Document rBwXZnwo1ZgMMqEDJrMBQBjR0

Mr. Carl Reid B.F. Goodrich Co. P.O. Box 1026 Niagara Falls, Ontario L2E 6V9 July 12,1988 Dear Mr. Reid, Thanks for reminding me to send you a copy of Que Lan's M.Sc. thesis in draft form for your comments. I have enclosed her thesis draft with this letter but I would ask you to return it to me in one week if possible so that she can incorporate your comments into her thesis in time for her upcoming thesis defence. Following our phone conversation last week I realized that I was truely puzzled by a number of apparent discrepancies between what you have told me on the phone and in private about the composition of your B.F. Goodrich waste stream and the information which I received from MOE and from Que Lan's thesis research. For example, you told me repeatedly that the ammonium nitrogen which you (B.F. Goodrich) add to your discharge prior to secondary treatment is always below 2 mg/1 and that this is diluted 4 to 1 by the time it reaches the Welland River. This is not consistent with the MOE Report (enclosed) which indicates that on 7 separate occasions in 1987 the B.F. Goodrich ammonium nitrogen concentrations were above the 2 mg/1 level (mean = 4.95 mg/1) and in one month (July 1987) it was even above the MOE guidelines of 24.8 mg/1. I would appreciate it if you would try and explain this apparent contradiction as I suspect that the inhibition of alkaline phosphatase activity in the SOS Chromotest (discussed below) was a result, in part, of the B.F.Goodrich ammonium ion toxicity. The SOS chromotest for alkaline phosphatase activity which Que, Lan performed on your sediments (i.e. those near your discharge pipe) indicated that these sediments contained substances which were inhibitory to alkaline phosphatase activity. We concluded on this basis that these sediments displayed low levels of toxicity during the test series carried out by Que Lan. More important, however, was her repeated observations that the sediments located at the end of your pipe under the road along the Welland River were contaminated with genotoxic materials. This resulted in the highest SOS chromotest genotoxicity levels anywhere in the Welland River (7 stations). These results were further corroborated by the findings 21309007 BFG12765 that the labial plates of Chironomids in your sediments had the highest frequency of abnormalities of any of the seven test sites in the Welland River. You will note in the the thesis that all samples were coded to avoid any unconscious bias in chironomid deformity frequency analyses. We note that vinyl chloride which is listed in the attached paper published in the Proceedings of the U.S. National Academy of Sciences is a known mutagen and carcinogen. In 1987, B.F. Goodrich discharged over 807 million litres of water containing an average of 0.041 mg/1 of vinyl chloride. Thus over 33 Kg of this known carcinogen was released into the natural environment by the B.F. Goodrich Co. in Niagara Falls, Canada during 1987. Because vinyl chloride is lipophilic it binds with organic material in the sediments of rivers and lakes. Thus the concentration of vinyl chlorides in sediments is often a thousand times higher than it is in the water column. Is it any wonder, therefore, that nearly 15% of the midge larvae (chironomids) in your discharge receiving stream displayed deformities while the norm was always less than 5%? I suggest that the onus of proof should now be on B.F. Goodrich to show why this unusually high frequency of chironomid deformities associated with the end of your discharge pipe was not induced by the release of vinyl chloride (a known mutagen and carcinogen) into the Welland River. As you and I agreed initially, I have npt circulated these results to anyone but yourself. By so doing I have afforded you the opportunity to critically review.the enclosed thesis draft. However, I would ask that this draft is not to be circulated to anyone other than yourself until it is officially defended at the thesis defence to be scheduled in shortly at Brock University. I will look forward to your comments. 2 enc. c.c. Mrs. Que Lan Mike Dickman, Professor. 21309008 BFG12766 21309009 ao UJ HI <4 ae o (0 n *4 * o r*4 1040 Nf) to <4 r-to 4n <4 to to > oN zo ot -- f) \f- to 3 O CD 4 <4 OC4 O at <o to tO (4 ato n c 14 to n 44 4 to rt OB 9 to to to to <4 <4 O 01 c to n to 01 m C4 to 4 to at f4 OtO rt O (4 14 o Ot t(J 01 o oi 4 C4 a. i o IO o *4 v tO j a 04 4 r* n p> at CO r*> 4 <4 0 to -- to >*9 tO 4n no n nn r 0101 r- at 4 <4 o to at 4 r* n <4 rt CO rt to to n 0 O <4 r* to at ob rt r> <4 f(0 o at ot 4 <4 tO rt o O C4 O 3s 4 r>* 01 Ko -- r* n9 r-> <4 a --K t C o rto OB (4 to 4 O 4 4 k at O f* at o 3 3-- 4 to n-l o 3 CO -Vh * r- ol 4 N >*N 01 r* 4 1 h* 4 I- <4 <4 rt <4 CO 4 1 C4 n o r (4 to o r- rt o rt rt to r- o-- CO 4 C4 Z 3 01 o at at 4 "1 f \oo r- f N > to -- N rt h 00 N > 4n 2O 4 -- 4 to vr*0 ^ Ot NN N kn O 0t O4 <4 to a: A. at 44 14 4 at --4 n rt n to at 4 I*. N c <4 to to o to 4 O N 4 0t to to nn OB 4 c O to 0t rt (4 (4 to rt to to n to at 4 ct -- <4 n to rt tO (4 4 <r4t <4 rt to r** to 4 at O 1- 14 o CD rt o at 4 O 4 at - 4 O <4 K z UI z z o u UI Ml to Cl to C 4 Z o HI U. Z 4 3 <0 m ot x uc mO C 4. O O4 OHl 0<<K OW IL. K o O HI az --4 O < a ---t4Z W ^ 43 Oo Z i a mo O4 * JO r* o -l n or *. OK* n 010 vf at 01 > re a 104 4 r \oo di -- no N t0 M n o nn (4 to 90 r* -- n HI JZ 4 * 3-1 hll ua 4 3 O HI Jz 4M 3-1 hit uo 4 +* 3 O os J M. K b. > 4 o *s ~ to O |0 n 4| al> |4 tHo W nn to r* -- 01 <4 n c- at 4-- C4 <4 N f* 4 tO 4 O mi Jz 4 *3 -i H Ml UO 4 *- 3 O K t_l U. 44 Ml X or z 4Z M. L> 2fO it - co to K to 4 to Ml -J Z 4M 3 -l tr Ml UO < *4 3 O to rt f*1 h OB c> (4 N r-- Ol Ct <4 tO <4 O to O C4 ui JZ a 3 -1 tel ci o 4 3 O t<3 3 ja a ( < >> 4 O lO V at to r- at to to 4 4 OB to N Ml 01 at n (4 rt rt o Ot to n to Mi -J z 4 3 -1 H Ml ua < 3 O Cl HX ar 0 4r a - Ml 3c o a r 4 <4 to f-. o to to r (4 to f* Ml -1 Z 4 *4 3 -1 h Ml UQ 4 *- 3 O -1 4 tK A o or t- W tel > 34 ao O rt rot <4 rt f*. n ot o 14 rt f4 tO O <4 Ml .1 z 4M 3J t- Ml ua 4 3 O tel _l O Cl > ac -- OX -J w X o> 4 .1 o tel o a: 4 X u to a a ui O tel Ml Cl X tel ui X 4 K Z tO 111 t4 U O Z a to ot I in at t- Z _i Z a VOX-' < | BFG12767 89ZlOdg *!PW ,f i _ QT-QSQgTZ I/ll V 1110 /ll V tttt I/I * two 0009/04* H00> oeot/044 100'> 0M/04* n*o* o*i/n * oot/m ft 001/912 . i/i 10 001/004 1*4 f *4 * , mtm (84 t i"> m mwuM (u , III m |MN> I4l| 0|>4>-*|4/lM*f|4MNO|i*|*l (92 -|l.l*il.H|<|l-t*()-|-l<--!*( IK vote i/n V 040 X/t| V 040 i/n V 040 X/ll 1/91 **I/*I to 1 to 0001/04* no'O* Jftt/04* ooo*o> .01/04* tOQO`0* 0001/04* 100*0* 044/041 ro m/m no Hll/tU *00*0 1*1/11 * 18 02/0921 it I't/ll - OS ai/ocn s*o n/n to cot/un 4t*0 t*i/ii * 1 4/044t ft i/i ytsco .i/w* iooo'o> t/tt > i*x/n 11/09*9 ft V/9l V 040 004/04* tn> VMI v tuo Ot/04* to*o> 1*1/11 - ov l*a/ll * . *uu - 1 Mli/lll taro to rlt/K<tl /n . 10 oa/a CfO </n V 040 0001/04* 400*0* o*i/*t V tuo t01/04 noo*o 1/It V Of0 X/I 0040 I/9I 0 ($40 4001/04* 001/04* 0001/04* *> ioft* 10*0* ft) |N)i.ir|t<MKHi (44 | (wfttniM) (if 0 ft! % niMvu |4n* (02 0 * t **Min Ui , nwMnM,l`l,l It! j w9MlMft*|-*K*4*| (4| 4* U , >*-{t |<| , InAmm 4 (51 ,{l*IJf|) (|| 40> *P (Cl , ll* "XM-t'l (It I f (|l t (oi j (4 t t j ( t "W*l (I t |A|fl*|i (t |i<n* (s 4 4 l*w*g)an |4<n* (> * 9 mniiMMwn it<na u 0 0 (ilU |ilM*4|2 0 ft t WJW |4|* (| WO 'illMNJll MMiftlt- 'tMli) (4 0M 0 0 , lIWlw*| (K , (If 4 *W*U (tc `1/U V 040 001/04* t*0* *l/lf V MO t/om 1*1/11 0 040 M/04* / 01/0441 I/I i/I - i l/tll t/M t/ " 4 r/4t K/tt V 040 1/04* */tt V 040 0001/04* l/l 0 040 0001/001* i/ll V 0 ot/oai i/li V 40 02/DC4 H > tc It f* M ft* tO'O* 400 It * 0 p nM*i>a>|An*>f^Mgii^i*tl (St 9| t) ^`nMii>aio*a>ii*4uiA'iii^i*tt (k 0 4 a t wmxwiM (tf U01 (|t (tf RiMMnM|Ai)<HMNi*Ai*|S0| (of a ( 4 MM*M|l>WiallK<lt4)f,|,| (u 0 0 t MlMRti (44 ft o 4 , (12 | tu*(3)i|An`"[-of/ lu 4* 1 t4 l/lt i/l M 0 i/i n 1/101 1/099 i/it z 4 . ()<|ffn.i|.|i>niM<i-i (n tit . (II % NMUflH m |t)tnii^ii-iffojj(i./ (it l/i( 0 I*9/ 0 8440 i/n y e i/a i*n*i/n V 40 t/n 10 i/n V 40 ri/n v in t/n - 4 f/n V 140 1/11 V 040 i/n v io x/n V 0 m it l/it -V 49 i/I */ **/n W*i/n n/n 48 vn V 4040 V MO t i/m tt/nit ll/IU oc it 44 4S/OH4 0l/0S t/8411 l/0itl 1/04 t/IO 1/004* t/m 0/f>l 01/444 i/m l/DOSt i/m imct 04/Oil t a 4*2 C K 1*1 11 in t 9*0 2 lit 144 042 141 04 4 4 4 4 *(8 ,WI 'w It *>ww imiMMi `in (t)?>5tI*MIl-|AO^*|-I (si i , Mxit>(l)M5lWai>-llai ut * J > t (it Mrm()ii (II % MMM)M(*)Mt (*| 1 , Wi1'<|Kff (il - J *^AI> (M , >MWlll*0| (tl t5 (II a *)ii (ii , M>JI|>>>|>'>)M|P (01 ( Miou(r'>)nmo (i j Nn4I()MM (ft ^ aM4ft(t)MwMpllft^| (! 04 | m/()m(HM*iMv( ( (5 ^ w>ili(t)iH|i<nw4--Mil (> * 9 MU(t)mi (f (i 'wNlfll'IJUVHO (1 S/41 v nu s/n V 4140 29*I/I $0 ip*9/* - MO 9/91 - ft 9/42*91 01 jfl/04* O0`ft> 0M1A4* lOO'ft* lAit2 M I/O2ft t2 002/DC2 20*0 fttftl/OK 90*0 ft . ft * * ft * 1* *2 I * f *)>|<|> |ihN 9 Pft|4t> w - hv s'/m 1/ft 2 1/019* |i) in oi ,(04l*3l) |XinMj|v{ M2 (0t*ft)l) >4***>J**'l'l- l/ft i/or - 1/04*91 V 4 K ft 1/0991 04 002/ltt ~tt*0 1/402 ft 4/12 y M OOftl/tOOt 92*4 ~ /***" OS * 00t/4!0l~ *! TMI " t Oftl/iSI 0f4 1*1/9! " OS 9/ - ot 41/41 **9C/K >8 > ft (*l/4t V 4940 ftimt mu? rt 4*4 0001/04* 00*0* f/K # to > ft'U'R/tl 9 l*t/IC - $ nomi 10*0 1/41 V to j0l/04* 100*0* l/ll * 0 9 ri/i V Oi 4*I/lt V 4IM t*CI/ll v n t/ll V 4140 X/*t V 4140 ft*I/t*4l V 4*4ft 1/41 V 4140 fl/tl 1 4140 I/ll V 4140 t/llftl 0002/04* Oft lft*0 001/042 tfO t/U> |0QO'0> 0001/04* too*o* 0001/04* 100*0* eoi/M* 18*0* 001/02* Cft`0* 0001/04* 10*0* * 4 42 4 0* ft 1f*t* l" * 4 4* ft 2 2 ft * ft0 i# 02 04 00 ft i ft 0 2)*i5-)-4-*>iy-9-i****t i f |j*4*u*|g? {> # N|BKtfug(|}<) U *f > |5*-* { | f (mim Mlaj)ty) tiM cr t [KM) JW1* it |i tft .yt # p|>t |ar|g> 11 NMIJW tl f #TJU[Vi "F*** t* , *m*|4gi* (` *<ir /g: JJ* t*||W i*- , HinM .t. f *W|U (( a **|010>M|P-** u* 9 **k*l** 111 $ (Cl f lit t (tf 1 atW|4Mpan*2 (si (W|||n4.4lj !>'M| I |Uf| */4l V 4140 0001/01* 20*0* i/n ft ooi/otot 1*2/11 2 ft t/tftft 4't 22 f*2/Sl 4 ft Ot/OOft it 1/02 V 4110 001/04> CD*8 0 l/lt V 1404 ft2/41 4 0001/04* DS/112 20*0* ft 0 1/11*11 y 0 fl/tl y on 2*1/41 y 400 002/901 01/0>2f 01/0114 It ft 14 IX 1* 4*1/11 * 001/201 ftt'ft 1/41 - 40 fl/ll toco l/4ttl 1/149 422 12 X/ll y ft 2*i/n V so 001/041 0IA4S 29*4 4*0 i*i/n 4 4 V*f!/lt 4 004 02/BOa 8/IS2S 22 Ott 1* IA1 y ft 1/804 M VIA1 r 440 02/009 99 t/1 t 2*1/1 V 4004 MIAMI M/D4C1 a 1*2/01 V 4048 I/M9 4 *!/! - M 02/19X2 n4 I/ll 8 ft l/fttt 1*4 flAt 8 ft 002/242 CO # 0*8*1/11 y 40ft 1*4*IA1 V 4M0 001/9ft ooi/Di* n*ft 10*8* r 0 0*4*1/41 V 4140 0001/84* *0* o*i/n 8 04ft OS2/9T9 ft*f/ll ft I2V22II fllAl ft |`/009( ircim -y M 1/9001 **i/n' 8 4040 " tAm I/O I- 8 41 biAbciC" 4*1A l M 1/M * 0901 * m * m T M* l|8 |M t (v0 **iy>-2-Tt-***i4vj**-,* (k 4% |({ 0# , (4f ( (*,(( (If 02 v Mimw*4|/a*t`n*}~.**.ii'*if ( # *mwi (44 # iWllNiw.! (v * t l<MK<|iaia.n|A(jaip-(`,! {it * , Itouuwix-t (I m ^**|nnw8*wotajit>2 {(/ A ut<M|4a*JpAnAnMani.a (>2 4 W|a|llNN|iV)<MM( {t 0> 1 W|r|/ir*rtiw* ( # M|M|igiKaM-ft (\! * 9 (0! % w9*jgawa|*-! (( 2 1 *>|i>*rf8 (t # ftftutj* Mipiis* (n 4 t (91 2 , (si , (>| | MM|>M|OT|i*|*I |(| t MlLMU*|A*>*>TIi-4*2 Ul 0 (U aMi>uai|aa|iMM>t-iinjl^ |0| 0 (4 (0 aMMOTUai|a<lOnfl-An{i>|-i 0 (t # MMftn|>M*OM-! (p m (f 4 4 4 , (2 4 `pi #f iwii jm mjj***i 'in `pj iwi p//Jt/ou//o' sqi ut ftiaiuahtnui cnuaA XiiatuaSoutaioQ ( |q* (9m) u vsn ps pv >w *><</ ]B )9 UUBQDJAJ :SOOU3(D<; |CDIj, SEDIMENT GENOTOXICITY AND ITS RELATIONSHIP TO THE FREQUENCY OF CHIRONOMID LABIAL PLATE DEFORMITIES by QUE LAN, B.Sc. (Hons.), China, A Thesis submitted to the Department of Biological Sciences in partial fulfilment of the requirements for the degree of Master of Science June 1988 Brock University St. Catharines, Ontario C Que Lan, 1988 BFG12769 21309011 I hereby declare that I sun the sole author of this thesis. I authorize Brock University to lend this thesis to other institutions or individuals for the purpose of scholarly research and further authorize Brock University to reproduce this thesis by photocopying or by other means, in total or in part, at the request of other institutions or individuals for the purpose of scholarly research. Signature r BFG12770 21309012 3. ACKNOWLEDGEMENTS I am greatly indebted to Dr. M.D. Dickman, my thesis supervisor, for suggesting the study of the relationship between sediment genotoxicity and the frequency of benthic invertebrate abnormalities. This thesis benefited from his encouragement, critical comments and support. I am grateful to Dr. A.W. Bown, Dr. A. Castle, and Dr. C. Lewis, member of my committee, for their helpful advice and encouragement. 1 thank Dr. B.J. Barclay for his helpful discussion and advice, especially during the genotoxicity tests which were done in his laboratory at Brock University. I wish to thank Dr. B. Dutka of the Canada Centre for Inland Waters for his help in guiding me through the genotoxicity tests and for kindly providing the test strains which I used in my SOS chromotest study. I am also grateful to Dr. K. Coates of the Royal Ontario Museum for her help in identifying oligochaete species, and Mr. B. Morton of Guelph, Ontario, for the long hours he spent helping me identify my invertebrate species. I wish to thank Miss K. Veltri, T, Huang, N.K. Ondrusek, B. f Shannon, M.D. Agbeti, B. Solymar, and M.M. Saavedra Alvarez for their encouragement and support. I express my deep gratitude to Mr. H. Xu for his encouragment thoughout my thesis research. Finally, I thank my family for their strong support thoughout my academic career. BFG12771 21309013 1. 4. Abstract Acknowledgements Table captions Figure captions Introduction Literature Review Genotoxicity of environmental samples Deformities in benthic invertebrates Mechanisms of deformity induction by genotoxins Toxicity vs genotoxicity of environmental samples Measures of generic richness of benthicinvertebrates Materials and Methods Study locations Sample preparation for the SOS chromotest The SOS chromotest Calculation of results * Sampling benthic invertebrates Preservation of invertebrate specimens Classification of the invertebrates Statistical analyses Results * Genotoxicity of sediment samples Frequency of deformities in chironomids Correlation between genotoxicity and frequency of deformities Benthic invertebrate community page 1. 3. 6. 6. 8. 12. 17. 19. 20. 22. 24. 29. 30. 34. 36. 36. 37. 38. 40. 55. 63. 66. 21309014 BFG12772 c. Generic richness and diversity 79. Discussion Sediment genotoxicity Frequency of deformities in chironomids Correlation between genotoxicity and the frequency of chironomid labial plate deformities Toxicity vs genotoxicity of the sediments The Benthic invertebrates, community 83. 87. 89. 92. 95. Conclusions 101. References 103. Appendices 1. Industrial discharges in the Welland River (MOE, 1987) 110. 2. The percent alkaline phosphatase activity in the SOS chromotest. ^ 112. 3. The percentage frequency of deformities in chironomid labial plates 114. ST060ETZ BFG12773 6. Tables page 1. The SOS chromotest induction factor of sediment extracts from seven locations in the Welland River. 43. 3. Relative abundance of benthic invertebrate taxa at seven locations in the Welland River. 70. 3. Benthic invertebrate taxa at seven locations in the Welland River. 72. 4. Abundance of benthic invertebrates at seven locations in the Welland River. 74. 5. Generic diversity of benthic invertebrates at seven locations in the Welland River. 81. Figures 1. Map of the study area (Modified from Dickman et al. 1983). . 27. 2. SOS chromotest induction factor and colour development time. 33. 3. The relationship of dose-response in the SOS chromotest. 45. 4. The percent alkaline phosphatase activity in the SOS chromotest of 10% DMSO extracts with S9. 47. 5. The percent alkaline phosphatase activity in the SOS chromotest of 10% DMSO extracts without S9. 49. 6. The percent alkaline phosphatase activity in the SOS chromotest of distilled water extracts with S9. 51. 7. The percent alkaline phosphatase activity in the SOS BFG12774 21309C16 7 chromotest of distilled water extracts without S9. 8. Normal structure of chironomid labialplate. 9. Deformed chironomid labial plate. 10. The frequency (%) of deformities in chironomid labial plates. 11. Correlation between genotoxicity and frequency of deformities in chironomid labialplates. 12. Generic richness of benthic invertebrates at seven locations in the Welland River. 53. 57. 59. 61. 64. 79. 21309017 Jirrivrr BFG12775 REPORT ON THE 1986 INDUSTRIAL DIRECT DISCHARGES IN ONTARIO October, 1987 BFG12776 21309013 8. INTRODUCTION Pollution from carcinogenic materials has become a serious problem as large amounts of contaminated municipal and industrial wastes annually enter natural waterbodies. For example, over the years, the government of Canada has poured tens of millions of dollars into the Sydney Steel Corporation in Nova Scotia (McMillan, 1988). The technology which was used to produce steel, in this case created an enormous lagoon of highly carcinogenic materials that constitutes the largest chemical carcinogenic industrial landfill in eastern Canada. It is now costing the Canadian and Nova Scotian tax payers 49 million dollars to excavate and incinerate the Sydney Steel Corporation landfill wastes. This represents the second biggest site clean-up in the history of North America (Mcmillan, 1988). Since aquatic communities are sensitive to mutagens, many ecologists have attempted to. use invertebrates to quantify the level of carcinogens in aquatic sediments (Warwick, 1985). To the best of my knowledge, my thesis research is the first study of the relationship between the frequency of benthic invertebrate abnormalities and sediment SOS chromotoxicity. The correlation between these two independent tests of genotoxicity constitutes the main focus of my thesis research. Although many studies have been done on the genotoxicity of environmental samples and the frequency of deformities in chironomids, this is the first study to use the SOS'chromotest in conjunction with the study of the frequency of chironomid labial plate deformities. The genotoxicity of environmental samples and BFG12777 21309019 9. the frequency of deformities of chironomids were both studied in terms of their relationship to industrial and agricultural discharge sources. The Welland River was chosen for this study because of its status as a provincially significant wetland area. The Welland River flows east through the City of Welland to the Niagara River. Upstream of the City of Welland, the river is mainly influenced by agricultural runoff. Within and downstream of the City of Welland, the major discharges to the river are from industrial and domestic sources (Steele, 1981; Dickman et al., 1983). The Welland river has been studied since the 1960's (e.g. Johnson, 1964). Serious shock loading of industrial waste materials in the Welland River has resulted in aquatic plant dead zones below some industrial discharge sites in the Welland River (Dickman et al., 1983, 1986 and 1988). Several industrial discharge effluents contained detectable genotoxic organic compounds, such as chloroform, carbon tetrachloride and dibromomethane (Kaiser and Comba, 1983). These organic compounds have been described as carcinogens (McCann and Ames, 1976). Discharges of heavy metals were also found at industrial sites, those, metals included Cr, Pb, Ni (Dickman et al. , 1986) some of which are both toxic and genotoxic (Flessel, 1979). Previous studies on the Welland River indicated that the frequency of gonadal neoplasms in wild carp-goldfish hybrids was higher in the industrial influenced portion of the Welland River than in the upstream agricultural portion (Dickman and Steele, BFG12778 21309020 10. 1986). This report prompted me to attempt to determine whether there were any genotoxic materials being discharged into the Welland River. The seven locations selected for this study were divided into two groups: 1). A control location (site A) which was located in an agricultural area upstream of the city of Welland, and 2). Six "treatment" locations (sites B, C, D-l, D-2, E, and F) which were all located in an industrial area downstream from the city of Welland with the single exception of station B which received insecticides and herbicides from an agricultural area near station A (Fig. 1). It should be noted that similar herbicides and insecticides only reached station A after they had passed through an extensive aquatic plant lined streamcourse. These aquatic plants serve to remove pesticides from the water (Bingham, 1973). The goals of this study were: 1. To determine the genotoxicity of sediment samples from the Welland River, using the SOS chromotest and coded samples to avoid unconscious biases. 2. To sample benthic invertebrates from the Welland River, at the identical locations that the SOS chromotest sediment i mutagenicity test samples had been taken. 3. To observe the frequency of chironomid labial plate deformities at these locations, after first again coding the samples to prevent unconscious bias. 4. To determine the correlation between the level of SOS BFG12779 21309021 11. chromotest genotoxicity and the frequency of chlronomid labial plate deformities. 5. To determine the diversity of benthic invertebrates (at the generic level) at each of the seven study sites in the Welland River. BFG12780 ZZ0606TZ Literature Review 12. Aquatic toxicological studies of freshwater include two major areas of study: 1.) field studies of species pattern, species diversity, species abundance, etc. (Guafin and Tarzwell, 1952). These field studies have been carried out with bacteria, aquatic plants and invertebrates, etc. (Cairns et al., 1972; Buikema and Herricks, 1978; Dickman et al., 1983; Pettibone and Cooney, 1986). 2.) the determination of pollutants in environmental samples by using some sensitive, inexpensive, convenient and short-term methods to estimate genotoxic pollutant loading (de Serres and Matsushima, 1987). Both areas have developed rapidly, as genotoxic pollution problems have become more and more evident (Sato et al., 1983; Samolloff et al., 1983; Xu and Dutka, 1986). In fact, t,, contaminated drinking water was considered at one time as a potential cause of cancer in humans (Velema, 1987). Genotoxicity of environmental samples Genotoxins include mutagens, clastogens, carcinogens, and teratogens (Quillardet et al., 1985). Mutagens cause permanent modifications in DNA structure in both prokaryotes and eukaryotes (Starr and Taggart, 1987). Clastogens result in chromosomal changes in eukaryotes (Adler, 1984). Carcinogens cause gene mutations leading to cancer in eukaryotes (Starr and Taggart, 1987). The differences between these genotoxins are not absolute. B Z & G m iZ BFG12781 For example, 90% of the carcinogens were also known mutagens tested in the Ames test of McCann and Ames (1976). Theoretically, mutagens cause gene mutations, and consequently they may result in chromosomal changes, and finally they may result in the appearance of abnormalities. Because there is little difference between genotoxic groups, especially when one considers eukaryotes and prokaryotes, the grouping of genotoxins is quite confused. Some authors limit mutagens to DNA damaging agents in bacteria (McCann et al., 1975), while others expand their definition of mutagens to include carcinogenic potentiality in higher eukaryotes (Quillardet et al., 1985; Metcalfe et al., 1985). I preferred to use the term "genotoxicity", in a restricted sense (e.g. DNA damaging agents) as a large range of organisms from bacteria to insect larvae were analyzed in my study. For the purpose of evaluating the genotoxic potentiality of waste materials in the environment, genotoxicologists have been looking for a widely available and uncomplicated method of detecting potential genotoxic compounds. Different methods for detecting potential genotoxic materials have been developed (Venitt and Parry, 1984; de Serres and Matsushima, 1987). The yeast "Saccharomyces cerevisiae'' has been used as a simple eukaryotic system for detecting genotoxicity of chemicals (Parry and Parry, 1984). Cultured mammalian cells have also been used in estimating genotoxicity of chemicals and environmental samples (Dean and Danford, 1984). However, bacteria are the most widely used test organisms in genotoxic studies (Hollstein and McCann, BFG12782 14. 1979). The Ames test The Ames test is a well-known mutagenicity test which has been widely used since 1975 to detect drug genotoxicity as well as chemical and environmental genotoxicity (Ames et al., 1975; Walker, 1983; Outka et al., 1981; Samolloff et al., 1983; Sato et al., 1983). The Ames test employs Salmonella bacteria which carry a mutation at an easily detected locus. An increase in the frequency of revertants is related to increasing dosage levels of genotoxic compounds. Fly ash collected from an oil-burning power plant was demonstrated using the Ames test to contain mutagens (Wei et al., 1984). The acid, base and neutral fractions of samples from petrochemical plant wastes were also found to be mutagenic using the Ames test (Brown and Donnelly, 1984). Metcalfe et al. found that the genotoxic materials associated with the discharge of particulate material in oil refinery effluents were present in the most polar silica gel fraction (Metcalfe et al., 1985). Some extensively used pesticides such as organphosphorous insecticides (Usha Ranni et al., 1980) were also demonstrated to be genotoxic using the Ames test. Many freshwater bodies have been used as duropsites for waste materials. Metcalfe et al. (1985) observed a positive relationship between the genotoxicity of particulate materials in petroleum refinery effluents and the concentration of oil and grease samples by using the Ames test and the carcinogenicity test. Samolloff et al. (1984) reported that neutral compounds BFG12783 szo e o rrg 15. extracted from sediments of Tabin Lake (Canada) which was contaminated with agricultural runoff, mining, petrochemical industries, and paper mills and municipalities showed mutagenic activity when tested with the Ames test. Sato et al. (1983) using the Ames test found that some sediments which they removed from the Niagara River and its feeder streams were mutagenic. The SOS Chromotest The SOS Chromotest is both more sensitive and more specific than the Ames test (Quillardet et al., 1982), and was therefore adopted in this study as the principle genotoxicity test. The first report on the SOS Chromotest was made by Quillardet et al. in 1982. The SOS chromotest is based on the "SOS" response to DNA-damaging agents in JL coli (Walker, 1984; Little and Mont, 1982). The test strain (IS. coli PQ 37) carries a gene fusion of the SOS repair operon (lacZ gene). The test strain has a deletion for the normal lac region so that beta-galactosidase (coded for by lacZ) activity is strictly dependent upon sifA expression. Thus, the extent of any DNA deunage is measured by quantifying the activity of beta-galactosidase in test cells (Quillardet et al., 1982). It has been shown that the results of the SOS chromotest are closely correlated with those of the Ames test (Quillardet et al., 1982; Quillardet and Hofnung, 1985; Dayan et al., 1987). The SOS chromotest was first used to measure genotoxicity of pure chemicals which were mutagenic and/or carcinogenic in other test systems (Quillardet et al., 1982). These studies revealed BFG12784 21309026 16. that the SOS chromotest was reliable in detecting genotoxicity. Genotoxicity studies of 83 compounds were made in order to compare the results of the SOS chromotest and the Ames test with the carcinogenicity test. It was concluded that there were no false positives with the SOS chromotest (e.g. non-carcinogen but SOS inducer) among the 83 tested chemicals. The Ames test on the other hand produced a number of false positives (38%) (Quillardet et al., 1985). Three out of 42 chemicals which displayed mutagenicity in the Ames test did not induce the SOS response in the SOS chromotest (Ohta et al., 1984). Although the SOS chromotest has been used in examining pure chemicals (Quillardet et al., 1985; Ohta et al., 1984; Olivier and Marzin, 1987), only a few environmental studies using the SOS chromotest have been carried out (Xu and Dutka, 1987). Six samples taken from contaminated areas along Prince Edward Island were tested with the SOS chromotest, five of the six samples displayed weak genotoxicity (Xu and Dutka, 1987). The SOS chromotest has a major advantage over the Ames test. In the SOS chromotest, the test strain does not need to survive for as long a time (only two hours) as it does in the Ames test (48 hours). Therefore, the SOS chromotest can'be used for detecting toxic mutagens which are undetectable in the Ames test because they kill the bacteria (Quillardet et al. 1982). Since the SOS chromotest has some major advantages over the Ames test (ie. it gives a quantitative response within a few hours and it is more sensitive than the Ames test), I used it as my primary mutagenicity screening tool in my thesis research. BFG12785 21309027 17. Deformities of benthic invertebrates exposed to mutagenic substances Because it is well recognized that some pollutants cause mutations, the studies pf freshwater pollution were expanded to consider the frequency of benthic invertebrate deformities (Hamilton and Saether, 1971). Genotoxic pollution is a concern because such pollutants frequently accumulate without causing the death of organisms. Instead, long term disabilities occur (Baumann, 1984). Studies in France revealed that in drinking water, mutagenic substances could be detected by using a micronuclear test (Jaylet et al., 1987). Milbrink (1983) found that the frequency of chaetal deformities in oligochaetes was significantly correlated with mercury pollution in Lake Vanern, Sweden. Benthic invertebrate chironomid larvae represent some of the most widely distributed gen.era in freshwater (Roback, 1978). In addition, their larvae represent a sensitive stage in their life cycle. Research on the frequency of deformities of chironomid larvae in polluted freshwater (Hamilton and Saether, 1971) led to the use of deformities of mouth parts, antennae and chironomid body wall thickness (Hamilton and Saether, 1971; Wiederholm, 1984; Warwick et al., 1987). The mouthparts of chironomid larvae normally are symmetrical. Deformed specimens are characterized by asymmetrical labial plates and mandibular structures. Unusual thickenings of the chironomid larval body wall were also considered as a deformity by Hamilton and Saether (1971). It was suggested that BFG12786 ezoeoerz 18. different types of deformities may be caused by different types of pollutants (Hamilton and Saether, 1971). The susceptability to these malformations seemed to differ among the various chironomid species (Wiederholm, 1984). Some species were far more sensitive to mutagens than others. In a study of Lake Erie, it was found that deformed chironomid larvae were generally restricted to locations near known sources of industrial pollution (Hamilton and Saether, 1971). Sediment core studies of the Bay of Quinte in Lake Ontario, revealed that the presence of deformed chironomid larvae increased inversely with depth (in most cases, the age of sediment core is determinated by depth, the deeper the older). The increase in deformed chironomid larvae was associated with increased pollution in the bay (Warwick, 1980). The studies of heavy metal (Hg, Zn, Cd, Ni, Pb, Cu and Cr) polluted lakes in Sweden concluded that in these lakes there were much higher occurrences of deformed chironomids than in unpolluted lakes (Wiederholm, 1984). Experimental pond studies showed that pollution by a synthetic, coaltar-derived oil could induce a high frequency of deformities in Chironomus decorua (Cushmam, 1984). It was suggested that antennal deformities in chironomid larvae could also be used as a biological screening marker for the detection and evaluation of mutagenic contaminants in aquatic ecosystems (Warwick, 1985). It was found that the number of deformed antennae in Chironomus sp. was higher in samples from Tobin Lake (contaminated with chemical pollutants and toxic residues of agricultural and industrial waste) than ezoGoexs 19. Last Mountain Lakes which were comparatively uncontaminated. In the study of Chironomid larvae collected from sediments in the harbour at Port Hope, Ontario, it was found that a direct relationship between the degree of sediment radioactivity and the frequency of deformities in Chironomus spp. existed (Warwick et al., 1987). Hamilton and Saether (1971) reported that aberrant mouth parts, thickened body walls and head capsule malformations might be caused by pollutants from agricultural as well as industrial sources. Mechanisms of deformity induction by genotoxins The mechanisms of deformity induction include gene mutations, interference with transcription and translation, disruption of cell division, and metabolic disturbance (Weis and Weis, 1987; Jaylet et al., 1987). Mutagenic or genotoxic compounds cause deformities in animals by disrupting genes (Metcalfe and Sonstegard, 1985). Genotoxic pollutants which are discharged into water systems as waste materials accumulate in the associated sediments which comprise habitats of many benthic invertebrate taxa (Buikeman and Herricks, 1978; Lafont, 1984; Wetzel, 1983). Although the absolute concentration of genotoxic pollutants may be very low in water and sediment, bioconcentration and accumulation will serve to magnify them in the food chain (Buikema and Herricks, 1978; Tarkpea et al., 1985). For example, PCBs in the water were 8.2 ppt, while in Lake Trout (Salvelinus namycush) they were detected at 28.0 ppm (Metcalfe, 1977). RFG12788 20. To locate the sources which cause the highest occurrence of deformities in chironomids, Hamilton and Saether (1971) tested several pesticides (including some known genotoxins) on Lab cultures of Chironomus spp. The authors failed to observe the same high frequencies of deformed chironomid larvae as they obtained in their field study. They concluded that synergistic effects occur in the feild which they could not duplicate in the lab. The studies in experimental ponds revealed that a coal oil derived product did increase the frequency of deformities in Chironomus decorus labial plates (Cushman, 1984), while in other studies, it was found that petroleum refinery effluents (Metcalfe et al., 1985) and some crude oils (Vandermeulen et al. 1985) were genotoxic. It was suggested that genotoxic sediments in water bodies cause a high frequency of deformities in benthic invertebrates (Warwick et al., 1987). To the best of my knowledge, there are no published studies of sediment genotoxicity using the SOS chromotest and the frequency of benthic invertebrate deformities for the same sample locations. Toxicity vs genotoxicity of environmental samples Some genotxins act alos as general toxins, e.g. KaCr207 (Olivier and Marzin, 1987), and neocarcinostatin (Quillardet et al., 1982). It was noted that some environmental samples might contain these so-caaled toxic mutagens. Vandermeuleu et al. (1985) found that crude oils and oil products were toxic to the BFG12789 21309031 21. Ames -test bacteria. Chemical fractions of sediments from contaminated Tobin Lake demonstrated in biological assay systems that major toxic constituents of the sediments were neutral compounds eluted from the Florisil columns by 1:1 hexane-dichloromethane (Samolloff et al., 1983). Sediment extracts of samples from the Niagara River and its feeder streams appeared to have high levels of toxicity (Sato et al., 1983). Therefore, genotoxic pollutants could exert their effects on the benthic invertebrate community in two ways: 1.) DNA-damaging activity. 2.) direct toxicity to the organisms. Underestimation of the genotoxicity of environmental samples can be due to the toxic effects of contaminated samples (Vandermeulen et al. 1985; Metcalfe et al. 1985). The SOS chromotest has another advantage over the Ames test; the SOS chromotest can also detect the toxicity of samples by their suppression of alkaline phosphatase activity (Quillardet et al. 1982). In the SOS test strain, E_. coli K-12 PQ 37, alkaline phosphatase is noninducible by genotoxins. It is constantly synthesized by living cells. Monitoring the alkaline phosphatase^ activity can screen for the survival of the bacteria. If a tested sample causes a decrease of alkaline phosphatase activity, it can be considered as a protein biosynthesis inhibitor and therefore, the sample is toxic to the bacteria (Quillardet et al., 1982). In my study, the toxicity of samples was estimated since their toxicity could have serious impacts on the aquatic benthic invertebrate community. BFG12790 21309032 22. Measures of generic richness Changes in species pattern in a benthic invertebrate community can be related to the level of toxic materials entering the environment (Hart and Fuller, 1974; Buikema and Herricks, 1978; Wiederholm, 1984). Earlier studies of freshwater toxins in the environment were mainly field studies. Species diversity and species richness are two ways to characterize community structure. In a normal aquatic system, the number of individuals of each species is comparatively low, and the number of species is high. As the pollution levels increase, a reduction in the complexity of the community is observed as a reduction in the the number of species. At the same time the abundance of a few species generally increases (Cairns et al., 1972). Cushman (1984) reported that a coal-derived oil product reduced the species diversify of benthic insects in his experimental study ponds. In Lytle Creek, U.S.A., an aquatic invertebrate community survey was conducted, and it was found that upstream of a sewage outfall, higher species richness occurred than at the discharge site, and with distance downstream the species richness recovered (Gaufin et al., 1952). Thus, species richness generally decreases when toxic pollutants enter an aquatic system (Cairns et al., 1972; Cushman, 1984). In the Welland River, aquatic macrophytes and attached microbiotic species richness decreased downstream of a number of industrial discharge sites, and then recovered with distance (Dickman et al. 1983 and 1988; ; Albanese et al., 1988). Fish populations BFG12791 21309033 23. declined in species richness where waste materials from agricultural and industrial effluents in the Welland River were high (Steele, 1981). By comparing the species pattern of benthic invertebrates in normal (control areas) with that of polluted areas, it has been found that not only do species patterns change, but a shift in the dominant species also occurs (Buikema and Herricks, 1978). Therefore, it was concluded that those dominant species surviving in polluted environments were tolerant of the sort of pollutants to which they were exposed. It was found that a high relative abundance of tolerant species in a particular location was related to the specific pollutants in each location (Hart and Fuller, 1974). BFG12792 21309034 24. MATERIALS AND METHODS The Welland River The Welland River is a slow flowing, shallow river and it is a provicially significant class 1 wetland in Niagara. The river begins in Ancaster, flows east to the Niagara River which brings water into Lake Ontario Upstream of the City of Welland the river passes through agricultural areas while downstream of Welland the river passes portion in an industrialized areas (Fig. 1). Sampling sites Site A: the upstream Welland River control site was located at the confluence of Beaver Creek and the Welland River, in the agricultural area, upstream of the City of Welland. The emergent aquatic vegetation at this site was dominated by Potamogeton spp., Nuphar spp., Nymphaea. spp., Typha spp., Phraflmitea spp., Sagittaria spp., Pontederia spp., Polygonum sp., Vallisnaria spp. and Cladophora spp. Site B: Welland River entensive agricultural site was located at the confluence of the OWMC Swale and the Welland River, in acore and wheat agricultural area, downstream of site A, and upstream of the City of Welland. The aquatic emergent vegetation at this site was similar to those at site A. Site C was located at the confluence of the discharge pipe of the Atlas Specialty Steels Co. and the Welland River, downstream of the City of Welland. The mean discharge from the Atlas-mansfield storm sewer from which their waste stream entered BFG12793 >7>r` 25. the Welland River was 2458.9 m3/day (Ontario Ministry of the Environemt (MOE), 1987). Their effluent contained high levels of cadmium, nickel, chromium, lead, zinc, copper, cobalt and iron (Appenix 1). The aquatic emergent vegetation at this site was dominated by Sagittaria spp., and Phragmites spp. Site D-l was located below the discharge pipe of the B.F. Goodrich Co. which produces polyvinyl chlorides. The mean discharge from the sewer was 2213.58 m3/day (MOE, 1987). Biological Oxygen Demand (BOD), a measure of the quantity of oxidizable material present in a water sample), values of the effluents were 8 out of 12 months higher than the MOE requirement, and the discharge contained ammonium ion, vinyl chloride and phosphate. The emergent vegetation at this site was dominated by Potamogeton spp., and Typha spp. Site D-2 was located at the confluence of the discharge stream of the B.F. Goodrich^Co. and the Welland River just downstream of site D-l. The emergent vegetation at this site was dominated by Sagittaria spp., Cladophera spp., and Typha spp. Site E was located in Thompson Creek which was used as the main discharge stream of the Cyanamid Chemical Co. The company produces organic and inorganic nitrogen and phosphorus products. The mean discharge to this creek from Cyanamid was 27342 m3/day (MOE, 1987). The effluents contained detectable concentrations of cyanide, ammonia, urea, nitrate, phosphorus, chromium, nickel, and zinc. There was no aquatic vegetation at this sample site due to the high toxicity of their ammonia waste discharges during frequent shock loading events (Dickman et al., BFG12794 21309036 26. discharges during frequent shock loading events (Dickman at, al. , 1983 and 1988). Site F was located at the confluence of the discharge channel of the Ford Glass Co. with the Welland River, downstream of site D-2. The discharge from their sewer was 8,554 m3/day (MOE, 1987). The effluents contained iron, ammonium ion and phosphate. The dominant emergent aquatic plant species at this site were Typha spp., Sagittaria spp., and Pontederia spp. g'FG 12^95 21309037 Figure 1. Map of the study area (modified from Dickman et al., 1983). 2i30903 BFG12796 0 .28 LA K E O N TA R IO i i BFG12797 f \ L # i: *. i i 3 i I Jf w obl CoD CM ! CO 29. Sample preparation for genotoxicity -tests Seven sediment samples were collected from the Welland River at the same sites as previously described for the benthic invertebrate sampling (Fig.l) The sediments for genotoxicity testing were placed into sterilized plastic bags and within 2 hours they were returned to the laboratory and stored at 4c in a refrigerator. Extraction of samples was made within 24 hours of sample collection. The dimethyl sulfoxide (DMSO) sediment extracts were made by adding 25 g (the wet weight of sediment was weighed out and placed in a clean bottle) to 25 ml of 10% DMSO in distilled water. The bottle was then stoppered and vortexed for 2 minutes to make the contents homogeneous. The mixture was then'poured into a sterilized centrifuge tube and centrifuged for 20 minutes at 2000 rpm at 4*C (IEC CENTRA-7R refrigerated centrifuge). The suspension was next filtered through a 0.22um filter. This was done for purposes of sterilization to permit its use in the SOS chromotest. The distilled water extract procedure was similar to the DMSO extraction procedure except that ultra-pure distilled water replaced the 10% DMSO solution. The E.coli K-12 PQ 37 strain was kindly provided by Dr. B.J. Dutka of the Canada Centre for Inland Waters. The genetic markers of this strain are thr, leu, his, pyr D, thi, gal E, gal K, lac U169, srl300::Tnl0, rpo B, rps L, sfiA::Mud (Ap lac) cst, rfa, uvrA, trp: :Muc* and Pho' (Qui'llardet et al. , 1982). Media and Buffers for the SOS chromotest BFG12798 2X309040 30 L medium: 1% Bacto tryptone, 0.5% Bacto yeast extract, 1% NaCl, pH 7.2. Bacteria were cultured in L medium supplemented with 20 ug/ml ampicillin. B buffer: Na^PO*. 161. lg, NaHj,PQ^. 2Hxo 5.5g, sodium dodecyl sulfate(SDS) lg, beta-mercaptoethanol 2.7ml per 1 double distilled water, adjusted to pH 7.0 with HC1 or NaOH. P buffer: tris(hydroxymethyl)aminomethane 121g, SDS lg per 1 double distilled water, adjusted to pH 8.8 with HC1. ONPG solution (4 mg/ml): 400Mg 0-nitrophenyl-b D-galactopyranoside (ONPG) per 100 ml of 0.1 M phosphate buffer pH 7.0. PNPP solution (4 mg/ml): 400 mg p-nitrophenyl phosphate disodium (PNPP) per 100 ml of P buffer. Activation mixture (S9 mixture): per 10 ml of rat liver S9 (purchased from Litron Laboratories Lit.), salt solution (1.65 M KC1 + 0.4 M MgC1.6H*o) 0.2 ml; G6P(1 M) 0.05 ml; NADP( 0.1 M ) 0.15 ml; P buffer 2.5 ml; L medium 6.1 ml; S9 1 ml. The SOS Chromotest The SOS chromotest test procedure was based on the published methods of Quillardet et al (1985). A fraction of 0.1 ml (overnight culture) was reconstituted in 5 ml of fresh L medium at 37for 2 hours, One ml of reconstituted culture (OD 600 = 0.15-0.2) was then i diluted in 9 ml of fresh L medium (without metabolic activation) or 9 ml of fresh S9 mixture ( with metabolic activation). Fractions of 0.3 ml were placed into a series of sterilized test tubes (ice bathed) containing 20 ul of sample extract to be tested. The mixture was incubated by shaking for 2 hours at 37*C. BFG12799 T&060CT2 31. After incubation, the fractions were used for beta-galactosidase and alkaline phosphatase assays. Two parallel series of test tubes were set up at the same time and under the same condition for these two enzyme assays. In order to screen the pro-genotoxic materials, an S9 mammalian microsomal fraction was used in the SOS chromotest. Optimal chromotest development time To determine the optimum SOS chromotest time, several tests for induced colour development time were conducted. The results of the optimum SOS chromotest time test showed that induced colour development of 60 minutes was the optimum time (Fig. 2). Therefore, the results of this study were reported as 60 minutes colour development times expcet where otherwise noted. Assays for beta-galactosidase To each test tube 2.7 ml of B buffer was added to the beta-galactosidase series and incubated at 374C for 15 minutes. The enzyme assay was then initiated by the addition of 0.6 ml of ONPG solution per tube. The assay of color development was made at 30 minutes, 60 minutes, and 90 minutes after addition of the substrate. The absorbance at 420 nm was read against a colorimeter -blank consisting of an assay in which the bacterial culture had been replased by either L medium in experiments performed without metabolic activation or activation mixture (S9 mix) in experiments performed with metabolic activation. Assays of alkaline phosphatase BFG12800 21309043 Figure 2. The SOS chromotest induction factor and colour development time (test concentration = 20 mg wet weight /assay). i BFG12801 21309043 33 O station 0-1 O station F station 0-2 'station C A station E X station A SOS CHROMOTEST INDUCTION FACTOR AND THE COLOUR DEVELOPMENT TIME 21309044 BFG12802 3A. The alkaline phosphatase assay was similar to that of the beta-galactosidase assay except that instead of B buffer, P buffer was used and instead of ONPG solution, 4mg/ml PNPP solution was used. Controls for the SOS chromotest Positive controls in each set of assays were included. 4NQ0 (4-nitroquinoline-N-oxide) was used in the experiments without metabolic activation and 2AA (2-aminoanthrancene) was used in experiments with metabolic activation. The results obtained from the positive controls were compared with previously published values Quillardet et al., 1985; Xu and Dutka, 1987). Negative controls in each 3et of assays were also included. 10% DMSO solution and double distilled sterilized water were used in all experiments using DMSO extract and ultra-pure distilled water extracts. The induction factor for each sample was compared with the negative control to determine the statistical significance of the test. Calibration of the SOS chromotest The units of enzyme activity were calculated according to Quillardet and Hofnung's (1985): 1000 x A420 Enzyme units= ---------------------------------- t In this formula, A420 is the optical density at 420 nm read from the incubation mixture, and t is the length of time for incubation in the presence of the substrate (ONPG or PNPP) in BFG12803 21309045 35. minutes. The ratio of beta-galactosidase units to alkaline phosphatase units reflects the induction of the sfiA gene (Quillardet et al, 1982): A420 B X tP R=---------------------------- A420 PXtB where A420 B and A420 P represent the optical density at 420 nm read from the incubated mixture. Where tB and tP represent the reaction time from beta-galactosidase and alkaline phosphatase. The induction factor 1(c) is R(c) I(c)= ---------------- R(0) where R(c) and R(0) represent the ratio (R) at concentrations (C) and (0), respectively. The induction factor of each sample was compared with the negative control in order to evaluate the statistical significance of the test. BFG12804 21309046 36. Ekman dredge samples Invertebrate samples were collected from the bottom of the Welland River (10 to 40 cm depth) at seven locations during August and September of 1987. An Ekman Dredge (15.3 x 15.3 x 23.0 cm) was used to collect the sediment samples. The dredge was set up and placed on the surface of the sediment and carefully pressed into the mud to a depth of approximately 4 cm. Before I lifted the dredge out of the sediment, I checked to see that the jaws were tightly closed. At least three replicate dredges were randomly collected at each location and then mixed together to form a single composite sample which was then placed into a plastic bag and returned to the laboratory. Three of these composite samples were taken at each location (ie. 9 Ekman samples) and these were returned to the laboratory for sorting. Sample preservation and mounting techniques K.A.A. solution (Pimentel,1967): kerosene 10 ml acetic acid 20 ml 95% ethanol 100 ml 5% formalin solution: 10% formalin 50 ml distilled water 50 ml Ammans Lactophenol (Pimentel, 1967): kerosene 20 ml lactic acid 20 ml glycerol 40 ml 21309047 BFG12805 37. distilled water 20 ml 20% Glycerin solution (Pennak, 1953): glycerin 20 ml 70% ethanol 80 ml 70% ethanol: 95% ethanol 70 ml distilled water 25 ml Separation of the invertebrates Separation of invertebrates from the sediment sample was made within 12 hours of sample collection. The collected sample, a mixture of mud or clay and water, was poured into a large white enamel plate and gently mixed with clean water so that the movement of invertebrates could be easily observed. The invertebrates were carefully separated from the sample with forceps and an eye dropper. The separated invertebrates were preserved in K.A.A. solution (Pimentel, 1967). After one day, I replaced the K.A.A. solution with 5% formalin, and stored the invertebrates in vials at room temperature. All vials were coded by Dr.M. Dickroan (my thesis supervisor) to avoid unconscious bias before classifying and counting the specimens at each station. Classification of the invertebrates Two days before the identification of the specimens, I replaced the 5% formalin solution with a 70% ethanol solution. For Annelida, one day before identification', I placed the specimens in Ammans Lactophenol solution. For Diptera, one day before observation, I placed the specimens into 20% glycerine 21309C48 BFG12806 38 solution. The Annelids were carefully picked from the vials, and placed on clean glass slides, mounted in Ammans Loctophenol solution and covered with a glass cover slip. Next, they were each examined under a Leitz compound microscope at 400 x magnification. Dichotomous keys (Pennak, 1953; Pimental, 1967; Brinkhurst and Cook, 1974) were used for identifying the collected Annelid specimens. The specimens of Diptera were mounted in pure glycerin solution, observed under a Leitz microscope at 400 x magnification. A dichotomous key (Pennak, 1953) was used for classifying the specimens (Wiederholm, 1983). Large specimens of other classes were observed under the dissecting microscope. Smaller ones mounted in 70% ethanol solution were identified by means of Leitz compound microscope. Dichotomous keys to the betjiic invertebrates were in Pennak (1953). Evaluation of symmetry patterns and abnormalities The symmetry of each invertebrate was observed (ie. labial plates of each chironomid specimen was checked for symmetry). If a deformed structure was found, it was recorded (see results section). The number of chironomids and the number of malformations observed were listed and were later transcribed into tables (see results section). Statistical Analyses The Student's "t" test was used to determine the difference BFG12807 21309049 39 between controls and tested samples. The Sperman-Kendall rank correlation analysis was used for non-parametric tests and the relationship between the frequency of Chironomid larvae labial plate deformities and the SOS chromotest induction factor level. Correlation coefficients were used for parametric tests. Calculation of Species Diversity Indices Simpson's diversity index: N( N-1 ) D =----------------------------- En ( n - 1 ) where N was the total number of specimens, n was the individual number of different species. Shannon-Wiener's diversity index: k H= -E p i=l log p where p was the proportion of individual species, and k was the number of species. 21309050 BFG12808 40. RESULTS Genotoxicity of sediment samples Distilled water extracted sediment samples were tested using the SOS chromotest. All samples gave a negative response to the SOS chromotest with or without metabolic activation (Table 1). The 10% DMSO extracts of sediment samples yielded a higher induction factor value with metabolic activation than did the sample without metabolic activation (Table 1). With metabolic activation, the samples from site D-l, collected under the discharge pipe of the B.F.Goodrich Co. showed the highest induction factor (I.F = 2.053 0.361), and the samples from station B, located in the agriculturally influenced portion of the Welland River, showed the lowest induction factor (I.F = 1.062 0.066). The DMSO extract with metabolic activation for station D-l gave a significant positive response (P<0.01), stations D-2 and E gave a weaker but still significant positive response (p<0.05). The genotoxicity of sediment extracts was higher in the industrial influenced portion than it was in the agriculturally influenced portion of the river (Table 1). Without metabolic activation, all DMSO extracts of samples gave negative responses to the SOS chromotest (Table 1). To confirm these results, another set of samples was taken at the same locations in the river four months after the first set of samples was taken. At this time, only the DMSO extract of the sediment samples from station D-l gave a significant positive SOS chromotest response with metabolic activation (P<0.01). BFG12809 21309051 Table 1. The SOS chromotest induction factor of sediment extracts from seven locations in the Welland River (test concentration = 20 mg wet weight/ assay). BFG12810 21309052 42. Table 1. Induction Factor of seven samples sample sites A B C D-l D-2 F 10% DMSO extract +S9 10% DMSO - S9 (December,1986) (April,1987) mean n S. D. mean n S. D. (December ,1986) mean n S.D. 1.173 1.062 1.664 1.638 1.272 1.450 1.049 6 0.123 0.854 4 0.066 0.915 10 0.429 1.072 4 0.135 2.053 13 0.361 1.231 4 0.070 1.537 9 0.427 1.200 4 0.168 1.209 10 0.126 0.995 4 0.032 1.179 11 0.209 0.978 4 0.119 4 0.098 2 0.007 3 0.140 3 0.240 3 0.140 6 0.058 Table 1. (continuous) Distilled Water Extract+S9 Distilled Water Extract -S9 sample sites (December, 1986) mean n S.D. (December, 1987) mean n S.D. A B C D-l D-2 E F 1.064 3 0.086 0.993 4 0.019 0.984 4 0.010 1.285 4 0.041 1.059 4 0.084 1.024 4 0.097 0.964 4 0.026 0.873 4 0.033 0.827 4 0.094 0.806 3 0.066 1.062 3 0.088 1.031 3 0.078 0.827 3 0.031 1.200 6 0.441 21309053 BFG12811 43. Of the samples assayed, only samples from station D-l were pro-genotoxic in DMSO extract. To construct a dose-dependent response pattern for the samples which gave a positive response to the SOS chromotest, the enzyme activities of beta-galactosidase and alkaline phosphatase were assayed. The dose-response curve for samples from station D-l showed that the induction factor rose to a maximum at a concentration of 20mg/assay, and then decreased (Fig. 3). The results of the alkaline phosphatase assay for DMSO extracts with S9 for samples from station D-l indicated that the enzyme units decreased with increasing sample concentration (Fig. 4). In DMSO extracts with S9 added, almost all samples except for those from station A had slight inhibition effects on general protein biosynthesis when doses were increased (Fig. 5). According to the alkaline phosphatase activity levels (as percentage of initial level!, DMSO extracts from stations C and D-l showed a significant (p<0.05) inhibition effect on protein biosynthesis (Fig.5). DMSO extracts without S9 in the SOS chromotest did not appear to inhibit protein biosynthesis (Fig. 6), though station F did display a slight inhibition effect (0.05< p <0.10). Among the distilled water extracts, only samples from stations C and D-l showed a significant (p<0.05) inhibition of protein biosynthesis when tested with S9 (Fig. 7). Distilled water extracts of samples from stations F without S9 slightly inhibited protein biosynthesis (0.05<p<0.10) (Fig. 7). These results implied that some water soluble contaminants in samples BFG12812 21309054 frpm stations C and D-l were toxic activated by metabolic activation. BFG12813 21309055 Figure 3. The dose-response relationship for the SOS chromotest. BFG12814 9soG orrg 46 SAMPLE D-1 DOSE-RESPONSE O AP UNITS beta-G UNITS SAMPLE D-1, DMSO S9 INDUCTION FACTOR OF SOS CHROMOT^-. r j AS0602T2 BFG12815 Figure 4. The percent alkaline phosphatase activity in the SOS chromotest of 10% DMSO extracts with S9 (vertical bars represent one standard deviation). BFG12816 1309058 DMSO +S9 48 21303059 BFG12817 Figure 5. The percent alkaline phosphatase activity in the SOS chromotest of 10% DMSO extracts without S9 (vertical bars represent one standard deviation). BFG12818 09060STS Si:--;* AP ACTIVITY (PERCENTAGE) AP ACTIVITY (PERCENTAGE) AP ACTIVITY (PERCENTAGE) DMSO -S9 CONCENTRATION mg/assay BFG12819 T9060ST2 Figure 6. The percent alkaline phosphatase activity in the SOS chromotest of distilled water extracts with S9 (vertical bars represent one standard deviation). BFG12820 21309062 DW +S9 52. 69060273 BFG12821 Figure 7. The percent, alkaline phosphatase activity in the SOS chromotest of distilled water extracts without S9 (vertical bars represent one standard deviation). BFG12822 SJ.309064  SAMPLE A DW -S9 t aSAMPLE B 54 O SAMPLE C n SAMPLE D-1 *SAMPLE D*2 t 2JL309065 BFG12823 n 55. The frequency of labial plate deformities in Chironomids The appearance of normal mouth parts in the chironomid labial plate is shown in Fig. 8. The structure was symmetrical and well defined. The deformed chironomid labial plates included various types of asymmetry, such as mild asymmetry in the numbers of lateral teeth (Fig. 9), fused processes on the median teeth, gaps in the labial plate. Deformities of mandibles and antennae were not analyzed, although some deformed mandibles were occasionally noted. Six out of 15 chironomid genera which were found in the agricultural influenced portion of the Welland River appeared to have deformed individuals, while 6 out of 10 genera in the industrial influenced portion of the river had deformed specimens (Table 3). Labial plate deformities of chironomid larvae showed that the highest frequency of deformed labial plates was associated with the industrial influenced portion of the Welland River (station D-l, 10.90% 3.23%, Fig. 11). The control site A displayed the lowest frequency of deformities (3.84% 1.32%) (Fig. 10). The frequency of deformed chironomid labial plates (6.42% 3.30%) was higher at station D-2 than the agricultural site A. Site E displayed a slightly higher frequency of deformities<(4.20% 0.28%) than site A. Site B (6.29% 1.71%), located at the confluence of the Welland river and an old discharge pipe carring farm field runoff yielded a higher frequency of deformed Chironomid labial plates than the nearby upstream site A (Fig. 10), though the difference was not significant (p> 0.05). Station F displayed a very low abundance BFG12824 99060STZ 56. of chironomids as only 3 specimens were found in three composite samples. Therefore, site F was not included in the chironomid deformity analyses. The difference in the frequency of deformities in Chironomid labial plates was statistically significant between site A and D-l, though there were no significant differences between other stations. 3FG12825 21309067 Figure 8. "Normal" structure of chironomid labial plate (modified from Wiederholm, 1983). < | 'r i4 31309068 BFG12826 58 BFG12827 21309069 Figure 9. Deformed chironooiid labial plate '> :: x SV x * 5 :* A I 21309070 BFG12828 hr: "T 60 BFG12829 21309071 Figure 10. The frequency (percentage) of deformities in chironoroid labial plates at 5 sample sites in the Welland River (vertical lines represent one standard deviation). BFG12830 21309072 % Frequency of deformities in chironomld labial plates 62 BFG12831 21308073 63. The correlation between the genotoxicity of sediments and the frequency of deformities in chironomid labial plates The highest genotoxicity of sediments and the highest frequency of deformities in chironomid labial plates occurred at industrial site D-l which was at the end of the discharge pipe of a company which produces polyvinyl chlorides (Fig. 1). The correlation between the genotoxicity of the sediment samples and the frequency of deformities of labial plates of chironomid larvae showed a significant positive correlation ( r = 0.88, p < 0.05). However, when tested using a non-parametric test (Sperman-Kendall Rank Test) the correlation was not significant at the p < 0.05 level because a high correlation coefficient of 0.9 is required by the non-parametric test for five or less degrees of freedom when using the less powerful non-parametric test. It is interesting that station B showed a relatively high frequency of deformities in chironomid labial plates, but the sediment samples had the lowest SOS chromotest induction factor (negative responses). Reasons for this are offered in the discussion section. The standard error for the frequency of deformities was very high (Fig.11). As a result, the differences between most sites were not significant. However, the values of sites A and D-l were significantly different (p<0.05). BFG12832 21309074 Figure 11. Correlation between the genotoxicity and the frequency of deformities in chironomid labial plates (vertical bars represent one standard deviation of % deformity frequency estimates, and horizontal bars represent one standard deviation in SOS induction factor estimates) BFG12833 21309075 % frequency of deformities in chironomid labial plates SOS induction factor r--| 2.6 BFG12834 21309076 66. Benthic invertebrates Invertebrates were sampled at the same seven locations as described for the genotoxicity tests (Fig 1). During a one month period of sampling, it was found that the generic richness was higher at the control station and lowest in the industrial influenced portions of the Welland River *(Fig. 12). The most abundant benthic invertebrates in the agricultural control sections were Oligochaetes, Crustaceans and Chironomids (Table 2). In the industrial sections, the most abundant species were Oligochaetes and Chironomids; Crustaceans were not found. It is noteworthy that site C "which was polluted by heavy metals from a steel company's effluents was essentially bereft of all benthic invertebrates (Table 3). Because Crustaceans and Ephemeropterans were essentially absent in the industrial influenced portion of the Welland river during the entire sampling period, I concluded that they were pollution sensitive taxa (Table. 3). There were 13 Crustacean genera in my samples from the agricultural area, only 1 genus was found in the industrial area, a single specimen of a Cyclopoid copepod. Mayfly nymphs (abundant upstream) were essentially absent in the industrial area during the sampling period. The highest abundance of benthic invertebrates was at the B.F. Goodrich site D-2, (11,899 2,721 mlsurface area of sediment), while the lowest abundance of benthic invertebrates was at the Atlas Steels site C, (114 85 m* Table 2). The agricultural (control) site had higher benthic invertebrate abundance than the industrial sites except site D-l and D-2 where BFG12835 21309077 Table 2. Abundance of benthic invertebrates at seven locations in the Welland River ( number of specimens /m*) (mean standard deviation). 21309078 <,*<:; rjr* :;r; ,'j idl BFG12836 68 Table 2. Benthic Invertebrate Abundance (number of specimens /ma ). sample site Total Oligochaetes Chironomids A B C D-l D-2 E F 4654 219 5610 1352 114 85 8329 1842 11899 2721 1325 584 3860 1377 138 42 306 80 109 81 7944 1990 11510 2806 7 7 3794 1390 551 241 4932 1257 0.0 347 126 375 153 1318 591 14 8 Table 2 (continued) sample Oligochaetes Chironomids site ------------------------------------------------------------------------------------------------------------------------------------------------------ Limnodrilus sp. Tanypus sp. Ablabesmvia sp. Chironomus sp A B C D-l D-2 117 42 299 81 33 10 2051 1068 11466 2806 0.0 3272 1251 0.0 634 634 0.0 237 163 223 163 64 64 55 223 107 14 0.0 19 i 0.0 0.0 0.0 0.0 14 i 6 0.0 24 24 142 58 0.0 0.0 Table 2. (continued) . ample Chironomids site ----------------------------i----------------------------------------------------------------Isopods Pseudochironomus sp. Tanytarsus sp. Arophipods 152 + 98 36 27 0.0 -1 5 5 2 0.0 0.0 0.0 , 104 50 2119 950 0.0 0.0 0.0 0.0 0.0 332 104 50 32 0.0 0.0 0.0 0.0 0.0 124 33 11 11 0.0 0.0 0.0 0.0 0.0 **i w a o o w > i u i i i j m 21309079 BFG12837 Figure 12. Generic richness of benthic invertebrates at seven locations in the Welland River (number of genera per 700 cm ) (vertical lines represent one standard deviation). BFGUS38 J8060STZ GENERIC RICHNESS OF Bl 22~ 20 18- 16" 14" c 12- a io- 8" 6" 4- 2- rh rii B C D-1 D-2 E SAMPLE LOCATIONS IN THE WELLAND RIVER 21309081 BFG12839 Table 3. Benthic invertebrate taxa at seven locations in the Welland River ( number of specimens per 700 cma). BFG12840 29060ETZ s2 I a 2a a 2X309083 72 ! rirtliiu ||. :---------- 'rf' BFG12841 i n;smu ilatrl if U H llliD il U utiU <j. ntoiicouou 21309084 ;a.3Sl.i 2s 3 BFG12842 73 74. substantial quantities of organic matter were discharged (MOE, 1987). Site D-2 showed the highest oligochaete abundance, (11,510 + 2,806 m ,Table 2). Sites D-l, D-2 and F had higher oligochaete abundances than that of the agricultural control section, while sites C and E appeared lower than the control in oligochaete abundances (Table 2). The chironomid abundance was higher in the agricultural section, especially site B. Chironomid abundance at site B (OWMC Swale) was over 10 times higher than at any other sites (Table 2). At the Atles industrial site C, all chironomid species were absent. Although the number of Oligochaete genera in the agricultural and industrial areas was 5 and 6, respectively, Aulophorus spp. (Dero spp.) and Audrilus spp. were only found in the industrial influenced portions of the river. The most abundant species of Oligochaetes in both agricultural and industrial influenced sections was Limnodrilus spp., but the relative abundance of Limnodrilus spp. was much higher in the / industrialized influenced sections of the Welland River (Tables 2 and 4). At sites A and B, the relative abundance of Limnodrilus spp. ranged from 5.24% 3.53% to 5.43% 1.18% of the total benthic invertebrates, while in the industrial sections it was 34.92% 23.39% to 95.50% 2.56% except at station E where ammonia toxicity resulted in the virtual elimination of all benthic invertebrates. (Tables 3 and 4). In the agricultural area, 15 Chironomid genera were found, while in the industrialized sectionsof the Welland River only 10 Chironomid genera were observed. Tanytarsus spp., BFG12843 21309085 Table 4. Relative abundance of benthic invertebrate taxa at seven locations in the Welland River (percentage abundance) (mean + standard deviation). BFG12844 21309086 76 Table 4. Relative abundance of benthic invertebrates ( percentage abundance) sample Oligochaetes iste Chironomids Isopods Arophipods A B C D-l D-2 E F 5.80 3.38 5.56 1.13 98.33 1.67 93.67 4.07 95.71 2.58 0.96 0.96 97.58 1.04 11.82 5.14 87.25 1.03 0.0 5.67 3.43 4.20 2.61 99.04 0.96 0.55 0.28 14.28 9.15 0.75 0.37 0.0 0.0 0.0 0.0 0.0 4.39 1.60 0.11 0.11 0.0 0.0 0.0 0.0 0.0 Table 4. (continued) Oligochaetes Chironomids sample -------------------------------------- ----------------------------------------------------------- site Limnodrilus sp. Tanypus sp. Ablabesmyia sp. Chironomus sp A B C D-l D-2 E F 5.24 3.53 5.43 1.18 71.67 28.33 34.92 23.39 95.50 2.56 0.0 83.28 2.07 0.0 4.45 2.16 15.61 15.61 0.25 0.09 0.0 0.0 3.88 2.33 0.24 0.06 2.95 2.56 0.0 3.36 3.36 0.0 0.24 0.24 0.0 0.0 0.25 0.16 0.0 0.25 0.25 1.15 0.25 0.0 0.0 Table 4. (continued) Chironomids sample site Pseudochironomus sp. Tanytarsus sp. A B C D-l D-2 E F 2.54 0.88 37.81 12.63 0.0 0.04 0.04 0.0 0.0 0.0 2.52 1.45 0.82 0.66 0.0 0.0 0.0 0.0 0.0 213090S7 BFG12845 77. Cryptochironomus spp. and Coalotanvuus spp. were not found in the industrial influenced portions of the river. Cricotopus spp., Psectrocladius spp. and Natarsia spp. were not found in the agricultural areas. The most abundant species of Chironomids in the agricultural sections were Tanytarsus spp., Ablabesmyia spp., while in the industrial sections, Tanypus spp., Pseudochironoms spp. and Chironomus spp. dominated (Tables 2 and 4). The relative abundance of Chironomids was higher in the agricultural areas, at stations A and B (11.82% 5.14% and 87.25% 1.03%, respectively). In the industrial areas, the relative abundance of Chironomids was low (0.55% 0.28% to 5.67% 3.45%) except at station E (99.04% . 0.96%) where ammonia shock loads eliminated nearly every invertebrate except a few tolerant chironomid taxa. It is interesting that stations A and B displayed substantial differences in their species pattern. Station B which I belive receives more agricultural chemicals (discussed later) t than station A had slightly lower species richness and diversity than station A (Fig. 8 and Table 5), while site B displayed a slightly higher abundance of some of the more pollution tolerant benthic invertebrates (Table 2). There was a great difference in the relative abundance of chironomid and crustacean species between stations A and B. The relative abundance of chironomids was greater at station B (87.25% 1.03%) in comparison with station A (11.82% 5.14%). The roost abundant chironomid species shifted from Ablabesmyia spp. at station A to Tanvpus spp. and Pseudochironomus spp. at station B (Table 5). At the same time, the relative abundance of isopods and amphipods was lower at BFG12846 21309068 78, station B (0.75% 0.37% for isopods and 0.11% 1 0.11% for amphipods) as compared with station A (14.28% 9.15% for isopods and 4.39% 1.60% for amphipods). Comparing the relative abundance of Oligochaetes and Chironomids along the river, it was found that a significant negative correlation (Sperman-Kendall Rank test) (r = -1, p< 0.01) was observed. In other words, as the relative abundance of Oligochaetes increased the relative abundance of Chironomids decreased (Table 4). This is suggestive of competitive exclusion, an hypothesis which was not tested in this study. 21309089 T............. ................. ... BFG12847 - ... .......... n 79. Species diversity and species richness of bethic invertebrates The species diversity at the generic level was higher in the agricultural influenced portions of the Welland River than in the industrialized areas. The difference in generic diversity between the agricultural and industrialized areas was statistically significant (p< 0.05). The lowest generic diversity was recorded at site C (Simpson's index was 0.85 0.45, Shannon-Wiener's index was 0.09 0.09) (Table 5). The highest generic diversity was observed at the agricultural control site A (Simpson's index was 6.15 2.20, Shannon-Wiener' s index was 0.86 0.13). The differences in generic diversity were consistent with the generic richness data set. Locations displaying high generic richness also displayed high generic diversities. The agricultural control site A displayed the highest generic richness (20 3 per 700 cm2, surface area of sediment) while the industrial sample station C had the lowest generic richness (2 1 per 700 cm1) (Fig. 8). The difference in generic richness between the agricultural and industrialized sections of the Welland River was statistically significant (p< 0.05). At site D-l, there was a relatively high generic richness (8 1 per 700 cm1) and diversity (Simpson's index 1.75 0.32) than at any other industrial sites (Fig. 12). This was due to the 2 additional oligochaete species found at D-l, Auldrilus spp. and Aulophorus spp. (Table 3). The decrease in generic richness and diversity in the industrial influenced portion of the Welland River was mainly the result of absent Crustacean species and Mayfly nymphs (Table 3). At station F, generic richness and .... ___ BFG12848 21309090 80. diversity increased significantly compared with the upstream stations D-2 and E (Fig. 12 and Table 5). This was due to the increase in the number of oliogochaete species and dragonfly nymph species at station F (Table 3). BFG12849 TS060STZ N: Table 5. Generic diversify of benthic invertebrates at seven locations in the Welland River (mean standard deviation). BFG12850 2J.309092 82. Table 5 Species diversity of benthic invertebrates at generic level. sample site Simpson's index Shannon-Wiener's index A B C D-l D-2 E F 6.15 2.20 3.06 0.18 0.85 0.45 1.75 db 0.32 1.10 0.06 1.11 k 0.03 1.42 0.07 0.86 db 1.23 0.66 0.04 0.09 k 0.09 0.35 k 0.08 0.10 k 0.04 0.10 k 0.01 0.28 0.04 21309093 BFG12851 DISCUSSION Sediment genotoxicity Each extract of the sediment samples was tested more than three times using the SOS chromotest. Absolute values for the induction factors varied slightly between experiments. This was a function of the initial number of bacteria and the degree of activity of the S9 mixture which was used (Quillardet et al., 1985). The DMSO extract of samples from station D-l gave a significant positive response ( p < 0.01 ) in both the December and April samples. I inferred from this that there were some potential genotoxic materials at station D-l under the discharge pipe of the B.F. Goodrich Co. The presence of vinyl chloride, a known genotoxin, in their discharge water (MOE, 1987) was the most causitive agent. Although sample D-2 and sample E gave weak positive responses in the SOS chromotest in the first collection of samples (p < 0.05, December, 1986), they were not positive in the second collections (April, 1987). I therefore considered the genotoxicity at sample sites D-2 and E to be seasonal and less strong than D-1. DMSO is widely used as an organic solvent in genotoxicity tests (Ames et al., 1975; Quillardet et al., 1982). DMSO sample extracts were the only fraction that gave a positive response to the genotoxicity test. Therefore, it was concluded that some organic materials were genotoxic in the sediments of sample D-l. The industrial MOE (1987) reported that the B.F. Goodrich BFG12852 21309094 84. effluent at station D-l contained detectable concentrations of vinyl chloride (0.09 kg per day or 0.041 ppm). Vinyl chloride has been demonstrated to be a potential genotoxin when activated by the S9 mixture (liver extract) (McCann et al., 1975). Therefore, vinyl chloride in the sediments at station D-l may have induced the observed SOS response at that site. Although DMSO extracts of sample D-l yielded a positive response in the SOS chromotest, the induction factor was not very high ( 2.053 0.361 at 20 mg wet weight/assay) compared to the positive control 2AA ( 29.11 + 5.01 at 10 ug/assay). The genotoxicity of the B.F. Goodrich sediments was therefore not strong. Thi3 may be due to the low concentration of potential genotoxic compounds in the sediments at this site. Low levels of mutagenic contaminants in sediments from Prince Edward Island were also reported by Xu and Dutka (1986). Their genotoxicity studies of sediment samples from Prince Edward Island revealed that the induction factor (positive response to the SOS chromotest) was also quite low (the highest induction factor for their samples was 1.65) (Xu, and Dutka, 1986). Short-term assays of genotoxicity for environmental samples have been developed in a number of studies. The SOS chromotest was recommended as a sensitive genotoxicity test (Quillardet et al., 1982 and 1985). In my study, it was found that the SOS chromotest was more sensitive than the Ames test. At concentrations of 20 mg per assay sample D-l yielded a positive response to the SOS chromotest, while the Ames test was negative even at 100 mg per assay. BFG12853 21309095 85. There are three possible explanations why the Ames test yielded negative results at station D while the SOS test was positive. First, the SOS chromotest is more sensitive than the Ames test (Quillardet et al., 1982). Aquatic ecosystems in close proximity to human activity will contain literally hundreds of contaminants and their by-products. The concentration and dissolution rates of each of these chemicals vary in different aquatic systems (Samolloff et al., 1983; Dutka et al. , 1981). Generally, the concentrations of organic lipophilic compounds are lower in the water phase than in surface sediments (Wetzel, 1983). A previous study conducted in our lab (World Wildlife Report, 1987) showed that the liquid samples which were taken at the same locations as the sediment samples gave negative responses in both the SOS chromotest and the Ames test (Veltri, personal comm.). It appeared that negative genotoxicity responses from water samples and positive responses from sediment samples at the same test stations were the result of the higher concentrations of DMSO soluble materials in the sediments. Therefore, it may be speculated that the concentrations of the potential genotoxic compounds were very low in the water of the Welland River and were lower than the detection level of the Ames test. Second, the inducible SOS repair in EL coli K-12 PQ 37 responds to mutagens and indirect genotoxic agents (Quillardet et m al., 1985). The SOS system is inducible when a cell's DNA is damaged or its DNA replication is inhibited (Walker, 1984). In the Ames test, the reversions have to be caused by DNA base-mutagen actions. For example, the TA98 strain in the Ames BFG12854 21309096 86. test detects mutagens that cause base-pair substitutions, while the TA100 strain detects various frameshift mutagens (Haron and Ames, 1983). The different mechanisms in the SOS chromotest and the Ames test may result in different responses to the same genotoxin. For example, hydroxyurea, has been showed to have teratogenic and carcinogenic effect's on mammals, and is a strong inhibitor of DNA synthesis (Timson, 1975). Hydroxyurea gave a positive response to the SOS chromotest and a negative response to the Ames test (Quillardet et al., 1985). The results of my research indicated that the potential genotoxic compounds in the samples from the Welland River were indirectly genotoxic and would be expected to be mutagenic only when metabolically activated. The third factor is that the Ames test needs low toxicity levels if the test strain is to survive. Therefore, the Ames test cannot be used to detect mutagenic compounds if they are so toxic that they kill the Ames test bacteria (Ames et al., 1975; Quillardet et al., 1985). The results of my study indicated that the extracts from station D-l were toxic (Fig. 3), probably as a result of their B.F. Goodrich ammonia discharges. The toxicity of the samples might therefore have resulted in the negative responses observed in the Ames tests which K.Veltri and I carried out (World Wildlife Report, 1987). It is also worthy of note that the positive response of the organic compounds extracted by DMSO in the SOS chromotest were obtained only in the presence of the S9 metabolic activator. The S9 mixture was prepared from rat livers and contained enzymes BFG12855 ~T 21309097 87. that metabolize pro-mutagenic substances converting them to mutagens (Ames et al., 1975; Venitt and Parry, 1984). Thus, the data obtained in this study implied that some organic compounds in the samples tested may be pro-genotoxic. The present study suggested that one site in the industrialized area in the Welland River was contaminated with potential genotoxic waste materials. Further work of impacts of genotoxic pollution on the aquatic ecosystem in the Welland River is warranted. Deformities of chironomid labial plates Deformities in chironomid larvae occur naturally. In Parry Sound, Georgian Bay, 0.0-1.3% of the chironomus cucini collected were deformed (Haer and Carter, 1976). Wiederholm (1984) reported that in the unpolluted (control) areas the frequency of deformities in chironomid larvae ranged from 0.7-0.8%. In my study, it was found that the frequency of natural occurrence of deformities in chironomid labial plates was 3.84% 1.32% (station A, Fig. 10). Thus, in the Welland River, the frequency (%) of naturally deformed chironomid labial plates was slightly higher when compared with other studies. Observations of deformed labial plates in chironomids are common because these deformities are very easily detected (Hamilton et al., 1971; Wiederholm, 1984; Warwick et al., 1987; Hamilton et al. (1971). Fifteen Chironomid genera were found in the agricultural areas of the Welland River and 10 in the industrial sections. Six out of 15 genera in the control sections appeared to have BFG12856 1309098 88. deformed individuals, six out of 10 genera in the industrial sections had deformed specimens (Table 1). Therefore, it was concluded that in the industrialized areas of the Welland River chironomids had higher proportions of deformed taxa than in the agricultural areas. This may have indicated that industrial pollution in the Welland River was more mutagenic than the pesticides and related agricultural chemicals released into the river in the agricultural areas. In most cases, deformed chironomid labial plate teeth were shorter than normal ones. In some cases, there were extra teeth. Deformed labial plates were invariably asymmetrical. The highest percentage of deformities in chironomid labial plates was found at station D-l (10.90% 3.23%). This was significantly higher (p<0.05) than at station A (3.84% 1.32%).Although chironomids at station D-2 had higher frequencies of deformed labial plates than chironomids at station *A, the difference was not significant at the p<0.05 level (Fig. 10). Station D-l was affected by B.F. Goodrich Co. effluent which according to the MOE (1987) and my SOS chromotest analyses contained some progenotoxic materials. The frequency of deformities in chironomid labial plates in the agricultural areas was not very high compared to the industrial sections. Wiederholm (1984) found that heavily polluted lakes had higher frequencies of deformities in chironomids than did less polluted lakes ( 4.1%-10.7% in polluted lakes, 1.4% to 1.6% in slightly polluted lakes and 0.7% to 0.8% in control lakes). In heavily polluted lakes the frequency of labial plate deformities ranged from 3.8% to 25.0%. At Port Hope, BFG12857 21309099 89. Ontario, Chironomus sp. showed a very high frequency frequency of deformities within the heavily polluted inner harbour area (83%) compared with the outer harbour (14%) where levels of radioactivity were much lower. From the results of the frequency of deformities analyses, the high percentages of deformed chironmid labial plates at station D-l may be associated with the genotoxic pollution at this site. Station B was located in the agricultural influenced portion of the river. At this station, it was found that a higher frequency of deformites in chironomid labial plates (6.29% 1.71%) occurred than at the upstream station A (3.84% 1.32%). Both stations were affected by agricultural effluents. Station A was located in an aquatic plant lined stream course while station B was located at the end of a pipe draining agricultural runoff from a storm sewer near the proposed site of the Ontario Waste Management Corporation. The observed difference in the frequency of chironomid labial plate deformities between these two locations may be because aquatic plants play an important role in removing hazardous waste materials from the water (Bingham, 1973). Few aquatic plants occurred at station B while many occurred at station A. Chironomids from station E had a slightly higher frequency of deformities in labial plates (4.20% 0.28%) compared with the upstream control station A, but the difference was not statistically significant (p>0.05). Correlations between genotoxicity and the frequency of BFG12858 00T60ST 90. deformities in chironomid labial plates. The cause-effect relationship between high chironomid deformity frequencies and certain effluents of industrial discharges was discussed by Warwick et al. (1987). Theoretically, deformities have to be caused by interruption of cell physiological activities including DHA-damaging substances (Warwick et al., 1987). Warwick (1980) found that in the sediment cores taken from the Bay of Quinte the frequency of deformed chironomid larvae increased from 0.09% in the pre-European sediments to 1.06% in the 1950's sediments and to 1.99% in the present day (1972) chironomid population of the bay. It was concluded that the increase in the frequency of chironomid deformities was associated with the increased level of pollution in the bay. Warwick et al. (1987) suggested that genotoxins induce an elevated frequency of deformities in chironomids. Wiederholm (1984) related the high frequencies of deformed chironomids to the presence of heavy metals including Ni, Pb, Cr etc. which were demonstated to be genotoxic (Fleesel, 1979). However, no studies have been published on sediment genotoxicity using the SOS chrorootest and the frequency of deformities in chironomid labial plates. My study revealed that the highest sediment genotoxicity and the highest frequency of deformities in chironomid labial plates both occurred at the same site (station D-l) (Table 2 and Fig. 10). The correlation between sediment genotoxicity and the frequency of deformities in chironomid labial plates was tested statistically. In the parametric test, the correlation BFG12859 T0T60ET l&Zi^tliliiiiiL'iiiili: 91. coefficient was 0.88, which was a statistically significantly positive correlation (p<0.05). However, this correlation was not significant when the non-parametric test was employed (p<0.05). The reason for adopting the non-parametric test was that the sample size (number of replicated and stations, Appendix 3) was small. It would not be safe to use a non-parametric test with so few samples (Ward, 1978). Other studies have concluded that there were detectable carcinogenic chemicals in the industrial influenced portion of the Welland River (Kaiser and Comba, 1983). At station D, water samples contained chloroform (75 ng/1), carbon tetrachloride (15 ng/1) and dibromomethane (10 ng/1). Those compounds were reported to be carcinogens (McCann et al., 1975; Quillardet et al., 1985). There were detectable levels of vinyl chloride (0.09 kg/day or 0.041 ppm. MOE, 1987) in the water samples taken at station D. Vinyl chloride was demonstrated to be genotoxic (McCann et al., 1975). At station E, water samples contained even higher concentrations of chloroform (1800-3750 ng/1, in 1980; 150- 300 ng/1, in 1981), carbon tetrahloride (120 ng/1, in 1980; 15-60 ng/1, in 1981) and dibromonethane (15-20 ng/1, 1980; 5 ng/1, 1981) (Kairser and Comba, 1983). The DMSO extracts of sediment from station D-l were pro-genotoxic, while DMSO extracts of sediments from stations D-2 and E were possibly pro-genotoxic. It was therefore inferred that genotoxic pollutants from industrial sources in the sediments of the Welland River ma^. have caused the high frequency of deformities in chironomids which I observed. BFG12860 21309102 92. Sediment from station B had the lowest SOS chromotest induction factor (negative response), but site B displayed a relatively high frequency of deformities in chironomids (Table 1 and Fig. 8). This may be due to the agricultural effluent which contained genotoxic compounds. Some genotoxins are undetectable in bacterial tests. The best example of this is that PCB (Arochlor 1254) is a carcinogen but does not give a positive response in either the SOS chromotest or the Ames test (Quillardet et al., 1985). The possibility that agricultural chemicals were responsible for the high frequency of deformities in chironomid labial plates at station B cannot be ruled out. Ecologically, the main difference between stations A and B is that station A was located at a bigger creek whose water passed through an extensive aquatic plant lined stream course, whil.e station B was located at the end of a small sewer and its water flowed directly into the Welland River through this pipe. Aquatic plants play an important role in removing pollutants from the water (Bingham, 1973). Station A was a more natural site in terms of its generic richness (Fig. 12) and the relative abundance of individual benthic invetebrate genera (Table 2). Therefore, station B may be slightly polluted by agricultural effluents. < Toxicity vs genotoxicity of the sediment samples Sample extracts from stations C and D-l were found to be toxic according to the alkaline phosphatase test which is part of the SOS chromotest. In the SOS chromotest, alkaline phosphatase was used to check protein biosynthesis activity. Alkaline BFG12861 21309103 93. phosphatase was noninducible by DNA damaging agents. During the test period, alkaline phosphatase activity should be maintained at its initial level or increased if the test samples are not toxic (Quillardet et al., 1982). A decrease in the enyzme activity of alkaline phosphatase is considered to be an inhibititory response to a toxin and protein biosynthesis of the . coli test strain is reduced (Quillardet et al., 1982). Comparing the alkaline phosphatase activity in DMSO extracts with and without the S9 mixture, major differences were noted (Figs. 4 and 5). When tested with S9, samples from stations C and D-l showed a significant decrease in alkaline phosphatase activity (p< 0.05). In contrast, when tested without S9, none of the samples except those from station F (0.05< p <0.10) inhibited protein biosynthesis. Although it was reported that the S9 mixture displayed a slight inhibitory effect on protein biosynthesis (Quillardet et al., 1985), the results of the inhibition of protein biosynthesis in this study were obviously not because of the S9 mixture itself as indicated by my controls (Appendix 2). Thus the S9 (liver extract) converted some materials into toxins. It was concluded, therefore, that potential toxic contaminants occurred in the sediments of the Welland River, especially near the discharge pipe of the B.F. i Goodrich Co. and the Atlas-Mansfield storm sewer. Those potential toxic organic compounds might be associated with agricultural and industrial effluents. As the effluents contained many compounds which were not identified, it was impossible to determine which compounds caused the toxicity responses. I concluded, however, BFG12862 21309X04 94. that in many cases, chemical toxicity prevented positive Ames test mutagenicity analyses from being detected with the presence of S9 mixture. The distilled water extracts of the Welland River sediments in the presence of the S9 (liver extracts) caused alkaline phosphatase activity to decrease significantly (p< 0.05) in samples taken from stations C and D-l, while without S9, only sample F showed a slight (p>0.05, p <0.10) inhibition of protein biosynthesis. The results of the distilled water extracts of samples from stations C and D-l implied that the toxicity of some DMSO and water soluble compounds was activated by the S9 mixture. For station F, the addition of the S9 (liver extract! mixture mixture seemed to detoxify the sedimnet DMSO extracts (Figs. 5 and 7). Several other studies were found in which the addition of S9 increased the toxicity of sample materials , and decreased toxicity of other compounds (Vanderneulen et al., 1985; Sato et al., 1983). Mr. Brindle's studies of the Welland River showed that the sediments from site C (near the discharge location of the Atlas Steels Co.) contained very high concentrations of heavy metals, such as Pb, Zn, Ni, and Cr etc. (Dickman et al., 1988). Cr, Ni, and Pb have been reported to be both toxic and genotoxic (Fleesel, 1984; Olivier et al., 1987). Site E was contamined with ammonia and nitrates at toxic levels (Dickman et al., 1988). Benthic invertebrate community structure In my reasearch, only live benthic invertebrates (ie. those which moved during sorting) were preserved. Although the BFG12863 21309105 95. classification of thses benthic invertebrates was at the generic level, the data from the agricultural and industrialized areas of the Welland River watershore are comparable. The generic richness and. diversity was significantly lower in the industrial influenced portion of the Welland River especially at station C, where only a few oligochaete and dragonfly nymph specimens were found. In the agricultural influenced portion of the Welland River, there were 13 genera of crustaceans. At station A, the relative abundance of isopods and amphipods were 14.28% 9.15% and 4.39% 1.60%, respectively. At station B, the relative abundance of isopods was 0.75% 0.37%, and 0.11% 0.11% for amphipods. In the industrialized area, the crustacean species decreased dramatically. Crustacean species are very sensitive to many types of pollution (Gaufin and Tarzwell, 1952; Buikeman et al., 1978). The results of my study were in accord with Gaufin's observations. Gaufin and Tarzwell (1952) reported that in the domestic sewage polluted zone of Lytle Creek there were no crustacean species. Crustaceans had lower Lc50 values for various pollutants including pesticides and heavy metals than any other invertebrate species (Buikeman et al., 1978). For example, the Lc50 for PCB was 10-200 ug/1 for Gammarus spp., while PCB toxicity for dragonflies was 800-1000 i ug/1 (Buikeman and Herricks, 1978).A concentration of 0.025 ppm of Baytex (fenthion) was lethal to chironomid larvae, while 0.01 ppm of Baytejf exceeded the toxic limits for the isopod (Palomonetes) and the amphipod (Hyalella) (Muirhead-Thomson, 10 1971). 90T60ET BFG12864 96. Sediment type also affects the distribution of benthic invertebrates (Wetzel, 1983). In the Welland River, the structure of bottom sediment from upstream to downstream was fairly uniform. A clay base was found at all stations in both the industrial and the agricultural sites in the river. Therefore, differences of sediment type at the industrial sites were attributed to the impacts of industrial effluents and not to substrate differences. At station C, the discharge of heavy metals resulted in a "reef" structure in front of the confluence of the Atlas Steels Co. discharge stream and the Welland River. The observed changes in invertebrate species composition at this site was probably caused by industrial discharges of heavy metals at this site. The seasonal changes of crustacean species pattern in the Welland River were not studied. Although during the sampling period few crustaceans were found in the industrial influenced portions of the river, it was not possible to conclude that crustaceans were absent from the industrial section of the river because benthic invertebrate samples were only taken in August and September. In the agricultural sections of the Welland River, stations A and B displayed slight differences in genera. Generic richness i and diversity decreased slightly (0.05< p <0.10) at station B (16 1.1 per 700 cm2). The remarkable difference was the relative abundance of individual species (genera). At station A, the relative abundance of chironomids was 11.82% 5.14%, while at station B it increased to 87.25% 1.03% (Table 5). Isopod and BFG12865 21309107 97. \ amphipod relative abundances at station A were 14.28% 9.15% and 4.39% 1.60%, respectively, while at station B it was 0.75% 0.37% for isopods and 0.11% 0.11% for amphipods. Therefore, it implies that station A had a more "normal" aquatic assemblage than did station B. It may be because station A was located below a dense aquatic plant stand which may have played a role in removing hazardous agricultural chemicals from the water (Bingham, 1973). At station C, there were no chironomid larvae found. Sediment heavy metal concentrations at station C were high (3927 600 ppm Nickel, 3712 130 ppm Chromium, 800 200 ppm Lead, 1088 190 ppm Zinc, 753 77 ppm Copper, and 237 + 43 ppm Cobalt) (Dickman et al., 1986). The studies on avoidance response of Chironomus sp. to sediment containing heavy metals revealed that the threshold avoidance was 213-422 ppm Cadmium, 4385-8330 ppm Zinc, and 799-1513 ppm chromium (Wentsel et al., 1977). Therefore, the absence of chironomids at station C is likely due to the high concentrations of sediment heavy metals at this station, especially Nickel and Chromium. At station D-l, the generic richness and diversity were significantly higher than at any other industrial site, though they were significantly lower than at the agricultural control site. The high species richness at station D^l was apparently due to the increase in oligochaete species at this station. The discharge effluent of the B.F. Goodrich Co. contained high levels ^ of organic materials (BOD was 43.37 kg/day. MOE, 1987). the BFG12866 1309108 98. oligochaete species, Limnodrilus spp. and Dero spp. are tolerant of high BOD and high organic pollution (Hart et al., 1972; Lafont, 1984). Therefore, more oligochaete tolerant taxa appeared at station D-l due to the organic pollution there. Another possibility to explain the higher species richness at station D-l is that site D-l was located in a branch stream. Station D-l was not affected by the main current of the river which carries industrial effluents from upstream industrial areas. This may be more obvious when comparing site D-l with D-2 which was located at the confluence of the Welland River and the discharge stream of the B.F. Goodrich Co. At site D-2, species richness and diversity were significantly lower than at site D-l, while site D-2 was affected by both the B.F. Goodrich effluent and the upstream materials carried in the current of the river. Additionally, sediment structure was different at site D-l and D-2. Site D-l was more rocky than other parts of the Welland River. The difference in substrate may also account for the observed species differences. Oligochaete abundance was significantly higher at most industrial discharge sites in the Welland River (Table 4). At stations A and B, the relative abundance of oligochaetes was 5.80% 3.38% and 5.56% 1.13%, respectively, while in the industrial sites it exceeded 90% except at station E where ammonia concentrations were so high that they were likely inhibitory. My research results showed that in the industrial influenced portions of the river, generic richness and diversity were lower BFG12867 21309109 99. than at the upstream control site (Fig. 12) and at the same time the relative abundance of oligochaetes was greater (Table 5). This is consistent with the findings of other researchers. Brinkhurst and Cook et al. (1974) concluded that amount of organic effluents tend to increase the individual number of oligochaetes but the number of other benthic species often decline. It was proposed that a bottom invertebrate community containing 80% or more tubificids indicated a high degree of organic enrichment or organic pollution (Aston, 1973). Tubificid relative abundance at stations C, D-2, and F were in excess of 95%. Limnodrilus spp. were suggested to be pollution indicators (Brinkhurst and Cook, 1974). The relative abundance of Limnodrilus spp. increased significantly in the industrial influenced portion of the Welland River. At the agricultural i stations, the relative abundance of Limnodrilus spp. was 5.24% 3.53% and 5.43% 1.18% for station A and B, respectively. In the industrial stations, it ranged from 34.92% 23.39% to 95.50% 2.56% except at station (Table 5). The lower benthic invertebrate generic richness and diversity in the industrial influenced portion of the Welland River may be due to toxic pollution. As discussed in the previous section, the river may be contamined with potential toxic organic pollutants, especially at stations C and D-l. Stations C and E had very low abundances of benthic invertebrates (114 85 mzand 1325 534 m* respectively) compared with the agricultural area stations A and B (4654 219 m and 5610 1352 m2-). The toxicity of samples from station C was evident from both DMSO and BFG12868 0TT60STZ 100.. distilled water extracts (Figs. 4 and 6). The high concentration of toxic heavy metals in sediments at station C was also noted. At station C, Nickel was about 90 times higher in its concentration than at upstream sites (3929 600 ppm), Chromium about 120 times higher (3712 Jt 130 ppm), Lead about 11 times higher (800 200 ppm), Zine about 8 times higher (1088 i 390 ppm), and Copper about 27 times higher (753 77 ppm) (Dickman et al., 1986). The high content of cyanide (2.3 kg/day or 0.092 ppm. Niagara River Toxics Committee, 1984) and ammonia at site E may be one of the reason for the observed low species richness and low abundances of benthic invertebrates at station E. Another reason was that at stations C and E serious shock loading was found (Dickman et al., 1983 and 1988). Shock loads are defined as short term sporadic discharges. At station C, it was recorded that the specific conductivity increased about 8 times when sporadic discharges occurred, and at station E the specific conductivity increased about 4 times during shock loading events (Dickman et al., 1986). Shock loads damaged the aquatic plants in the Welland River, and it was concluded that shock loads were a more serious pollution problem than heretofore belived (Dickman et al., 1988). Invertebrate community structure at stations C and t E may be in part a response to these shock loads which altered their habitat by eliminating many of the natural aquatic plants at these sites. iO 1309111 BFG12869 1 101. CONCLUSIONS 1. The SOS chromotest revealed that organic soluble (DMSO soluble) contaminants in the sediments from station D-l (an industrial discharge site in the Welland River near a polyvinyl chloride plant) resulted in significantly higher genotoxicity at this site. 2. Sediment genotoxicity was higher in the industrialized sections of the river than in the agricultural areas. 3. The highest frequency of deformities in chironomid labial plates was found at station D-l. The lowest frequency of deformities in chironomids was found at the control site (station A). 4. The correlation between genotoxicity of sediments and the frequency of deformities in chironomid labial plates was statistically significant (P<0.05) using a parametric test. The non-parametric correlation test (Sperman-Kandell Rank Test) was not significant (p>0.05). 5. The generic richness and diversity of benthic invertebrates was lower in the industrial influenced portions of the Welland river than in the agricultural areas. 6. The sediment toxicity of samples extracted in the DMSO was high for samples from stations C and D-l. Toxicity was also found in the distilled water extracts of sample sediments from stations C ans D. 7. The most abundant benthic invertebrate genera in the agricultural section of the Welland River were chironomids, oligochaetes, and crustaceans, while in the industrialized BFG12870 2TT60STZ . 102. section of the river, oligochaetes and chironomids dominated and the crustaceans were extremely rare. BFG12871 21309113 103. REFERENCES Adler, I.-D. 1984. Cytogenetic tests in mammals, in "Mutagenicity testing --.a practical approach" Venitt, s and J.M. Parry. IRL Press, Oxford, Washington DC. pp 275. Albanese, P., J. Middleton, and J. Russell. Unpublished manuscript. Effect of industrial effluent on the aquatic macrophytes of the Welland River near Niagara Falls, Canada. Institute of Urban and Environmental Studies, Brock University, St. Catharines, Ontario, L2s 3A1, Canada. Ames, B.N., J. McCann and E. Yamasaki. 1975. Method for detection carcinogens and mutagens with the Salmonella/ mammalian-microsome mutagenicity test. Mut. Res. 31: 347-364. Aston, R.J. 1973. Tubificids and water quality: a review. Environ. Pollut. 5: 1-10. Baumann, P.C. 1984. Cancer in wild freshwater fish populations with emphasis on the Great Lake. _J_. Great Lakes Res. 10: 251-253. Bingham, S.W. 1973. Improving water quality by removal of pesticide pollutants with aquatic plants. Virginia Polytechnic Institute-Water Resources Research CenterBulletin. 58: 1-95. Brinkhurst, R.O. and D. G. Cook. 1974. Aquatic earthworms (Annelidia : oligocheata). In " Pollution ecology of freshwater invertebrates" C.W. Hart and S.L.H. Fuller Academic Press, New York and London, pp 143-156. (Ed.) Brown, K.W. and K.C. Donnelly. 1984. Mutagenic activity of the liquid waste from the production of acetonitrile. Bull. Environ. Contam. Toxicol. 32: 742-748. Buikeman, A.L.Jr. and E.E. Herricks. 1978. Effects of pollution on freshwater invertebrates. _J. WPCF. 50:.1637-1648. Cairns, J. Jr., G.R. Lanza and B.C. Parker. 1972. Pollution related structural and functional changes in aquatic communities with emphasis on the freshwater algae and protozoa. Proc. Acad. Natl. Scl. Phil. 124: 79-127. Coates, K. and D. V. Ellis. 1980. marine pollution indicators. No. 6: 171-174. Enchytraeid oligocheates as Marine Pollution Bulletin. 11, Cushman, R.M. 1984. Chironomid deformities as indicators of pollution from a synthetic, coal-derived oil. Freshwater Biology. 14 : 179-182. BFG12872 V lT S O E T Z 104. Dayan, J., S. Deguingand, C. Truzman and M. Cherron. 1987. Application of the SOS chromotest to 10 pharmaceutical agents. Mut. Res. 187: 55-66. Dean, B.J., A. and N. Danford. 1984. Assays for the detection of chemically-induced chromosome damage in cultured mammalian cells, in " Mutagenicity testing -- a practical approach" Venitt, S. and J.M. Parry. IRL Press, Oxford, Washington DC. pp 187-232. Dickman, M.D. and P.0. Steele. 1986. Gonadal neoplasms in wild carp-goldfish hybrids from the Welland River near Niagara Falls,Canada. Hydrobiologia. 134:257-263. Dickman, M.D., J. Smol and P. Steele. 1980. The impact of industrial shock loading on selected biocoenoses in the Welland River, Ontario. Water Research Jounal of Canada. 15: 17-31. Dickman, M.D., C. Prescott and K.L.E. Kaiser. 1983. Variations the aquatic vegetation of the Welland River (Ontario, Canada) above and below an industrial discharge. 3_^ Great Lake Res. 9:317-325. in Dickman, M.D., F. Johnson, and I. Brindle. 1986. The development of a threshold activated pump sample in an industrial shock load study. Submitted to Water Res. June, 1988. Dickman, M.D. 1988. Impact of industrial shock loading on the aquatic plant community of a class one wetland in regional Niagara, Ontario, Canada. Proceedings of the international wetland conference. C.Rubec (Ed.) pp. 307-315. Dutka, B.J., A. Jova, J. Brechin. 1981. Evaluation of four concentration/extraction procedures on water and effluents collected for use with the Samonella typhimurium screening procedure for the mutagens. Bull. Environ,. Contam. Toxicol. 27: 758-764. Edmondson, W.T. 1966. Freshwater Biology. 2nd Ed. New York. London. John Wiley & Sons, Inc. 1. Fleesel, C.P. 1979. Metals as mutagens, in " Trace metals in health and disease" N. Kharash (E.d.). Roven Press, New York, pp 117-128. Gaufin, A.R. and C.M. Tarzwell. 1952. Aquatic invertebrates as indicators of stream pollution. Public Health Reports. 67, No. 1: 57-65. Hamilton, A.L. and 0. Saether, 1971. The occurrence of characteristic deformities in the Chronomid larvae of several Canadian lakes. Can. Ent. 103: 363-368. 21309115 BFG12873 105* Hare, L. and J.C.H. Carter. 1976. Distribution of Chironomus (s.s.)? cucini (salinarius group) larvae (Diptera: Chironomidae) in Parry Sound, Georgian Bay, with particular reference to structural deformites. Can. J. Zool. 54: 2129-2134. Hart, C.W. and S.L.H. Fuller. 1974. Pollution ecology of freshwater invertebrates. Academic Press, New York and London. Hayes, S. and A. Gordon. 1984. Validating RK test: correlation with Salmonella mutatest and SOS chromotest assay results for reference compounds and influence of pH and dose response on measured toxic, mutagenic effects. Mut. Res. 130: 107-111. Hoi1stein,M. and J.McCann. 1979. Short-term test for carcinogens and mutagens. Mut. Res. 65:133-226. Jaylet, A., L. Gauthier and M. Fernandez. 1987. Detection of mutagenicity in drinking water using a micronucleus test newt larvae (Pleurodeles waltl). Mutagenesis. 2, No.3: 211-214. in Johnson, M.G. 1964. Biological survey of the Welland River. Ontario Water Resources Commission Hepoirt. Kairser, L.E. and M.E. Comba. 1983. Volatile contaminants in the Welland River watershed. J_. Great Lakes Res. 9:274-280. 1. Lafont, M. 1984. Oligocheata communities as biological descriptors of pollution in the fine sediments of rivers. Hydrobiologia. 115 : 127-129. Little, J.W. and D.W.Mont. 1982. The SOS regulatory system in E_. coli. Cell. 29:11-22. Maron, Dorothy M and Bruce N. Ames. 1983. Revised methods for the Salmonella mutagenicity test. Mut. Res. 113:173-215 McCann, J., E. Choi, E. Yamasaki, and B.N. Ames. 1975. Detection of carcinogens as mutagens in the Salmonella/microsome test:assay of 300 chemicals. Proc. Nat. Acad. Sci. USA 72:5135-5139. McCann, J. and B. Ames. 1976. Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals: discussion. Proc. Nat. Acad. Sci. USA 73:950-954. McMillan, T. 1988. The quqdrangular forum, 26 April 1988. Available from Environment Canada Parliament Buildings, Ottawa, Ontario, KIN 6N5. Mellanby, H. 1965. Animal life in freshwater. A guide to BFG12874 21303116 106. freshwater invertebrates. London Methnen & Co. Ltd. Metcalfe, C.D., R.A. Sonstegard, and M.A. Qulliam. 1985. Genotoxic activity of particulate material in petroleum refinery effluents. Bull. Environ. Contain. Toxicol. 35:240-248. Metcalfe, C.D. and R.A. Sonstegard. 1985. Oil refinery: of co-carcinogenic activity in the Trout embryo microinjection assay. JNCI, 75: 1091-1097. evidence Metcalfe, C.D. and R.A. Sonstegard. 1986. Relationship between anaphase aberrations and carcinogenicity in the Trout embyro microinjection assay. JNCI, 77: 1299-1302. 1. Metcalfe, R.L. 1977. Biological fate and transformation of pollutants in water, in " Fate of pollutants in the air and water environments " Part 2. Suffet, 3.H. (E0d). 1977. A Wiley-Interscience Publication. New York/ London/ Sydney/ Toronto, pp 195-221. Milbrink, G. 1983. Characteristic deformities in tubificid oligochaetes inhabiting polluted bays of Lake Vanern, southern Sweden. Hydrobiologia. 106: 169-184. Ministry of the Environment, 1987. Report on the 1986 industrial direct discharges in Ontario. Muirhead-Thomson, R. C. 1971. Pesticides and freshwater fauna. Academic Press, London and New York. Niagara River Toxics Conmmittee, 1984. Report of the Niagara River Toxics Committee, p 2-39. Ohta,T., N. Nakamura, M. Moriya, Y. Shirasu and T. Kada. 1984. The SOS-function-inducing activity of chemical mutagens in Escherichia coli. Mutat. Res. 131:101-109. Olivier, P. and D. Marzin. 1987. Study of the genotoxic potential of 48 inorganic derivatives with the SOS chromotest. Mutat. Res. 189 : 263-269. Parry, E.M. and J.M. Parry. 1984. The assay of genotoxicity of chemicals using the budding yeast "Saccharomyces cerevisiae". in "Mutagenicity testing--a practical approch" Venitt, S and J.M. Parry (E.d.). IRL Press, Oxford, Washington DC. pp 119-148. Pennak, R.W. 1953. Freshwater invertebrates of United States. The Ronadl Press Co. Pettibone, G.W., J.J. Cooney. 1986. Effect of organotins on fecal pollution indicator organisms. Appl. Environ. Microbiol. 52, No. 3: 562-566. BFG12875 21309117 107. Tarkpea, M. , I. Hagen, G.E. Carlberg, P. Kolsaker, and H. S-torflor. 1985. Mutagenicity, acute toxicity, and bioaccumulation potential of six chlorinated styrenes. Bull. Environ. Contam. Toxicol. 35: 525-530. Timson, J. 1975. Hydroxyurea. Mut. Res. 32 : 115-132. Usha Rani, M.V., 0. S. Reddi and P.P. Reddy. Mutagenicity studies involving Aldrin, Endosulfan, Dimethoate, Phosphamidon, Carbaryl and Ceresan. Bull. Environ. Contam. Toxicol. 25 : 277-282. Vandermeulen, J.H., A. Foda and C. Stutlard. 1985. Toxicity vs mutagenicity of some crude oils, distillates and their water soluble fractions. Water Research. 19, No. 10: 1283-1289. Venitt, s. and J.M. Parry. 1984. Mutagencity testing, a practical approach. IRL Press. Oxford. Washington DC. Velema, J.P. 1987. Contaminated drinking water as a potential cause of cancer in humans. Environ. Carcino. Revs. 5(1): 1-28. Walker, G. C. 1984. Mutagenesis and inducible responses to deoxyribonucleic acid damage in J2. coli. Microbiological Reviews. 48. No. 1:60-93. Ward, D. V. 1978. Biological environmental impact studies: and methods. Academic Press, New York, San Fransisoc, London, pp 113-119. theory Warwick, W.F. 1985. Morphological (Diptera) larvae as measures ecosystem: indexing antennal meigen. Can. J.. _Fish. Aouat. abnormalities in Chironomidae of toxic stress in freshwater deformities in chironomus Sci. 42 : 1881-1914. Warwick, W.F., 1987. The Port Hope 88-92. J. Fitchko, P.M. McKee, D.R. Hart and A.J. Burt. incidence of deformities in Chironomus spp. from Harbour, Lake Ontario. J_. Great Lake Res. 13 (1) : Weis, J.S. and P. Weis. 1987. Pollutants as developmental toxicants in aquatic organisms. Environmental Health Perspectives. 71: 77-85. Wei, C-i., O.G. Raabe and B.J. Kimble. 1984. Mutagenicity studies of size-fractioated oil fly ash in the Ames Salmonella typhimurium assay. Bull. Environ. Contam. Toxicol. 32 : 179-186. Wentsel, R., A. Malntosh, W.P. McCafferty, G. Atchison and V. Anderson. 1977. Avoidance response of midge larvae (Chironomus tentans) to sediments containing heavy metals. Hydrobiologia. Vol. 55 : 171-175. BFG12876 8TT60STZ 'S'-!: 1;u 108. Wetzel, R.G. 1983. Limnology. Second edition. Saunders College Publishing, pp. 646-654 and 713-714. Wiederholm, T. 1984. Incidence of deformed chironomid larvae (Diptera: Chironomidae) in Swedish lakes. Hydrobiologia. : 243-249. 109 Wiederholm, T. 1983. Chironomidae of the Holarctic region. Keys and diagnoses. Part 1. Larvae. Entomological Scandinavica. Supplement. No. 19. World Wildlife Report, 1987. Environmental toxicology of the Welland River. World Wildlife Toxicology Fund. 60 St. Clair Ave. , East Suite 201, Toronto, Ontario, M4T 1N5 Canada. Xu, H and B.J. Dutka. 1987. Genotoxicity studies on sediments using a modified SOS chromotest. Toxicity Assessment: International* Quarterly Vol. 2: in press. 213091 BFG12877 :*-u . Irtr-r Appendix 1. Industrial discharges in the Welland River. (Moe, 1987) (kg/day). 21309120 BFG12878 110 Appendix 1. Industrial discharges in the Welland River (MOE, 1987) ( kg/day). sample sites C DE F Cadmium 0.085 -- -- -- Chromium 15.6 -- 2.28 -- Iron Phosphate 47.7 -- -- 0.89 -- 20.14 ^ 0.703 3.2 Ammonium ion i Nitrogen -- -- 4.95 -- -- 2106.59 3.28 -- Kjeldahl organic. -- -- 10.79 11.9 Oil and grease 31.0 -- -- 24.1 Vinyl chloride -- 0.09 -- Suspending solids 1148.3 24.76 6.67 145.10 BOD flow (m3/day) -- 24584.9 43.37 2213.6 16.63 27342.0 47.6 8554.0 TZT60STZ BFG12879 Appendix 2. The percent alkaline phosphatase activity in the SOS chromotest of seven sediment samples taken from the Welland River. BFG12880 1309122. 0 .0 1 0 .0 2 0 .0 0 .0 1 0 .0 2 0 .0 0 .0 1 0 .0 2 0 .0 0 .0 1 0 .0 2 0 .0 0 .0 1 0 .0 12 2 0 .0 9 o- -<r -cr ^ ^^ ^ CD C/5 d co CM CM CM CO <: o 2 <O LU o to o CO o O o O o r- o co -M ooo r* T"* T~ ^ <r~ rf CO CO CM CM O CO CO o oo M- CO CO CO CO O 1- CM ooo M- CO CO CO CO OOO O 1- T- in 10 in OfflN O O CO -M- CO Tf ^ 'f d C/5 Q CO CO CO -r- W 5 Q U) O CM OO m O o O o 00 o> o co co O O CD t" 1- rf 'M* XT CM CM O o o o> M' M- M1- CO O CD CO O O) O T-- r-- M1 M M 1-- T" o CD CD O CD CD T" co co CO CO CO Or (O oo o CM CM CM t* M1 "<r Tj* co ^ CD C/5 o CO -- o CO CM CM CO CM C/5 <5 Q UJ O CO oo 00 o ONt- o m CO 0^0 O CD O *r~ r" TJ- CO CO CO M- o CO CO ooo n- co co CM CM O CD CO ooo tT CO CO Tf CO O O CM O r- -r- to t/5 in -M- CM o CD CD O O CD Ci-M OJ TO- M- -cr "M- CM CM CM O) CO + Q o CO CO CM CM -- CM CM S5 0 O 0 O 2 o o o CD CD CO CD o cm in o CD CD T* T-- "T* CM CM CM 1-- T-- CM CM o in to o"T" o> o> CM CM CM CM CM o co co o CD CD t-- T- CM CM T-- T-- CM CM ONN O CD CD CM CM CM CM CM omn o CD CD T* > m C/l CD ooo odd O) r- CM E ooo odd r- CM CO , UJ cto: < m o Q CM O LU UL 21309123 '' ' > 112 (: Appendix 3. The % frequency of deformities in chironomid labial plates at five stations in the Welland River. 10 BFG12882 ZT60T replicates Appendix 3. The >% frequency of deformities in chironomid labial plates sample sites A B D-l D-2 E 1 5.66 13.37 7.32 2.27 3.92 2 0.0 4.26 4.76 0.0 4.40 3 3.39 1.95 9.09 7.14 4 7.69 3.17 20.0 7.69 5 2.44 8.43 21.43 6 6.54 0.0 mean S.D. 3.84 1.32 6.29 1.71 10.90 3.23 6.42 3.30 4.20 0.28 21309125 114.