Effects of Sublethal Concentrations of Cadmium on Adult Palaemonetes pugio Under Static and Flow-through Conditions W. B. Vernberg, P. J. DeCoursey, M. Kelly, and D. M. Johns Belle W. Baruch Institute for Marine Biology and Coastal Research, Department of Biology, and School of Public Health University of South Carolina Columbia, S.C. 29208
M o r t a l i t y studies of a d u l t organisms over a given period of time have g e n e r a l l y been the only standard bioassay techniques in p o l l u t i o n studies. Consequently, such techniques have played a major r o l e in s e t t i n g standards. I t has also been shown t h a t subl e t h a l , thermal or chemical p o l l u t i o n can modify p h y s i o l o g i c a l and behavioral responses of organisms (DECOURSEY and VERNBERG 1972; HOSS e t a l . 1974; VERNBERG e t a l . 1973). These sublethal e f f e c t s are often more d i f f i c u l t to assess than immediate l e t h a l i t y , but the long-term chronic exposure to sublethal s o l u t i o n s may markedly a l t e r the normal f u n c t i o n i n g o f organisms and thus destroy a popul a t i o n as e f f e c t i v e l y as a s i n g l e l e t h a l dose. Hence, i t i s imperative that techniques also be developed f o r detecting the e f f e c t s of a sublethal concentration of a t o x i c a n t . In measuring the impact of sublethal concentrations of a p o l l u t a n t on organisms, i t is e s s e n t i a l to standardize the bioassay. Factors such as environmental regimes, stage in l i f e h i s t o r y , sex, and body size may e i t h e r s i n g l y or s y n e r g i s t i c a l l y a f f e c t t o x i c i t y (DECOURSEY and VERNBERG 1972 ; VERNBERG and VERNBERG 1972; VERNBERG e t a l . 1973; BOOKHOUT e t a l . 1972; STRUHSAKER e t a l . 1974). Therefore, in measuring the impact of sublethal concentrations of a p o l l u t a n t on organisms, a l l these f a c t o r s must be considered. To e s t a b l i s h a uniform bioassay procedure f o r sublethal concentrations of cadmium in a t y p i c a l estuarine species, a series of studies was carried out with the grass shrimp Palaemonetes pugio, a common euryhaline estuarine species with a wide d i s t r i b u t i o n a l range from Massachusetts to the Gulf Coast. S u r v i v a l , molting time and metabolism were the measurement parameters f o r a d u l t male grass shrimp maintained under d i f f e r e n t t h e r m a l - s a l i n i t y regimes in e i t h e r a s t a t i c or a f l o w through system, using CdCI2 at d i f f e r e n t concentration l e v e l s as the t o x i c a n t . MATERIALS AND METHODS Shrimp were c o l l e c t e d from the North I n l e t Estuary near Georgetown, South Carolina during December, January, and February then transported immediately to the l a b o r a t o r y and housed in p l a s t i c boxes. Males were used in these experiments to e l i m i n a t e possible sex d i f f e r e n c e in responses. Shrimp were acclimated to e i t h e r cold (15~ or warm (25~ c o n d i t i o n s f o r not less than I0 days, nor more than 3 weeks.
16 Bulletin of Environmental Contamination & Toxicology, Vol. 17, No. 1 9 1977 by Springer-Verlag New York Inc.
For the s t a t i c series, shrimp were housed individually in compartmentalized plastic boxes at either 15~ or 25~ in 5, I0, 15 and 30 O/oo S on a 12L-12D l i g h t schedule. The water was changed every other day and the shrimp were fed newly-hatched Artem~ nauplii at that time. The i n i t i a l 50 ppb concentration of Cd was replenished with each water change. Flow-through chambers were designed and b u i l t capable of accommodating twenty animals for either control or experimental situation (Fig. I ) . CADMIUM ~RESKNVOtR
~ , --"
~
EXPE RIMENTAL j~HAMBER
/
,o.
E•
couoMN
--
"~~~
CHAHBER
Figure i. Flow-through System
The feeding regime paralleled that in the s t a t i c system. Concentrated CdCI2 (2.2 x I0 -~ M) was dispensed from the cadmium reservoir by a Buchler polystatic pump and maintained the cadmium concentration in the flow-through vessels at approximately 50 ppb through the experiments. Temperatures were held at either 25~ or 15~ in s a l i n i t i e s of 5, 15 and 30 o/oo S for 7 days. Oxygen uptake rates of 10-18 shrimp (see Table 1 for group size) in s t a t i c and flow-through systems were measured individually in a Gilson Respirometer after 7 days exposure to each experimental condition: 25o or 15~ acclimated at 5, 15 and 30 o/oo S. Subsequently the animals were rinsed with d i s t i l l e d water, then dried to a constant weight at 80oc. Results are expressed as ~I 02/hr/gm dry weight. Since a r e l a t i v e l y wide size range of shrimp were used (0.007-0.07) respiration data were analyzed using linear regression techniques, and respiration rates were calculated from the linear regression curves for medium-sized (0.032 gm) shrimp.
i?
TABLE 1
Oxygen consumption rates of control and cadmlum-exposed Palaemoneces pu$io from static and flow-through system, after warm or cold acclimation. Rates were determiRed after 7 days in test conditions. The data were calculated from the linear regression curves for medlum-slzed (0.032 gm) shrimp. Significant differences between static and flow-through linear regressions are noted: a) i0% level, b) 2.5% level.
warm-acclimated (25aC)
COI~ROL Static u l s O2/hT/
Salinity
~
#Det.
gm dry wE.
#Det.
5 15 30
12 17 7
2282 2446 1279
9 18 20
5 15 30
16 9 7
1409 818 1504
5 15 30a
14 16 8
1850 2269 1243
5b 15b 30
17 9 16
644 1493 1089
Flow-thro~ ~ l s O2/hr/ gmdry wt. 2674 2448 1829
cold-accllmated (15"C)
11 16 10
1078 1268 1192
warm-acclimated (25~
CADMIUM-EXPOSED
9 22 24
3058 2404 1916
cold-accllmated (15"C) 5 13 15
1178 1408 1147
Significant differences were calculated between l i n e a r regressions of control and experimental regimes as well as between s t a t i c and flow-through regimes. Survival of shrimp maintained in the s t a t i c system at 25~ was monitored under s a l i n i t i e s of 5, I 0 , 20 and 30 ~ S using 20 freshly-caught shrimp for each s a l i n i t y . Molting frequency for these shrimp was recorded, then analyzed with the Student's "t-test". Cadmium concentration in shrimp tissues was analyzed using the dithizone method (STANDARD METHODS 1971). Three or four shrimp were used for each determination, and results expressed as ppm dry weight. Under static conditions, tissue concentration was measured a f t e r 3, 7, 14 and 21 days exposure at 25~ in 5, I0, 15, 20 and 30 ~ S, and a f t e r 7 days exposure in 15~ at 5, 15 and 30 ~ S. In the flow-through system, cadmium levels were determined a f t e r 7 days at 15~ or 25~ in 5, 15 and 30 ~ S. Linear regression analysis was used to present the data graphically.
18
RESULTS AND DISCUSSION M o r t a l i t y f o r shrimp under control c o n d i t i o n s in the s t a t i c system was very low in a l l s a l i n i t i e s . The increased m o r t a l i t y of the cadmium-exposed shrimp was r e l a t e d to the s a l i n i t y regime; as s a l i n i t i e s decreased, m o r t a l i t y increased. The l e v e l o f Cd used in these experiments (50 ppb) i s higher than t h a t found in many marine waters. However, the level of cadmium reported from p o l l u t e d estuaries have ranged as high as 78 ppm in Gulf Coast waters (HOLMESet al. 1974) and up to 9 ppb in the Bristol Channel in England (ABDULLAH 1974). P. pugio is able to tolerate relat i v e l y high levels of Cd (Fig. 2). Even at concentration levels of approximately 23 ppm, mortality rates were only I0%. Not all marine species are so resistant to Cd. Survival of Uca pugilator larvae, for example, was shortened upon exposure to a Cd concentration of l ppb (VERNBERG et al. 1974). B 5%,S. ond 50ppb Cd ~;~.10 %, S. I 20%,S. i 30 ."., S. D CONTROLS {all ~oIinitiol pooled)
MORTALtTY 25~ Static 50
>.. I-- 2o ._1 0
Io
m
3
14
7
21
DAYS CADMIUM 25~
UPTAKE Static
"~ 3C
(5 Z
20
C]I I0
b 3
14
7 DAYS
Figure 2. Palaemonetes pugio
19
h 21
Tissue accumulation of cadmium under both static and flowthrough conditions was proportional to salinity at 25~ (Fig. 3). Highest cadmium concentrations for the static system occurred at 5 and 10 ~ S; for example, after 21 days the experimental shrimp in 5 ~ S contained approximately 35 ppm, while those at 10 ~ contained only 20 ppm. Cadmiumuptake rates in the static system differed from the flow-through system in some respects. Uptake levels were greater in shrimp from the flow-through system at 25~ after 7 days, although differences in the slopes indicating uptake levels are not s t a t i s t i c a l l ~ significant. At 15~ less cadmium is concentrated than at 25uc in both static and flowthrough systems, but shrimp in the flow-through system accumulated much more cadmium than those maintained under static conditions (Fig. 4), and the slopes indicating accumulation at this temperature were significantly different in the 2 systems. Uptake levels STATIC 2O
18
!
o
16
,,J
o,9
jo
--.....,~o,
FLOW- THROUGH
22 20
~
tu
1~
,,~
101
u
~
| x,
5
10
15
20
25
30
5
x x
|0
SALINITY (ppt)
Figure 3.
20
25
30
7-day cadmium uptake levels vs s a l i n i t y at 25~ STATIC
22
FLOW- THROUGH
2: 2C
T6
w
w
~
I5
I.~4
SALINITY ( p p t )
20
~ -J
Y, -o.o~sex.
12
~: ~o
10
Ig a o
y , o.ossX § ~.s
o
o
~0(2 $
y,
10
15 SALINITY
Figure 4. 15uc.
O. O Z 3 X
M o r t a l i t y studies of a d u l t organisms over a given period of time have g e n e r a l l y been the only standard bioassay techniques in p o l l u t i o n studies. Consequently, such techniques have played a major r o l e in s e t t i n g standards. I t has also been shown t h a t subl e t h a l , thermal or chemical p o l l u t i o n can modify p h y s i o l o g i c a l and behavioral responses of organisms (DECOURSEY and VERNBERG 1972; HOSS e t a l . 1974; VERNBERG e t a l . 1973). These sublethal e f f e c t s are often more d i f f i c u l t to assess than immediate l e t h a l i t y , but the long-term chronic exposure to sublethal s o l u t i o n s may markedly a l t e r the normal f u n c t i o n i n g o f organisms and thus destroy a popul a t i o n as e f f e c t i v e l y as a s i n g l e l e t h a l dose. Hence, i t i s imperative that techniques also be developed f o r detecting the e f f e c t s of a sublethal concentration of a t o x i c a n t . In measuring the impact of sublethal concentrations of a p o l l u t a n t on organisms, i t is e s s e n t i a l to standardize the bioassay. Factors such as environmental regimes, stage in l i f e h i s t o r y , sex, and body size may e i t h e r s i n g l y or s y n e r g i s t i c a l l y a f f e c t t o x i c i t y (DECOURSEY and VERNBERG 1972 ; VERNBERG and VERNBERG 1972; VERNBERG e t a l . 1973; BOOKHOUT e t a l . 1972; STRUHSAKER e t a l . 1974). Therefore, in measuring the impact of sublethal concentrations of a p o l l u t a n t on organisms, a l l these f a c t o r s must be considered. To e s t a b l i s h a uniform bioassay procedure f o r sublethal concentrations of cadmium in a t y p i c a l estuarine species, a series of studies was carried out with the grass shrimp Palaemonetes pugio, a common euryhaline estuarine species with a wide d i s t r i b u t i o n a l range from Massachusetts to the Gulf Coast. S u r v i v a l , molting time and metabolism were the measurement parameters f o r a d u l t male grass shrimp maintained under d i f f e r e n t t h e r m a l - s a l i n i t y regimes in e i t h e r a s t a t i c or a f l o w through system, using CdCI2 at d i f f e r e n t concentration l e v e l s as the t o x i c a n t . MATERIALS AND METHODS Shrimp were c o l l e c t e d from the North I n l e t Estuary near Georgetown, South Carolina during December, January, and February then transported immediately to the l a b o r a t o r y and housed in p l a s t i c boxes. Males were used in these experiments to e l i m i n a t e possible sex d i f f e r e n c e in responses. Shrimp were acclimated to e i t h e r cold (15~ or warm (25~ c o n d i t i o n s f o r not less than I0 days, nor more than 3 weeks.
16 Bulletin of Environmental Contamination & Toxicology, Vol. 17, No. 1 9 1977 by Springer-Verlag New York Inc.
For the s t a t i c series, shrimp were housed individually in compartmentalized plastic boxes at either 15~ or 25~ in 5, I0, 15 and 30 O/oo S on a 12L-12D l i g h t schedule. The water was changed every other day and the shrimp were fed newly-hatched Artem~ nauplii at that time. The i n i t i a l 50 ppb concentration of Cd was replenished with each water change. Flow-through chambers were designed and b u i l t capable of accommodating twenty animals for either control or experimental situation (Fig. I ) . CADMIUM ~RESKNVOtR
~ , --"
~
EXPE RIMENTAL j~HAMBER
/
,o.
E•
couoMN
--
"~~~
CHAHBER
Figure i. Flow-through System
The feeding regime paralleled that in the s t a t i c system. Concentrated CdCI2 (2.2 x I0 -~ M) was dispensed from the cadmium reservoir by a Buchler polystatic pump and maintained the cadmium concentration in the flow-through vessels at approximately 50 ppb through the experiments. Temperatures were held at either 25~ or 15~ in s a l i n i t i e s of 5, 15 and 30 o/oo S for 7 days. Oxygen uptake rates of 10-18 shrimp (see Table 1 for group size) in s t a t i c and flow-through systems were measured individually in a Gilson Respirometer after 7 days exposure to each experimental condition: 25o or 15~ acclimated at 5, 15 and 30 o/oo S. Subsequently the animals were rinsed with d i s t i l l e d water, then dried to a constant weight at 80oc. Results are expressed as ~I 02/hr/gm dry weight. Since a r e l a t i v e l y wide size range of shrimp were used (0.007-0.07) respiration data were analyzed using linear regression techniques, and respiration rates were calculated from the linear regression curves for medium-sized (0.032 gm) shrimp.
i?
TABLE 1
Oxygen consumption rates of control and cadmlum-exposed Palaemoneces pu$io from static and flow-through system, after warm or cold acclimation. Rates were determiRed after 7 days in test conditions. The data were calculated from the linear regression curves for medlum-slzed (0.032 gm) shrimp. Significant differences between static and flow-through linear regressions are noted: a) i0% level, b) 2.5% level.
warm-acclimated (25aC)
COI~ROL Static u l s O2/hT/
Salinity
~
#Det.
gm dry wE.
#Det.
5 15 30
12 17 7
2282 2446 1279
9 18 20
5 15 30
16 9 7
1409 818 1504
5 15 30a
14 16 8
1850 2269 1243
5b 15b 30
17 9 16
644 1493 1089
Flow-thro~ ~ l s O2/hr/ gmdry wt. 2674 2448 1829
cold-accllmated (15"C)
11 16 10
1078 1268 1192
warm-acclimated (25~
CADMIUM-EXPOSED
9 22 24
3058 2404 1916
cold-accllmated (15"C) 5 13 15
1178 1408 1147
Significant differences were calculated between l i n e a r regressions of control and experimental regimes as well as between s t a t i c and flow-through regimes. Survival of shrimp maintained in the s t a t i c system at 25~ was monitored under s a l i n i t i e s of 5, I 0 , 20 and 30 ~ S using 20 freshly-caught shrimp for each s a l i n i t y . Molting frequency for these shrimp was recorded, then analyzed with the Student's "t-test". Cadmium concentration in shrimp tissues was analyzed using the dithizone method (STANDARD METHODS 1971). Three or four shrimp were used for each determination, and results expressed as ppm dry weight. Under static conditions, tissue concentration was measured a f t e r 3, 7, 14 and 21 days exposure at 25~ in 5, I0, 15, 20 and 30 ~ S, and a f t e r 7 days exposure in 15~ at 5, 15 and 30 ~ S. In the flow-through system, cadmium levels were determined a f t e r 7 days at 15~ or 25~ in 5, 15 and 30 ~ S. Linear regression analysis was used to present the data graphically.
18
RESULTS AND DISCUSSION M o r t a l i t y f o r shrimp under control c o n d i t i o n s in the s t a t i c system was very low in a l l s a l i n i t i e s . The increased m o r t a l i t y of the cadmium-exposed shrimp was r e l a t e d to the s a l i n i t y regime; as s a l i n i t i e s decreased, m o r t a l i t y increased. The l e v e l o f Cd used in these experiments (50 ppb) i s higher than t h a t found in many marine waters. However, the level of cadmium reported from p o l l u t e d estuaries have ranged as high as 78 ppm in Gulf Coast waters (HOLMESet al. 1974) and up to 9 ppb in the Bristol Channel in England (ABDULLAH 1974). P. pugio is able to tolerate relat i v e l y high levels of Cd (Fig. 2). Even at concentration levels of approximately 23 ppm, mortality rates were only I0%. Not all marine species are so resistant to Cd. Survival of Uca pugilator larvae, for example, was shortened upon exposure to a Cd concentration of l ppb (VERNBERG et al. 1974). B 5%,S. ond 50ppb Cd ~;~.10 %, S. I 20%,S. i 30 ."., S. D CONTROLS {all ~oIinitiol pooled)
MORTALtTY 25~ Static 50
>.. I-- 2o ._1 0
Io
m
3
14
7
21
DAYS CADMIUM 25~
UPTAKE Static
"~ 3C
(5 Z
20
C]I I0
b 3
14
7 DAYS
Figure 2. Palaemonetes pugio
19
h 21
Tissue accumulation of cadmium under both static and flowthrough conditions was proportional to salinity at 25~ (Fig. 3). Highest cadmium concentrations for the static system occurred at 5 and 10 ~ S; for example, after 21 days the experimental shrimp in 5 ~ S contained approximately 35 ppm, while those at 10 ~ contained only 20 ppm. Cadmiumuptake rates in the static system differed from the flow-through system in some respects. Uptake levels were greater in shrimp from the flow-through system at 25~ after 7 days, although differences in the slopes indicating uptake levels are not s t a t i s t i c a l l ~ significant. At 15~ less cadmium is concentrated than at 25uc in both static and flowthrough systems, but shrimp in the flow-through system accumulated much more cadmium than those maintained under static conditions (Fig. 4), and the slopes indicating accumulation at this temperature were significantly different in the 2 systems. Uptake levels STATIC 2O
18
!
o
16
,,J
o,9
jo
--.....,~o,
FLOW- THROUGH
22 20
~
tu
1~
,,~
101
u
~
| x,
5
10
15
20
25
30
5
x x
|0
SALINITY (ppt)
Figure 3.
20
25
30
7-day cadmium uptake levels vs s a l i n i t y at 25~ STATIC
22
FLOW- THROUGH
2: 2C
T6
w
w
~
I5
I.~4
SALINITY ( p p t )
20
~ -J
Y, -o.o~sex.
12
~: ~o
10
Ig a o
y , o.ossX § ~.s
o
o
~0(2 $
y,
10
15 SALINITY
Figure 4. 15uc.
O. O Z 3 X
