d'

INVESTIGATION OF THE EFFECTS

COMPOUNDS PRODUCED I N COOLING SYSTEMS AND PROCESS

EFFLUENTS ON AQUATIC ORGANISM:

J. R. Trabalka and M. B. Burch -

Environmental Sciences D iv is ion Oak Ridge National Laboratory Oak Ridge, Tennessee 37830

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INTRODUCTION

Both p u b l i c hea l th and environmental concern has been generated by

the i d e n t i f i c a t i o n o f an assemblage of halogenated. o rgan ic compounds

1-3 ' produced i n the c h l o r i n a t i o n o f d r i n k i n g water, . sewage, 4-7 and

power p l a n t coo l i ng waters. 8'g E a r l y eco log i ca l concern was r e i n -

f o rced by the f i r s t t o x i c i t y t e s t s on aquat ic biotalO'll which seemed

t o i n d i c a t e t h a t a t 1 east two ch l oro-organics ( 5 - c h l o r o u r a c i l and

4 -ch lo ro reso rc ino l ) were t o x i c a t low i g / ~ concent ra t ions 'comparable t o

those produced i n t reatment o f sewage and coo l i ng waters. 5 '9 Both

compounds s i g n i f i c a n t l y 1 owered hatch ing success o f carp (Cyprinus . .

c a r p i o ) eggs a t 5 pg/!L1O and 5 -ch lo rou rac i l s i g n i f i c a n t l y delayed and

re'duced i n i t i a1 product ion o f o f f s p r i n g i n Daphnia magna a t 10 pg/R. 11

Later , however, t e s t s o f a seventeen compound composite mixture,

c o n t a i n i n g chloro-organics ( 1 n c l ud ing. 5-chlur.our.aci 1 and ..

4 - c h l o r o r e ~ o r c i n o l ' ) i n the p ropb r t i ons o r i g i n a l l y i d e h t i f i e d by J o l l e y 5

from, sewage e f f 1 uent, produced no .sign i f i c a n t e f f ' ec t s on Daphnia magna

a t concentrat ions 1 mg/R or carp embryos at. concent ra t ions . .

< 10 rng/!L.l2 This occurred desp i te the f a c t t h a t bo th compounds were

present a t concentrat ions up t o 3 orders o f magnitude h ighe r than those

a t which t o x i c e f f e c t s were p r e v i o u s l y repor ted. The i n v e s t i g a t o r s .

suggested t h a t a complex an tagon is t i c i n te rac t - i on might be requ i red t o

e x p l a i n the apparent d i f fe rences. " Our ob jec t i ves were t o determine

. . which compounds 'cont r ibu ted s i g n . i f i cant l y t o t he t o x i c i t y o f such

. ch loro-organ i c mixtures and whether s i g n i f i c a n t i n t e r a c t i o n occurred.

METHODS AND MATERIALS . .

T o x i c i t y s tudies were conducted us ing the embryo o f t he carp . .

. .

(Cypr inus c a r p i o ) , and t h e c l adoceran Daphnia pulex. The c h a r a c t e r i s t i c s

s tud ied are l i s t e d i n Table 1. While we consider the measurement o f

chron ic e f f e c t s an important . component o f a t o x i c i t y t e s t i n g regime,

c o n s t r a i n t s o f t ime and space r e q u i r e t h a t we r e p o r t the s p e c i f i c

r e s u l t s elsewhere.

Grav id carp were c o l l e c t e d and spawned a r t i f i c i a l l y i n the labo-

r a t o r y as described prev ious ly . l3 Gametes were in t roduced d i r e c t l y . .

i n t o t e s t so lu t ions . A f t e r 30' minutes the so lu t i ons were decanted and

r e p l aced by f r e s h s o l u t i ons o f the appropr iate chemi c a l concentrat i on .

Thereaf te r , so lu t i ons were changed tw ice d a i l y u n t i l a l l eggs had e i t h e r

hatched o r perished. Embryos were incubated i n . c o n t r o l l e d environment

chambers a t ~ 3 . 5 ~ ~ under a 14 h r l i g h t - 10 hr dark cyc le . Under t h i s

regime, eggs hatched i n 4 t o 7 days. The percent s u r v i v a l o f ' the. eggs

a t ha tch ing i s used t o generate an es t ima te o f the 96 h r LC5,.,

Tests were conducted f o r t h ree syn the t i c e f f l u e n t s i n decade con-

c e n t r a t i on steps from lo-' m g / ~ t o 100 mg/a o f . t o t a l organ i c s p lus

c o n t r o l s . The chloro-organic. composit ion o f t h e t h r e e s y n t h e t i c

ch l 'o r ina ted e f f l u e n t s c.hosen f o r t e s t i n g i s shown i n Table 2. The

mix tures were based o n . i d e n t i f i c a t i o n s from sewage e f f l u e n t and c o o l i n g

waters repor ted by J o l l ey -- e t a ~ . ~ Synthet ic mix tures were prepared

f rom - 1 adoratory stocks o f t h e i ndi v i dual compounds 1 i sted. Contro l . .

r e p l i cates were poured ( i .e., spawned) s e q u e n t i a l l y throughout a t e s t

Table 1. . Toxicity t es t ing regime

Organ ism Mode of exposure Character i s t i c s measured

Carp embryo Acute Hatching success (Cyprinus carpio)

Daphni a pulex Acute

Chronic

Abnormality frequency

LC5'o f o r predetermined time inte.rva1

Life span . .

Average reproduction r a t e adjusted . for mor ta l i ty

Total reproduction

. . Date of f i r s t reproduction

Table 2. Concentrations of chloro-organic cons t i tuen t s i n syn the t i c chlor inated e f f l uen t s a t 100 mg/R t e s t concentrat ion.

Concentrat ion a (mg/l)

Secondary Mississippi Watts Bar sewage River Reservoir

I e f f l u e n t cooling cool i ng Oak Ridge wa'ter water .-

I Phenols 1 4-Chl oro-3-methyl phenol . .

. . . 2-Chl orophenol

I . . 3-Chlorophenol 4-Chl orophenol . .

I I 4-Chi ororesorci no1 .

Aromatic ac ids 2-Chl orobenzoic ac id 1 .5 . . 3-Chlorobenzoic acid 2.9 4-Chl orobenzoic ac id 5.4 3-Chl oro-4-hydroxybenzoic acid 6.4 4-Chl oromand.el i c ac id 5.4 4-Chlorophen,ylacetic a c i d 2.0 5-Chlorosal icy1 i c acid 1 . O

~ ~ r i m i d ' i ne 5-Chl orouraci 1

Purines 6-Chloro-2-aminopurine 4.4 2.8 5.6 8-Chlorocaffeine 8.3 5 . 5 . 6.. 2

. 8-Chloroxanthine 7.4 --- 17

Nucl eosi de 5-Chlorouridine

a Proportions based on determinations by Jo l l ey -- e t a1 1976. Actual t o t a l concentrat ions observed were: secondary sewage e f f l uen t s , . 20.4 yg/!L; Mississippi River cooling water , 108.7 pg/R.; and Watts Bar Reservoir . cool i ng water , 17.8 pg/R.

so tha t changes in egg and sperm v iab i l i t y during the a r t i f i c i a l

spawning process could be monitored . .Three rep1 i cates (100-300 eggs

each) were used for each t e s t concentration and six repl icates for con-

t r o l s . Concentrations were based on ser i a1 di 1 uti ons prepared daily

from refrigerated stocks in order to minimize qual i ta t ive and (o r )

1 quantitative changes i n the t e s t medium. Test solutions were prepared

from the same spring water sources used by Gehrs e t a l . 10,11,12 --

Cladoceran studies were conducted in 100-ml beakers containing

80 ml' of the appropriate concentration of the t e s t solutions in spring . .

water. Ten replicates were used a t each concentration with two indi-

viduals (12 k 12 hr o l d ) added to each beaker t o i n i t i a t e a t e s t . The . .

i n i t i a l water temperature was 20°c. Beakers 'were kept a t 20 5 1°c in a . '

constant temperature chamber having a 14 hr l ight - 10 hr dark cycle

durinj- the t,est,ing period. ~n i m a l s were cens,js,ed daily f nr mortali ty '

and reproduction. Animals were fed 0.1 ml of a t rout chow and water

mixture twice weekly.14 A t t h i s time animals were transferred t o

fresh solution; young were counted and removed. Chronic exposure

studies were conducted for an 8-week period, whi 1 e LCs0 determinations

were conducted over 96 hours. Acute LCs0 values were calculated by

probit analysis using log concentration values.

A 'combi necl acute exposure screen ing-chron i c exposure regime

covering the concentration range described previously fo r carp embryos

was used for a number of rep'resentative compounds in individual t e s t s .

Concentration ranges fo r individual . tes ts . of other compounds and for

the synthetic mixtures were more res t r ic ted based ei ther on resu l t s

f r om p r e v i o u s work1' o r on our . judgment o f expected t o x i c i t y . Acute

t o x i c i t y sc reen ing t e s t s were conducted on a l l compounds p resen t i n t h e

s y n t h e t i c e f f 1 uents. Acute 96 h r LC50 va lues were determined f o r a1 1

t h r e e s y n t h e t i c e f f l u e n t s -and t h e most t o x i c i n d i v i d u a l compounds.

As an a i d i n e v a l u a t i n g t h e observed acute t o x i c i t y o f the

s y n t h e t i c m i x tu res t o Daphni a pu lex , we used a r e 1 a t i v e l y s imp le index,

t h e T o x i c i t y Index, which sums the f r a c t i o n a l LC50 c o n t r i b u t i o n s o f

t h e i n d i v i d u a l t o x i cants assuming s imple a d d i t i . v i t y . Background mate-

r i a1 f o r t h e d e r i v a t i o n o f t h i s Index has been r e p o r t e d p r e v i o u s l y . 15

TOX I CITY Concent ra t ion o f Compound i = C INDEX i = l LC50 o f Compound i

The ~ o x i b i t y I n d e x may be c a l c u l a t e d u s i n g any r e p r e s e n t a t i v e concent ra -

t i o n o f a m i x t u r e and i s equal t o t h e number o f m u l t i p l e s o f an LCs0

p resen t a t t h a t c o n c e n t r a t i on. Our v a l ues have. been c a l c u l a ted u s i n g

t h e 100 m g / ~ concen t ra t i on da ta - repor ted i n Table 2 i n o rde r t o f a c i l i-

t a t e comparisons. The T o x i c i t y Index a l lows a r a n k i n g o f expected . .

t o x i c i t y o f complex m ix tu res f o r comparison w i t h b o t h observed t o x i c i t y

and c o n c e n t r a t i o n o f i n d i v i d u a l t o x i c a n t s . S i g n i f i c a n t c o n t r i b u t i o n s

f r om i n d i v i d u a l compounds o r c lasses o f compounds a re a l s o r e a d i l y

observed.

When t h e T o x i c i t y Index . i s c a l c u l a t e d f o r t he c o n c e n t r a t i o n o f a

m i x t u r e which corresponds t o i t s LC50 va lue, t h e T o x i c i t y Index

becomes an A d d i t i v i t y Index. The A d d i t i v i t y Index i s equal t o t h e

number o f f r a c t i o n a l LCs0 va lues, c o n t r i b u t e d by i n d i v i d u a l corn-

pounds, p resen t i n a m i x t u r e a t i t s LC50 conckn t ra t i on . I f t o x i c i t y

o f t h e i n d i v i dual compounds i s pu re l y add i t i ve , t h e . A d d i t i v i t y .Index

I should be equal t o u n i t y . Values less than one i n d i c a t e greater than

I a d d i t i v e c o n t r i b u t i o n s (<< 1 i n d i c a t e s synergism). Corresp,ondingly,

when the A d d i . t i v i t y Index i s g rea ter than u n i t y , t o x i c i t y i s less than

a d d i t i v e ' ( > > l , s i g n i f i c a n t negat ive i n t e r a c t i o n ) . The T o x i c i t y Index,

c a l c u l a t e d fpr a concentrat ion other than the LCSO o f a mixture, may

be conver ted t o an A d d i t i v i t y ' Index by m u l t i p l i c a t i o n by t h e quo t i en t

o f t he m ix tu re LCs0 ' concent ra t ion w i t h the m ix tu re concent ra t ion used

t o c a l c u l a t e t h e T o x i c i t y Index. , ' I

RESULTS

The LCSU-96 hr values we obta ined f o r Daphnia pulex are shown i n

Table -3. Note t h a t t h e pheno l ic compounds are by f a r the most t o x i c

compounds tes ted , A value f o r bromoform has been inc luded because we

were a l so i n t e r e s t e d i n t he p o t e n t i a l f o r v o l a t i l e compounds such as

the haloforms (he re to fo re no t i d e n t i f i e d ' f rom c o o l i n g waters) t o con-

tri bute t o t h e t o x i c i t y o f ch l oro-organic mix tures . Bromof orm spec.i-

f i c a l l y ' was chosen as the l e a s t vo1,a t i le o f the haloforms and because

i t may a l s o predominate i n c h l o r i n a t e d e ' f f l uen ts i n areas w i t h a h igh

n a t u r a l bromi de content .' Resorcinol. has been inc luded because o f

i t s r o l e as a probable precursor f o r 4 -ch lo ro reso rc ino l . 9

The m a j o r i t y o f t he 1 7 i n d i v i d u a l compounds t e s t e d have been shown

t o p e r s i s t i n ' so lu t i on over t ime i n t e r v a l s comparable t o those i n o u i

t e s t s (Reference 16 and George Southworth, unpubl ished data) . However,

some degradat ion o f 4-chlorores,orc inol and v o l a t i l i z a t i o n o f bromoform

Table 3. Acute t ox i c i t y of halogenated. organic compounds . .

t o Daphnia .pulex . .

x Daphn i.a pul'ex 96 h r LC,,

compound . . ( m g / &

2-chl orophenol

3-ch 1 oropheno 1

4-chl orophenol

4-ch.1 oro-3-met hyl phenol

4-chl ororesorci no1

resorcinol

.8-chl orocaff e i ne 3 2

I . bromof orm 44 1

3-chl urobenzoic acid.

Others - i

1. I a ~ o mor ta l i ty a t 96 hr.

. .

i s expected over 96 h r . Thus, abso lu te values f o r the 96 h r LC50 f o r

these two compounds cou ld be s ign i f i cant l y 1 ower under exper imenta l

condi ' t i ons whereby constant concent ra t ions are maintained . .The r e s u l t s o f t e s t s o f s y n t h e t i c c h l o r i n a t e d e f f l u e n t s on carp

embryos a l lowed o n l y est imates o f 96 h r LC50 values (Table 41 , b u t d i d

show t h e t r e n d i n comparat i ve t o x i c i t y (LCs0 f o r Secondary Sewage

Eff .1 uent. < ~ i s s i s s i ~ ~ i R i ve r Coo l ing Water < Watts Bar .Reservo i r Cool ing

Water). The r e s u l t s were a l so impor tan t because t h e y showed t h a t t h e

96 h r LC5g o f a l l t h ree mix tu res i s approx imate ly 3 orders o f magni-

tude g r e a t e r than the concent ra t ion found i n t h e ac tua l c h l o r i n a t e d

e f f l u e n t s (Tab le 4 ) . I n no case d i d we f i n d a s i g n i f i c a n t de t r imen ta l

e f f e c t a t concent ra t ions 5 10 mg/R i n our t e s t s .

The 96 h r LC50 values f o r Daphnia pu lex exposed t o t he t h r e e

s y n t h e t i c e f f l u e n t s f o l l o w e d t h e p a t t e r n es tab l i shed i n t h e carp embryo

t e s t s ( T o x i c i t y o f t he Syn the t i c Secondary Sewage E f f l u e n t > M i s s i s s i p p i

R i v e r Coo l ing Water > Watts Bar Reservo i r Cool ing Water). It seemed

apparent t h a t the c h l o r i n a t e d phenols. i n t h e mix tu res were respons ib le

f o r t h e observed t o x i c i t y o f t h e s y n t h e t i c e f f l u e n t s . Phenol con ten t i n

t h e m ix tu res dec l ines i n a f ash ion cons i s ten t w i t h the decrease i n acute

t o x i c i t y (Tab le 4 ) . Other c lasses o f compounds appear t o be excluded . .. '

e i t h e r because they do no t d e c l i n e as the t o x i c i t y o f s y n t h e t i c

e f f l u e n t s .dec l ine (Table 2 ) o r because they are no t demonstrably h i g h

i n t o x i c i t y co able 3 ) . A cons i s ten t dec l i ne i s a l so observed f o r t he

T o x i c i t y Index (Table 4) , whose o n l y s i g n i f i c a n t c o n t r i b u t i o n s are

p rov ided by the ch~o ropheno ls . Est imated c o n t r i b u t i o n s f rom o ther

Tab le 4. Ana l ys i s o f t o x i c i t y o f s y n t h e t i c c h l o r i n a t e d e f f l u e n t s t o t he ca rp embryo and Daphnia pu lex

Concent ra t ion (ng/R)

. Secondary M i s s i s s i p p i Watts Bar sewage R i ve r Reservo i r

e f f 1 uent ' c o o l i n g ' c o o l i n g Oak Ridge water water r 2

a 96 h r LCs0 Carp embryo

Ac tua l E f f 1 uent Concent ra t ion

96 h r LC,,~ Daphn i a pu 1 ex

1 phenols a t 100 mglR

T o x i c i t y Index a t 100 mg/ iC

A d d i t i v i t y Index

a ~ o s i g n i f i cant d e t r i m e n t a l e f f e c t s observed a t concen t ra t i ons < 10 mg/!L.

b ~ i n e a r r eg ress ions ( l e a s t squares f i t ) performed aga ins t corresponding va lues o f 96 h r LC50 f o r -,-.-- Daphnia - pulex .

' ~ a s e d on concen t ra t i ons i n Tab le 2 and 96 h r LC50 va lues f o r Daphnia pu lex i n Table 3.

. .

ch1oro'-organics are l ess than. 7% of the t o t a l in a l l three. cases and

have been omitted here.

. Linear regressions of 96 hr LCs0 f o r t h e t h r ee synthet ic . .

e f f 1 uents against the corresponding values f o r phenolic content of t h e

1& mg/l t e s t so lu t i ons (again, chosen' f o r comparative, purposes) and

the Toxici ty Index are both highly s ign i f ican t ( P < 0 . 0 1 ) ~ r2 = 0.982

and r2 = 0.999,. respectively. Further, the Addi t iv i ty Index for the

mixtures i s approximately 2 in a l l th ree cases, which indicates t h a t no

substant i a1 nega t ive in teract ion (of the type c i t ed e a r l i e r ) 1 2 between , .

compounds occurs. If a l l compounds were as e f f ec t i ve i n producing mor-

t a l i t y a t concentrations below t h e i r i n d i v i d ~ a l , L C ~ ~ values as they

are a t LC50 f igure , t ox i c i t y in mixtures wou'ld be purely addi t ive and

the Addi t iv i ty Index should be equal t o unity. Since t h i s assumption

may be unreal in many cases, values greater than uni ty could be a

p rac t ica l expectation.

Acute t ox i c i t y of the three syn the t ic e f f luen ts t o Daphnia pulex

i s evidenced a t .concentrations approximately 3 orders of magnitude

greater than those reported i n actual e f f luen ts . In addi t ion, no

s ign i f ican t detrimental e f f ec t s were observed on any o f the charat-

t e r i s t i c s previously described in Table 1 when ~ a p h n i a pulex were

chronical ly exposed to the th ree mixtures a t concentrations 1 10 mg/l .

DISCUSS ION

We conclude t ha t the t o x i c i t y of the chloro-organic mixtures t e s t ed

was primarily a t t r i bu t ab l e t o i t s chlorophenol content and t ha t acute

t o x i c i t y was demonstrable i n t h e carp embryo and Daphnia pulex a t l e v e l s . .

approximate ly 3 orders o f magnitude. g rea ter than those expected i n

actual. e f f l uen ts . It would a l so appear t h a t a t l e a s t the c o n t r i b u t i o n s

t o acute t o x i c i t y o f some complex mix tures o f compounds can be s a t i s f ac-

. t o r i l y exp la ined by a r e l a t i v e l y simple procedure, i .e., by us ing t h e . .

T o x i c i t y and A d d i t i v i t y Ind ices p r e v i o u s l y described. Whi le we recog-

n ize. t ha t . it would be h i g h l y des i rab le t o have more data p o i n t s w i t h ,

which t o con f i rm the r e s u l t s o f our regress ion o f ,LC50 on T o x i c i t y

Index f o r t h e , complex mixtures, we d i d no t observe s u b s t a n t i a l i n t e r - . .

a c t i o n which m i gh t have compromised a .comparison. The absence o f

. s i g n i f i c a n t i n t e r a c t i o n i n . our t e s t s s t r o n g l y suggests t h a t e a r l i e r

r e s u l t s which. i nd i ca ted chloro-organ i c t o x i c i t y at. 1 ow pg./l l e v e l s were

,' anomal ous . \. . . .

The s y n t h e t i c e f f l u e n t s we t e s t e d conta ined l ess than 1/3 o f the

compounds on a numerical .basis and l e s s than 1 /4 o f the c h l o r i n e by

weight t h a t i s associated w i t h c h l o r i n a t e d organics i n ac tua l c h l o r i -

nated e f f 1 uents. 5 9 9 Thus, many iompounds remain u n i d e n t i f i e d and

could prove more t o x i c than those a l ready tes ted . Such compourids do

e x i s t . i n c h l o r i n a t e d p u l p m i l l e f f luents . . l7 However, ' un 1 ess other

aquat ic organisms are much more s e n s i t i v e and/or s i g n i f i c a n t (bu t

unexpected) bioaccumulat ion o f these 17 water-so-lub'le ch lo r i na ted

organic compounds can occur ( i n compet i t ion w i t h chemical and

b i o l o g i c a l degradat i on), t he p a r t i c u l a r assemblage - o f compounds t e s t e d

may .not prove t o be an important 'environmental problem. I n t h i s

regard, i t i s a lso s i g n i f i c a n t t h a t 4-chlororesorcinol , which

contributed a s ign i f ican t proportion t o the t o x i c i t y of a l l three

syn the t ic e f f l uen t s , was not more t ox i c than resorc ino l , a potential .

precursor..

We believe t ha t development of concentrates of actual chlorinated

e f f l uen t s (see paper by R . ' L. . Jo l ley -- e t a1 . , t h i s symposium) for 'use i n

t o x i c i t y t e s t i n g and determinations of bioaccumulation potent ia l should

be given ser ious consideration. Such mater ia ls , i f made. avai lable i n

su f f i c i en t quan t i t i es , could s i gn i f i c an t l y reduce the current complexity

of t o x i c i t y t e s t i ng involving mu1 ti-component chloro-organic e f f luen ts

and help overcome the current absence of iden t i f i ca t ions of the bulk of . .

the compounds produced.

ACKNOWLEDGMENTS . .

wish t o g ra te fu l ly acknowledge the technical advice provided by

R . L. Jo l l ey and the assistance of J . E. Thompson in procuring labora- . .

tory stocks of compounds used in t h i s study. , 'We thank G. R . Southworth, . .

R. B. ~ c ~ e a n , and J . S. Mattice fo r technical comments on the rnanu-

s c r i p t .

Research sponsored by the Department of Energy .under contract w i t h

Union Carbide Corporation. Publication No. , Environmental

Sciences Division, ORNL.

. . REFERENCES

1. Rook, J . J . , "Formation of Haloforms During Chlorinat ion of

Natural Waters," Water Treat,. Exam. 23:234-243 (1974).

2 . B e l l a r , ,T. A. , J . J.. Lichtenburg; and R . C . Kroner, "The

Occurrence of Organohalides in Chlorinated Drinking Water," - J .

Amer. Water Works Assoc. 66:703-706:(1974).

3 . Stevens, A . A . , C. J . Slocum, D . R . Seeger, and G . G . Robeck,

" ~ h l o r i n a t i o n . of Organics in Drinking Water." In: Water

Chlor ina t ion: Environmental Impact and Health Ef fec t s , Vol'. 1, R .

L . . J o l l e y , Ed., (Ann Arbor, Michigan., Ann Arbor Science

Pub1 i s h e r s , 1978). pp. 85-114. . .

4. Glaze, W . H . , and J . E . Henderson, ."Formation of Organochlorine

Cpmpounds from t.he Chlorinat ion of a Municipal Secondary

' E f f l u e n t , " J . Water Pol l u t . Control Fed. 47:2511-2515 (1975).

5. J o l l e y , R . L . , "Chlorination E f f e c t s on Organic Const i tuents i n

Eff 1 uents from Domestic S a n i t a r y Sewage Treatment P lan t s , "

ORNL-TM-4290 (Oak Ridge, Tennessee: Oak Ridge National

Laboratory, 1973).

6 . J o l l e y , R . L . , "Determination of Chlorine-Containing Organics i n . . . .

Chlorinated sewage Eff luents by Coupled 3 6 ~ 1 Tracer-High

Resolut ion Chromatography," Environ. L e t t . 7:321-340 (1974).

7. Jo l ley . , R . L., "Chlorine-Containing o rgan ic Const i tuents in Sewage . .

Eff luen t s , " J . Water Pol l u t . Control Fed. 47:601-618 (1975).

8 . Jo l ley , R . L . , C . W . Gehrs, and W . W . P i t t , "Chlorination of

Cooli ng Water: A Source of Chlorine-Containing Organic' Compounds

with Possible Environmental Significance.. " In: Radioecology and

Ener'gy Resources, C . E . Cushing, Ed., (Stroudsburg, Pennsylvania,

Dowden, Hutchinson and Ross, Inc., 1976). pp. 21-28. . .

9 . Jo l l ey , R . L . , G . o ones, W . W . P i t t s , and J . E . Thompson,

"Chl or i nati on of Organics in cooling Waters and' Process

E f t ' l uents." In: Water Chlorination: Environmental Impact and

Health Effects , Vol'. 1, R . L . Jo l l ey , E d . , (Ann Arbor, Michigan,

Ann Arbor Science Publishers, 1978).

'10. Gehrs, C . W . , L. D . Eyman, R . L . Jo l l ey , and J . E . Thompson,

"Effects of Stable Chlorine-Containing Organics on Aquatic

Environments," Nature 249 (5458) : 675-676 (1974').

I 11. Gehrs, C . W . , and R . L . Jo l ley, "Chlorine-Containing Stable I

I Organics: New Compounds of Environmental Concern," Verh.

In te rna t . Verein. Limnol. 19:2185-2188 (1975).

12. Gehrs, C . W., and G . R . Southworth, " Invest igat ing t he Effects of

Chlorinated Organics." In: Water Chlorination: Environmental . .

Impact and Health Effects , Vol. 1, R . L. Jo l l ey , Ed . , (Ann Arbor,

M i ch igan , Ann Arbor Sci.ence Pub1 i shers , 197'8). .pp. 347-362.

13. Blaylock, 13. ' G . , and' N . A . G r i f f i t h , "A Laboratory Technique f o r

Spawning Carp," Prog. Fish. Cult. 33:48-50 (1971).

14. Gehrs, C . W., "Aspects of the Population Dynamics of the ~ a l a n o i d

Copepod Diaptomus clavipes," P h . D . Thesis, Univ. of Oklahoma,

Norman, 1972.

15.. Sprague, J. B., "Measurement o f P o l l u t a n t T o x i c i t y t o F ish . 11.

U t i 1 i z i n g and App ly ing Bioassay Resu l t s , " Water Research 4:3-32

(1970).

16. Southworth, G. R., and C. W. Gehrs, " P h o t o l y s i s o f 5 - C h l o r o u r a c i l

i n N a t u r a l Waters," Water Research 10:967-971 (1976).

17. Leach, J. M., .and A. N. Thakore, " I s o l a t i o n and I d e n t i f i c a t i o n o f

C o n s t i t u e n t s Tox ic t o J u v e n i l e Rainbow T r o u t (Salrno g a i r d n e r i ) i n "

c a u s t i c E x t r a c t i o n E f f l u e n t s f r om K r a f t Pu lp rn i l l Bleach. Plants,"

J . F i s h . Res. Board Can. 32:1249-1257, (1975).