Bull. Environ. Contam. Toxicol. (1985) 35:133-142 9 1985 Springer-Verlag New York Inc.
• CE no vnitraomnimn ae tnitoanl
~and Toxicology
Aquatic Bioassay of 11 Pesticides Using Larvae of the Mosquito, Wyeomyia smithii (Diptera: Culicidae) Daniel Strickman 1 USAF Occupational and Environmental Health Laboratory, Brooks AFB, TX 78235
One of the uses of aquatic bioassays is to d e t e r m i n e the presence or absence of toxic contaminants in water f r o m the field. Our L a b o r a t o r y has d e v e l o p e d a bioassay system for this purpose using larvae of the pitcher-plant mosquito, Wyeomyia smithii (Coquillett) (Strickman 1984). The major application of this system is the investigation of minor fish kills which may occur at any U.S. Air Force base. The system was developed around a number of requirements, including: (1) Simplicity so that it may be taught to technicians who rotate from their jobs frequently; (2) E c o n o m y of space and time; (3) Tolerance to u n c o n t a m i n a t e d water from a wide variety of sources; (4) Capability of testing small samples of water in order to reduce s h i p p i n g costs; and (5) C a p a b i l i t y of d e t e c t i n g toxicants at levels at or below the LCso for c o m m o n species of fish. The Wyeomyia bioassay ~@oved to be a practical tool in the investigation of over 20 fish kills during 1983 and 1984. Toxicants detected by the bioassay were later identified as engine l u b r i c a t i n g oil, copper, paint stripper, and high levels of free ammonia. Water from the sites of many of the fish kills produced no effect on larvae, indicating that the fish kills had not involved toxic contaminants or that contaminants had dissipated by the time of sampling or bioassay. Such negative results were important because they indicated that chemical analysis would probably not have been productive. Elimination of unnecessary analyses saved over $15,000 during a single year. We have tested the W y e o m y i a smithii sens~ivity--o--~ results of larval
effect of known chemicals on larval in order to establish the limits of bioassay system. This paper reports mosquito bioassays of ii pesticides
iCurrent address: Unit, NHB 165, National Washington, DC 20560
W a l t e r Reed B i o s y s t e m a t i c s M u s e u m of Natural History, 133
in 4 c h e m i c a l groups. The p e s t i c i d e s were selected b e c a u s e of their c o m m o n o c c u r r e n c e in d r a i n a g e w a t e r (U.S. Air Force O c c u p a t i o n a l and E n v i r o n m e n t a l Healtb Laboratory, u n p u b l i s h e d data) or b e c a u s e of their common use in or near water (Anonymous 1983). MATERIALS AND METHODS Samples of pesticides were obtained from manufacturers or the U.S. E n v i r o n m e n t a l P r o t e c t i o n Agency. The s a m p l e s v a r i e d in p u r i t y f r o m 93% to 100%. The chemical categories and common names of the compounds w e r e as follow: O r g a n o c h l o r i n e s : DDT, h e p t a c h l o r epoxide; O r g a n o p h o s p h a t e s : chlorpyrifos, malathion, temephos; C a r b a m a t e s : carbaryl, m e t h o m y l , propoxur; Pyrethroids: permethrin, phenothrin, resmethrin. Colony maintenance, production of larvae for bioassays, and the b i o a s s a y p r o c e d u r e are d e s c r i b e d in detail in p r e v i o u s papers (Lillie et al. 1980, S t r i c k m a n ]983, S t r i c k m a n 1984). Briefly, larvae for b i o a s s a y s w e r e reared under u n c r o w d e d c o n d i t i o n s from eggs r e m o v e d from a m a i n colony. Eggs w e r e r e m o v e d daily so that their age never varied by m o r e than 24 hours. Seven days after the date of d e p o s i t i o n of the eggs (i.e. 3 to 4 days after eggs hatched), s e c o n d - i n s t a r larvae w e r e selected for use in a bioassay. Each r e p l i c a t e for a b l o a s s a y c o n s i s t e d of 30 ml of l a b o r a t o r y w a t e r (aged tap water, see Strickman 1984 for specifications) in a clear, glass jar (50-ml capacity). The d e s i r e d a m o u n t of p e s t i c i d e was added to each r e p l i c a t e by pipetting 0.i ml of an a c e t o n e solution at an appropriate concentration. The concentrations for the b i o a s s a y s w e r e selected on the basis of i or 2 rangefinding tests for each chemical. Laboratory-water control treatments received acetone with no pesticide added. Following addition of the acetone solution, i0 larvae and the a p p r o p r i a t e a m o u n t of food ( T e t r a m i n Baby Fish Food "E," Tetra W e r k e Mille, W e s t G e r m a n y ) were placed in each replicate and the jars covered with paraffin film to prevent evaporation. Bioassays were maintained in an environmental chamber at 27~ with a 16-hour daily photophase. Larvae were observed daily for survival, stage of d e v e l o p m e n t , and b e h a v i o r a l s i g n s of i n t o x i c a t i o n . F o o d w a s a d d e d at e a c h o b s e r v a t i o n in p r o p o r t i o n to n u m b e r of s u r v i v o r s and their stages of development. Bioassays lasted 7 days, d u r i n g w h i c h time m o s t larvae in l a b o r a t o r y w a t e r c o m p l e t e d d e v e l o p m e n t to the fourth instar. For each pesticide, larvae from eggs d e p o s i t e d on the same day w e r e used in 9 r e p l i c a t e s of each of 3 c o n c e n t r a t i o n s and the laboratory-water control.
134
The SAS Statistical Analysis System (SAS Institute, Inc. 1979) was used to perform statistical analyses of survival and development. Survival was analyzed by calculating means of total survivors on each day of a bioassay for each concentration. Differences among daily means for each concentration were determined at the 95% level using Duncan's multiple range test. D e v e l o p m e n t was analyzed by e x a m i n i n g the n u m b e r of third-instar l a r v a e on each day of the b i o a s s a y . Previous studies (Strickman 1984) had indicated that the third instar is representative of d e v e l o p m e n t a l effects throughout the juvenile life of W~l~p_myia smithii. Adjustments of number of third-instar larvae total survival within the replicate (the number of third instars m u l t i p l i e d by i0, then the product divided by the total number of survivors in that replicate) allowed comparison of d e v e l o p m e n t independent of survival. This adjustment was possible only in concentrations where at least i larva remained a l i v e in each r e p l i c a t e . R e s u l t s of the e n t i r e concentration treatment were eliminated from analysis when any of its replicates contained no living larvae. Means of adjusted number of third instars were analyzed similarly to means of number of survivors. RESULTS AND DISCUSSION The b i o a s s a y s y s t e m d e t e c t e d c o n c e n t r a t i o n s of pesticides within the range of levels toxic to fish. A c o m p a r i s o n of the concentrations detectable by the bioassay to LC~o values presented in the literature (Table i) indic~a~ted that the larval mosquito bioassay was within the same range of sensitivity as fish to all of the pesticides tested except DDT and resmethrin. Delayed development was a more sensitive indicator than mortality for d e t e c t i o n of 6 of the p e s t i c i d e s (heptachlor epoxide, methomyl, propoxur, permethrin, phenothrin, and resmethrin). Patterns of the survival curves (Figs. 1-4) suggested a relationship between m o r t a l i t y late in the bioassays and persistence of the chemicals. The slope of the survival curve during the last 3 days of the bioassay was nearly horizontal (indicating cessation of further mortality) for those larvae affected by malathion, carbaryl, permethrin, phenothrin, and resmethrin. The slope was negative (indicating continuing mortality) for DDT, heptachlor epoxide, chlorpyrifos, temephos, methomyl, and propoxur. Literature (Eichelberger and Lichtenburg 1971, Sharom et al. 1980, M i y a m o t o 1976, Kottkamp el al. 1981, Chapman and Cole 1982) documents
135
Table 1. Comparison of sensitivity of larval mosquito (Wyeomyia smithii) bioassay and toxic levels to fish a.
Chemical
Lowest conc. (ppb) detected by bioassay Survival
Lowest LC50 (ppb) to fish and species c
Highest LC50 (ppb) to fish and species c
Development
DDT
50b
50b
Heptachlor epoxide
10
LB
21 CC
5
5 BG
20 RT
1
1
2 BG
280 CC
Malathion
100
100
Temephos
5
5
Chlorpyrifos
2
62
RS
12900 BB
1 CT
34 FM
Carbaryl
I000b
I000b
690
LT
20000 BB
Methomyl
5000
1000b
530 CC
6800 CT
Propoxur
1000
500b
4800 BG
25000 FM
3 BT
Only 1 sp. tested
Permethrin
5
Ib
Phenothrin
20
10b
Resmethrin
200
50b
41
KF
2 LT, BG
Only 1 sp. tested 17 CC
aAll data for f i s h from Johnson and Finley (1980) except f o r t o x i c i t y of phenothrin, which was from Miyamoto (1976). bLowest concentration bioassayed. CLB = ]argemouth bass (Micropterus salmoides), CC = channel c a t f i s h ( I c t a l u r u s p u n c t a t u s ) , BG = b l u e g i l l (Lepomis macrochirus), RT = rainbow t r o u t (Salmo 9 a i r d n e r i ) , RS = redea-~ sunfish (Lepomis microplus), BB = black bullhead ( I c t a l u r u s melas), CT = cutthroat trout (Salmo clarki), FM = fathead minnow ~-#imephales promelas), LT = lake trout ~Salvelinus namaycush), BT = brook t r o u t (Salvelinus f o n t i n a l i s ) , KF = k i l l i f i s h (Oryzias latipes).
136
10
91
9
8
$
7
7
DDT
6
6
S
5
~-
9 LAB WATER
A.---- -- -,L 50PPB o~-- ----o 100 PPB o---+---.e, 200 PPB
4 3
3 t~ z-g
z
0
i
i
1
'
1
2
3
4
5
6
00
7
m
1
2
$
4
5
6
7
1 z
9
~
"--
s 7
\
s
\
"]:'S '~"
<~
\ \
\\
.EPrACHtOR EPOXIDE
: ~
5 4
~
_- LAB WAI"ER 5 PPB 10 PPB 20 PPB
\ 2
\.
, 0
'x I
i
..L
l
I
t
1
2
3
4
5
6
I 0
0
F i g u r e 1.
1
2
3
4
5
6
7
DAY OF BIOASSAY
DAY OF BiOASSAY
Results of bioassays of organochlorine p e s t i c i d e s u s i n g l a r v a l m o s q u i t o e s (Wyeomyia smithii). Data for heptachlor epoxide previously reported in a different format ( S t r i c k m a n 1984).
137
10
\
\ \
.-.-~
~ , ' ~ \ \
CHLORPYRIFO5
*
\
6
3
~
o.6,,,
~
I PPB 5
\
PPR
\\
\ \,
"-
LAB WATER
.....
/
'
"
.....
/ \
1C"
\ \
\
2
:
\
\ \
1
\
4
\
5
0
7
1
2
3
4
5
6
7
--:----.i,.--.~---~,----i
!\, i
MALATHION =
\
~ ~---.~
|
~ LAB WATER 50 PPB 100 PPB 200 PPB
i
|
=. 1
2
3
4
5
6
7
1
2
3
4
5
6
7
9
\.
TEMEPHO$ 9
~---~ ~ ~----~
1
2
3
4
5
OAY~BIO~Y
Figure 2.
6
7
1
2
3
4
5
6
9
LAB WATER IPPB 5PPIB 10 PPB
7
OAYOFBtOA~AY
Results of bioassays of organophosphate pesticides using larval mosquitoes (Wyeomyia smithii). Data for malathion previously reported in a different format (Strickman 1984).
138
IL.~..,IL CARBARYL .,:
~ l A B WkTER
~ --------~ 10OO PPR o . - , - -----o .T~O0 PPB ~.------,.-~ 10000 PPB
,
~
,
1
2
:%,
1C
3
4
-'-
5
~
6
1
7
" --_:~.__.,,
2
3
4
5
6
7
10
9
- - - ~'',, ,
~
z
O
i'
'i
S
METHOMYL : : LAB WATER b - - - - - - - ~ 1COOPPB o . - - - - - . - o 5000 PPB ----~ 10000 PPB
| z
"~x
1
1
2
3
4
\
5
6
7
1
2
\..
3
/
4
5
6
7
,1",\ -'~'-.~ \ \
\
\
pROPOXUR
,1
E:Z
\
4
: LAB wATER
.~-.
1oo0 ~ 2000 PPB
\
\ 2 l 0
0
i
i
1
2
i
i
i
i
i
3
4
S
6
7
1
DAY OF 81OASSAY
Figure 3.
2
3
4
5
6
7
OAY OF BIOASSAY
Results of bioassays of carbamate pesticides using larval mosquitoes (Wyeomyia smithii).
139
10c
10
9
\ \
\ \
8
7
PERMETHRIN
\
6
9
II LAB WATER - - - ~ | PPB ~---o5 PPR ~-~ 10 PPR
\ '\
5
\ \
i 1
i 2
i 3
i 4
;~ I
i 5
i 6
i 7
0
i._ __-; _..1'
~> 10
0
1
2
3
4
5
6
7
\,~\
9
PHENOTHRiN
z
i |
9 ILAB WATER ~ - - - ~ 1 0 PPB ~ ~ 2 0 PPB ~-~30 PPR
3
! c~
! 0
1
2
3
4
7
1
2
3
4
5
6
7
10
\
i
/ ~ ~ , I
_._~k, \
RESMIETHRIN '\ 9 9 LAB WATER ~----,J 50 PPe ~ ~ 1 0 0 PPB ~-~200 PPB
t
'~
o S'/!i'' I
0
1
Figure 4.
I
I
I
3 4 5 DAY OF IIIOASSkY
I
I
6
7
0
1
2
3
4
S
6
7
DAY OF BIOASSAY
Results of bioassays of pyrethroid pesticides using larval mosquitoes (Wyeomy!a smithii).
140
that the latter group of chemicals is more persistent than the former at neutral pH similar to that of the bioassays. Behavioral signs of intoxication by pesticides included uncoordinated movement, jerky movement, difficulty unflexing, inactivity, and tonic c o n t r a c t i o n of longitudinal muscles resulting in shortening and thickening of larvae. Although all of the toxicants tested caused s o m e or all of t h e s e signs of intoxication, the following distinctions between the chemicals were possible: heptachlor epoxide caused a q u i v e r i n g p a r a l y s i s of larvae, but n e v e r t o n i c contraction of longitudinal muscles; pyrethroids caused a more pronounced inability to unflex than the other chemical groups; and, organophosphates caused tonic contraction of longitudinal muscles more uniformly than the other toxicants. Aquatic bioassay using larval W y e o m y i a smithii was capable of detecting a wide varTety-o--~ pestTc-i~s at levels comparable to the LCso of various fish species. Consideration of practical a ~ l i c a t i o n of the technique s u g g e s t s s e v e r a l t o p i c s for a d d i t i o n a l research. First, the influence of natural water c h e m i s t r y on toxicity would contribute to more accurate detection of toxicants. Second, careful testing of heavy metal salts, solvents, and other c o m m o n contaminants would expand the ability to interpret bioassay results. Finally, systematic evaluation of signs of intoxication could lead to a means of identifying contaminants from responses of the larvae. Acknowledgments. The author appreciates donation of chemicals by American Cyanamid Co., Dupont & Co., Mobay Chemical Corp., Penick Corp., M c L a u g h l i n Gormley King Co., Fairfield American Corp., and t h e U.S. Environmental Protection Agency. The U. S. Air Force provided material support for the study. REFERENCES Anonymous (1983) Farm chemicals handbook. Meister Publishing Co., Willoughby, Ohio. Chapman R, Cole CM (1982) Observations on the influence of water and soil pH on the persistence of insecticides. J Environ Sci Health B17:487-504 Eichelberger 3M, Lichtenberg JJ (1971) Persistence of pesticides in river water. Environ Sci Technol 5:541-544 Henry RA, Schmit JA, Dieckman JF, Murphey FJ (1971) Combined high speed liquid chromatography and bioassay for the evaluation and analysis of an organophosphorus larvicide. Anal Chem 43:1053-1057 141
Johnson WW, Finley MT (1980) Handbook of acute toxicity of chemicals to fish and aquatic invertebrates. U.S. Fish and Wildlife Service, Resource Publication 137 Kottkamp WB, Roberts RH, Meisch MV (1981) Efficacy of three pyrethroids as larvicides against riceland mosquito larvae in field plots. Mosq News 41:382-383 Lillie TH, Campbell JM, Thalken CE, Lang JT (1980) The pitcher plant mosquito, Wyeomyia smithii, a recent introduction to the bioassay laboratory. Trace Subst in Environ Health Symp 14:383-389 Miyamoto J (1976) Degradation, metabolism and toxicity of synthetic pyrethroids. Environ Healtb Perspect 14:15-28 SAS Institute Inc. (1979) SAS I User's Guide, 1979 edition. SAS Institute Inc., Raleigh, North Carolina Sharom MS, Miles JRW, Harris CR, McEwen FL (1980) Persistence of 12 insecticides in water. Water Res 14:1089-1093 Strickman D (1983) An improved method for rearing Wyeomyia smithii. Mosq News 43:499-501 Strickman D (1984) A new aquatic bioassay technique using Wyeomyia smithii, the pitcher-plant mosquito. In: Cardwell RD, Purdy R, Bahner RC (eds) Aquatic toxicology and hazard assessment: Seventh symposium, A S T M STP 854. A m e r i c a n Society for Testing and Materials, Philadelphia, pp 104-116 Received May Ii, 1984; accepted August I0, 1984.
142