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Ecotoxicology Studies
John A. McCann U.S.
Environmental Protection Agency, EN-342, Washington, DC 20460
Ecotoxicology involves the study of the effect of toxic substances on mammals and birds in their environment, and aquatic organisms in fresh and salt water. This paper will discuss the quality assurance aspects of studies involving direct application of potentially toxic materials to test organisms. It will stress the need for chemists to become more involved in ecotoxicology testing by assisting biologists in documenting the identity of test substances, the exposure levels and the stability of the test material in water, air and/or food, and in measuring residue levels in the test organisms and their surrounding environment. Good Laboratory Practice and Quality Assurance procedures required for acute and chronic health effect studies can be used for acute and chronic mammalian, aquatic and avian ecotoxicology studies. Publication of the Environmental Protection Agency's Good Laboratory Practice (GLP) regulations governing the conduct of studies submitted to the Agency in support of The Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) and the Toxic Substances Control Act (TSCA) has caught the attention of the regulated public. An important part of these regulations assures that the laboratory and the studies meet GLP requirements by describing the activities of the Quality Assurance Officer and Quality Assurance Unit at the laboratory. This paper discusses the quality assurance procedures for ecotoxicology laboratories. It will attempt to concentrate on those areas that are of particular concern to those individuals determining the toxicity of chemicals to fish and wildlife (ecotoxicology testing). This chapter not subject to U.S. copyright Published 1988 American Chemical Society
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A minimum amount of time w i l l be spent on those a r e a s t h a t are common to a l l s t u d i e s and which w i l l have been covered more e x t e n s i v e l y by other authors. The r e q u i r e m e n t s f o r e c o t o x i c o l o g y s t u d i e s vary l i t t l e from the r e q u i r e m e n t s f o r h e a l t h e f f e c t s t u d i e s . L a b o r a t o r i e s t h a t have adequate f a c i l i t i e s , equipment, s t a f f , and procedures f o r h e a l t h e f f e c t s t u d i e s , w i t h adequate t r a i n i n g of t h e i r s t a f f i n s p e c i f i c a r e a s of e c o t o x i c o l o g y , c o u l d conduct these t e s t s . For example, the r e q u i r e m e n t s f o r t r a c k i n g the r e c e i p t and use o f the t e s t substances and t e s t a n i m a l s are the same. The need f o r the c a l i b r a t i o n of the t e s t equipment, s t o r a g e , and a r c h i v e s are the same. E x t r a e f f o r t s h o u l d be expended, however, t o determine t h a t adequate good q u a l i t y water i s a v a i l a b l e a t a q u a t i c t e s t i n g f a c i l i t i e s by more f r e q u e n t a n a l y s e s of incoming w a t e r . Special arrangements s h o u l d a l s o be made to handle l a r g e volumes of waste water such as p r e t r e a t m e n t of the water u s i n g c h a r c o a l f i l t e r s b e f o r e d i s c h a r g i n g the water from the f a c i l i t y . Because the a q u a t i c s t u d i e s r e q u i r e a more p r e c i s e e v a l u a t i o n of the q u a l i t y of the i n c o m i n g water and the use of s p e c i a l i z e d equipment, I w i l l spend more time d i s c u s s i n g t h i s a r e a . Aquatic
Environments
F i s h and a q u a t i c i n v e r t e b r a t e s are exposed to t o x i c substances i n the l a b o r a t o r y by one of t h r e e types of systems: s t a t i c , f l o w through and renewal, as d e s c r i b e d below. Stati c In s t a t i c t e s t s the t e s t m a t e r i a l i s mixed w i t h the w a t e r . Then the a q u a t i c organisms are p l a c e d i n the t e s t s o l u t i o n and remain t h e r e f o r the d u r a t i o n of the t e s t or u n t i l they d i e . The t e s t s n o r m a l l y l a s t f o r two days f o r small i n v e r t e b r a t e s and f o u r days f o r f i s h , amphibians and l a r g e r i n v e r t e b r a t e s . The experiments are g e n e r a l l y c o n s i d e r e d "quick and d i r t y " t e s t s t h a t g i v e a r e a s o n a b l e e s t i m a t e of the t o x i c i t y of the t e s t substance w i t h a minimum of e f f o r t . In the p a s t , they d i d not r e q u i r e a n a l y s i s of t e s t m a t e r i a l c o n c e n t r a t i o n s u n l e s s s o l u b i l i t y l i m i t s were b e i n g exceeded. Flow-through In f l o w - t h r o u g h t e s t s , t h e t r e a t e d w a t e r i s c o n t i n u a l l y r e p l a c e d e i t h e r by a c o n s t a n t f l o w o r by a d d i t i o n s of small volumes of t r e a t e d water a t 1-10 minute i n t e r v a l s . V a r i o u s d e l i v e r y systems have been d e s i g n e d to e i t h e r s u p p l y measured amounts of newly mixed t e s t c o n c e n t r a t i o n s or to add premixed s o l u t i o n s to the t e s t chambers. Depending on the p h y s i c a l c h a r a c t e r i s t i c s of the c h e m i c a l s i n v o l v e d and the r e l i a b i l i t y o r accuracy of the d e l i v e r y s y s t e m s , t h e r e i s ample o p p o r t u n i t y w i t h t h i s method f o r something t o go wrong i n the d e l i v e r y of the t e s t c h e m i c a l . However, because t h e water i s c o n s t a n t l y r e p l a c e d and the organisms can be f e d , i t i s p o s s i b l e i n t e s t s conducted i n f l o w - t h r o u g h systems, t o expose t e s t
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organisms to the t e s t s o l u t i o n s f o r s e v e r a l y e a r s . Because of the importance put on these t e s t s by r e g u l a t o r y agencies and the p o t e n t i a l f o r f l u c t u a t i o n of t e s t c o n c e n t r a t i o n s , i t i s g e n e r a l l y c o n s i d e r e d necessary to measure d a i l y and document the c o n c e n t r a t i o n o f the t e s t substance i n the water i n a t l e a s t the h i g h , medium and low c o n c e n t r a t i o n s . I f c o n t r o l and s o l v e n t c o n t r o l s are a l s o i n c l u d e d i n the sampling scheme, t h e number of a n a l y s e s c o u l d i n v o l v e over 1500 samples per y e a r per s t u d y . Flow-through systems can be used f o r acute s t u d i e s but g e n e r a l l y they are used f o r uptake and d e p u r a t i o n s t u d i e s or complete l i f e cycle studies. Renewal The renewal system i s a c o m b i n a t i o n of the above systems. The t e s t organisms are removed from the o l d t e s t s o l u t i o n s and are p l a c e d i n new, f r e s h l y prepared s o l u t i o n s of the same c o n c e n t r a t i o n s t h r e e times each week. A r e p r e s e n t a t i v e sample of the o l d and new s o l u t i o n s a t h i g h , medium and low c o n c e n t r a t i o n s are a n a l y z e d each time the t r a n s f e r s o c c u r . T h i s system i s f r e q u e n t l y used f o r the daphnia life-cycle tests. I t i s a l s o used when the t e s t chemical has a s h o r t h a l f - l i f e i n water o r i f the t e s t a n i m a l s must be f e d d u r i n g the t e s t . Length o f T e s t s Another area I want to d i s c u s s i s the l e n g t h of e c o t o x i c o l o g y s t u d i e s . T h i s g e n e r a l l y i s dependant on the type of t e s t . Acute S t u d i e s A c u t e a q u a t i c t e s t s n o r m a l l y l a s t 2 t o 4 days depending on the t e s t organisms. Chronic t e s t s f o r i n v e r t e b r a t e s , l i k e daphnia, l a s t f o r 21 t o 28 days and may i n v o l v e s e v e r a l g e n e r a t i o n s of o f f s p r i n g . We are always l o o k i n g f o r t e s t organisms t h a t w i l l reach m a t u r i t y f a s t e r so we can e v a l u a t e the e f f e c t of the t e s t substance on m u l t i p l e g e n e r a t i o n s i n a s h o r t e r p e r i o d of t i m e . C h r o n i c and S u b c h r o n i c S t u d i e s F u l l l i f e - c y c l e o r c h r o n i c f i s h s t u d i e s may take two y e a r s o r more. C o n s e q u e n t l y , b i o l o g i s t s and r e g u l a t o r s are r e q u e s t i n g more s u b c h r o n i c s t u d i e s where the t e s t organisms are s u b j e c t e d t o t o x i c c o n c e n t r a t i o n s d u r i n g t h e i r e a r l y l i f e s t a g e s (egg t o f r y ) f o r g e n e r a l l y a 30-day exposure p e r i o d . I f t h e r e are m u l t i p l e t e s t c o n c e n t r a t i o n s and the t u r n o v e r r a t e f o r t h e water i s r a p i d enough, a l a r g e number of a n a l y s e s and e x t e n s i v e r e c o r d k e e p i n g may be required. For chemical a n a l y s e s t o be u s e f u l to the b i o l o g i s t , a n a l y s e s must be t i m e l y . A s i g n i f i c a n t delay i n s u p p l y i n g a water or food a n a l y s i s t o a b i o l o g i s t c o u l d i n v a l i d a t e a study i f the d e v i a t i o n from nominal c o n c e n t r a t i o n i s shown to be too d r a s t i c or f o r t o o long a d u r a t i o n . Most of the e c o t o x i c o l o g y t e s t s i n v o l v i n g b i r d s and/or small
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mammals have the same r e q u i r e m e n t s as the r a t and mouse t e s t s i n v o l v e d i n r o u t i n e t o x i c i t y t e s t i n g and r e q u i r e the same a n a l y s i s o f the food and t i s s u e . Special Considerations Now t h a t we have d i s c u s s e d the t e r m i n o l o g y and the types of t e s t s i n v o l v e d , I want to emphasize some areas t h a t s h o u l d be w e l l d o c u mented i n an e c o t o x i c o l o g y s t u d y . I am not g o i n g t o go i n t o the more o b v i o u s q u a l i t y assurance program r e q u i r e m e n t s t h a t are a p p l i c a b l e to a l l l a b o r a t o r i e s . These general r e q u i r e m e n t s i n c l u d e the p r o v i s i o n f o r a Q u a l i t y A s s u r a n c e U n i t , the a v a i l a b i l i t y of a q u a l i f i e d s t a f f , the presence of f a c i l i t i e s and equipment adequate to p e r m i t the type and number of s t u d i e s b e i n g p e r f o r m e d , a master schedule of ongoing and completed s t u d i e s , s t o r a g e a r e a s f o r a c t i v e and used samples, and a r c h i v e s f o r the r e t e n t i o n of r e p o r t s and raw d a t a . With the e x c e p t i o n of the s p e c i a l h a n d l i n g r e q u i r e d f o r the t e s t w a t e r , w i l d b i r d s and f i s h , f a c i l i t i e s t h a t conduct r o u t i n e mammalian t o x i c i t y s t u d i e s s h o u l d be adequate to conduct e c o t o x i cology t e s t i n g . S i n c e we are t a l k i n g about t e s t i n g t h a t i s f r e q u e n t l y conducted on w i l d s p e c i e s t h a t have not been r o u t i n e l y t e s t e d i n the l a b o r a t o r y i t i s i m p o r t a n t to s t r e s s t h a t Q u a l i t y A s s u r a n c e personnel be aware of s p e c i a l r e q u i r e m e n t s , such as temperature c o n t r o l , l i g h t c o n t r o l , cage or tank s i z e , water q u a l i t y , e t c . T h i s i n f o r m a t i o n s h o u l d be a v a i l a b l e i n the p r o t o c o l and t h e SOP. Again, the environmental and t e s t i n g c o n d i t i o n s f o r many w i l d mammalian and a v i a n s p e c i e s a r e c o m p a t i b l e w i t h the domestic mammalian s t u d i e s t h a t have been done i n some l a b o r a t o r i e s f o r y e a r s . S i n c e some of the e c o t o x i c o l o g y s t u d i e s c o n c e n t r a t e on the n o n l e t h a l e f f e c t s of the t e s t substance on the t e s t organisms, i t i s i m p o r t a n t t h a t the t e s t c o n d i t i o n s and e v a l u a t i o n c r i t e r i a be a c c u r a t e l y d e s c r i b e d and the s t a f f be very aware of s u b l e t h a l e f f e c t s of the t o x i c a n t on the t e s t s p e c i e s . F o r a q u a t i c s t u d i e s , i t i s i m p o r t a n t t o f r e q u e n t l y document the q u a l i t y of the incoming w a t e r , the q u a l i t y of the water i n which t h e t e s t organisms are h e l d and a c c l i m a t e d , and the q u a l i t y of the w a t e r i n which the a n i m a l s are t e s t e d . The t e m p e r a t u r e , a l k a l i n i t y , pH, h a r d n e s s , s a l i n i t y , e t c . , may be w e l l w i t h i n the c r i t e r i a t h a t i s a c c e p t a b l e f o r mammals and b i r d s but not a q u a t i c a n i m a l s . F o r example, the a b r u p t changes t h a t c o u l d occur when f i s h and/or i n v e r t e b r a t e s are t r a n s f e r r e d from one water q u a l i t y to a n o t h e r , c o u l d e i t h e r s t r e s s or k i l l the a n i m a l s . Low c h l o r i n e or m i n e r a l l e v e l s i n water might be a c c e p t a b l e f o r b i r d s and mammals but may be deadly to f i s h o r i n v e r t e b r a t e s . Raw water coming i n t o an a q u a t i c l a b o r a t o r y s h o u l d be a n a l y z e d f o r water q u a l i t y and c h e m i c a l r e s i d u e s a t l e a s t q u a r t e r l y , u n t i l a data base has been e s t a b l i s h e d t h a t demonstrates t h a t t h e water q u a l i t y f a l l s w i t h i n a c c e p t a b l e parameters and c o n t a i n s no s i g n i f i c a n t c o n t a m i n a n t s . Semi-annual checks s h o u l d then be used to c o n f i r m the c o n t i n u e d a c c e p t a b i l i t y o f the w a t e r . The l a b o r a t o r y s h o u l d r o u t i n e l y document the q u a l i t y o f water b e i n g used to h o l d , a c c l i m a t e and t e s t the a q u a t i c organisms t o a s s u r e t h a t the water i s a c c e p t a b l e f o r each t e s t o r g a n i s m .
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Types o f E c o t o x i c o l o g y S t u d i e s There a r e a p p r o x i m a t e l y 15 t o 20 t y p e s of e c o t o x i c o l o g y s t u d i e s t h a t are n o r m a l l y r e q u e s t e d by EPA. I am n o t g o i n g to d e s c r i b e them a l l , but I do want to f a m i l i a r i z e you w i t h some of them (Table 1 ) . T a b l e 1. R e p r e s e n t a t i v e L i s t o f Toxicology Studies 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
Environmental
Daphnid acute t o x i c i t y t e s t Daphnid c h r o n i c t o x i c i t y t e s t ( l i f e - c y c l e ) M y s i d shrimp acute t o x i c i t y t e s t Mysid shrimp c h r o n i c t o x i c i t y t e s t O y s t e r acute t o x i c i t y t e s t Oyster b i o c o n c e n t r a t i o n t e s t Oyster s h e l l growth t e s t Penaeid shrimp t o x i c i t y t e s t Acute f i s h t e s t ( c o l d and warm water) f r e s h and salt F i s h b i o c o n c e n t r a t i o n t e s t s ( f r e s h water) F i s h e a r l y l i f e s t a g e t e s t ( f r e s h and s a l t water) A v i a n d i e t a r y t e s t ( c o l d and warm water) f r e s h and salt A v i a n r e p r o d u c t i o n t e s t ( m a l l a r d , bobwhite q u a i l ) W i l d r o d e n t 5 day f e e d i n g t e s t W i l d mammal t e s t s ( s k u n k s , w o l v e s , f o x e s , r o d e n t s ) S p e c i a l avian and mammalian t e s t s Algal acute t o x i c i t y t e s t s Seed g e r m i n a t i o n / r o o t e l o n g a t i o n t o x i c i t y t e s t P l a n t uptake and t r a n s l o c a t i o n t e s t Small p e n / f i e l d s t u d i e s
B i o l o g i s t s f r e q u e n t l y use daphnia i n f r e s h w a t e r s h o r t and l o n g term t e s t s , w h i l e shrimp and o y s t e r s are used to e v a l u a t e the p o t e n t i a l t o x i c i t y of c h e m i c a l s to s a l t w a t e r i n v e r t e b r a t e s . Freshwater f i s h t e s t s a r e g e n e r a l l y conducted on b l u e g i l l , a warm water f i s h , and rainbow t r o u t , a c o l d water f i s h . Catfish, fathead minnows and sometimes c a r p are a l s o used depending on the e x p e c t e d r o u t e o f exposure. Sheepshead minnow i s the commonly used saltwater f i s h . C h r o n i c s t u d i e s are conducted on the f a s t - m a t u r i n g f a t h e a d minnow and sheepshead minnow. S u b c h r o n i c s t u d i e s are conducted on those s p e c i e s r e l a t i v e l y e a s i l y r a i s e d i n the l a b o r a t o r y ; e . g . , f a t h e a d minnow, t r o u t and sheepshead minnow. The q u a l i t y of the incoming w a t e r i s of p a r t i c u l a r concern t o i n d i v i d u a l s c o n d u c t i n g a q u a t i c s t u d i e s . P o t a b l e water i s g e n e r a l l y r e c o g n i z e d as poor q u a l i t y water f o r f i s h and i n v e r t e b r a t e s . A good f r e s h water i s one i n which daphnia w i l l l i v e and s a t i s f a c t o r i l y reproduce. A v i a n LD50, d i e t a r y LC50, and r e p r o d u c t i o n s t u d i e s are n o r m a l l y conducted on young or a d u l t m a l l a r d ducks o r bobwhite q u a i l , depending on the r e q u i r e m e n t s of the t e s t . There has not been the same demand f o r e c o t o x i c o l o g y t e s t i n g o f mammals because the Agency has r o u t i n e l y extended the r e s u l t s of
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r o u t i n e ( h e a l t h e f f e c t ) t o x i c o l o g y t e s t s to w i l d mammals. A f i v e day f e e d i n g t e s t and o t h e r s p e c i a l t e s t s have been designed to g i v e the environmental t o x i c o l o g i s t a b e t t e r i d e a of f i e l d exposures of mammals and b i r d s to t o x i c m a t e r i a l s . B o t a n i c a l t e s t s would be e v a l u a t e d u s i n g s i m i l a r c r i t e r i a as those used on o t h e r e c o t o x i c i t y t e s t s . Analytical
Support
There are s e v e r a l a r e a s i n a q u a t i c t o x i c o l o g y where c h e m i s t r y s u p p o r t would be very h e l p f u l to the b i o l o g i s t c o n d u c t i n g the study. In o t h e r a r e a s , c h e m i s t r y s u p p o r t i s e s s e n t i a l , e . g . , a n a l y s e s of t e s t c o n c e n t r a t i o n s and r e s i d u e s i n t e s t o r g a n i s m s , f e e d and w a t e r . I f c h e m i s t r y support i s not a v a i l a b l e to the b i o l o g i s t , the b i o l o g i s t s h o u l d have c o n s i d e r a b l e e x p e r t i s e i n these chemistry o r i e n t e d areas. I hope to encourage e c o t o x i c o l o g y t e s t i n g f a c i l i t i e s to supply the r e q u i r e d c h e m i s t r y s u p p o r t to t h e i r b i o l o g i s t s . Test Material I am going t o s t a r t the d i s c u s s i o n by t r a c k i n g a t e s t chemical from i t s a r r i v a l i n the l a b o r a t o r y to i t s f i n a l a r c h i v i n g and s t o r a g e . However, as you w i l l s e e , t h e r e q u i r e d a n a l y s e s are not l i m i t e d to a n a l y s i s of the t e s t s u b s t a n c e . When the t e s t m a t e r i a l a r r i v e s a t a f a c i l i t y , t h e r e s h o u l d be a f a c t sheet w i t h i t t h a t l e a v e s no doubt as to the i d e n t i t y of t h e t e s t m a t e r i a l , l o t or batch number, p e r c e n t a c t i v e i n g r e d i e n t , storage c o n d i t i o n s , e t c . The w e i g h i n g of the i n c o m i n g sample can be determined by a t e c h n i c i a n . The w e i g h t of the sample w i l l serve as the s t a r t i n g p o i n t f o r the use l o g f o r the c h e m i c a l . In s e v e r a l l a b o r a t o r i e s , I have seen c h e m i s t s go s e v e r a l s t e p s f u r t h e r . They sometimes c o n f i r m the i d e n t i t y and p u r i t y of the sample. They sometimes i n d i c a t e the s o l v e n t s of p r e f e r e n c e and the s o l u b i l i t y of the m a t e r i a l i n the v a r i o u s s o l v e n t s and w a t e r . T h i s a i d s the b i o l o g i s t i n s e l e c t i n g t e s t c o n c e n t r a t i o n s and a p p r o p r i a t e s o l v e n t s and does not c r e a t e unnecessary problems w i t h chemical d e t e r m i n a t i o n s l a t e r because the wrong s o l v e n t s were u s e d . O b v i o u s l y , a l l the c a l c u l a t i o n s , measurements, e t c . , must be a d e q u a t e l y r e c o r d e d to s a t i s f y GLP r e q u i r e m e n t s . The adequately l a b e l e d c o n t a i n e r s of t e s t m a t e r i a l must be s t o r e d i n a p p r o p r i a t e l y l a b e l e d areas under c o n d i t i o n s t h a t w i l l have no adverse a f f e c t on the c h e m i c a l ' s s t a b i l i t y , c o m p o s i t i o n , e t c . Water A n a l y s i s I f s u r f a c e scums or p r e c i p i t a t e s are observed i n an acute study o r the p r o t o c o l r e q u i r e s i t , the t e s t c o n c e n t r a t i o n s need to be measured and documented. T h i s r e q u i r e s t a k i n g a p p r o p r i a t e samples. The i n v e s t i g a t o r must have c o n s i d e r a b l e e x p e r t i s e i n t a k i n g and a n a l y z i n g the samples i f the c o n c e n t r a t i o n s are low o r i f s o p h i s t i c a t e d a n a l y t i c a l t e c h n i q u e s and/or equipment are needed. In a r e n e w a l - s t u d y o r a c h r o n i c study r e q u i r i n g a f l o w - t h r o u g h system, i t i s important to t a k e and measure t e s t c o n c e n t r a t i o n s on a d a i l y
18. McCANN
Quality Assurance for Ecotoxicology Studies
137
b a s i s so t h a t t i m e l y c o r r e c t i o n s can be made i n the d e l i v e r y system to m a i n t a i n t e s t c o n c e n t r a t i o n s . I f the samples are not a n a l y z e d i n a t i m e l y manner, the b i o l o g i s t may not be a b l e to m a i n t a i n the t e s t concentrations. I f the d e v i a t i o n from the i n t e n d e d c o n c e n t r a t i o n o c c u r s f o r too l o n g o r i s too severe i t c o u l d i n v a l i d a t e the s t u d y . Many of these chemical a n a l y s e s may be too d i f f i c u l t f o r the average biologist. Test Solutions Some c h e m i s t r y u n i t s support the b i o l o g i s t s by p r e p a r i n g the t e s t solutions. Whoever prepares the a l i q u o t s of t e s t m a t e r i a l and/or makes the stock s o l u t i o n s s h o u l d a d e q u a t e l y document the removal of the a p p r o p r i a t e amounts of t e s t m a t e r i a l from the b u l k c o n t a i n e r . The b a l a n c e r e a d i n g s used t o weigh out the samples s h o u l d become a permanent r e c o r d and s h o u l d be r e t a i n e d as o r i g i n a l raw data i n t h e archives. The a r c h i v e s s h o u l d be adequate to m a i n t a i n the i d e n t i t y and i n t e g r i t y of the sample. In some c a s e s , t h i s c o u l d r e q u i r e f r e e z i n g t h e sample. Residue A n a l y s i s Chemistry support i s f r e q u e n t l y needed to measure the r e s i d u e s of p o s s i b l e t o x i c substances such as p e s t i c i d e s i n each l o t of t e s t organisms b e f o r e they are used i n t o x i c i t y t e s t s . The a n a l y s i s s h o u l d be e x t e n s i v e enough t h a t the data w i l l document any r e s i d u e s t h a t might i n t e r f e r e w i t h the u s e f u l n e s s of the t e s t . I n s p e c t o r s and q u a l i t y assurance personnel s h o u l d r o u t i n e l y ask t o see the chemical a n a l y s e s o r r e s i d u e a n a l y s e s performed on each l o t of t e s t organisms used i n b i o a s s a y s t h a t were r e p o r t e d t o t h e Agency. I f the t e s t organisms were fed w h i l e i n the l a b o r a t o r y , t h e data base f o r the study s h o u l d i n c l u d e documentation t h a t the food was f r e e of r e s i d u e s of c h e m i c a l s t h a t c o u l d a d v e r s e l y a f f e c t the r e s u l t s of the t e s t . There s h o u l d be a complete r e c o r d of these a n a l y s e s i n the a r c h i v e s . Water
Quality
Another area of concern to an a q u a t i c b i o l o g i s t i s the q u a l i t y of t h e water b e i n g used to h o l d the organisms d u r i n g r e a r i n g , a c c l i m a t i o n and t e s t i n g . F r e q u e n t l y b i o l o g i s t s or t e c h n i c i a n s can conduct t e s t s to document pH, d i s s o l v e d oxygen, a l k a l i n i t y , h a r d n e s s , and s a l i n i t y i n the incoming w a t e r and t e s t w a t e r . However, i t f r e q u e n t l y r e q u i r e s a r e a l commitment of the c h e m i s t r y s u p p o r t to o b t a i n d e t a i l e d a n a l y s e s of the t o x i c chemical and m i n e r a l s i n the incoming water used to h o l d and/or t e s t a q u a t i c o r g a n i s m s . Many of the a n a l y ses s h o u l d be conducted on the incoming w a t e r a t l e a s t semi-annua l l y (1). In the case of new water s u p p l i e s , these a n a l y s e s s h o u l d be conducted monthly o r q u a r t e r l y u n t i l the l a b o r a t o r y s t a f f i s a b l e to document t h a t no seasonal or p e r i o d i c changes i n water q u a l i t y occur that could adversely a f f e c t t e s t r e s u l t s . This h i s t o r i c a l database s h o u l d be r e t a i n e d by the l a b o r a t o r y as raw d a t a .
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GOOD LABORATORY PRACTICES
The Agency's s c i e n t i f i c s t a f f w i l l e v a l u a t e t h e e f f e c t the c h e m i s t r y f i n d i n g s might have on the outcome of the s t u d y . P o t a b l e water c o n t a i n i n g c h l o r i n e o r copper i s g e n e r a l l y c o n s i d e r e d t o x i c t o many i n v e r t e b r a t e s . Y e t , the l a c k o f m i n e r a l s , e t c . , i n d i s t i l l e d water makes i t o s m o t i c a l l y u n a c c e p t a b l e t o many a q u a t i c o r g a n i s m s . The f a c t t h a t t h e water i s f i t f o r human consumpt i o n does not mean the w a t e r i s a c c e p t a b l e to a q u a t i c o r g a n i s m s . Chemical
Stability
In some cases i t i s necessary to a n a l y z e t h e w a t e r sample immediately a t t h e t e s t f a c i l i t y because of the r a p i d breakdown of the chemical i n w a t e r , p a r t i c u l a r l y a t some of the low c o n c e n t r a t i o n s t h a t might be t o x i c to the t e s t o r g a n i s m . I f samples a r e to be s t o r e d f o r any length of time, or shipped to another f a c i l i t y f o r a n a l y s i s , the l a b o r a t o r y and/or sponsor s h o u l d be a b l e to document the t e s t m a t e r i a l s t a b i l i t y i n w a t e r a t the c o n c e n t r a t i o n s b e i n g used i n t h e b i o a s s a y . The data s h o u l d be a v a i l a b l e t o the t e s t i n g f a c i l i t y before a t e s t i s i n i t i a t e d . Inadequate documentation of the s t a b i l i t y of the c h e m i c a l under t e s t c o n d i t i o n s c o u l d r e s u l t i n the study b e i n g u n s a t i s f a c t o r y f o r t h e purpose i n t e n d e d . Agency i n s p e c t o r s s h o u l d document t h e a v a i l a b i l i t y of the s t a b i l i t y data when water and t i s s u e samples a r e taken f o r r e s i d u e a n a l y s e s d u r i n g t h e s t u d y . When a n a l y s e s on u n s t a b l e t e s t s o l u t i o n s a r e n o t conducted i n a t i m e l y f a s h i o n , the t e s t r e s u l t s are unusable. Many l a b o r a t o r i e s have the t e s t chemical examined by the chemi s t r y s t a f f b e f o r e i t i s t e s t e d i n the l a b o r a t o r y . The c h e m i s t s f r e q u e n t l y i n d i c a t e the s o l v e n t s to be u s e d , the r e l a t i v e s o l u b i l i t y o f the t e s t m a t e r i a l , and any s p e c i a l i n s t r u c t i o n s c o n c e r n i n g t h e h a n d l i n g o r a n a l y s e s of the t e s t m a t e r i a l o r t e s t s o l u t i o n s . An e a r l y i n v o l v e m e n t o f a c h e m i s t i n the conduct of the b i o a s s a y can save t h e b i o l o g i s t many hours of wasted e f f o r t i n t r y i n g t o prepare t e s t s o l u t i o n s o r i n a n a l y z i n g them f o r r e s i d u e s . Some very poor h a n d l i n g o f very t o x i c c h e m i c a l s o c c u r s because the s t a f f i s not aware of the t o x i c i t y o f t h e t e s t c h e m i c a l . A t some l a b o r a t o r i e s , e x c e s s i v e s a f e t y procedures i n v o l v i n g n o n t o x i c c h e m i c a l s a r e used because the s t a f f do not know a n y t h i n g about the t e s t m a t e r i a l . The l a b o r a t o r y s t a f f s h o u l d be knowledgeable about any chemical they a r e t e s t i n g . F o r no o t h e r r e a s o n , they s h o u l d know the c h a r a c t e r i s t i c s of the chemical b e f o r e the t r e a t e d water i s r e l e a s e d from t h e f a c i l i t y .
Literature Cited 1. U.S. Environmental Protection Agency, Committee on Methods for Toxicity Tests with Aquatic Organisms. 1975. Methods for acute toxicity tests with fish, macroinvertebrates, and amphibians. Ecol. Res. Ser. EPA-660/3-75-009. RECEIVED
January 29, 1988
