The Toxicity of RDX to the Freshwater Green Alga Selenastrum Capricornutum

8/3/2019 The Toxicity of RDX to the Freshwater Green Alga Selenastrum Capricornutum

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T H E T O X I C I T Y O FH E X A H Y D RO - 1 , 3 , 5 - T RI N I T RO - 1 , 3 , 5 . T RI A Z I N E

(R D X ) T O T H E F R E S H W AT E R G R E E N A L G AS E L E N A S T R U M

C A P R I C O R N U T U M

DENNIS T. BURTON,* STEVEND. TURLE Y and GREGO RY T. PETERS**The University of Maryland System, Agricultural Experiment Station, Wye Research and Education

Center, PO. Box 169, Queenstown, MD 21658, U.S.A.

(Received 2 June, 1993; accepted 23 September, 1993)

Abs t rac t . RDX was not acutely toxic to the green alga Selenastrum capricornutum when tested at

the solubility limit of the compound in algal assay media. A maximum reduction of 38% in celldensity occurred after a 96-h exposure to 36.7 mg L- l ; thus, an EC50 could not be determined. Thelowest-observed-effect concentration (LOEC) and no-observed-effect concentration (NOEC) for thegreen alga were 4.8 and 0.5 mg L -1 (reduction in cell density), respectively, when chronic end pointswere used to analyze the data. A comparison of the data in this study with RDX toxicity data from theliterature for the diatom Navicula pelliculosa, and the blue-green algae (cyanobacteria) Microcystisaeruginosa and Anabeanaflos-aquae shows that S. capricornutum is the most sensitive to RDX. AU.S. Environmental Protection Agency (EPA) numerical water quality criterion Final Plant Valuebased on the green alga data should protect the above algal groups.

In troduct ionHexah ydro-1 ,3 ,5 - t rin i tro - l ,3 ,5 - t r iaz ine (RDX) is an exp los ive used ex tens ive ly bythe m i l it a ry fo r va r ious app l ica t ions . The c om pou nd m ay be re leased to the aqua ti cenv i ronm ent at m uni t ions p lan t s dur ing man ufac tu r ing and b lend ing ope ra tions .RD X m ay a l so be re leased to the aqua t ic env i ronm enta l a t load , a s sembly, and pack(LAP ) p lan t s w here the com pou nds is loaded in to bom bs and p ro jec t il e s (E tn ie r,1986).

One l abora to ry s tudy has been c ondu c ted on the tox ic i ty o f RD X to aqua ti c a lgae

(Ben t leye t a l . , 1977). N o EC 50s (ce ll dens i ty and ch lo rophy l l a ) were ob ta ined fo rfour spec ie s expo sed to nom ina l RD X concen t ra tions up to 32 m g L -1 fo r 96-h .Su l l ivane t a l . (1979) re -ana lyzed the da ta ob ta ined by B en t leye t a l . (1977) for thefour a lga l spec ie s and conc luded tha t RD X inh ib it ed the g row th o f the a lga l spec ies .A f ie ld su rvey a t an Arm y am m uni t ion p lan t has been con duc ted to eva lua te theinpac t o f m uni t ions was te e ff luen t on f re shwa te r b io t ic com m uni t i e s . The su rveysho we d tha t RD X was p resen t in the d i scha rge f rom the m uni t ions p lan t ; however,i t was n o t c l ea r ly e s tab l i shed whe the r o r no t RD X caused a de t r imen ta l e ffec t top e r i p h y t o n a n d p h y t o p l a n k t o n c o m m u n i t ie s (Su ll iv a ne t a l . , 1977).

Th e acu te and chron ic tox ic ity o f RDX to inver teb ra te s have been s tud iedby Pe te r s e t a l . (1991). Th ey fo und tha t RD X was no t acu te ly tox ic to s eve ra l

* To whom all correspondence should be addressed.** Present Address: Ciby-Geigy Corporation, Environmental Fate and Effects Department, Box

18300, Greensboro, NC, U.S.A.

Water, Air and Soil Pollution 76: 449-457, 1994.(~) 1994 Kluwer Academic Publishers. Printed in the Netherlands.



4 5 0 DENNIS T. BURTON ET AL.

s p e c i es o f i n v e r t eb r a t e s a t c o n c e n t r a ti o n s n e a r t h e s o lu b i li ty l im i t o f t h e c o m p o u n din wa te r. Ch ro n ic to x ic i ty was o b se rv ed in o n e o f two in v er t eb ra tes s tu d ied b y

P e te r s e t a l . ( 1 9 9 1 ). T h e L O E C a n d N O E C f o r a r e d u c t i o n i n n e o n a t e p r o d u c t io ni n t h e c l a d o c e r a nC e r i o d a p h n i a d u b i aw ere 6 .0 a n d 3 .6 m g L -1 , r e sp ec t iv e ly.N o c h r o n i c to x i c i ty w a s f o u n d i n a li fe c y c l e s t u d y o f th e m i d g eP a r a t a n y t a r s u sp a r t h e n o g e n e t ic u s .

I n c o n t r a s t t o a l g a e a n d i n v e r te b r a te s , R D X h a s b e e n s h o w n t o b e b o t h a c u t e l ya n d c h r o n i c a l l y t o x ic t o f is h . A c u t e t o x ic i ty 9 6 - h L C 5 0 s r a n g e f r o m 3 .6 m g L - 1f o r j u v e n i l e b l u e g i l l (L e p o m i s m a c r o ch i ru s )t o > 1 0 0 m g L - 1 f o r f a t h e a d m i n n o w( P i m e p h a l e s p r o m e l a s )e m b r y o s ( B e n t l eye t a l . , 1 9 7 7 ). Bu r to ne t a l . ( 1 9 9 2 ) c o n -d u c t e d a 2 8 - d a y e a r l y l if e s ta g e t es t w i th t h e f a t h e a d m i n n o w a n d o b t a i n e d a L O E Ca n d N O E C b a s e d o n b o t h w e t a n d d r y w e i g h t o f 2. 4 a n d 1 .4 m g L - 1 , r e p e c ti v e ly.

A n i n t e r im E PA n u m e r i c a l w a t e r q u a li ty c r it e ri o n F in a l A c u t e V a lu e o f 5 .1 8 m gL - 1 w a s e s t a b li s h e d f o r R D X b y E t n i e r ( 1 98 6 ). A p p r o p r i a te d a t a w e r e n o t a v a i la b l eto es t ab l i sh a F in a l P lan t Va lu e (S tep h ene t a l . , 1985) .

T h e o b jec t iv es o f th e p re sen t s tu d y w ere to ( a ) v e r i fy th e p o ten t i al t o x ic i ty o fR D X t o f r e s h w a t e r al g a e s i n c e t h e o n l y d a t a a v a il a b le f o r a lg a e a r e b as e d o n n o m i n a lt e s t co n cen t ra t io n s an d (b ) p ro v id e q u an t i ta t iv e d a ta to e s t ab l ish an E P A n u m er ica lwa te r q u a l i ty c r i t e r io n F in a l P lan t Va lu e . T h e g reen a lg aS . c a p r i c o r n u t u mw as

se lec ted fo r s tu d y b ec au se i t i s a ' s t an d ard ' a lg a l t e s t sp ec ies an d was u s ed in th eB e n t l e y e t a l . ( 1 9 7 7 ) s t u d y a s o n e o f f o u r a l g al s p e c ie s e x p o s e d t o R D X .

M a t e r ia l s a n d M e t h o d s

EXPERIMENT AL PROCEDURES

A g r e e n a lg a ( S . c a p r i c o r n u t u m )s t a r t e r cu l tu re was o b ta in ed f ro m th e cu l tu rec o l l e c ti o n a t t h e U n i v e r s i t y o f N o r t h Te x a s , D e n t o n , T X . S t o c k a l g a l c u lt u r es w e r e

rea red in 2 .5 L P y rex cu l tu re f l a sk s co n ta in in g 1 L o f s t e r i l i zed d o u b le s t r en g th'A A P ' a lg a l a s say m ed iu m , w i th su ff i c ien t P ad d ed to ach iev e a 2 0:1 N :P ra t io a sd e s c r i b e d i n M i l le re t a l . (1 9 7 8 ) . Cu l tu res we re main ta in ed in a co n s tan t t em p era tu rei n c u b a t o r u n d e r c o n s t a n t c o o l - w h i t e f l u o r e s c e n t l ig h t s ( ~ 4 .7 5 W m - E ; ~ 3 0 0 f o o tcan d les ) a t a t em p era tu re o f 2 0 (+ 1 ) ~ o n a sh ak er t ab le o sc il l at in g a t 1 0 0 (+ 1 0 %)r p m .

T h e t o x i c i t y ( 9 6 - h E C S 0 f o r c e ll d e n s i ty ) o f R D X t o t h e g r e e n a l g a w a s d e t e r-m i n e d b y t h e p r o c e d u r e s g i v e n i n E PA To x i c C o n t r o l S u b s t a n c e A c t ( T S C A )M e t h o d 7 9 7 . 1 0 6 0 ( U .S . E PA , 1 9 8 5 a n d 1 9 86 ). D e v i a t i o n s f r o m t h e t e s t m e t h o d

a r e d i s c u s s e d w h e r e a p p r o p r i at e . T h e 9 6 - h E C 5 0 a lg a l te s t f o r g r o w t h is c o n s i d -e r e d t o b e a n a c u t e te s t b y E PA ' s T S C A O ff ic e . E PA ' s O ff ic e o f R e s e a r c h a n dD e v e l o p m e n t c o n s i d e r s t h e t e st t o b e a c h r o n i c t e st f o r d e t e r m i n i n g t h e to x i c i ty o fe f f lu e n t s ( H o m i n g a n d We b e r, 1 9 85 ; W e b e re t a l . , 1 9 8 9 ) as d o o th e r in v es t ig a to r sf o r e v a l u a t i n g s i n g l e c h e m i c a l s ( f o r ex . , se e H u g h e se t a l . , 1 9 88 ). W e a n a l y z e d t h ed a t a u s i n g b o t h a c u t e a n d c h r o n i c t e s t e n d p o i n t s s o a c u t e a n d c h r o n i c c o m p a r i s o n sc o u l d b e m a d e w i t h t h e li m i te d R D X d a t a b a se .



THE TOXICITY OF HEXAHYDRO- 1,3,5-TRINITRO- 1,3,5-TRIAZINE 451

T h e n u t r i e n t m e d i u m u s e d f o r t h e b i o a s s a y w a s t h e s a m e m e d i u m u s e d f o rc u l t u r i n g t h e o rg a n i s m . A l g a l t e s t s o l u ti o n s w e r e p r e p a r e d b y d i lu t io n o f th e s t o c k

so lu t io n s w i th f i l t e r s t e r i l i zed assay med ia w i th in a s t e r i l e t r an s fe r ro o m. Tes ts o l u ti o n s ( 1 0 0 m L t o t a l v o l u m e ) w e r e d i s p e n s e d in t o 2 5 0 m L D e l o n g f l as k s a n di n o c u l a t e d w i t hS. capricornutum ce l l s in lo g g ro wth to ach iev e a d en s i ty o f1 0 0 0 0 ce l ls m L -1 . Tr ip l i ca tes w ere p rep ared fo r each t r ea tmen t . T h e f l a sk s werep laced o n a sh ak er t ab le in an in cu b a to r se t a t t h e cu l tu r in g co n d i t io n s d esc r ib eda b o v e ; t h e m e a n t e m p e r a t u r e d u r i n g t h e b i o a s s a y w a s 2 0 . 0 ( R a n g e = 1 9 . 9 t o2 0 . 1 ) ~ G r o w t h m e a s u r e m e n t s ( c e ll d e n s i t y ) w e r e m a d e f r o m al l r e p l ic a t es i neach t r ea tm en t a t 0 , 2 4 , 4 8 , 7 2 , an d 9 6 -h . A lg a l ce l l d en s i ty was d e te rm in ed f ro m a1 m L s a m p l e w i t h a M o d e l Z B I C o u l t e r C o u n t e r ( C o u l t er E l ec t r o n i cs I n c ., H i a l ea h ,F L ) . T h e i n s t r u m e n t w a s c a l ib r a t e d w i t h e a c h u s e v ia h e m o c y t o m e t e r c o u n t s.

TOXICANT

R D X ( C A S N o . 1 2 1 - 8 2 -4 ) w a s o b t a i n e d f r o m t h e H e a lt h E ff e c t s R e s e a r c h D i v i s io no f U .S . A r m y B i o m e d i c a l R e s e a r c h a n d D e v e l o p m e n t L a b o r a t o ry ( U S A B R D L ) ,F t. D e t r i c k , F r e d e r i c k , M D . T h e c o m p o u n d w a s r e c r y s t al l iz e d t o a p u r i ty o f > 9 9 %a s m e a s u r e d b y H P L C a n d s t o r e d i n a m b e r bo t tl es i n th e d a r k a t r o o m t e m p e r a t u r e .A s a t u r a t e d s to c k s o l u t io n o f R D X w a s p r e p a r e d f o r t h e a l ga l t e st b y a d d i n g R D Xto a lg a l a s say med iu m, s t i r r ed a t ro o m temp era tu re fo r 2 4 -h , an d f i l t e r s t e r i l i zedpr io r to tes t in i t ia tion .

ANALYTI CAL CHEMISTRY

A q u e o u s c o n c e n t r a ti o n s o f R D X w e r e d e t e r m i n e d b y t h e i so c ra ti c H P L C m e t h o d o fB r u e g g e m a n ( 1 9 8 3 ) w i th m i n o r e x c e p t i o n s in t h e o p e r at in g c o n d i ti o n s as d e s c r i b e db e l o w. A W a t er s H P L C s y s t e m ( Wa t e rs A s s o c i a t es , M i lf o r d , M A ) w a s u s e d w h i c h

c o n s i s te d o f t h e f o ll o w i n g c o m p o n e n t s : d u a l M 4 5 p u m p s w i th M o d e l 6 8 0 g r a d ie n tco n t ro l l e r, M o d e l 7 8 0 d a ta mo d u le ( in t eg ra to r ) , U 6 K in jec to r, M o d e l 48 1 v a r i ab lew a v e l e n g t h U V d e te c to r , Z - M o d u l e r a d ia l c o m p r e s s io n c o l u m n s y s te m , a n d M o d e l7 1 2 W a t e rs i n t e l li g e n t s a m p l e p r o c e s s o r ( W I S P ) . A W a t er s # B o n d a p a k C 18 r e v e r s ep h a s e c o l u m n w a s u s e d . T h e m o b i l e p h a se , d e l iv e r e d at a r a te o f 1 .0 m L m i n - 1 , w a s5 5 % m e t h a n o l : 4 5 % w a t e r. T h e e f f l u e n t w a s m o n i t o r e d at 2 4 0 n m , 0 .1 a b s o r b a n c eu n i ts f u l l s c a le ( a .u .f .s .) . T h e i n j e c ti o n v o l u m e w a s 2 - 2 0 0 # L d e p e n d i n g o n t h eco n cen t ra t io n o f ma te r i a l i n so lu t io n .

H P L C g r a d e m e t h a n o l w a s u s e d ; t h e w a t e r w a s d e i o n i z e d g la s s di st il le d . S t a n -

d a r d s o l u ti o n s o f R D X w e r e p r e p a r e d b y d i l u ti o n o f a s t o c k s o lu t io n f r e s h l y p r e p a r e de a c h d a y o f a n a ly s is . P r e c is i o n a n d a c c u r a c y a n a ly s e s o f th e H P L C m e t h o d w e r ec o n d u c t e d p r i o r t o t h e s ta r t o f t h e b i o a ss a y. P r e c is i o n w a s e v a l u a t e d b y i n j ec t in ga s a m p l e t h r e e t i m e s o n t h r e e s e p a r a t e d a y s . M e a n r e l a t i v e s t a n d a r d d e v i a t i o n sf o r t h e l o w a n d h i g h c o n c e n t r a t i o n s o f R D X r o u t i n e ly u s e d to g e n e r a t e s t a n d a r dcu rv es fo r ev a lu a t in g t e s t co n c en t ra t io n s were 2 .0 1 an d 0 .4 3 %, resp ec t iv e ly. T h ea c c u r a c y o f t h e m e t h o d w a s e v a l u a te d b y q u a n t i fy i n g th e c o n c e n tr a ti o n o f R D X i n




a n u n k n o w n s a m p l e p r e p a r e d b y a n i n d e p e n d e n t c h e m i s t. A c c u r a c y, e x p r e s s e d a sp er cen t e r ro r, was 0 .9 3 % .

T h e aq u eo u s samp les were in j ec ted d i rec t ly in to th e HP L C a f t e r f i l t r a t io n tor o m o v e p a r ti c le s > 0 .4 5 # m . R D X c o n c e n t r a ti o n s w e r e m e a s u r e d a t t h e b e g i n n i n gan d en d o f th e t e s t ( i. e. , 0 an d 9 6 -h ) in a l l r ep l ica tes a t each t r ea tmen t . T h e an a ly t i ca lm e a s u r e m e n t s w e r e m a d e t h e d a y t h e s a m p l e s w e r e t a k e n f r o m t h e s a m p l e f la sk s .R D X c o n c e n t r a t i o n s d e c r e a s e d s l ig h t ly f r o m t h e s t a rt o f t h e t es t to t h e e n d o f th etes t at 9 6 h o u rs . T h e av e rag e d ec re ase fo r a ll t r ea tmen t s was 4 .7 %; th e r an g e was2 .6 to 8 .0%.

S TAT I S T IC A L A N A LY S E S

A l l c o n c e n t r a t i o n d a ta u s e d i n t h e s t at is ti ca l a n a l y s e s w e r e m e a n m e a s u r e d c o n c e n -t ra ti o ns . T h e t e s t e n d p o i n t f o r t h e t o x i c it y o f R D X t o t h e g r e e n a l g a w a s g r o w t h ,m e a s u r e d a s c e ll d e n s i t y (c e ll s m L - 1 ) . A n a c u t e 9 6 -h E C 5 0 f o r r e d u c t io n in a l g a lg r o w t h c o u l d n o t b e c a l cu l a te d b e c a u s e a m a x i m u m r e d u c ti o n o f 3 8% o c c u r r e din ce l l d en s i ty a t t h e so lu b i l i ty l im i t o f th e co m p o u n d in a lg a l a s say me d ia ( seeb e l o w ) .

T h e L O E C a n d N O E C w e r e d e t e r m i n e d b y a t- te s t w i th B o n f e r r o n i a d j u s tm e n to f e r r o r r at e b e c a u s e t h e n u m b e r o f re p l ic a te s w a s n o t c o n s t a n t a m o n g t r ea t m e n t s .

O n e o f th e th ree co n t ro l r ep l i ca tes w as s t a ti s ti ca lly e l im in a ted as an o u t l i e r (d ec i s io nb e a s e d o n a r e g r e s s i o n a n a ly s i s a n d c o m p a r i s o n o f r e s id u a l s) a n d w a s n o t i n c l u d e din th e g ro wth ca lcu la t io n s an d su b se q u en t s t at is t ica l an a ly ses . T h e B o n fe r ro n i t- t e s tc o n s i s ts o f a n a n a l y s i s o f v a r i a n c e ( A N O V A ) t o d e t e r m i n e t h e e r ro r t e rm , w h i c h i st h e n u s e d i n a m u l t i p le c o m p a r i s o n t es t fo r c o m p a r i n g e a c h o f th e t r e a t m e n t m e a n sw i t h t h e c o n t r o l m e a n . T h e a s s u m p t i o n s u p o n w h i c h t h e u s e o f th e B o n f e r r o n it - t e s t a re co n t in g en t a re th a t th e o b se rv a t io n s w i th in t r ea tmen t s a re in d ep en d en ta n d n o m a l l y d i s tr ib u t e d , w i t h h o m o g e n e i t y o f v a ri a n ce . T h e c h i - s q u a re t es t f o rn o r m a l i ty a n d B a r t l e tt 's t e s t f o r h o m o g e n e i t y o f v a r i a n c es w e r e p e r f o r m e d b e f o r eth e Bo n fe r ro n i t - t e s t was u sed . T h e ab o v e s t a ti s ti cal t e s ts we re p e r fo rm ed u s in gTo x s t a t ( G u l l e ye t a l . , 1 9 8 9 ). A m in im u m p ro b ab i l i ty l ev e l o f 0 .0 5 w as u sed .

R e s u l t s a n d D i s c u s s io n

T h e t o x i c i t y d a t a f o rS . c a p r i c o r n u t u me x p o s e d t o R D X a r e g i v e n i n Ta b l e I .A m a x i m u m r e d u c t i o n o f 3 8 % i n ce l l d e n s i t y o c c u r r e d a f te r a 9 6 - h e x p o s u r e t o3 6 .7 m g L - l ; t hu s , a n E C 5 0 c o u l d n o t b e d e t er m i n e d . T h e m a x i m u m r e d u c ti o n in

ce l l d en s i ty o cc u r re d a t th e so lu b i l i ty l im i t o f RD X in a lg a l a s say med ia a t t h e assayt e m p e r a t u r e o f 2 0 ~ T h e s a t u ra t io n co n c e n t r a t io n o f 36 .7 m g L - 1 o b t a i n e d i n t h iss tu d y in a lg a l a s say m ed ia is s l ig h t ly lo w er th an th e v a lu es r ep o r t ed in th e l i te ra tu refo r aq u eo u s so lu t io n s . S ik k ae t a l . ( 1 9 8 0 ) a n d t h e U . S . A r m y ( 1 9 8 4 ) r e p o r t e dsa tu ra ted co n cen t ra t io n v a lu es a t 2 0 ~ o f 4 2 .3 an d 5 0 m g L -1 , r e sp ec t iv e ly.

B e n t l e y e t a l . ( 1 9 7 7 ) s t u d i ed t h e a c u t e to x i c it y o f R D X t o t h e g r e e n a l g a S.c a p r i c o r n u t u m ,t h e d i a t o mN . p e l l i c u l o s a ,a n d t h e b l u e - g r e e n a l g a e ( c y a n o b a c t e r i a )



THE TOXICITY OF HEXAHYDRO-1,3,5-TRINITRO-1,3,5-TRIAZINE 453

TABLE I

Green alga mean cell density (cells mL -1) after 96 hours of exposure to RDX

Conc. Rep(mg L -1) 0-h

Mean cell density24-h 48-h 72-h 96-h

control 1 10 699 16 256 26 597 83 213 269 507 ~2 9 093 13 395 50 195 220 883 1 0449283 9 568 14 571 63 989 261 008 1 186 997

0.47 1 8 477 11 656 91 688 220 040 989 5002 9 523 13 419 48 744 215 171 1 003 753

3 8 848 12 037 47 064 237 661 1 033 027

4.81 1 10 600 13 269 56 651 259448 901 000 b2 10 317 15 328 46 224 176 947 802 9133 10 203 16 043 46 181 160 723 787 680

7.95 1 10 227 22 896 46 475 215 797 928 147 b2 10 531 17 216 59 595 270 280 1 017 2133 10 219 13 389 49 837 232 611 915 880

13.02 1 10 707 16 339 56 029 229 739 895 780 b

2 11 493 12 736 53 909 252 917 8476273 9 643 13 104 49304 232 821 840587

21.80 1 12 144 12 024 55 877 189 949 670 067 b2 11 163 15 091 48 888 181 339 683 8473 10 651 17 109 46 363 204 784 769 940

36.69 ~ 1 13 624 14 709 52 251 214 413 767220 b2 10 693 15 664 49 152 187 171 638 5873 9 520 14 197 48 056 183 629 662 040

Replicate 1 of the control was treated as an outlier (decision based on aregression analysis and comparison of residuals) and was not includedin the growth calculations and subsequent statistical analyses.b Mean cell density of the three replicates was significantly differentfrom the mean cell densi ty of the control at alpha = 0.05.

Saturation concentration at 20 ~

M . a e r u g i n o s a , A . f lo s - a q u a e .N o 9 6 -h EC 5 0 s fo r c el l d en s i t y o r c h l o ro p h y l l a c o u l d

be de t e rmi ned fo r the fou r speci .es exposed to nomia l concen t r a t ions up to 32 m g

L -1 . Thus , Ben t ley e t a l . (1977) conc l uded tha t RDX had no e ffec t on the fou r

spec ies expo sed fo r 96 -h to 32 m g L -1 (nomina l ) . Harvey e t a l . (1991) found

tha t concen t ra t i ons o f RDX up to 10 mg L -1 (h ighes t concen t ra t ion t es ted ) d id

no t appear to a ffec t the g rowth o f the d ico t y ledon P h a s e o l u s v u l g a r i s(bush bean)




w h e n g r o w n f o r 7 d a y s u n d e r h y d r o p o n i c co n d i t io n s ; a 1 0 - f o ld u p t a k e o f R D X d i do c c u r in t h e l e av e s .

S ta t is t i ca l ly s ig n i f ican t ( a = 0 .0 5 ) r ed u c t io n s in ce ll d en s i ty we re o b se rv ed wh enc h r o n i c e n d p o i n ts w e r e u s e d i n t h e a n a l y s is o f th e d a t a in t h is s t u d y. T h e L O E C a n dN O E C f o r r e d u c t i o n in g r o w t h w e r e 4 .8 a n d 0 .5 m g L - 1 , r e s p ec t iv e l y. T h e N O E Cis a p p r o x i m a t e l y a n o r d e r o f m a g n i t u d e l o w e r t h a n t h e L O E C . I t s h o u l d b e n o t e dt h a t in c h r o n i c s t u d ie s , th e N O E C is a l w a y s d e p e n d e n t o n t h e c h o i c e o f e x p o s u r eco n c en t ra t io n s (u su a l ly a d ilu t io n fac to r o f 5 0 -6 0 % ) s in ce th e ca lcu la t io n s in v o lv eda r e n o t b a s e d o n a p o i n t e s t im a t e f r o m a r e sp o n s e c u r v e b u t o n ' h y p o t h e s i s ' t es tso f sp ec i f i c t e s t co n cen t ra t io n s . T h e ac tu a l ' s a fe ' o r ' n o e ffec t co n cen t ra t io n ' f al l ss o m e w h e r e b e t w e e n t h e L O E C a n d N O E C d e t e r m i n e d i n a n y g iv e n s tu d y. T h efu r th e r th e t e s t co n cen t ra t io n s a re ap e r t , t h e l e s s ex ac t i s th e es t ima te . T h u s , i tis p o s s i b le t h a t t h e N O E C m a y n o t b e a s l o w a s th a t d e t e r m i n e d i n th i s s t u d y i fa s m a l l e r c o n c e n t r a t io n i n t er v a l h a d b e e n u s e d i n t h e e x p e r i m e n t a l c o n c e n t r a t i o nseries.

I n t h e B e n t l e ye t a l . (1 9 7 7 ) s tu d y o f RD X to x ic i ty to fo u r a lg a l sp ec ies , t h ein v es t ig a to r s r ep o r t ed th a t th e re was n o s ig n i f i can t ( a = 0 .0 5 ) d i ff e ren ce in ce l ld e n s i t y o r c h l o r o p h y l l a f o r a n y o f t h e s p e c ie s b a se d o n p r o b i t E C 5 0 a n a l y s e s.B e n t l e y e t a l . (1 9 7 7 ) d id n o t an a ly ze th e i r d a ta fo r an y ch ro n ic en d p o in ts . A s

was th e case in th e p resen t s tu d y fo r ce l l d en s i ty, r ed u c t io n s in ce l l d en s i ty an dc h l o r o p h y l l a c o n c e n t r a t i o n s o c c u r r e d f o r v a r io u s s p e c i e s i n t h e B e n t l e ye t a l .(1 9 7 7 ) s tu d y. S u l l iv ane t a l . ( 1 9 7 9) r e a n a l y z e d th e n o m i n a l c o n c e n t r a t io n d a t a o fB e n t l e ye t a l . (1 9 7 7 ) an d fo u n d th a t R D X s ig n i f ican t ly ( a = 0 .0 5 ) in h ib i t ed g ro wthin a l l sp ec ies an d to a l e s se r ex ten t ch lo ro p h y l l a co n cen t ra t io n s in a l l fo u r sp ec ies .B a s e d o n t h e a n a l y s e s o f S u l l i v a ne t a l . ( 1 9 79 ) a n d t h e r a w d a t a o f B e n t le y.e ta l . ( 1 9 7 7 ) , t h e L O E C ( c e l l d e n s i t y ) f o rA . f l o s - a q u a ea n d N . p e l l ic u l o s aw o u l db e 1 0 m g L - 1 ( n o m i n a l ) a n d 3 2 m g L - 1 ( n o m i n a l ) f o rM . a e r u g i n o s a ;n o L O E Cc a n b e d e t e r m i n e d f o rS . c a p r i c o r n u t u m . T h eN O E C s f o r re d u c ti o n i n g r o w t h a sr e p o r t e d b y S u l l iv a ne t a l . (1 9 7 9 ) w ere < 0 .3 2 m g L -1 (n o m in a l ) fo r th e g ree n a lg a ;3 .2 m g L - 1 ( n o m i n a l ) f o r th e d i a t o m a n dA . f l o s -a q u a ea n d 1 0 m g L - 1 ( n o m i n a l )f o r M . a e ru g i n o s a .

S u l l iv an e t a l . ( 1 9 7 9) d e t e r m i n e d t h a t t h e N O E C f o rS . c a p r i c o r n u t u mw as< 0 . 3 2 m g L - 1 ( n o m i n a l ). T h e y p o i n t o u t w i th r e g a r d t o t h e g r e e n a l g a th a tt h e i n h i b it io n o f g r o w t h a n d c h l o r o p h y l l a a t th e c o n c e n t r a t i o n o f 0 . 3 2 m g L - 1( n o m i n a l ) i n t h e B e n t l e ye t a I . (1 9 7 7 ) s tu d y were o n ly 2 an d 3 %, r esp ec t iv e ly.S u l l iv an e t a l . (1 9 7 9 ) s t a te th a t th e s t a ti s ti cal m e th o d s th ey u se d in th e i r an a ly ses

w ere ex t rem ely sen s i tiv e an d th a t th e smal l ch an g e o f 2 to 3 % wh ich was s t a t is t ica l lys ig n i f i can t m ay n o t p ro v e to b e b io lo g ica l ly s ig n i f ican t. T h e s t at is t ica l me th o d s u sedi n t h e p r e s e n t s t u d y a r e a p p a r e n t l y m o r e c o n s e r v a t iv e th a n t h o s e u s e d b y S u l l iv a ne t a l . (1 9 7 9 ) . We co u ld n o t d e tec t a 9 .6 % red u c t io n in g ro wth b e tween th e ce l l se x p o s e d t o 0 .5 n ag L - 1 a n d t h e c o n t r o l c e il s. B a s e d o n t h e L O E C ( c e ll d e n s i t y ) o f4 .8 m g L - 1 a n d N O E C o f 0 .5 m g L o b t a i ne d in th is s t ud y, t h e N O E C o f < 0 .3 2 m g



THE TOXICITY OF HEXAHYDRO-1,3,5-TRINITRO-1,3,5-TRIAZINE 4 5 5

L -1 ( n o m i n a l ) r e p o r t e d b y S u l l iv a ne t a l . (1 9 7 9 ) ap p ear s to b e lo w fo r th e g ree nalga .

T h e d a t a in t h e B e n t l e ye t a l . ( 1 9 7 7 ) s t u d y a n d s u b s e q u e n t r e a n a ly s i s b y S u l l iv a ne t a l . ( 1 9 7 9 ) a r e b a s e d o n n o m i n a l R D X c o n c e n t r a t i o n s ; t h e r e f o r e , t h e t o x i c i t yv a l u e s s h o u l d b e v i e w e d w i t h s o m e c a u ti o n . A l t h o u g h t h e t o x ic i ty v a l u e s a r e b a s e do n n o m i n a l c o n c e n t r a t i o n s , t h e d a t a s h o w t h a t s o m e s p e c i e s a r e m o r e s e n s i t i v et h a n o t h e r s to R D X e x p o s u r e . B a s e d o n t h e a n a ly s i s o f S u l li v a ne t a l . (1979) , theg r e e n a l g aS . c a p r i c o r n u t u mi s t h e m o s t sen s i t iv e sp ec ies ; th e b lu e -g reen a lg a A .f l o s - a q u a e a n d t h e d i a t o mN . p e l l i c u l o s aa re in t e rmed ia te in sen s i t iv ity fo l lo w edb y t h e b l u e - g r e e n a l g aM . a e r u g i n o s awh ich i s th e mo s t to l e ran t . T h e b u sh b ean(P. vu lgar i s ) , w h e n g r o w n u n d e r h y d r o p o n i c c o n d i ti o n s , a ls o a p p e ar s to b e m o r et o l e r a n t t o R D X r e la t iv e t oS . c a p r i c o r n u t u m ;1 0 m g L - 1 ( h i g h e s t c o n c e n t r a t i o ns t u d ie d ) h a d n o t o x i c e f f e c t o n t h e b u s h b e a n a f t e r a 7 - d a y e x p o s u r e . T h e q u a n t it a ti v ed a t a o b t a i n e d i n t h is s t u d y f o r t h e g r e e n a lg a c a n b e u s e d f o r t h e E PA n u m e r i c a lw a te r q u a l i ty c r i t e r io n F in a l P lan t Va lu e . T h e c r i t e r io n b ased o n th e g reen a lg a d a tas h o u l d p r o t e c t t h e a l g a l g r o u p s s t u d i e d b y B e n t l e ye t aL (1977) .

T h e t o x i c i ty o f R D X t o n a tu r a l ly o c c u r r i n g a q u at ic p e r i p h y t o n a n d p h y t o p l a n k -to n co mmu n i t i e s i s n o t c l ea r. S u l l iv ane t a l . ( 1 9 77 ) c o n d u c t e d a n a q u a t ic i m p a c ts t u d y a t t h e H o l s t o n A r m y A m m u n i t i o n P l a n t (K i n g s p o rt , T N ) a n d f o u n d e f f e c ts

o n p e r i p h y t o n i n w a t e r c o n t a i n i n g R D X a t c o n c e n t r a t i o n s a s l o w a s 2 0 # gL -1i n th e Ho ls to n R iv er. Ho wev er, t h ey co u ld n o t a t t r ib u te th e e ffec t s o b se rv ed toR D X b e c a u s e o t h e r m u n i t i o n s a n d c o n v e n t i o n a l c o n t a m i n a n t s w e r e p r e s e n t in t h ee ff lu en t d i sch arg e d b y th e p lan t . Co n ce n t ra t io n s o f RD X as h ig h as 1 .9 an d 1 .7 m gL - 1 w e r e f o u n d i n w a s t e w a t e r s f r o m A r e a s A a n d B , r e s p e ct iv e l y, o f th e H o l s t o nA r m y A m m u n i t i o n P l an t b y S ti lw e lle t a l . (1977) .

I n s u m m a r y , th e l a c k o f R D X a c u t e t o x ic i ty to t h e g r e e n a l g aS . c a p r i c o r n u t u mw a s c o n f i r m e d . R D X is t o x i c t o t h e g r e e n a lg a w h e n c h r o n i c e n d p o i n ts a r e u s e dt o a n a l y z e t h e d a t a . T h e L O E C a n d N O E C , b a s e d o n c e l l d e n s i t y, w e r e 4 . 8 a n d0 .5 m g L -1 , r e sp ec t iv e ly. O f fo u r a lg a l sp ec ies s tu d ied to d a ta ,S . c a p r i c o r n u t u mi s t h e mo s t sen s i t iv e sp ec ies ; th e b lu e -g reen a lg aA . f l o s - a q u a ea n d t h e d i a t o mN . p e l l i c u l o s aa re in t e rmed ia te in sen s i t iv i ty fo l lo wed b y th e b lu e -g reen a lg a M.a e r u g i n o s aw h ich i s th e l eas t sen s it iv e . T h e q u an t i ta t iv e d a ta o b ta in ed in th is s tu d yf o r t h e g r e e n a l g a c a n b e u s e d f o r t h e E PA n u m e r i c a l w a t e r q u a l it y c ri te r io n F i n a lP lan t Va lu e. A c r i t e r io n b ased o n th e g reen a lg a d a ta sh o u ld p ro tec t th e ab o v e a lg a lg ro u p s . I t sh o u ld b e p o in ted o u t , h o w ev er, th a t a c r i te r io n b ased o n th e g reen a lg am a y n o t n e c e s s a r i l y p r o t e c t p e ri p h y t o n .

Acknowledgements

T h i s w o r k w a s s u p p o r t e d b y t he U .S . A r m y M e d i c a l R e s e a r c h an d D e v e l o p m e n tC o m m a n d . T h e o p i n i o n s , i n te r p r et a ti o n s , c o n c l u s io n s , an d r e c o m m e n d a t i o n s o f t h es t u d y a r e t h o s e o f t h e a u t h o r s a n d a r e n o t n e c e ss a r i ly e n d o r s e d b y t h e U . S . A r m y. Wea c k n o w l e d g e t h e R e s e a r c h M e t h o d s B r a n c h /H e a l th E f f ec t s R e s e a r c h D i v i si o n o f



456 DEN NIS T. BURTONET AL.

the U .S. Arm y B iome dical Research and Dev elopm ent Laboratory for supportingthe study through U.S. Army Contract No. MIPR 88MM8501 via U.S. Navy

Contract No. N 00 039 -89-C -000 1 to The Johns Hop kins University Ap plied Ph ysicsLaboratory (JHU/APL). W e ackn ow ledge JH U/A PL for providing a subcontractto the U niversity o f M aryland Agricultural Exp eriment Station, W ye R esearch andEduca tion C enter to com plete the study. This paper is Sc ientific Article No. A6 556 ,Contribution No. 87 67 from the Maryland Agricultural Exp erim ent Station.

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THE TO XICITYOF HEXA HYD RO-1,3,5-TRINITRO-1,3,5-TRIAZINE 457

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