of organic and inorganic contaminants on

crocodilians. Campbell reported only 26 studies related to the bioaccumulation of

organic contaminants, with just 35% (8/23) of crocodilian species being represented. Of

the 26 studies, 38% involved American alligators (Alligator mississippiensis), 26%

involved Nile crocodiles (Crocodylus niloticus), 13% involved American crocodiles

(Crocodylus acutus), and 13% involved Morolet's crocodile (Crocodylus moreletii).

Slightly more studies were found that investigated effects of organic contaminants. With

respect to these 39 studies, only 13% (3/23) of crocodilian species were represented,

consisting of the American alligator (91% of studies), the Nile crocodile (5%), and the

African dwarf crocodile (Osteolaemus tetraspis, 4%). Of these studies, American









alligators are the only species in which an effort has been made to determine the

relationship between OCPs and depressed hatch rates, with most of this work involving

populations in central Florida.

Reproductive Problems in Florida Alligators

In the early to mid 1980s, studies showed that the population of juvenile alligators

inhabiting the aquatic ecosystem of Lake Apopka, Florida, declined by 90%. This

decline was preceded by a 1980 chemical spill and decades of OCP contamination via

anthropogenic activities described earlier. The loss of juveniles was attributed primarily

to a dramatic decrease in clutch viability (the proportion of eggs in a clutch that produce a

live hatchling) (Woodward et al., 1993).

Alterations in sexual differentiation, sex steroid hormone concentrations, and

metabolism were also documented among Lake Apopka alligators. For example,

testosterone was lower in male alligators from Lake Apopka as compared to those of

control sites. Ovaries of female juvenile alligators from Lake Apopka showed

abnormalities, suggesting that reproductive alterations were occurring in both sexes

(Gross et al., 1994; Guillette et al., 1994; Gross, 1997). In addition, high concentrations

of OCPs were measured in egg yolk, but concentrations were not clearly associated with

increased mortality (Heinz et al., 1991).

Later studies suggested that the cause for the population decline was potentially

more complex than previously suggested. First, poor egg viability for Lake Apopka

alligators was more closely associated with muck farm reclamation (wetland restoration)

sites than with tissue and egg concentrations of the predominant pesticide residue (DDE)

(Giroux, 1998). Second, altered endocrine function and decreased egg viability were

documented among alligators at another site, Lake Griffin, where tissue and egg









concentrations of residues such as DDE are modest or intermediate compared with those

of Lake Apopka. However, like Lake Apopka, Lake Griffin is highly eutrophic and has

adjacent muck farms and muck farm reclamation areas (Marburger et al., 1999). Third,

poor reproductive success among Lake Apopka alligators appeared to result from both

decreased proportions of fertile eggs that produce a live hatchling and decreased

proportions of hatchlings that survive through the first 20 days of life (which is the

toxicant-sensitive organogenesis period); and decreased proportions of unbanded eggs

(i.e., eggs that are nonviable on initial examination) (Masson, 1995; Wiebe et al., 2001).

Unbanded eggs show no evidence of embryo-eggshell attachment (as indicated by

the presence of an opaque spot or band that results from fusion of extraembryonic

membranes to the dorsal portion of the inner eggshell membrane). Unbanded eggs may

result from very early embryo mortality (fertilization has been confirmed in many cases

by the presence of paternal DNA, via DNA microsatellite analysis); or may result from

infertile eggs (Rotstein, 2000).

The last similarity between alterations in alligator populations of Lake Griffin and

Lake Apopka is increased mortality among adult Lake Griffin alligators (Schoeb et al.,

2002), which is similar to increased adult mortality on Lake Apopka in the early 1980s.

These data indicate that alligator populations are adversely affected at each of several life

stages. Although anatomic and endocrinologic effects of exposure to

endocrine-disrupting OCPs could account for many of these effects, additional

underlying mechanisms are almost certainly involved. Overall, these data point to a

complex process involving the introduction of OCPs into these aquatic ecosystems from









chemical spillage or from muck farming and reclamation activities; possibly leading to

developmental toxicity, in addition to endocrine disruption.

Specific Aims

The overall objective of our study was to determine the causes of decreased hatch

rates among alligators in contaminated sites, and to determine if causal links could be

established between specific adverse effects and exposure to individual OCPs or

combinations of OCPs. The project consisted of epidemiological field studies, which

evaluated embryonic development and mortality as a function of maternal and

environmental exposure to OCPs and egg nutrient composition; and controlled laboratory

experiments to test hypothesized links between decreased hatch rates, altered egg

composition, and exposure to selected OCPs.

Specific aim 1: Conduct field epidemiological studies to determine the relative

contributions of unbanded eggs, embryonic mortality in banded eggs, and decreased

perinatal mortality to the overall decreased reproductive success in alligators at OCP-

contaminated sites, to determine which OCPs or combinations of OCPs are most closely

associated with adverse effects at each life stage, and to examine the relationship between

OCP burdens in maternal tissues and eggs. For Specific Aim 1, the hypotheses were

H1a: Adverse effects at early life stages are associated with muck farm
environments, exposure to specific OCPs or OCP combinations, or both;

Hlb: Specific OCPs found in maternal tissues are highly correlated to those present
in eggs indicating maternal transfer of OCPs and that maternal size is correlated with
OCP burdens and hatch rates;

H1c: Eggs in which embryonic and perinatal mortality occur result from
developmental abnormalities, altered structure or composition of the egg, or both.

Specific aim 2: Conduct controlled in ovo and in vivo experiments with alligators

to confirm causal links between decreased hatch rates and affected life stages as a






21


function of exposure to selected OCPs or altered egg qualities, or both. For Specific Aim

2, the hypotheses were

H2a: Exposing a captive breeding population of adult alligators to an
environmentally relevant mixture of OCPs will elicit OCP concentrations in eggs and
developmental effects similar to those observed in wild eggs from OCP-contaminated
field sites;

H2b: Exogenous in ovo alteration of egg nutrients based on data from field studies
will alter embryonic development.



























































Figure 1-1. Map of Ocklawaha Basin.














CHAPTER 2
EGG AND EMBRYO QUALITY OF ALLIGATORS FROM REFERENCE AND
ORGANOCHLORINE CONTAMINATED HABITATS

In the southeastern US, aquatic ecosystems have experienced habitat degradation,

alterations in water quality, and in some cases, important declines in biodiversity due to

increases in land development and associated anthropogenic impacts. A case-in-point is

the Ocklawaha River Basin in central Florida. Within this basin, American alligators

(Alligator mississippiensis) from impacted lakes have exhibited poor clutch viability

(number eggs that yield a live hatchling / total number of eggs found in clutch) (Masson,

1995), abnormal reproductive hormone concentrations (Gross et al., 1994), and

unexplained adult mortality (Schoeb et al., 2002). During the mid 1980s, clutches from

alligators on Lake Apopka experienced severe declines in clutch viability (declined from

50% to 4%), and alligator clutches from other impacted lakes had only moderate

viabilities (range of 40 to 60%). These rates were below those observed in other less

impacted Florida lakes (reference sites), including Lake Woodruff National Wildlife

Refuge (79%), Orange Lake (82%), and the Everglades Water Conservation Areas (65-

75%) (Woodward et al., 1993; Masson, 1995; Rice, 1996).

Possible causal factors for reduced hatch rates in alligator populations within the

impacted sites within the Ocklawaha River Basin include pesticides, algal toxins,

nutritional changes, density-related stress, and diseases. In one case, a chemical spill

from a chemical manufacturing plant in 1980 near Lake Apopka (EPA, 2004) was

temporally associated with the decline in reproductive success and consequent alligator









population decline on Lake Apopka during the early 1980s. However, decreases in

clutch viability for Lake Apopka appeared to be more related to proximity to muck farm

restoration areas as compared to yolk concentrations (Giroux, 1998), which is consistent

with decreases in clutch viability on Lake Griffin and Emeralda Marsh, Griffin's adjacent

muck farm restoration area (Sepulveda et al., 2001).

Poor reproductive success threatens the long-term conservation of alligators,

potentially altering the ecology of affected ecosystems, and substantially reducing the

aesthetic and economic values of alligators in affected areas. Understanding and

characterizing poor reproductive performance and determining associated factors is

needed so that efficacious mitigation strategies may be developed. Thus, the overall

objective of the present study was to determine the relative contributions of losses during

in ovo development in American alligators at impacted sites in central Florida, and to

evaluate whether organochlorine pesticides (OCPs) are associated with adverse

developmental effects and altered clutch characteristics.

Materials and Methods

Egg Collections and Incubation

Lakes Apopka (N 280 35', W 810 39'), Griffin (N 280 53', W 81 46'), Emeralda

Marsh Conservation Area ((N 280 55', W 810 47'), and Lochloosa (N 290 30', W 820

09') in Florida were selected as collection sites because prior studies indicate vastly

different levels of OCP exposure across these sites (Gross unpublished data, (Masson,

1995).

Alligator nests were located via aerial (helicopter) and ground surveys airboatt),

and clutches were subsequently collected by ground crews. The top of each egg was

marked before eggs were removed from the nest to ensure proper orientation; thus,









preventing embryo mortality due to inversion. Embryo mortality due to inversion occurs

if an embryo has attached to the top of the egg, inversion may either break embryonic

attachment or cause the yolk mass to settle on top of the embryo, crushing it.

After marking each egg and placing about 5 cm of nest substrate in a uniquely

numbered polypropylene pan (43 cm x 33 cm x 18 cm), all eggs found in each clutch

were placed in the pan in five rows with six eggs per row. If a clutch contained more

than 30 eggs, a second layer of nest substrate was added and the additional eggs were set.

The top layer of eggs was covered with nest substrate so that there was no space left

between the top of the pan and the top of the eggs (approximately 10 cm). Clutches were

transported to the US Geological Survey's Center for Aquatic Resources Studies,

Gainesville, Florida (CARS). Upon arrival, clutches were evaluated for embryonic

viability using a bright light candling procedure. Viable eggs (i.e. having a visible band)

were nested in pans containing moist sphagnum moss and incubated at 30.5C and -98%

humidity, in an incubation building (7.3 m x 3.7 m). This intermediate incubation

temperature will normally result in a 1:1 male/female sex ratio, since alligators have

temperature dependent sex (or gender) differentiation. One or two eggs were opened

from each clutch to identify the embryonic stage of development at the time of collection,

and to collect yolk samples for later measurement of OCP burdens. From each clutch,

information on the following parameters was collected: total number of eggs found per

nest (fecundity); number of unbanded eggs, number of damaged eggs, number of dead

banded eggs, number of live banded eggs, total clutch mass, and average egg mass of

clutch.









For years 2001 and 2002, some clutches were involved in an embryo development

study. For these clutches, each clutch was evenly divided between two pans, with half of

the clutch left relatively undisturbed (except for weekly monitoring of embryo mortality)

to determine clutch viability (the number of live hatchlings / fecundity), and the other

half of the clutch used to study embryo development and morphometry (Chapter 5).

Analysis of OCPs in Yolk

Analytical grade standards for the following compounds were purchased from the

sources indicated: aldrin, alpha-benzene hexachloride (a-BHC), P-BHC, lindane, 6-BHC,

p,p '-dichlorodiphenyldichloroethane (p,p '-DDD), p,p '-dichlorodiphenyldichloroethylene

(p,p '-DDE), dichlorodiphenyltrichloroethane (p,p '-DDT), dieldrin, endosulfan,

endosulfan II, endosulfan sulfate, endrin, endrin aldehyde, endrin ketone, heptachlor,

heptachlor epoxide, hexachlorobenzene, kepone, methoxychlor, mirex, cis-nonachlor,

and trans-nonachlor from Ultra Scientific (Kingstown, RI, USA); cis-chlordane, trans-

chlordane, and the 525, 525.1 polychlorinated biphenyl (PCB) Mix from Supelco

(Bellefonte, PA, USA); oxychlordane from Chem Service (West Chester, PA); o,p '-

DDD, o,p '-DDE, o,p '-DDT from Accustandard (New Haven, CT, USA); and toxaphene

from Restek (Bellefonte, PA, USA). All reagents were analytical grade unless otherwise

indicated. Water was doubly distilled and deionized.

Egg yolk samples were analyzed for OCP content using methods modified from

Holstege et al. (1994) and Schenck et al. (1994). For extraction, a 2 g tissue sample was

homogenized with -1 g of sodium sulfate and 8 mL of ethyl acetate. The supernatant

was decanted and filtered though a Btchner funnel lined with Whatman #4 filter paper

(Fisher Scientific, Hampton, NH, USA ) and filled to a depth of 1.25 cm with sodium

sulfate. The homogenate was extracted twice with the filtrates collected together. The









combined filtrate was concentrated to ~2 mL by rotary evaporation, and then further

concentrated until solvent-free under a stream of dry nitrogen. The residue was

reconstituted in 2 mL of acetonitrile. After vortexing (30 s), the supernatant was applied

to a C 18 solid phase extraction (SPE) cartridge (pre-conditioned with 3 mL of

acetonitrile; Agilent Technologies, Wilmington, DE, USA) and was allowed to pass

under gravity. This procedure was repeated twice with the combined eluent collected in a

culture tube. After the last addition, the cartridge was rinsed with 1 mL of acetonitrile

which was also collected. The eluent was then applied to a 0.5 g NH2 SPE cartridge

(Varian, Harbor City, CA, USA), was allowed to pass under gravity, and collected in a

graduated conical tube. The cartridge was rinsed with an additional 1 mL portion of

acetonitrile which was also collected. The combined eluents were concentrated under a

stream of dry nitrogen, to a volume of 300 [tL, and transferred to a gas chromatography

(GC) vial for analysis.

GC/MS Analysis

Analysis of all samples was performed using a Hewlett Packard HP-6890 gas

chromatograph (Wilmington, DE, USA) with a split/splitless inlet operated in splitless

mode. The analytes were introduced in a 1 [iL injection and separated across the HP-5MS

column (30 m x 0.25 mm; 0.25 [tm film thickness; J & W Scientific, Folsom, CA, USA)

under a temperature program that began at 600 C, increased at 10 C/min to 2700 C, was

held for 5 min, then increased at 250 C/min to 3000 C and was held for 5 min. Detection

utilized an HP 5973 mass spectrometer in electron impact mode. Identification for all

analytes and quantitation for toxaphene was conducted in full scan mode, where all ions

are monitored. To improve sensitivity, selected ion monitoring was used for the









quantitation for all other analytes, except kepone. The above program was used as a

screening tool for kepone which does not optimally extract with most organochlorines.

Samples found to contain kepone would be reextracted and analyzed specifically for this

compound.

For quantitation, a five-point standard curve was prepared for each analyte (r2 >

0.995). Fresh curves were analyzed with each set of twenty samples. Each standard and

sample was fortified to contain a deuterated internal standard, 5 [iL of US-108 (120

[g/mL; Ultra Scientific), added just prior to analysis. All samples also contained a

surrogate, 2 [g/mL of tetrachloroxylene (Ultra Scientific) added after homogenization.

Duplicate quality control samples were prepared and analyzed with every twenty samples

(typically at a level of 1.00 or 2.50 [g/mL of y-BHC, heptachlor, aldrin, dieldrin, endrin,

andp,p '-DDT) with an acceptable recovery ranging from 70 130%. Limit of detection

ranged from 0.1-1.5 ng/g for all OCP analytes, except toxaphene (120-236 ng/g), and

limit of quantitation was 1.5 ng/g for all analytes, except toxaphene (1500 ng/g).

Repeated analyses were conducted as allowed by matrix interference and sample

availability.

Data Analysis

Specific OCP analytes were removed from analysis if measurable concentrations

were found in < 5% of all clutches. Numerical data were log-transformed [ln(x)], while

proportional data were arcsine square root transformed to meet statistical assumptions.

ANOVA (PROC GLM; SAS Institute Inc., 2002) was used for inter-site

comparisons of adult female and clutch characteristics, and the Tukey test was used for

multiple comparisons among sites (a = 0.05). Because relationships between response

variables and explanatory variables (Table 2-1) in ecological studies are often complex









with interactions occurring, an indirect gradient multivariate analysis method, Detrended

Correspondence Analysis (DCA) (ter Braak, 1986) was used to initially evaluate

data structure. Two matrices were constructed for DCA, with the first representing the

response variables (clutch number x clutch parameters) and the second representing the

explanatory variables (clutch number x OCP burdens) (Table 2-2). DCA results

indicated that a direct gradient, multivariate linear analysis, redundancy analysis (RDA)

(Rao, 1964), was appropriate since the gradient lengths of the DCA ordination axes were

equal to or less than 2 standard deviations (ter Braak, 1995).

RDA is the canonical form of principal components analysis (PCA). In RDA, as in

PCA, a straight line is fitted to each the response variable (clutch survival parameters) in

an attempt to explain the data of all response variables. Similar to PCA, the lower the

residual sum of squares, the better the environmental variable is at explaining the

variation in response variables. RDA, unlike PCA, restricts the clutch scores (from the

response variables measured on each clutch) to a linear combination of the environmental

(explanatory variables). Because clutch scores are constrained to a linear combination of

environmental variables, RDA explains slightly less variance compared to PCA (ter

Braak & Tongeren, 1995; ter Braak, 1994). For RDA involving compositional data (i.e.,

clutch viability rates or percentages) and quantitative environmental variables,

compositional data is log-transformed (In (x + 1)) with correlation biplots being centered

by the response variables (i.e., unbanded egg percentage) and by the samples (i.e.,

clutches) (ter Braak, 1994). These correlation biplots provide a way to examine

relationships among a number of response variables and explanatory factors with

response variable arrows forming a biplot of correlations with each other, environmental









arrows forming a biplot among each other, and response variable arrows and

environmental arrows forming a biplot of correlations with each other (ter Braak, 1995).

For the RDA, separate matrices were constructed for response variables measured

as a percentage (i.e., clutch viability) and response variables measured as a number (i.e.,

clutch mass) because percentage data were ln(x+1) transformed and not standardized,

while continuous data were ln(x) transformed and standardized(ter Braak & Smilauer,

2002). Automatic forward selection of the best four explanatory variables was conducted

for both sets of RDA analyses and tested for significance by Monte Carlo permutation

tests. DCA and RDA were conducted using the program CANOCO (ter Braak &

Smilauer, 2002). Biplots of environmental variables and response variables were then

constructed to interpret relationship between clutch parameters (response variables) and

explanatory factors.

Results

Inter-Site Comparisons of Clutch Characteristics

From 2000-2002, 168 clutches were collected from Lakes Lochloosa (n = 44),

Apopka (n = 31), Griffin (n = 47), and Emeralda Marsh (n = 46). No significant

differences were determined among sites with respect to clutch mass (overall mean +

standard error: 3.7 0.08 kg), egg mass (83 + 1.4 g), or percentage of unbanded eggs (15

+ 1.7%) (Table 2-3).

In contrast, significant differences were determined among sites with respect to

fecundity, clutch viability, percentage of damaged eggs, percentage of early embryo

mortality, and percentage of late embryo mortality. Clutches from Lochloosa had lower

fecundity and late embryo mortality rates compared to all other sites. In addition,

Lochloosa clutches had greater clutch viability rates than all other sites and lower early









embryo mortality rates than all other sites, except for Apopka. Clutches from Emeralda

Marsh had greater incidence of damaged eggs than all other sites, except for those of

Lake Griffin (Table 2-3).

Organochlorine Pesticides Burdens and Clutch Characteristics

From 2000-2002, clutch characteristics and OCP burdens were measured on 115

clutches collected from Lakes Lochloosa (n = 19), Apopka (n = 23), Griffin (n = 42), and

Emeralda Marsh (n = 31). No significant differences were determined among sites with

respect to clutch mass (overall mean standard error: 3.8 0.09), clutch viability (50 +

3.1), percentage of damaged eggs (4 1), unbanded eggs (13 1.6), early embryo

mortality (21 2.3), and late embryo mortality (11 + 1.7) (Table 2-4). However,

significant differences were determined for fecundity and egg mass, with Lochloosa

clutches having lower fecundity than all other sites, and greater average egg mass

compared to those of all other sites, except for Lake Apopka. Furthermore, significant

differences were detected among sites with respect to individual OCP concentrations in

egg yolks, total OCP concentrations in egg yolks, and number of OCPs detected at

measurable levels. For total OCP concentrations and number of analytes detected at

measurable levels, egg yolks of Lake Lochloosa clutches had significantly lower total

concentrations and a lower number of analytes detected at measurable levels (Table 2-4).

Individual OCP analyte concentrations in egg yolks of Lochloosa clutches were

significantly less than those of the other sites, except for Lake Griffin with respect to

aldrin and trans-nonachlor. Aldrin and trans-nonachlor egg yolk concentrations of

Lochloosa clutches did not significantly differ from Lake Griffin, but egg burdens of

these analytes of both sites were significantly less than those of Lake Apopka and

Emeralda Marsh (Table 2-4).









Clutch Survival and OCP Burdens in Egg Yolks

Because a number of site specific factors may potentially affect clutch survival

parameters and since OCP burdens varied greatly among sites, relationships between

OCP egg yolk variables and clutch survival were evaluated on a site-by-site basis.

For Lake Lochloosa, redundancy analysis with Monte Carlo permutation tests for

significance indicated that none of the four extracted OCP variables (Table 2-5) were

found to be significantly correlated with the clutch survival variables. Number of OCPs

detected (NOC) approached significance (P = 0.07), was negatively associated with

clutch viability, positively correlated with percentage unbanded eggs and late embryo

mortality, and accounted for 11% of the variation in clutch survival parameters (Fig. 2-1).

For Lake Griffin, redundancy analysis with Monte Carlo permutation tests for

significance indicated that three of the four extracted OCP variables were found to be

significantly correlated with the clutch survival variables and together accounted for 21%

of the variance in clutch survival parameters. The extracted OCP variables were

concentration of p,p '-DDE, toxaphene, and p,p '-DDT, accounting for 8, 7, and 6%,

respectively, of variation in clutch survival variables (Table 2-5). Clutch viability was

positively associated with toxaphene and p,p '-DDE egg yolk concentrations, but had little

to no correlation with p,p '-DDT yolk burdens. Early embryo mortality rates were

negatively associated withp,p '-DDE and toxaphene. Late embryo mortality rates were

positively associated with toxaphene, and negatively associated withp,p '-DDT, andp,p '-

DDE. Unbanded egg rates were positively associated with p,p '-DDT and p,p '-DDE, but

negatively associated with toxaphene (Fig. 2-2).

For Lake Apopka, redundancy analysis with Monte Carlo permutation tests for

significance also indicated that three of the four extracted OCP variables were found to









be significantly correlated with the clutch survival variables. These OCP variables were

percentage dieldrin (lambda A = 17%), percentage trans-chlordane (12%), and percentage

aldrin (10%), and together accounted for 3% (Z lambda A's) of the variance in the clutch

survival parameters (Table 2-5). Clutch viability was positively associated with aldrin,

weakly associated with trans-chlordane, and negatively associated with dieldrin. Early

embryo mortality and unbanded egg rates were positively associated with dieldrin and

trans-chlordane, and negatively associated with aldrin. Late embryo mortality rates were

negatively with all three OCP variables (Fig. 2-3).

For Emeralda Marsh, redundancy analysis with Monte Carlo permutation tests for

significance also indicated that only percentage toxaphene was found to be significantly

correlated with the clutch survival variables, and it accounted for 9% of the variance in

the clutch survival parameters (Table 2-5). Percentage toxaphene was positively

associated with clutch viability, weakly associated with late embryo mortality, and

negatively associated with early embryo mortality and unbanded egg rates (Fig. 2-4).

Percentage of heptachlor epoxide showed a near significant association (P = 0.09) with

clutch parameters, being negatively correlated with clutch viability and positively

correlated with early and late embryo mortality rates.

Average Egg Mass, Clutch Size and OCP Burdens

For Lochloosa clutches, three of four OCP variables were determined (via RDA

analysis) to be significantly associated with egg and clutch size parameters and accounted

for 64% of the variation in egg and clutch size parameters. These OCP variables

included number of OCPs detected at measurable levels (NOC) (lambda A = 31%), p,p '-

DDT concentrations (20%), and trans-nonachlor concentrations (13%) (Table 2-6).









NOC and trans-nonachlor concentrations were negatively associated with average

egg mass but positively associated with fecundity and clutch mass. In contrast, p,p '-DDT

concentrations were positively associated with egg mass, negatively associated with

fecundity, and had little to no association with clutch mass (Fig. 2-5).

For Lake Griffin clutches, however, no significant associations were found between

OCP variables and egg and clutch size variables. In contrast, percentage o,p '-DDT in

Emeralda Marsh clutches was found to be positively associated with increasing egg and

clutch mass but negatively associated with fecundity. Lastly, Lake Apopka clutches were

somewhat similar to Emeralda clutches in that one OCP variable (p,p '-DDD

concentration) was found to be positively associated with egg and clutch mass and

negatively associated with fecundity (Table 2-6).

Discussion

Inter-Site Comparisons of Clutch Characteristics

The results of the present study suggested that the 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. Although not significantly different among sites,

infertility and/or embryo mortality before embryo attachment (unbanded eggs) also

appears to be a major constituent of reduced clutch viability among all sites. In order of

importance, major constituents of reduced clutch viability for all sites include early

embryo mortality, unbanded eggs, late embryo mortality, and damaged eggs. In addition,

clutches from OCP-contaminated sites had an average of 10 more eggs per clutch as

compared to the reference site, but average clutch mass was not significantly different,









making average egg mass of reference site clutches greater than that of clutches of OCP-

contaminated sites.

The reduced clutch viability, increased rates of unbanded eggs and embryo

mortality, and concurrent increase in fecundity without proportional increase in clutch

mass observed in clutches from OCP-contaminated sites, as compared to the reference

site (Lochloosa), suggest that females and their embryos from contaminated sites may be

responding to one or more environmental factors common to the three OCP-contaminated

sites. Although measurement of all environmental factors is impractical, the large

differences in OCP concentrations in alligator eggs between reference and OCP-

contaminated sites were found. Specifically, total OCP egg yolk burdens and number of

OCPs detected at measurable levels in Lake Lochloosa were significantly less than those

of Lake Griffin clutches, and OCP burdens in Lake Griffin clutches were, in turn,

significantly less than those of Lake Apopka and Emeralda Marsh.

Although Lake Apopka and Emeralda Marsh were not determined to be

significantly different with respect to total OCP concentrations in egg yolks, significant

differences were determined between these two high OCP exposure sites in regard to

certain analytes, as well as the total number of OCPs detected at measurable levels.

Clutches from Emeralda Marsh had a greater number of OCP analytes in their egg yolks

and contained higher concentrations of cis-chlordane, p,p'-DDD, o,p-DDD, trans-

chlordane, and toxaphene compared to those from Lake Apopka. Conversely, clutches

from Lake Apopka had higher concentrations of aldrin, dieldrin, heptachlor epoxide, and

oxychlordane compared to those of Emeralda Marsh.









The differences in OCP exposure profiles among sites likely reflect the differences

in historic land-use and OCP applications, as opposed to differences in xenobiotic

biotransformation among the different alligator populations inhabiting the respective

sites. Importantly, although Emeralda Marsh is separated from Lake Griffin by only a

levee easily traversed by alligators, large differences in OCP egg burdens were noted

between the two sites. Such differences in exposure suggest that the highly exposed adult

females which oviposite within Emeralda Marsh likely have established territories and

reside year round within Emeralda Marsh (a former agricultural property). Furthermore,

the relatively high egg burdens in clutches of Emeralda Marsh likely occurred over a

relatively short period since this 2,630 ha area was not flooded until the early 1990s

(Marburger et al., 1999).

In summary, the differences in OCP egg burdens between the reference site and the

contaminated sites support the hypothesis that OCP contaminants may be associated with

reduced clutch viability, given that OCPs have been causally linked to reduced

reproductive success in other oviparous species (Donaldson & Fites, 1970; Fry, 1995).

Clutch Survival Parameters and OCP Burdens

Results of redundancy analyses more directly addressed the question of whether

OCPs are associated with reduced clutch viability by relationships on a site-by-site basis

to control for potential site-associated confounding factors. For Lake Lochloosa, no

significant correlations were determined although significance might have been detected

given a greater sample size. The positive but insignificant correlations between increases

in unbanded egg and late embryo mortality percentage and number of OCPs may suggest

that increased OCP burdens in eggs play a role in clutch viability or it may simply

indicate that older females have increased levels of OCPs due to increased exposure time









and that decreased clutch viability is due to decreased egg quality associated with

senescence.

For Emeralda Marsh, the weak associations between OCP variables and clutch

survival variables suggests that other factors may be involved in reduced embryo survival

and increased rates of unbanded eggs. The weak associations for Emeralda Marsh are

surprising given that relatively stronger associations were determined for the other high

exposure site (Lake Apopka; Table 2-5), as well as the intermediate exposure site (Lake

Griffin, Table 2-5), with Emeralda Marsh being separated from Lake Griffin by only a

non-fenced levee.

Stronger associations were noted for Lake Apopka in contrast to the weak,

associations noted for Emeralda Marsh. The positive association between early embryo

mortality and unbanded egg rates and extracted OCP variables for Lake Apopka clutches

suggests that the percentages of dieldrin and trans-chlordane in eggs may play an

important role in altered egg fertility and/or early embryo survival Interestingly, the

percentage of aldrin, (dieldrin's parent compound) had a negative association with late

embryo mortality, a positive association with clutch viability, and near-zero correlations

with percentage unbanded eggs and early embryo mortality. However, dieldrin (a

metabolite formed from aldrin) had strong, positive correlations with percentage

unbanded eggs and early embryo mortality, and a negative correlation with clutch

viability, suggesting this metabolite has greater efficacy than its parent compound in

affecting embryo survival. The potential consequence exists that increasing a female

alligator's ability to biotransform aldrin to dieldrin may increase the risk of early embryo

mortality. Another important note is that the level of dieldrin in Apopka clutches was









two-fold greater than those of Emeralda Marsh, suggesting that OCP mixture

composition may be more important than sum OCP concentrations.

For Lake Griffin, the negative to near-zero association between early embryo

mortality rates and extracted OCP variables suggests that OCP burdens in eggs may not

play an important role in early embryo mortality. However, the positive association

between toxaphene burdens and late embryo mortality suggests that as toxaphene burdens

increase, so does the risk for increased embryo death during the last 35 days of

development. Furthermore, the positive association between p,p '-DDT concentrations

and unbanded egg rates suggests that these analytes may be involved in altered egg

fertility and/or embryo survival (prior to eggshell membrane attachment) (Fig. 2-2).

Egg and Clutch Size and OCP Burdens

For Lochloosa, the strong associations between OCP burdens and egg and clutch

size parameters suggest that, although a low OCP exposure site, certain patterns of OCP

exposure are strongly associated with egg and clutch size characteristics. The positive

associations p,p '-DDT concentrations have with clutch weight and average egg weight

and p,p '-DDT's negative association with fecundity may be potentially related to

senescent females, since older females have been reported to lay smaller clutches of

larger eggs (Ferguson, 1985) and would likely have higher OCP burdens due to extended

exposure period. In contrast, the positive associations that NOC and trans-nonachlor

have with fecundity and clutch mass, and the negative associations these OCP variables

have with egg mass, suggests that increased OCP exposure may have altered clutch and

egg size characteristics, as opposed to female age. Although these speculations are

interesting from a low exposure effect standpoint, they are irrelevant at the population-

effect level since clutch viability rates were unrelated.









Since the low exposure site had stronger associations between OCP variables and

egg and clutch size variables than intermediate and high exposure sites, one might

initially conclude that other factors are more important than OCP burdens in influencing

egg and clutch size characteristics. While this may be the case, the fact that the

intermediate and high exposure sites have significantly greater fecundity (averaging 10

more eggs per clutch compared to the low exposure site), significantly less average egg

mass, and similar clutch mass suggest that females attaining their maximum

physiological response in regard to number of eggs ovulated. These intermediate and

highly exposed females appear to be producing more ova but are unable to sequester

additional egg components (i.e., lighter eggs), in effect decreasing the amount of energy

and structural supplies available to each embryo and resulting in lighter eggs and higher

embryo mortality rates.

In summary, our results suggest that, over all sampled clutches, clutch survival

parameters and egg and clutch size parameters vary between the low OCP exposure site

(Lochloosa) and the intermediate-high OCP exposure sites. Furthermore, OCP burdens

do not appear to be related to clutch survival for the low exposure site but are associated

with clutch survival for the intermediate-high OCP contaminated sites. In contrast, egg

and clutch size parameters appear to be a sensitive endpoint in OCP response in alligators

due to the strong associations noted between OCP and clutch size variables for the low

exposure site and the lack thereof for the intermediate-high exposure sites, suggesting

attainment of maximum response. In order to better determine the role of OCPs in the

reduced reproductive efficiency of OCP-exposed alligator populations, suggested future

studies should examine the relationship between maternal OCP burdens and respective






40


egg burdens, presence of other environmental contaminants, maternal factors associated

with clutch survival and OCP burdens, and how egg composition relates to clutch

survival and OCP burdens.









Table 2-1. Reproductive, morphometric, and contaminant parameters measured on
clutches of alligator eggs collected during summer 2000, 2001, and 2002.


Clutch Parameter
Response variables
Fecundity
Clutch mass
Ave. Egg Weight
Unbanded eggs% a
Early embryo mortality%

Late embryo mortality%

Clutch Viability

Explanatory variables
[OCP analyte] in egg yolk
% OCP analyte


Definition

Total No. of eggs in one clutch
Total mass of eggs in one clutch
Clutch mass / Fecundity
No. of unbanded eggs / fecundity x 100
No. of deaths < dev. Day 35 / fecundity
x 100
No. of deaths > dev. Day 35
/ fecundity x 100
No. eggs yielding live hatchling
/ fecundity x 100

ng OCP analyte / g egg yolk wet weight
[OCP analyte] / 1 [OCP] x 100


aAn egg with no evidence of embryonic attachment


Measured
as

n
kg
g
Percentage
Percentage

Percentage

Percentage


ppb
Percentage









Table 2-2. Explanatory variables included in RDA with forward selection of four best
variables (a = 0.05).
Variable Code
Age Age
No. OCPs at measurable levels NOC
Z [OCP] TOC
% Aldrin ALD%
[Aldrin] [ALD]
% cis-Chlordane CC%
[cis-Chlordane] [CC]
% cis-Nonachlor CN%
[cis-Nonachlor] [CN]
% Dieldrin DL%
[Dieldrin] [DL]
% Heptochlor epoxide HE%
[Heptachlor epoxide] [HE]
%Lipid content LPC%
% Mirex MX%
[Mirex] [MX]
% o,p-DDT ODDT%
[o,p-DDT] [ODDT]
% o,p-DDD ODDD%
[o,p-DDD] [ODDD]
% Oxychlordane OX%
[Oxychlordane] [OX]
% p,p'-DDE PDDE%
[p,p'-DDE] [PDDE]
% p,p'-DDD PDDD%
[p,p'-DDD] [PDDD]
% p,p'-DDT PDDT%
[p,p'-DDT] [PDDT]
% trans-Chlordane TC%
trans-Chlordane [TC]
% trans-Nonachlor TN%
[trans-Nonachlor] [TN]
% Toxaphene TX%
[Toxaphene] [TX]












Table 2-3. Summary of clutch parameters and site comparisons for clutches of American alligator eggs collected during 2000-2002.


Parameter'a
N. Clutches
Fecundity (n)

Clutch mass (kg)

Egg mass (g)

Clutch viability (%)

Damaged eggs (%)

Unbanded eggs (%)

Early Emb. Mort. (%)

Late Emb. Mort. (%)


Lochloosa
44
36 1.2 B
(22-56)
3.4 0.15
(1.6-4.8)
87 + 2.2
(61-139)
70 + 3.9 A
(0-100)
2+ 1.4 B
(0-60)
11 2.2
(0-84)
12 2.7 B
(0-69)
6+ 1.7B
(0-34)


Apopka
31
46 1.3 A
(28-56)
4+ 0.13
(2.4-5.1)
86 +2
(62-120)
51 + 5.8 B
(0-98)
2+0.6B
(0-16)
21 4.9
(0-100)
15 + 4.2 AB
(0-94)
12 + 3.5 A
(0-77)


Emeralda Griffin
46 47
46 1.1A 45+ 1.2A
(27-64) (19-58)
3.8 0.21 3.6 0.13
(1.9-9.2) (1.5-5.2)
83 4 80 1.6
(58-180) (46-113)
48 5.5B 44 4.9B
(0-97) (0-92)
5+1.3A 4+ 1.8AB
(0-33) (0-63)
14 3.7 17 3.2
(0-100) (0-100)
23 3.9 A 22 3.9 A
(0-95) (0-100)
10 2.4A 13 3.1A
(0-82) (0-89)


Summary
168
43 0.7
(19-64)
3.7 + 0.08
(1.5-9.2)
83 1.4
(46-180)
53 + 2.6
(0-100)
3 0.7
(0-63)
15 1.7
(0-100)
19 2
(0-100)
11 + 1.4
(0-89)


aValues indicate mean standard error of mean with ranges in parentheses. Values with different letters (A-B) indicate significant
differences (a = 0.05); same letters indicate significant differences were not detected. Clutch viability = No. of eggs yielding a live
hatchling / Fecundity x 100, Damaged eggs = No. damaged eggs / fecundity x 100, Unbanded eggs = No. of unbanded eggs /
fecundity x 100, Early Emb. Mort. = No. of embryonic deaths on or before developmental Day 35 / fecundity x 100, and Late Emb.
Mort. = No. of embryonic deaths post dev. Day 35 / fecundity x 100).












Table 2-4. Organochlorine pesticide burdens and clutch parameters and site comparisons for clutches of American alligator eggs
collected during 2000-2002.
Parameters Loch. Apopka Emeralda Griffin Summary


N. Clutches
Fecundity (n)

Clutch mass (kg)

Egg mass (g)

Clutch viability (%)

Damaged eggs (%)

Unbanded eggs (%)

Early Emb. Mort. (%)

Late Emb. Mort. (%)

Aldrin (ng/g)

Methoxychlor (ng/g)


19
40 1.7B
(26-56)
3.6 + 0.17
(2.2-4.8)
90 + 2.9 A
(78-139)
65 + 5.5
(0-95)
4 3.1
(0-60)
11+2
(0-33)
13 3
(0-36)
8 2.5
(0-34)
0 +OC
(0-0)
0 0C
(0-0)


23
47 1.4 A
(31-56)
4 0.16
(2.5-5.1)
86 2.5 AB
(62-120)
52 + 6.4
(0-98)
2 0.8
(0-16)
17 4.2
(0-81)
15 4.2
(0-90)
14 4.6
(0-77)
4 0.3 A
(2.9-5.2)
8+ 1B
(5.7-16.4)


31
46 1.3 A
(27-64)
3.8 + 0.25
(2.1-9.2)
82 4.8 B
(58-180)
50 + 6.9
(0-97)
6+ 1.6
(0-32)
10 2.3
(0-58)
26 + 5.1
(0-95)
10+ 2.5
(0-61)
2+ 0.3 B
(1.5-4.3)
9+ 1B
(5.8-18.4)


42
46 1.2 A
(24-58)
3.7 + 0.13
(1.5-5.2)
79 1.5B
(46-105)
43 5.1
(0-92)
5+2
(0-63)
15 3.2
(0-100)
24 4.3
(0-100)
13 + 3.3
(0-89)
0+0C
(0-0)
17 + 0.3 A
(16.9-17.5)


115
45 + 0.7 A
(24-64)
3.8 + 0.09
(1.5-9.2)
83 1.6
(46-180)
50 3.1
(0-98)
4+ 1
(0-63)
13 1.6
(0-100)
21 + 2.3
(0-100)
11 + 1.7
(0-89)
3 0.3
(1.5-5.2)
9 0.8
(5.7-18.4)












Table. 2-4. Continued.


Parameter'a
Mirex (ng/g)

Dieldrin (ng/g)

Hep. Epoxide (ng/g)

cis-Chlordane (ng/g)

cis-Nonachlor (ng/g)

Oxychlordane (ng/g)

p,p'-DDE (ng/g)

p,p'-DDD (ng/g)

p,p'-DDT (ng/g)

o,p'-DDD (ng/g)

o,p'-DDT (ng/g)


Orange/Loch
2+ 0.4 B
(1.2-2.7)
4+ 0.5 D
(1.3-8.2)
3 0.8 C
(1.2-9.7)
2+0.2D
(1.2-4.1)
5 0.6 C
(2.4-12.5)
4+ I D
(1.2-17.8)
74 11.7 C
(28-231)
2+0.2 D
(1.2-3)
1 0C
(1.2-1.3)
0+0C
(0-0)
1 0C
(1.2-1.4)


Apopka
6+ 1.1 A
(1.1-17.2)
344 + 80.9 A
(12.5-1783.2)
17 5.6A
(1.2-135.5)
43 + 7.6 B
(6.6-179.2)
88 + 27.3 A
(10.5-656.2)
51 + 14.6 A
(3.9-353.8)
5794 1794.7 A
(18.3-42653.4)
42 + 8.5 B
(10.6-192.8)
9+ 2.1 AB
(1.2-45.6)
5+0.7B
(3.1-9.2)
11 + 1.9A
(1.2-38.5)


Emeralda
3 0.5 AB
(0.1-10.3)
142 20.4 B
(8.7-386.7)
7+ 1.4 B
(0.1-32.1)
90 13 A
(8.9-281)
66 9.7 A
(11.6-232.2)
23 3.8 B
(3.2-109.5)
8069 1402 A
(36.2-33554.8)
1289 196.1 A
(10.3-2962.8)
12 1.2 A
(5.8-25.5)
37 5.1 A
(0.1-104)
170 161.6 A
(4.2-4372.8)


Griffin
3 + 0.2 AB
(1.1-4.5)
23 3.8 C
(2.9-124)
7+ B
(1.1-29.6)
11 0.9C
(4.3-31.8)
18 1.6B
(6.5-54.2)
10 1.3 C
(1.1-41.9)
271 + 31.3 B
(62.9-979.1)
7+0.9 C
(2.7-28.9)
5 0.8 B
(1.1-7.2)
1 0B
(1.3-1.3)
4+ 0.3 B
(1.1-7.4)


Summary
4 0.4
(0.1-17.2)
118+ 20.9
(1.3-1783.2)
8 1.4
(0.1-135.5)
37 5
(1.2-281)
43 6.7
(2.4-656.2)
21 3.4
(1.1-353.8)
3445 + 610.6
(18.3-42653.4)
382 + 78.7
(1.2-2962.8)
10 1
(1.1-45.6)
29 4.5
(0.1-104)
48 + 42
(1.1-4372.8)












Table. 2-4. Continued.


Parameter'a
trans-Chlordane (ng/g)

Toxaphene (ng/g)

trans-Nonachlor (ng/g)

-OCPs (ng/g)


Orange/Loch
3 0.7 C
(1.2-3.7)
0 0OC
(0-0)
8 1.6 C
(2.5-24.6)
102 15.5 C
(42.7-289.4)


N. OCPs 9 0.3 D
(7-11)


Apopka
8 1.5 B
(1.3-27.4)
2738 224.5 B
(1896.1-3809.1)
212 + 66.9 A
(10.5-1569.2)
7582 2008.2 A
(472.5-47333.8)
13 + 0.3 B
(10-16)


Emeralda
25 + 3.3 A
(2.9-58.2)
6865 + 552.4 A
(2300.6-12975.4)
191 + 30.5 A
(14.2-718.6)
15480 2265.4 A
(269.6-53559.7)
14 + 0.2 A
(13-17)


Griffin
2+ 0.2 C
(1.1-8.7)
3043 + 425.9 B
(1927.9-4533.2)
36 4.7 B
(8.6-155.2)
1169 422.8 B
(101.5-16795.4)
11 + 0.1 C
(9-13)


Summary
11 + 1.5
(1.1-58.2)
5456 + 483
(1896.1-12975.4)
108 17.5
(2.5-1569.2)
6133 + 940.8
(42.7-53559.7)
12 + 0.2
(7-17)









Table 2-5. Results of RDA evaluating associations between clutch survival parameters


and OCP variables.
Site Variablea
Lochloosa NOC
[DL]
PDDT%
PDDE%

Apopka DL%
TC%
ALD%
LPC%

Emeralda Marsh TX%
HE%
ME%
[HE]

Griffin [PDDE]
[TX]
[PDDT]
[ODDD]


LambdaA
0.11
0.09
0.08
0.11

0.17
0.12
0.10
0.06

0.09
0.06
0.06
0.06

0.08
0.07
0.06
0.04


P
0.074
0.194
0.166
0.104

0.004
0.024
0.042
0.16

0.044
0.09
0.15
0.15

0.024
0.016
0.04
0.09


F
2.25
1.59
1.72
2.45

4.25
3.32
3.16
1.85

2.99
2.27
1.85
1.89

3.67
3.16
2.71
1.96


aSee Table 2-2 for definition of variable codes.









Table 2-6. Results of RDA evaluating associations between egg and clutch size
parameters and OCP variables.
Site Variable LambdaA P F
Lochloosa NOC 0.31 0.004 10.15
[PDDT] 0.20 0.042 4.29
[TN] 0.13 0.006 6.77
OX% 0.08 0.088 2.8

Griffin PDDD% 0.05 0.134 2.32
[ODDT] 0.03 0.406 0.91
[PDDT] 0.02 0.236 0.95
[CC] 0.01 0.54 0.33

Emeralda [ODDT] 0.22 0.01 8.07
CC% 0.05 0.146 2
ODDT% 0.05 0.182 1.82
LPC% 0.04 0.21 1.7

Apopka [PDDD] 0.24 0.01 6.51
[ME] 0.08 0.112 2.54
[PDDT] 0.05 0.218 1.5
PDDE% 0.05 0.294 1.29









(C
C6 Early Emb. Mort.



[DL]
PDDE% '


Clutch viability '

------------- -- 4 ------- -------- - ^ -- -- -- -- -- -- -- -- -
PDDTP




Unbanded egg% 0
B


N A Late Emb. Mort.
CO A


-0.8 0.6


Figure 2-1. Biplot of clutch survival parameters (solid lines) and organochlorine
pesticide variables (dashed lines) for clutches of alligator eggs collected from
Lake Lochloosa during summer 2001-2002. Arrows pointing in the same
direction indicate a positive correlation (e.g., clutch viability and PDDE%),
arrows that are approximately perpendicular indicate near-zero correlation
(e.g., late emb. mort. and [DL]), and arrows pointing in opposite directions
indicate negative correlations (e.g., clutch viability and [DL]. Arrow lengths
indicate rank order of correlations. For example, late emb. mort. has higher
positive correlation with NOC (A) compared to unbanded egg% (B). Cosine
of angle formed between individual clutch variables and individual OCP
variables (see Table 2-2 for code definitions) equals correlation coefficient (r)
(ter Braak, 1995). For example, arrows pointing in exactly opposite directions
have an angle of 1800, and since cos(180) = -1.0, the arrows are perfectly,
negatively correlated (r) (ter Braak, 1995).














CO
Late Emb. Mort.
Early Emb. Mort.


[TX]
\,-- [ODDD]









Clutch Viability /


[PDDT] Unhanded egg%
[PDDT]
00/
C) [PDDE]

-1.0 1.0



Figure 2-2. Biplot of clutch survival parameters (solid lines) and organochlorine
pesticide variables (dashed lines) for clutches of alligator eggs collected from
Lake Griffin during summer 2000-2002.










00 A4 AD%
6 Clutch Viability
\ TC%



















Late Emb. Mort LPC%
6

-1.0 1.0



Figure 2-3. Biplot of clutch survival parameters (solid lines) and organochlorine
pesticide variables (dashed lines) for clutches of alligator eggs collected from
Lake Apopka during summer 2000-2002.












CME%
/
/1




// Early Emb Mort


Late Emb Mot / ,-- HE%
TX% -/ / ------ -

Clutch Viability--- ------ ---










N1 Unbanded egg%
[HE]
(0



-0.8 0.6



Figure 2-4. Biplot of clutch survival parameters (solid lines) and organochlorine
pesticide variables (dashed lines) for clutches of alligator eggs collected from
Emeralda Marsh during summer 2000-2002.



















Fecundity



OX%/o Egg Mass



NOC y\




(D0 [PDDT]
0 Clutch Mass

-1.0 1.0



Figure 2-5. Biplot of egg and clutch size parameters (solid lines) and organochlorine
pesticide variables (dashed lines) for clutches of alligator eggs collected from
Lake Lochloosa during summer 2001 and 2002.














CHAPTER 3
MATERNAL TRANSFER OF ORGANOCHLORINE PESTICIDES

Studies have documented organochlorine pesticide (OCP residues) in eggs and/or

somatic tissues of several crocodilian species including the American alligator, Alligator

mississippiensis (Heinz et al., 1991), Morelet's crocodile, C. moreletti (Wu et al., 2000a),

the American crocodile, Crocodylus acutus (Hall et al., 1979; Wu et al., 2000b), and the

Nile crocodile, C. niloticus (Skaare et al., 1991). Indeed, alligator populations inhabiting

Lake Apopka, where an OCP spill occurred in the 1980s, and other central Florida lakes

contaminated with OCPs (through historic OCP use) produce eggs that contain

concentrations of total OCPs that are over 100 times higher than concentrations found in

eggs from reference lakes (Gross, unpublished data). In addition, the alligator

populations inhabiting the OCP-contaminated lakes experience increased (and highly

variable) rates of embryonic mortality, leading to reduced clutch success, and juvenile

alligators appeared to have abnormal sex hormone concentrations as compared to those of

reference sites (Masson, 1995; Rice, 1996; Woodward et al., 1993). However, a clear

dose-response relationship has not been established with respect to individual or total

OCP concentrations in egg yolks and reduced clutch success (Heinz et al., 1991). The

lack of a clear dose-response suggests other factors (e.g., diet, population dynamics, and

specific OCP mixtures) might be involved and/or that developmental effects result from

altered maternal physiology resulting from OCP exposure, as opposed to direct

embryotoxicity.









With respect to altered maternal physiology, alterations in steroid hormone levels

have also been shown in alligators inhabiting OCP-contaminated sites (Guillette et al.,

1994). Furthermore, maternal exposure suggests that OCPs may be maternally

transferred from the adult female alligator to her offspring, as has been reported in other

oviparous vertebrates (Russell et al., 1999). Assuming OCPs are maternally transferred,

the possibility exists that yolks could be used as predictors of maternal exposure. A

noninvasive method such as this would aid ecological risk assessments in understanding

exposure levels for rare/endangered crocodilian species without having to capture and/or

remove adults from the breeding population. Therefore, the objectives of the present

study were to examine maternal transfer as a potential route for embryonic OCP

exposure, and to evaluate the use of yolk burdens for predicting OCP burdens in maternal

tissues in alligators. Our hypothesis was that OCP burdens in maternal tissues and yolks

would be strongly correlated, which would allow yolk burdens to be used to predict

maternal body burdens and suggest maternal transfer of OCPs as the major route for

embryonic OCP exposure.

Materials and Methods

Site descriptions

Lakes Apopka (N 280 35', W 810 39'), Griffin (N 280 53', W 81 49'), and

Lochloosa (N 290 30', W 820 09') in Florida were selected as collection sites because

prior studies by our laboratory indicate vastly different levels of OCP exposure across

these sites. All three lakes are part of the Ocklawaha Basin. Lake Lochloosa (which is

connected to Orange Lake) was selected as a low exposure (reference) site. Four years

(1999-2002) of data indicate mean total OCP concentrations in egg yolks from the

reference sites (Lakes Orange and Lochloosa) were 231 30 ppb (mean standard









deviation [SD], n = 56 clutches) with a concurrent mean clutch viability rate (number of

live hatchlings/total number of eggs in a nest) of 71 21% (Gross, unpublished data).

Lake Griffin was selected as an intermediate exposure site since yolk concentrations

averaged 4,414 + 617 ppb (n = 47 clutches) and Lake Apopka was selected as a high

exposure site since yolk concentrations averaged 15,911 + 1,786 ppb (n = 42) for the

same time period (Gross, unpublished data). Furthermore, mean clutch viability rates

during this time period for Lakes Apopka (51 31%, n = 42) and Griffin (44 33%, n =

47) have been below rates observed for the reference site.

Animal Collections

Adult female alligators and their corresponding clutches of eggs were collected

from Lakes Apopka (n = 4), Griffin (n = 8), and Lochloosa (n = 3) over the course of two

nesting seasons (June 2001 and June 2002). Nests were located by aerial survey

(helicopter) and/or from the ground airboatt). Once nests were located, all eggs were

collected, and the nest cavity was covered. A snare-trap was set perpendicular to the tail-

drag in order to capture the female as she crossed over the nest. After the traps were set,

one member of the trapping crew subsequently transported the eggs to the Florida Fish

and Wildlife Conservation Commission's Wildlife Research Unit (FWC; Gainesville, FL,

USA) and placed the eggs in a temperature-controlled incubator. Snare-traps were

checked later in the evening and early the next morning.

Trapped females were secured and transported from each lake to the United States

Geological Survey's Florida Integrated Science Center (USGS; Gainesville, FL, USA).

Upon arrival, the animals were weighed, measured, and blood samples were collected

from the post-occipital sinus. Adult alligators were then euthanized by cervical

dislocation followed by double pithing. A full necropsy was performed on each female.









Bile, liver, adipose (composite of abdominal fat and the abdominal fat pad), and tail

muscle samples were collected for later determination of OCP burdens. Liver, adipose

tissue, and muscle were wrapped in aluminum foil, while bile and blood were placed in

scintillation vials. All samples were grouped according to nest identification number

(ID), placed in plastic bags labeled with the appropriate ID, and stored in a -80 C

freezer. Each female's corresponding clutch of eggs was then transferred from FWC to

USGS where yolk samples were collected (two eggs/clutch) and stored with the

corresponding maternal tissues. The remaining eggs were set for incubation in a

temperature/humidity-controlled incubator (31-33 C, 88-92% relative humidity) located

at USGS.

Analysis of OCPs in Maternal Tissues and Yolk

Analytical grade standards for the following compounds were purchased from the

sources indicated: aldrin, alpha-benzene hexachloride (a-BHC), P-BHC, lindane, 6-BHC,

p,p '-dichlorodiphenyldichloroethane (p,p '-DDD), p,p '-dichlorodiphenyldichloroethylene

(p,p '-DDE), dichlorodiphenyltrichloroethane (p,p '-DDT), dieldrin, endosulfan,

endosulfan II, endosulfan sulfate, endrin, endrin aldehyde, endrin ketone, heptachlor,

heptachlor epoxide, hexachlorobenzene, kepone, methoxychlor, mirex, cis-nonachlor,

and trans-nonachlor from Ultra Scientific (Kingstown, RI, USA); cis-chlordane, trans-

chlordane, and the 525, 525.1 polychlorinated biphenyl (PCB) Mix from Supelco

(Bellefonte, PA, USA); oxychlordane from Chem Service (West Chester, PA); o,p '-

DDD, o,p '-DDE, o,p '-DDT from Accustandard (New Haven, CT, USA); and toxaphene

from Restek (Bellefonte, PA, USA). All reagents were analytical grade unless otherwise

indicated. Water was doubly distilled and deionized.









Adipose, liver, bile, and yolk samples were analyzed for OCP content using

methods modified from Holstege et al. (1994 and Schenck et al. (1994). For extraction, a

2 g tissue sample was homogenized with ~1 g of sodium sulfate and 8 mL of ethyl

acetate. The supernatant was decanted and filtered though a Btichner funnel lined with

Whatman #4 filter paper (Fisher Scientific, Hampton, NH, USA) and filled to a depth of

1.25 cm with sodium sulfate. The homogenate was extracted twice with the filtrates

collected together. The combined filtrate was concentrated to ~2 mL by rotary

evaporation, and then further concentrated until solvent-free under a stream of dry

nitrogen. The residue was reconstituted in 2 mL of acetonitrile. After vortexing (30 s),

the supernatant was applied to a C 18 solid phase extraction (SPE) cartridge (pre-

conditioned with 3 mL of acetonitrile; Agilent Technologies, Wilmington, DE, USA) and

was allowed to pass under gravity. This procedure was repeated twice with the combined

eluent collected in a culture tube. After the last addition, the cartridge was rinsed with 1

mL of acetonitrile which was also collected. The eluent was then applied to a 0.5 g NH2

SPE cartridge (Varian, Harbor City, CA, USA), was allowed to pass under gravity, and

collected in a graduated conical tube. The cartridge was rinsed with an additional 1 mL

portion of acetonitrile which was also collected. The combined eluents were

concentrated under a stream of dry nitrogen, to a volume of 300 [tL, and transferred to a

gas chromotography (GC) vial for analysis.

Whole blood was analyzed for OCP content using methods modified from

Guillette et al. (1999). A 10 mL aliquot was transferred from the homogenized bulk

sample and extracted in 15 mL of acetone by vortex mixer. The mixture was centrifuged

for 5 min at 3000 rpm, after which the supernatant was transferred to a clean culture tube.









This process was repeated with the supernatants collected and concentrated under a

stream of dry nitrogen until solvent-free. The residue was re-extracted in 11.5 mL of 1:1

methylene chloride-petroleum ether. After mixing, the sample was allowed to settle and

the upper layer was transferred to a clean culture tube. This extraction was performed

twice with the extracts collected together. The combined extracts were then applied to a

prepared florisil cartridge (5 mL Fisher PrepSep, Fisher Scientific, Hampton, NH, USA).

The cartridge had been prepared by filling the reservoir to a depth of 1.25 cm with

anhydrous sodium sulfate and by prewashing the modified cartridge with 10 mL of 2:1:1

acetone: methylene chloride: petroleum ether. After the sample passed under gravity

with the eluent collected in a 15-mL graduated conical tube, the cartridge was eluted with

4 mL of the 2:1:1 solvent mixture which was also collected. The combined eluents were

concentrated under a stream of dry nitrogen, to a volume of 300 [tL, and transferred to a

GC vial for analysis.

GC/MS Analysis

Analysis of all samples was performed using a Hewlett Packard HP-6890 gas

chromatograph (Wilmington, DE, USA) with a split/splitless inlet operated in splitless

mode. The analytes were introduced in a 1 [iL injection and separated across the HP-5MS

column (30 m x 0.25 mm; 0.25 [tm film thickness; J & W Scientific, Folsom, CA, USA)

under a temperature program that began at 600 C, increased at 10 C/min to 2700 C, was

held for 5 min, then increased at 250 C/min to 3000 C and was held for 5 min. Detection

utilized an HP 5973 mass spectrometer in electron impact mode. Identification for all

analytes and quantitation for toxaphene was conducted in full scan mode, where all ions

are monitored. To improve sensitivity, selected ion monitoring was used for the









quantitation for all other analytes, except kepone. The above program was used as a

screening tool for kepone which does not optimally extract with most organochlorines.

Samples found to contain kepone would be reextracted and analyzed specifically for this

compound.

For quantitation, a five-point standard curve was prepared for each analyte (r2 >

0.995). Fresh curves were analyzed with each set of twenty samples. Each standard and

sample was fortified to contain a deuterated internal standard, 5 [iL of US-108 (120

[g/mL; Ultra Scientific), added just prior to analysis. All samples also contained a

surrogate, 2 [g/mL of tetrachloroxylene (Ultra Scientific) added after homogenization.

Duplicate quality control samples were prepared and analyzed with every twenty samples

(typically at a level of 1.00 or 2.50 [g/mL ofy-BHC, heptachlor, aldrin, dieldrin, endrin,

andp,p '-DDT) with an acceptable recovery ranging from 70 130%. Limit of detection

ranged from 0.1-1.5 ng/g for all OCP analytes, except toxaphene (120-236 ng/g), and

limit of quantitation was 1.5 ng/g for all analytes, except toxaphene (1500 ng/g).

Repeated analyses were conducted as allowed by matrix interference and sample

availability.

Data Analysis

OCP concentrations in maternal tissues and egg yolks were lipid-adjusted (wet

weight concentration / proportion of lipid in tissue), and lipid-adjusted tissue-to-egg yolk

ratios (maternal tissue OCP concentrations /egg OCP concentrations) were examined.

Predictive models were determined by linear regression analysis of OCP concentrations

in yolk against those of maternal tissues (log-transformed wet weight concentrations).

Each model's ability to fit the data was evaluated by examining the p-value (a = 0.05),

the r2 value, and the residual plots (SAS Institute Inc., 2002). ANOVA was used for









inter-site comparisons of adult female and clutch characteristics, and the Tukey test was

used for multiple comparisons among sites. The relationship between maternal mass (kg)

and concentrations of OCPs in eggs and maternal tissues (log-transformed wet weight

concentrations) were evaluated using linear regression to assess whether increasing mass

was associated with increasing concentrations of OCPs in eggs and maternal tissues,

which may suggest adult females continue to bioaccumulate OCPs as they grow

throughout their life. Adult females were grouped by site since the extreme differences

in OCP exposure among sites would likely confound results. Unless otherwise noted,

values are reported as mean standard deviation.

Results

Female Morphological and Reproductive Characteristics

For all females, mass and snout-vent length (SVL) averaged 74 20 kg (range:

44-114) and 135 11 cm (119-156), respectively. Clutch mass (mass of all eggs from a

single nest) and fecundity (number of eggs collected from a single nest) of these

individuals were 3.65 0.86 kg (1.84-4.82) and 43 10 eggs/nest (19-56), respectively.

No significant differences were detected across sites with respect to female mass (p =

0.14), total length (p = 0.90), SVL (p = 0.25), tail girth (p = 0.98), head length (p = 0.55),

clutch mass (p = 0.23), or fecundity (p = 0.40, Table 1).

With respect to lipid concentrations in egg yolk and muscle, no significant

differences were detected across sites (p > 0.05). However, lipid concentration in liver of

Lochloosa females was significantly higher (p < 0.05) than that of Apopka and Griffin

females (which were not significantly different from one another). Furthermore, lipid

concentration in abdominal adipose tissue of Apopka females was significantly less (p <

0.05) than that of Lochloosa and Griffin females (Table 1).










OCP concentrations in Yolk

Egg yolks from Lake Apopka females contained the highest total OCP

concentration (15,108 13,704) and greatest number of individual OCPs detected above

the limit of quantitation (n = 18) with p,p'-DDE (66%) and toxaphene (32%) being main

constituents. Lake Griffin females produced eggs with the next highest total OCP

burdens (393 300 ng/g; n = 13) being mainly composed of p,p'-DDE (69%), trans-

nonachlor (10%), and dieldrin (7%). Lake Lochloosa females produced egg yolks with

the smallest total OCP burden (124 53 ng/g, n = 9), with main constituents being p,p'-

DDE (73%), trans-nonachlor (10%), and cis-nonachlor (4%; Table 3-2). The OCP

analytes with the highest average egg yolk concentrations were toxaphene (4,862 + 4,177

ng/g), which was detected above the limit of quantitation in 3 of 15 clutches, followed by

p, p'-DDE (2,828 5,968 ng/g), dieldrin (191 474 ng/g), and trans-nonachlor (126 +

209 ng/g), which were above quantitation limit in all 15 clutches.

OCP concentrations in maternal tissues

Adipose tissue (a composite of abdominal fat and fat pad) contained the highest

concentration of total OCPs (12,805 31,678 ng/g wet weight) of all tissues. p,p'-DDE

(67%) composed the majority of the total burden, followed by dieldrin (5%), and trans-

nonachlor (3%). Although toxaphene was only detected in 3 individuals from Lake

Apopka, its average burden in adipose tissue was 13,463 1,267 ng/g (Table 3-2). In

liver, OCP analytes were detected above the quantitation limit in 9 of 15 individuals, and

total OCP concentrations averaged 1,008 1,245 ng/g. Liver burdens were primarily

composed of p,p'-DDE (76%) and dieldrin (6%). Total OCP concentrations in muscle

averaged 716 1,053 ng/g and were above quantitation limits in 10 of 15 individuals









with most of the burden being composed of p,p'-DDE (83%), dieldrin (6%) and trans-

nonachlor (6%). Total OCP burdens in bile (412 483 ng/g) were above quantitation

limits in five individuals with p,p'-DDE (86%) and dieldrin (6%) comprising the majority

of the burden. Total OCP concentrations in blood (43 + 21 ng/g) were above quantitation

limits in 4 individuals with p,p'-DDE (64%) and dieldrin (14%) comprising most of the

burden. Overall, Lake Apopka alligators exhibited the highest OCP concentrations in

maternal tissues and egg yolks, followed by Lakes Griffin and Lochloosa, respectively

(Table 3-2).

Relationships between Maternal Tissue and Yolk Burdens

Examination of lipid-adjusted maternal tissue-to-egg yolk burdens showed

differences among tissues. With respect to total OCPs, the adipose burden-yolk burden

ratio was close to 1 (95% confidence interval (CI), 0.76 K< K < 1.11). In contrast, the

liver-yolk ratio was significantly greater than 1 (95% CI, 1.49 < [ < 9.19), and muscle

ratios showed considerable variation (95% CI, -1.17 < K < 37.35). As would be expected,

most individual OCPs followed the above trend. However, cis-chlordane was an

exception as liver ratios (95% CI, 2.85 < [ < 6.75) and muscle ratios (95% CI, 1.78 < [ <

15.1) were greater than 1, while adipose ratios (95% CI, 0.59 < [ < 0.84) were less than

1. With respect to total OCP concentrations, significant linear relationships (predictive

models) were found for adipose, liver, muscle, and bile (p < 0.05, Fig. 1). With respect to

individual OCP analytes, predictive models were derived for 12 of 14 (78%) of the OCPs

co-detected in adipose tissue and egg yolk, followed by liver (9/12, 75%), bile (8/11,

73%), and muscle (2/12, 17%; Table 3-3). Although nine OCP analytes were

concurrently detected in blood of the females and their respective egg yolks, no

significant linear correlations were detected (p > 0.05).









As for individual OCP analytes, p,p'-DDE concentrations in yolk was significantly

correlated with those of liver, muscle, bile, and adipose tissue. Blood p,p'-DDE

concentrations did not exhibit a significant linear relationship (p > 0.05) with yolk p,p'-

DDE concentrations. Heptachlor epoxide, trans-chlordane, cis-chlordane, trans-

nonachlor, cis-nonachlor, mirex, and dieldrin concentrations in yolk were significantly

correlated to their respective concentrations in adipose, liver, and bile. With respect to

oxychlordane, significant correlations were only derived for liver and adipose tissue, and

significant correlations for p,p'-DDD concentrations were found only for adipose and

bile. Toxaphene and o,p'-DDT concentrations in adipose tissue were significantly

correlated with respective egg yolk concentrations (Table 3-3).

Relationships between Maternal Mass and OCP concentrations in Eggs and Tissues

For females collected from Lakes Apopka (n = 4) and Lochloosa (n = 3), no

significant correlations (p > 0.05) were found when maternal mass (kg) was compared

against either individual or total OCP concentrations (log-transformed wet weight) in

maternal tissues and eggs. However, significant correlations might have been difficult to

detect because of the small sample size. In contrast, a larger number of Lake Griffin

females (n = 8) were collected, and analyses indicated significant correlations between

maternal mass and OCP concentrations in tissues and eggs indicating that larger females

have higher concentrations of OCPs in their tissues and eggs, which may suggest females

continue to bioaccumulate OCPs as they grow (increase in mass). For Lake Griffin

females, OCP burdens in eggs had the greatest number of significant correlations (p <

0.05) with body mass (kg), which consisted of cis-nonachlor (r2 = 0.87), cis-chlordane (r2

= 0.75), trans-nonachlor (r2 = 0.73), dieldrin (r2 = 0.69), p,p'-DDE (r2 = 0.66), o,p'-DDT

(r2 = 0.61), heptachlor epoxide (r2 = 0.59), oxychlordane (r2 = 0.58), trans-chlordane (r2 =









0.57), and total OCPs (r2 = 0.71). Following egg concentrations, abdominal fat OCP

burdens-to-body mass correlations consisted of cis-nonachlor (r2 = 0.67), cis-chlordane

(r2 = 0.81), trans-nonachlor (r2 = 0.63), dieldrin (r2 = 0.62), p,p'-DDE (r2 = 0.58),,

heptachlor epoxide (r2 = 0.53), oxychlordane (r2 = 0.51), and total OCPs (r2= 0.64).

Although egg burdens of o,p'-DDT and trans-chlordane were correlated with body mass,

abdominal fat burdens were not. Lastly, liver OCP burdens-to-body mass correlations

included only trans-nonachlor (r2= 0.99) and p,p'-DDT (r2= 0.99). No significant

correlations were found for cis-chlordane, trans-chlordane, oxychlordane, dieldrin,

heptachlor epoxide, o,p'-DDT, and cis-nonachlor.

Discussion

The presence of OCPs in the eggs and tissues of alligators is not novel; however,

the value of our study was that OCP concentrations in maternal tissues and yolks

appeared to be strongly correlated with one another, allowing yolk burdens to be used as

predictors of OCP burdens in tissues of adult reproductive alligators, which may be a

useful noninvasive technique that would aid risk assessments involving endangered

crocodilians. Furthermore, our results are consistent with other studies that suggest OCPs

are maternally transferred in wild alligators (Rauschenberger et al., 2004).

Several OCP analytes were detected in both maternal tissues and yolk (Table 3-3)

suggesting that mixture composition may be an important consideration in risk

assessment. One reason for this is that different xenobiotic compounds may induce or

inhibit certain biotransformation enzymes. Specifically, alligators from Louisiana

express several different xenobiotic biotransformation enzymes (e.g., liver cytochrome P-

450 enzymes [CYP] such as CYP1A, CYP2B) in response to xenobiotic exposure (Ertl et

al., 1999). Furthermore, genetic partitioning has been reported in spatially separated









alligator populations (Ryberg et al., 2002). Therefore, the possibility exists that certain

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

particular biotransformation enzyme, which may lead to increased risk of xenobiotic-

induced toxicity. For example, certain populations of black-banded rainbowfish

(Melanotaenia nigrans) were able to tolerate copper exposures (96-hr EC50) that were 8.3

fold greater than the tolerance limits of other, spatially-separated populations of the same

species. Genetic analyses suggested that allozyme frequencies of tolerant and susceptible

populations were significantly different at AAT-1 and GPI-1 loci, suggesting differences

in allozymes of exposed fish may have assisted in the increased copper tolerance

(Woosley, 1996).

Examination of maternal tissue-to-egg concentration ratios (lipid-adjusted) showed

differences among tissues. The adipose-to-yolk concentration ratio was close to 1,

suggesting that OCPs reach equilibrium within abdominal adipose tissue, and that lipids

and OCPs are mobilized and subsequently incorporated into the developing yolks. In

contrast, liver-to-yolk concentration ratios were significantly greater than 1, and muscle-

to-yolk concentration ratios showed considerable variation. One suggested explanation

for the high liver-to-yolk ratios relates to one major function of the liver cells

hepatocytess), which is to accumulate and convert hydrophobic xenobiotics into

hydrophilic metabolites to facilitate detoxication, excretion, and elimination. In addition,

the low lipid content of liver (relative to the lipid content of adipose tissue and yolk,

Table 3-1) may have contributed to the marked differences. With respect to the muscle-

to-yolk ratios, the reasons for the large degree of variability are not as clear. One

possible explanation is that muscle lipids are not mobilized during yolk formation and, as









a result, OCP burdens may continually accumulate in muscle lipids. Another potential

explanation relates to the low lipid content of muscle when compared to yolk (Table 3-1).

Lastly, cis-chlordane's exceptional liver, muscle, and adipose ratios underscore the fact

that different OCP analytes may not always exhibit identical pharmacokinetics.

When compared to other vertebrates, adipose tissue-to-egg ratios in alligators are

similar to those reported in the freshwater catfish, Clarias batrachus, in that adipose-to-

egg ratios are approximately equal to 1. Furthermore, C. batrachus mobilizes lipids from

its abdominal adipose tissue during vitellogenesis (Lal & Singh, 1987), similar to what

this study suggests occurs in the American alligator. In contrast to adipose tissue OCP

concentrations, muscle-to-egg OCP ratios in alligators appear to be quite different from

fish. Alligator muscle-to-egg ratios were highly variable and, for the most part, greater

than 1, while fish ratios appear to be consistently close to 1. With respect to more closely

related species, muscle-to-egg OCP ratios are similar to those reported for the common

snapping turtle (Chelydra serpentina) and several bird species with ratios exhibiting a

great deal of variability and being greater than 1 (Russell et al., 1999). These differences

suggest that fish differ from terrestrial vertebrates in regards to lipid content of muscle

and/or lipid mobilization strategy (during vitellogenesis), which could lead to differences

in embryonal exposure given equivalent maternal exposure.

Evaluation of Predictive Models

Although significant linear models were found for most tissues with respect to total

OCP concentrations, caution should be used in the application of these "total OCP"

models since it is probable that the concentrations and ratios of individual OCP analytes

may vary across different locations. The greatest number of predictive linear models was

derived for adipose tissue. This was not surprising considering that (for most analytes)









adipose-yolk lipid normalized ratios were close to 1. Next, with respect to the number of

significant linear models, were liver and bile. The similarities between liver and bile

should be expected since the liver produces bile, which transports OCP analytes to the

intestinal lumen, leading to their eventual elimination from the body. However, OCP

analytes may be reabsorbed from the intestine and redirected back to the liver via the

portal vein through a process known as enterohepatic circulation, which may delay

elimination of lipophilic xenobiotics, increase hepatic exposure and bioaccumulation

(Stenner et al., 1997). For OCP concentrations in muscle, regression analysis indicated

that only two out of 12 mutually detected analytes could be predicted using OCP

concentrations in eggs. Lastly, nine OCP analytes were concurrently detected in blood

and egg yolk with none exhibiting significant relationships. Possible explanations for the

few significant linear relationships include the low lipid content of these tissues and thus

the relatively low concentrations of OCP analytes in these tissues, as well as the

possibility that each of these tissue burdens exhibit a nonlinear relationship with yolk

burdens. In addition, blood samples were collected after the female had oviposited.

Since blood was collected after eggs were excreted from the body, it is likely that the

overall maternal body burden decreased, which would in turn lower the steady-state OCP

concentrations in blood.

As for individual OCP analytes, predictive models for p,p'-DDE were derived for

four of the five maternal tissues. One likely reason for this is that p,p'-DDE was detected

in considerable concentrations in all eggs and in almost all tissues for all 15 females.

Similarly, predictive models were derived for commonly detected analytes such as

heptachlor epoxide, trans-chlordane, cis-chlordane, trans-nonachlor, cis-nonachlor,









mirex, and dieldrin for most tissues. Somewhat surprisingly, oxychlordane (a metabolite

of cis- and trans-chlordane) and p,p'-DDD (an intermediate metabolite of p,p'-DDT)

showed significant linear models only with respect to liver and adipose tissue. The fact

that linear models for toxaphene and o,p'-DDT were derived only for adipose tissue was

likely related to their low concentrations and infrequent detections in other tissues (Table

3-3).

Relationships between Maternal Mass and OCP concentrations in Eggs and Tissues

Although a portion of a female alligator's OCP body burden may be eliminated

through egg deposition, adult female alligators from Lake Griffin had increased OCP

concentrations in their tissues and eggs as they increased in mass, similar to size-related

OCP bioaccumulation in smallmouth bass inhabiting contaminated sites in Michigan

(Henry et al., 1998). Corresponding increases in OCP burdens and mass indicate that

larger and possibly older females accumulate OCPs faster than they can excrete them. In

addition, the relationship between OCP burdens in eggs and body mass was very similar

to the relationship between abdominal fat burdens and body mass.

The correlation between OCP burdens in liver and body mass was significant for

trans-nonachlor and p,p'-DDT; however the major metabolites of these compounds

(oxychlordane and p,p'-DDE, respectively) were not significantly correlated with body

mass. These results contrast those of egg and abdominal fat burdens and suggest that that

alligator liver may not sequester OCP metabolites to the same extent as abdominal fat or

egg.

Maternal body burdens: Toxicological Implications

Although our study's objective was to evaluate maternal transfer and prediction of

the maternal OCP body burdens carried by the American alligator, we would be remiss if









we did not discuss whether these reported body burdens were capable of eliciting harmful

effects. Although several studies report body and egg burdens in crocodilians, relatively

few studies directly relate body and egg burdens to acute toxicological effects (Campbell,

2003), so we will briefly discuss how p,p'-DDE burdens in maternal alligator liver

compare to reported p,p'-DDE burdens in liver of birds (birds were not from the present

study areas) that have been associated with mortality (Blus, 1996).

In previous studies, mean DDE liver residues in birds which died due to DDT

exposure ranged from 19,000-55,000 ng/g. When birds were exposed to DDE alone,

liver residues of dead birds averaged 3,883,000 ng/g (range 460,000-11,725,000 ng/g)

(Blus, 1996). When compared to the liver residues of the most contaminated alligators

(Lake Apopka, upper 95% CI < 7,000 ng/g), it appears that death due to DDT/DDE

exposure might be unlikely assuming bird and alligator susceptibilities are similar.

However, since p,p'-DDE liver concentrations in alligators are almost half of lethal liver

concentrations in birds, there is reason for some concern. In addition, the assumption that

bird and alligator susceptibilities are similar might be argued as unfounded considering

the variability in toxic responses between individuals of the same species, different

species, and different vertebrate classes (James et al., 2000). To account for these

uncertainties the risk assessment process identifies the different sources of uncertainty

and incorporates the uncertainty in attempting to determine a "safe" tissue concentration

based on levels associated with no adverse effects (NOAEL) or lowest observed adverse

effect levels (LOAEL). Typically, interspecies extrapolation is assigned an uncertainty

factor of 10, as are inter-individual uncertainty, uncertainty related to comparing different

study designs (e.g., acute doses related to experimental bird studies, in contrast to chronic









exposure studies in wild alligators), and uncertainty related to database quality since

DDE (p,p'-DDE + o,p-DDE) liver residues were reported, instead of p,p'-DDE. These

four uncertainty factors constitute an overall uncertainty factor of 10,000, which is an

order of magnitude greater than commonly used uncertainty factors (range: 300-1000)

(James et al,. 2000). Considering the high degree of uncertainty, we suggest that more

information is required before a "safe" level of p,p'-DDE exposure is determined for the

American alligator based upon actual or predicted liver concentrations.

Sublethal effects are another possible consequence of OCP exposure. For example,

exposure of the freshwater catfish, Clarias batrachus, to an OCP analyte (y-BHC) at

sublethal levels (2,000-8,000 ng/g) during vitellogenesis significantly decreased the

biosynthesis and mobilization of phospholipids from liver to the developing follicles (Lal

& Singh, 1987). Interestingly, alterations in fatty acid profiles of alligator eggs have

been associated with reduced clutch success. Specifically, fatty acid profiles from wild,

alligator eggs (normal hatch rates) showed considerable differences when compared to

those of eggs from captive alligators (reduced hatch rates). One suggested explanation

for this association between altered fatty acid profiles and reduced clutch success in

captive alligators was that certain fatty acids are critical for reproductive success and that

captive diets were deficient in essential fatty acids (Noble et al., 1993). Thus, the

possibility exists that exposure to OCPs may alter the liver's ability to synthesize

necessary fatty acids, leading to altered egg quality and decreased clutch success in wild

alligators that inhabit OCP-contaminated sites. Chronic exposure to low doses of OCPs

prior to and during vitellogenesis has been suggested as a cause for significant increases

in OCP concentrations in egg yolk, as well as significantly decreased hatch rates in









captive adult female alligators. Importantly, the doses did not appear to induce acute

toxicity in the adult females (Rauschenberger et al., 2004). Presently, we are using a

captive breeding population of adult alligators, as well as data from field studies, to

further evaluate the relationships between OCP exposure, altered fatty acid biosynthesis,

nutritional content of eggs, and embryonic mortality.

In summary, the significant levels of OCP analytes observed across such a wide

range of crocodilian species and geography suggests the need for a greater understanding

of xenobiotic metabolism and toxicological responses in crocodilians. Such

understanding would aid in the conservation of this ancient group by determining what

risks are posed by contaminants with respect to species survival and how contaminant-

related risks compare to other risks, such as habitat destruction. The results of the present

study provide some evidence suggesting that maternal transfer of OCP analytes is the

major route for embryonic exposure. In addition, it provides several models for the

prediction of OCP concentrations in maternal tissues of American alligators, which may

be extrapolated to other crocodilians. Hopefully, the present study will encourage new

investigations into the pharmacokinetics and pharmacodynamics of contaminants in other

crocodilian species.









Table 3-1. Morphological and reproductive characteristics of adult female alligators
collected during June 2001 and 2002 from Lakes Apopka, Griffin, and
Lochloosa in central Florida.
Parameter a,b Apopka Griffin Lochloosa
Number of females collected 4 8 3
Total Length (cm) 252 38 258 + 17 258 7
Snout-Vent Length (cm) 142 + 15 134 + 9 129 5
Mass (kg) 94 30 70 17 63 4
Clutch Mass (kg) 3.78 + 0.98 3.33 + 0.82 4.31 + 0.45
Fecundity (# eggs/clutch) 43 10 40 10 49 6
Lipid % Adipose 47.0 32.5 B 78.1 + 8.0 A 81.4 + 4.0 A
Lipid % Liver 1.3 1.0 A 0.8 0.2 A 5.0 2.3 B
Lipid % Muscle 0.8 + 0.9 1.3 + 0.9 0.2 0.02
Lipid % Yolk 19.9 1.1 18.1 + 1.7 18.2 1.6
a Values represent mean standard deviation. b Different letters indicate significant
differences (p < 0.05).












Table 3-2. Pesticide concentrations (ng/g wet wt.) in tissues and yolks of adult female alligators collected during June 2001 and 2002
from Lakes Apopka, Griffin, and Lochloosa in central Florida.


Lake a
Apopka
(4)


Chemical b,c
Aldrin
a-BHC
(3-BHC
cis-Nonachlor
cis-Chlordane
6-BHC
Dieldrin
Endosulfan I
Endosulfan II
Endosulfan Sulfate
Endrin
Endrin Aldehyde
Endrin Ketone
y-BHC
Heptachlor
Heptachlor Epoxide
Hexachlorobenzene
Kepone
Methoxychlor
Mirex
o,p '-DDD
o,p '-DDE
o,p '-DDT
Oxychlordane
p,p '-DDD
p,p '-DDE
Toxaphene
trans-Nonachlor
Total OCP


Bile
X
X
X
10 + 3.2
4 1.9
X
38 + 10.2
X
X
X
X
3 0.4
X
X
X
3 2.1
1 0
X
X
2 2.6
X
X
2 0.2
7 1.3
2 0.2
806 341
X
21 + 7.8
900 369.7


Blood
X
X
X
2 0.4
1 0.4
X
5 0.4
X
X
X
X
X
X
X
X
0.3 0
1+ 0
X
X
X
X
X

1 0.2
1 0.2
42 5.7
X
3 0.4
55 7


Adipose
X
X
7.5 6.7
521 + 602.7
190 + 241.2
X
2,376 + 3,770.9
X
X
X
X
X
X
X
X
67 + 81.5
10
X
X
19 + 13.1
3 3.4
52 + 55.8
27 26.4
247 + 336.4
43 + 67.5
29,840 34,366
13,436 + 12,670.2
1,153 + 1,378.7
44,650 + 53,230


Liver
X
X
X
31 6.7
11 9.1
X
105 80.2
X
21
X
X
X
X
X
X
6 2.2
1 0.0
X
5
7+ 9.0
X
X
4 1.8
17 9.9
11 10.3
1,846 918.1
X
65 22.7
2,140 1,024


Muscle
X
X
X
23 18.3
14.1 11.7
X
68 48.3
X
X
X
X
X
X
X
8 11.7
4 3.6
1
X
X
1 0.4
X
X
4 2.2
12 10.7
17 8.0
1,392 1,0782
X
68 57.0
1,610 1,226


Yolk
1
X
2 1.4
123 81.9
62 59.2
X
663 803.0
X
X
X
X
X
X
X
1 0.04
26 15.0
1 0.0
X
X
7 7.1
X
45 17.7
17 7.8
75 68.2
52 61.4
9,994 8,529
4,862 4,177
387 277.7
15,108 13,704













Table 3-2. (Continued)


Lake
Griffin
(8)


Chemical
Aldrin
a-BHC
(3-BHC
cis-Nonachlor
cis-Chlordane
6-BHC
Dieldrin
Endosulfan I
Endosulfan II
Endosulfan Sulfate
Endrin
Endrin Aldehyde
Endrin Ketone
y-BHC
Heptachlor
Heptachlor Epoxide
Hexachlorobenzene
Kepone
Methoxychlor
Mirex
o,p '-DDD
o,p '-DDE
o,p '-DDT
Oxychlordane
p,p '-DDE
p,p'-DDT
Toxaphene
trans-Chlordane
trans-Nonachlor
Total OCP


Bile
X
X
X
4 2.4
2 0.5
X
13 4.9
X
X
X
X
X
X
X
X
3 3.3
10
X
X
0.3 0
X
X
1 0.2
7 4.9
54 25.7
1
X
1 0.3
9 5.9
87 46.4


Blood
X
X
X
1 0.4
10
X
7
X
X
X
5
X
2
X
X
1
1
X
X
1
X
X
1 0.0
X
13 9.1
13
X
10
1 0.09
31 28.4


Adipose
X
X
2.1 + 1.1
75 + 74.9
30 + 10.8
X
109 + 133.4
X
X
X
X
X
X
2
X
34 45.7
1+0
X
X
5+ 3.6
X
X
106.9
56 + 84
1,030 931.3
3 1.5
X
3 1.7
171 213.5
1,533 1,439


Liver
X
X
X
8 4.4
2 0.7
X
17 + 8.0
X
X
X
X
X
X
X
X
5 3.6
1 0
X
X
1
X
X
X
8 6.2
75 46.6
29 0.9
X
1 0.3
18 13.2
153 78.1


Muscle
X
X
X
9 10.6
3 3.4
X
22 20.8
X
X
X
X
X
X
X
2 1.4
10 12.0
1
X
X
1 0.5
X
X
2 0.1
16 17.8
131 132.4
X
X
1
28 36.0
208 227


Yolk
X
X
X
14 7.6
11 3.7
X
26 25.7
X
X
X
X
X
X
X
X
8 8.8
1 0.0
X
2
1 0.2
X
3
3 1.8
12 14.9
273 204.0
3
X
2 1.0
40 38.3
393 299












Table 3-2. Continued.
Lake Chemical Bile Blood Adipose Liver Muscle Yolk
Lochloosa Aldrin X X X X X X
(3) ca-BHC NA X X X X X
(3-BHC NA X 1 X X X
cis-Nonachlor NA X 17 + 1.8 1 X 5 1.9
cis-Chlordane NA X 8 + 1.4 X X 3 + 0.1
6-BHC NA X X X X X
Dieldrin NA X 14 4.7 2.6 1.4 4 2.8
Endosulfan I NA X X X 15.6 X
Endosulfan II NA X X X X X
Endosulfan Sulfate NA X X X X X
Endrin NA X X X X X
Endrin Aldehyde NA X X X X X
Endrin Ketone NA X X X X X
y-BHC NA X X X X X
Heptachlor NA X X X 18 +9.1 X
Heptachlor Epoxide NA X 11 9.6 X X 3 2.9
Hexachlorobenzene NA X X X X X
Kepone NA X X X X X
Methoxychlor NA X X X X X
Mirex NA X 2.6 X X X
o,p'-DDD NA X X X X X
o,p'-DDE NA X X X X X
o,p'-DDT NA X 3 0.2 X 7.1 1 0.0
Oxychlordane NA X 17 11.0 1 X 5 4.3
p,p'-DDD NA X 1 0.1 X 1 20.9
p,p'-DDE NA X 297 +90.1 20 +20.9 11 6.7 91 32.5
p,p'-DDT NA X 1.4 0.1 X 1.4 X
Toxaphene NA X X X X X
trans-Chlordane NA X 1 + 0.1 X 1.4 X
trans-Nonachlor NA X 38 + 24.6 2.6 1.4 12 + 8.8
Total OCP NA X 407 143.6 28 +32.6 33 33.9 124 53.3
a Number of females and clutches collected noted in parentheses beneath name of lake. b Values represent mean + standard deviation










[SD], values without SD indicate a single measurement. X indicates values which were below limit of detection (LOD) or below limit
of quantitation (LOQ) and NA indicates not analyzed. LOD ranged from 0.1-1.5 ng/g for most OCP analytes (toxaphene LOD ranged
from 120-236 ng/g), and LOQ ranged was 1.5 ng/g for all analytes except for toxaphene (1500 ng/g). Percent recovery ranged from
70-130%. The following chemicals were neither detected in females nor their eggs: a-BHC, 6-BHC, endosulfan sulfate, and kepone.
BHC = Benzene hexachloride; DDD = Dichlorodiphenyldichloroethane; DDE = Dichlorodiphenyldichloroethylene; DDT =
Dichlorodiphenyltrichloroethane; Total OCP = organochlorine pesticide concentrations for all analytes.











Table 3-3. Regression equations for predicting organochlorine pesticide (OCP)
concentrations in maternal tissues, where LOG [Tissue-OCP] = bo + b LOG
[Yolk-OCP].
Tissue Chemicala bo bi n r2 p
Adipose Dieldrin 0.6624 0.8785 15 0.87 < 0.0001
cis-Nonachlor 0.6737 0.9136 15 0.75 < 0.0001
cis-Chlordane 0.4037 0.9633 15 0.69 0.0001
Heptachlor Epoxide 0.6294 0.8134 14 0.62 0.0008
Mirex 0.8217 0.6030 6 0.89 0.0028
o,p -DDT 0.5840 0.6040 14 0.41 0.0141
Oxychlordane 0.6694 0.8544 15 0.80 <.0001
p,p'-DDD 0.2375 0.7 597 14 0.50 0.0046
p,p'-DDE 0.6968 0.9216 15 0.93 <.0001
Toxaphene 0.0880 1.0928 3 0.99 0.0486
trans-Chlordane 0.1733 0.9397 12 0.58 0.0041
trans-Nonachlor 0.6430 0.8960 15 0.84 < 0.0001
Bile Dieldrin -0.6196 0.9559 4 0.90 0.0494
cis-Nonachlor -0.3863 0.7646 5 0.97 0.0017
cis-Chlordane -0.4308 0.6314 5 0.83 0.0301
Heptachlor Epoxide -0.3207 0.6959 5 0.79 0.0435
p,p'-DDD -1.1407 1.0748 4 0.95 0.0246
p,p'-DDE -0.6385 0.9472 5 0.94 0.0057
trans-Nonachlor -0.2919 0.6867 5 0.96 0.0039
trans-Chlordane -0.2245 -0.4531 5 0.87 0.0220
Blood NSb
Liver Dieldrin 0.0248 0.7162 7 0.98 <0.0001
cis-Nonachlor -0.2471 0.8448 8 0.92 0.0002
cis-Chlordane -0.5557 0.8876 7 0.97 <0.0001
Heptachlor Epoxide -0.3878 0.8323 6 0.85 0.0084
Mirex -0.0547 0.9557 5 0.89 0.0155
Oxychlordane -0.2855 0.8123 7 0.92 0.0005
p,p'-DDE -0.7696 1.0156 10 0.93 <.0001
trans-Chlordane -0.0722 0.3300 7 0.94 0.0003
trans-Nonachlor -0.2854 0.8263 8 0.98 <.0001
Muscle p,p'-DDE -0.3733 0.8153 10 0.54 0.0160
Mirex 0.1816 -0.2797 0.96 0.0040
a BHC = Benzene hexachloride; DDD = Dichlorodiphenyldichloroethane; DDE =
Dichlorodiphenyldichloroethylene; DDT = Dichlorodiphenyltrichloroethane. b NS = no
significant linear regressions were determined for the 9 chemicals which were detected
both in blood and in yolk.











106
10 -

) 105

0 104
.2
"o
S 103 -
C)
0 102

0) 101 -
o
_J


C. y = -0.4817 + 0.8342x
(r2 = 0.89,p <0.05)













100 101 102 103 104
Log Total OCPs in Yolk (ng/g)


D. y = -0.0865 + 0.6688x
(r2 = 0.55,p <0.05)





O
O *

O
*0 0


100 101 102 103 104
Log Total OCPs in Yolk (ng/g)


Figure 3-1. Linear regressions of total organochlorine pesticide (OCP) concentrations in
maternal tissues against total OCP concentrations in egg yolks. A. Adipose
tissue. B. Liver. C. Bile. D. Muscle.


A. y= -1338.60 + 3.318x
(r2 = 0.95,p <0.05)


100 101 102 103 104 105


100 101 102 103 104 105













CHAPTER 4
MATERNAL FACTORS ASSOCIATED WITH DEVELOPMENTAL MORTALITY
IN THE AMERICAN ALLIGATOR

Recent data suggested maternal organochlorine pesticide (OCP) body burdens and

OCP egg yolk concentrations are significantly correlated, and that significant

relationships between maternal size and maternal body burdens exist. Maternal age and

size has also been shown to have a strong relationship with clutch viability (number of

live hatchings / total number of eggs) and clutch size characteristics (i.e., fecundity,

clutch mass). Specifically, females between 15 and 30 years old (~ 2.3-2.8 m in total

length) produce larger clutches (35-40 eggs / clutch) with increased clutch viability

compared to younger females, which themselves produce smaller clutches (15-25 eggs)

with smaller eggs and have decreased clutch viability. Females older than 30 years tend

to produce clutches similar to 15-30 year old females, with the only exception being

smaller clutches (15-25 eggs) (Ferguson, 1985). Therefore, female size or age may be a

confounding factor when examining the relationship between OCP burdens in yolk and

reproductive performance. In addition, age (or size) and maternal OCP exposure could

cause interactive effects. For example, females of optimum reproductive age may be

more resistant to effects of OCPs; while, younger (or older) females may show increased

susceptibility. Therefore, the objective of the present study was to test the hypotheses

that reproductive efficiency, clutch viability, and mortality rates are significantly

correlated with maternal OCP body burdens, maternal size, or both; and (2) that clutch

size characteristics are significantly correlated with maternal OCP body burdens,

maternal size, or both.









Materials and Methods

The greatest difficulty in examining the relationship between maternal age and

OCP exposure and effects is that determining the age of an alligator requires either long

term monitoring or counting the rings that form in the femur as a result of annual calcium

deposition (Ferguson, 1985). However, this technique is not valid for reproductive

females since femoral bone resorption provides calcium necessary for eggshell formation

and egg yolk nutrition, and subsequently causes the removal of "bone rings" and

underestimation of age (Elsey & Wink, 1985; Wink & Elsey, 1986). In addition,

removing an alligator's limb simply to age it is ethically unacceptable. Given these

difficulties with assigning a chronological age, female size will be used lieu of age. One

potential limitation in using female size as an indicator of age class is that female growth

rates between lakes may differ since dietary composition has been suggested to differ

among OCP-contaminated sites and reference sites (Rice, 2004). Therefore, the

possibility exists that a female from a reference site may be smaller than one from a

contaminated site, even though both are of the same age. This is important since age, in

addition to size, has been shown to be an important determinant of sexual maturity in

alligators. Indeed, alligator ranchers are able to accelerate growth so that a female may

reach six feet in length in 3-4 years, however, these females do not seem to be able to

reproduce until they reach 8-10 years of age (Ferguson, 1985). To control for potential

confounding due to differential growth rates, relationships between female size and OCP

burdens and clutch viability will be evaluated using site and year as covariates. If the

effects of covariates are determined statistically negligible, female data will be grouped

together.









Site Descriptions

Lakes Apopka (N 280 35', W 810 39'), Griffin (N 280 53', W 81 49'), and

Lochloosa (N 290 30', W 820 09') in Florida were selected as collection sites because

prior studies by our laboratory indicate vastly different levels of OCP exposure across

these sites. All three lakes are part of the Ocklawaha Basin. Lake Lochloosa (which is

connected to Orange Lake) was selected as a low exposure (reference) site. Three years

(2000-2002) of data indicate mean total OCP concentrations in egg yolks from the

reference sites (Lakes Orange and Lochloosa) were 102 16 ppb (mean standard

deviation [SD], n = 19 clutches) with a concurrent mean clutch viability rate (number of

live hatchlings/total number of eggs in a nest) of 70 4% (Gross, unpublished data).

Lake Griffin was selected as an intermediate exposure site since yolk concentrations

averaged 1169 423 ppb (n = 42 clutches) and Lake Apopka was selected as a high

exposure site since yolk concentrations averaged 7,582 2,008 ppb (n = 23) for the same

time period (Chapter 2). Furthermore, mean clutch viability rates during this time period

for Lakes Apopka (52 6%, n = 23) and Griffin (43 5%, n = 42) have been below rates

observed for the reference site.

Animal Collections

Adult female alligators and their corresponding clutches of eggs were collected

from Lakes Apopka (n = 19), Griffin (n = 18), and Lochloosa (n = 3) over the course of

four nesting seasons (June 1999 to June 2002). Nests were located by aerial survey

(helicopter) and/or from the ground airboatt). Once nests were located, all eggs were

collected, and the nest cavity was covered. A snare-trap was set perpendicular to the tail-

drag in order to capture the female as she crossed over the nest. After the traps were set,

one member of the trapping crew subsequently transported the eggs to the Florida Fish









and Wildlife Conservation Commission's Wildlife Research Unit (FWC; Gainesville, FL,

USA) and placed the eggs in a temperature-controlled incubator. Snare-traps were

checked later in the evening and early the next morning.

In 1999 and 2000, trapped females were secured and measurements (total length,

snout-vent length, head length and tail girth) were collected along with a blood sample

and a scute for OCP analysis. These females were then immediately released. In 2001

and 2002, females were captured and transported from each lake to the United States

Geological Survey's Florida Integrated Science Center (USGS; Gainesville, FL, USA).

Upon arrival, the animals were weighed, measured, and blood samples were collected

from the post-occipital sinus. Adult alligators were then euthanized by cervical

dislocation followed by double pithing. A full necropsy was performed on each female.

Bile, liver, adipose (composite of abdominal fat and the abdominal fat pad), and tail

muscle samples were collected for later determination of OCP burdens. Liver, adipose

tissue, and muscle were wrapped in aluminum foil, while bile and blood were placed in

scintillation vials. All samples were grouped according to nest identification number

(ID), placed in plastic bags labeled with the appropriate ID, and stored in a -80 C

freezer. Each female's corresponding clutch of eggs was then transferred from FWC to

USGS where yolk samples were collected (two eggs/clutch) and stored with the

corresponding maternal tissues. The remaining eggs were set for incubation in a

temperature/humidity-controlled incubator (31-33 C, 88-92% relative humidity) located

at USGS.

Analysis of OCPs in Maternal Tissues and Yolk

Analytical grade standards for the following compounds were purchased from the

sources indicated: aldrin, alpha-benzene hexachloride (a-BHC), P-BHC, lindane, 6-BHC,









p,p '-dichlorodiphenyldichloroethane (p,p '-DDD), p,p '-dichlorodiphenyldichloroethylene

(p,p '-DDE), dichlorodiphenyltrichloroethane (p,p '-DDT), dieldrin, endosulfan,

endosulfan II, endosulfan sulfate, endrin, endrin aldehyde, endrin ketone, heptachlor,

heptachlor epoxide, hexachlorobenzene, kepone, methoxychlor, mirex, cis-nonachlor,

and trans-nonachlor from Ultra Scientific (Kingstown, RI, USA); cis-chlordane, trans-

chlordane, and the 525, 525.1 polychlorinated biphenyl (PCB) Mix from Supelco

(Bellefonte, PA, USA); oxychlordane from Chem Service (West Chester, PA); o,p '-

DDD, o,p '-DDE, o,p '-DDT from Accustandard (New Haven, CT, USA); and toxaphene

from Restek (Bellefonte, PA, USA). All reagents were analytical grade unless otherwise

indicated. Water was doubly distilled and deionized.

Adipose, liver, bile, and yolk samples were analyzed for OCP content using

methods modified from Holstege et al. (1994) and Schenck et al. (1994). For extraction,

a 2 g tissue sample was homogenized with ~1 g of sodium sulfate and 8 mL of ethyl

acetate. The supernatant was decanted and filtered though a Btichner funnel lined with

Whatman #4 filter paper (Fisher Scientific, Hampton, NH, USA) and filled to a depth of

1.25 cm with sodium sulfate. The homogenate was extracted twice with the filtrates

collected together. The combined filtrate was concentrated to ~2 mL by rotary

evaporation, and then further concentrated until solvent-free under a stream of dry

nitrogen. The residue was reconstituted in 2 mL of acetonitrile. After vortexing (30 s),

the supernatant was applied to a C 18 solid phase extraction (SPE) cartridge (pre-

conditioned with 3 mL of acetonitrile; Agilent Technologies, Wilmington, DE, USA) and

was allowed to pass under gravity. This procedure was repeated twice with the combined

eluent collected in a culture tube. After the last addition, the cartridge was rinsed with 1