SEASONAL EFFECTS OF LEACHED MIREX ON SELECTED ESTUARINE ANIMALS M. E. TAGATZ, P. W. BORTHWICK and J. FORESTER Environmental Protection Agency Gulf Breeze Environmental Research Laboratory Gulf Breeze, Fla. 32561
Four 28-day seasonal experiments were conducted using selected estuarine animals in outdoor tanks that received continuous flow of mirex-laden water. Mirex (dodecachlorooctahydro-l,3,4-metheno-2H-cyclobuta [cd] pentalene) leached from fire ant bait (0.3% mirex) by fresh water and then mixed with salt water was toxic to blue crabs (Callinectes sapidus), pink shrimp (Penaeus duorarum), and grass shrimp (Palaemonetes pugio) but not to sheepshead minnows (Cyprinodo~ variegatus), at concentrations less than 0.53 /zg/L in water. The amount of leachin~ was greatest in summer and least in spring. Greatest mortality occurred in summer at the highest water temperature and concentration of mirex; least mortality occurred in spring at next to the lowest temperature and at the lowest concentration. Earliest deaths of blue crabs occurred after six days of exposure and shrimps after two days. Small juvenile crabs were more sensitive to leached mirex than were large juveniles. Mirex did not appear to affect growth or frequency of molting in crabs. All exposed animals concentrated mirex. Among animals that survived for 28 days, sheepshead minnows concentrated mirex 40,800X above the concentration in the water, blue crabs 2,300X, pink shrimp 10,000X, and grass shrimp 10,800X. Sand substrata contained mirex up to 770X that in the water. Most control and exposed animals in samples examined histologically had normal tissues, but alteration in gills of some exposed fish and natural pathogens in some exposed and control crabs and shrimp were observed. The experiments demonstrated that mirex can be leached from bait by fresh water, concentrated by estuarine organisms, and can be toxic .to crabs and shrimps. This study was conducted to determine the seasonal effects, on various estuarine organisms, of mirex 1 leached from mirex fire and bait (84.7% corn cob grits, 15.0% goybean oil, and 0.3% mirex) by fresh water. Mirex is a chlorinated hydrocarbon insecticide applied in bait form (1.4 kg per ha) to control the imported fire ant, Solenopsis richteri Forel, in the southeastern United States. Field studies have shown translocation of mirex from treated land and high marsh areas to estuarine biota (Borthwick et al. 1973). Possible routes of entry into the estuarine environment include, but may not be limited to, biological transport, tidal action, or fresh water runoff containing .the bait or mirex leached from bait. Low concentrations of mirex (ng/L or ppt range) have been detected in natural waters. In three cases during periods of heavy runoff, a residue of 0.03 ~g/L (part 1Dodecachlorooctahydro-l,3,4,-metheno-2H cyclobuta [cd] pentalene. Archives of Environmental Contamination and ToxicologyVol. 3,371-383 (1975) 9 1975 by Springer-Verlag New York Inc.
371
372
M.E. Tagatz et al.
per billion) was found in samples of water from various streams of Mississippi after application of mirex bait in the watershed (Alley, personal communication2). In almost all studies on the effects of mirex on non-target aquatic organisms, experimental animals were exposed directly to the bait or to the technical compound dissolved in a water-miscible solvent (Butler 1963, Muncy and Oliver 1963, Van Valin et al. 1968, McKenzie 1970, Lowe et al. 1971, Ludke et al. 1971, Bookhout et al. 1972, Cooley et al. 1972, Collins et al. 1973, Redmann 1973). However, the study by Ludke et al. (1971) included exposing crayfish in small aquaria to mirex leached from bait enclosed in filter paper and screen wire. Our study considers the possibility that in field applications of mirex, estuarine organisms may not come into direct contact with the bait, but could be exposed to mirex leached from the bait and carried from treated areas by rainwater runoff into estuarine drainage systems. Because solvents (other than the constituent soybean oil) are not used during application of mirex bait in the field, they were not used in the experimental design of the present work.
Materials and methods Four 28-day replicate experiments were conducted, using caged animals in outdoor tanks that received a continuous flow of mirex-laden water from gravity-flow columns that contained mirex ant bait. Our experimental design (mixing of mirexladen tap water from the columns with salt water) was chosen to simulate conditions in an estuary, where fresh water runoff from the watershed may contain mirex leached from bait and the mirex could be introduced into the estuary when fresh water mixes with salt water. The experiments were conducted seasonally: spring (April 25 - May 23, 1973), summer (July 10 - August 7, 1973), fall (October 6 - November 13, 1973), and winter (January 15 - February 12, 1974). Six 2.4-m diameter fiberglass tanks and six 10-cm diameter glass columns were used (Figure 1). Three treated tanks received filtered (cartridges of one-/zpore size) tap water that had trickle-filtered through columns of mirex bait before being mixed with unfiltered sea water. Similarly, three control tanks received water from columns containing all components of the bait except mirex. Tap water contained no detectable chlorine (< 0.1 rag/L). Tap water (0.5 L/min) and natural seawater (I .0 L/min) siphoned from constant-head boxes were mixed in glass troughs, positioned below the columns, before entering the tanks. The water level in each tank was maintained at 30.5 cm by a standpipe opposite the site where water entered from the mixing trough. The tank area was partially enclosed by a fiberglass roof and back wall for protection from severe weather. Each column consisted of an outer cylinder containing three 15-cm-high and 9-cm-diameter cylinder inserts (Figure 2). Each insert contained a 50-g layer of bait 2E. G. Alley, Mississippi State Chemical Laboratory, State College, Miss. 39762.
Seasonal Effects of Mirex
373
(total of 150 g per column) which was soaked for 24 hr in fresh water to allow swelling before being placed in the columns. The bottoms of the inserts were covered by nylon screen (0.84 mm mesh) to retain the bait. A funnel attached to the bottom of the column concentrated the flow of tap water leaving the column. Because exploratory tests indicated relatively high concentrations (> 2 ~g/L) of mirex in column runoff during the first few days, we passed tap water through the columns (0.5 L/rain.) for 4 to 8 days (spring, 8 days; summer, 4 days; fall and winter, 6 days) before the tanks were filled. Tanks were filled a day prior to the start of animal exposure. The amount of bait used in the column and the water flow were chosen because they produced low but detectable residues of mirex in tank water (desired range, 0.01-0.50 ~g/L). Temperatures (12:00 Noon, mercury thermometer) and salinities (8:00 AM, temperature-compensated refractometer) of the tank water were measured at the start of each test and four times weekly thereafter. Temperature and salinity changes were gradual. Temperature responded to natural changes in air and sea water temperatures; salinity, to natural fluctuations in salinity of the sea water source (Santa Rosa Sound, Florida).
Fig. 1. Experimental system showing outdoor tanks and gravity-flowcolumns.
374
M.E. Tagatz et al.
Caged animals were placed on two cm of beach sand covering the bottom of the tanks. Each tank held four cages that contained, respectively, 25 adult sheepshead minnows, Cyprinodon variegatus; 14 juvenile blue crabs, Callinectes sapidus; 25 juvenile pink shrimp, Penaeus duorarum; and 50 (35 in the fall experiment) adult grass shrimp, Palaemonetes pugio. Fish ranged from 32 to 59 mm total length; crabs, 21 to 75 mm carapace width; pink shrimp, 42 to 92 mm rostrum-telson length; and grass shrimp, 22 to 32 mm rostrum-telson in the spring study and 33 to 36 mm in the summer, fall and winter studies. All animals were captured in local waters and acclimated for 3 to 16 hr to the initial salinity and temperature of each experiment by gradual addition of tap water to the salt water stock-aquaria. Cages for crabs (115.5 cm x 33.0 cm x 23.0 cm deep) were made of stainlesssteel screening (7.1 mm mesh) over wooden frames, and cages for fish and shrimps (76.0 cm • 76.0 cm x 30.5 cm deep) were made of nylon screening (3.3 mm mesh) over wooden frames. To prevent cannibalism, crabs were confined in indi-
Freshwater siphon (301/hr)
)
Constant-hea( (Filtered fres!
Constant-head I (Unfiltered seat
c bait)
Flow into tank (901/hr)
Fig. 2. Gravity-flow column
Seasonal Effects of Mirex
375
vidual compartments (16.5 cm x 16.5 cm x 23.0 cm deep). Pink shrimp were provided 7.5 cm of sand on the cage bottom for burrowing. Crabs were fed small fish, and pink shrimp were fed cubes of fish meat, weekly. Fish and grass shrimp were not given food but could consume plankton. To determine growth of crabs, carapace width (in mm between the tips of the lateral spines) was measured at the beginning, middle and end of the experiment. Percentage survival of animals was determined at the end of the experiment. The chi-square test was used to determine significant differences in numbers of dead and living animals between treated and control tanks. Samples of water, sand, or animals were analyzed by electron-capture gas chromatography to determine mirex concentrations. Sensitivity was 0.01/zg/g (ppm) for whole animals (wet-weight basis) and for sediment samples (air-dried weight basis), and 0.01 /zg/L (ppb) for water samples. Samples to which known amounts of mirex were added gave recovery rates greater than 85%, but concentrations were not corrected for percentage recovery. Techniques for most residue analyses were those of Lowe et al. (1971) and for tissue samples that weighed less than five g, those of Hansen et al. (in press). Samples of water from each tank were obtained at the start of the experiment (when the animals were placed in the tank) and twice a week thereafter. A water sample consisted of a composite collection from two sites in each tank. At 14 and 28 days, a composite sample of sand from four sites was obtained from each tank. Surviving animals (composite samples) were analyzed for mirex at the end of the experiment, and dead animals (individual or composite samples) were analyzed as deaths occurred. No residues of mirex were detected in pretest samples of sand and animals or in fish used as food. Samples of sheepshead minnows, blue crabs, or pink shrimp surviving at the end of the experiments, and samples of dead pink shrimp, were taken for histological examination. Surviving pink shrimp were sampled in all experiments; fish and crab, in all experiments except winter. Samples of survivors consisted of 10 or 15 individuals of a species from treated tanks and 10 or 15 individuals from control tanks. Samples of dead pink shrimp consisted of seven treated shrimp in fall and nine treated and three control shrimp in winter.
Results and discussion Measured concentrations of mirex (~g/L) in the three treated tanks averaged approximately 0.04 in spring, 0.12 in summer, 0.06 in fall, and 0.09 in winter (Table I). Analysis of variance showed" no significant differences ( o : = 0.05) among the three treated tanks in each test. Also, mirex residues did not increase or decrease statistically ( o: = 0.05) with time within individual tanks. Seasonal temperatures and salinities of tank water and average residues of mirex are summarized in Table II. Temperatures averaged 23.1~ in spring, 29.8~ in
0.07 0.03
0.03 0.09
0.03
Week 3
Week 4
Average
0.04
0.04 0.11
0.07 0.06
0.03 < 0.01 a
0.02 0.03
0.03
0.11
0.12
0.01-
0.04
0.03 0.08
0.05 0.02
0.04 0.01
0.12 0.02
0.02
a0.005 used for calculating average.
0.09
< 0.01-< 0.01-
0.02 0.01
Week 2
Range
0.02 0.02
< 0.01 a
Week 1
Start
Spring
Tank 1 Tank 2 Tank 3
Time
elapsed
.0.36
0.03"
0.09
0.05 0.09
0.03 0.05
0.04 0.04
0.14 0.05
0.36
0.23
0.04-
0.10
0.10 0.23
0.09 0.10
0.04 0.08
0.08 0.04
0.17
0.52
0.05-
0.16
0.11 0.11
0.10 0.19
0.05 0.07
0.20 0.06
0.52
Tank 1 Tank 2 Tank 3
Summer
0.20
0.03-
0.07
0.04 0.05
0.05 0.04
0.03 0.04
0.14 0.07
0.20
0.23
0.03-
0.06
0.03 0.04
0.03 0.04
0.03 0.03
0.09 0.04
0.23
0.12
0.01-
0.04
0.02 0.02
0.02 0.02
0.01 0.02
0.07 0.04
0.12
Tank 1 Tank 2 Tank 3
Fall
0.25
0.03-
0.08
0.04 0.05
0.05 0.03
0.03 0.05
0.11 0.07'
0.25
0.24
0.02-
0.07
0.05 0.04
0.04 0.03
0.03 0.02
0.09 0.05
0.24
0.52
0.03-
0.11
0.03 0.04
0.05 0.03
0.04 0.08
0.12 0.04
0.52
Tank 1 Tank 2 Tank 3
Winter
Table I. Concentrations of Mirex (gg/L ) in Treated Tank Water During Four 28-Day Seasonal Experiment~
S
0~
t-t1 ,-q
Seasonal Effects of Mirex summer, 23.4~ in fall, and 19. I~ 19 parts per thousand.
377
in winter. Overall salinity range was from 7 to
In mirex-contaminated tanks, survival of crabs and shrimps usually was significantly reduced; survival of fish was not affected (Table III). The number of deaths was greatest in the summer, followed by fall, winter and spring: Observed deaths among exposed shrimps occurred after 2 to 15 days in summer (all pink shrimp died in 15 days), after 8 to 28 days in fall, and after 17 to 28 days in winter. During summer and fall, significantly more exposed blue crabs died than did control crabs, deaths occurring after 6 to 28 days of exposure. At the end of the summer experiment, one-third of the crabs surviving in treated tanks were paralyzed or had lost equilibrium. Most deaths occurred among the smaller crabs. Of 12 deaths among 42 crabs exposed in summer, I I were among 19 crabs 28 to 40 mm carapace width, but only one died among 23 crabs 41 to 64 mm. Survival of sheepshead minnows was unaffected in all experiments. This species can spawn in the presence of mirex since each tank contained 50 to 100 young sheepshead minnows (up to 14 mm total length) at the end of the spring studies and 75 to 200 young (up to 26 mm) at the end of the summer studies. Lowe et al. (1971) also found that fish were relatively unaffected, juvenile pinfish (Lagodon rhomboides) living for five months on a diet that contained approximately 20 /zg/g of technical mirex. Mortality and delayed toxicity to crabs and shrimps exposed to mirex (technical grade) occurred in other studies. Lowe et al. (1971) found that a significant number of juvenile Penaeus duorarum died during a seven-day exposure to 1.0 /zg/L of mirex in sea water averaging 17~ but few died during a 21-day exposure to 0.1 /~g/L in sea water averaging 14~ All juvenile Callinectes sapidus died within three weeks after a 96-hr exposure to 0.1 mg/L (Lowe et al. 1970). Redmann (1973) reported a 40% mortality ofPalaemonetes pugio in 12 days from a 48-hr exposure to 0.01 ~g/g of mirex in sea water at 20~ McKenzie (1970) and Lowe et al. (1971) demonstrated that smaller blue crabs were more sensitive to mirex bait than were larger crabs. Table II. Temperature, Salinity and Average Concentration of Mirex in Tank Water During Four 28-Day Seasonal Experiments
Temperature(~
Salinity (Parts per thousand) Av. Range
Mirex /~g/L
Av.
Range
Spring
23.1
19.3-25.2
13.2
10-18
0.04
Summer
29.8
28.0-30.8
15.7
14-18
0.12
Fall
23.4
17.0-27.0
17.5
15-19
0.06
Winter
19.1
13.8-22.9
13.6
7-16
0.09
M . E . Tagatz et al.
378
The greater mortality observed in summer could be caused by increased leaching and by increased toxicity of mirex in warmer water. Analysis of variance showed significant differences ( cc = 0.05) in mirex residues in water between summer and spring and between summer and fall; other paired treatments were not significant. Most deaths of affected animals occurred in summer at the highest water temperature and concentration of mirex; fewest in spring at next to the lowest temperature and at the lowest concentration. McKenzie (1970) reported that survival time and rate in juvenile blue crabs exposed to mirex bait decreased as temperatures increased from 20 to 27~ Mirex had no marked affect on growth of juvenile blue crabs exposed in these studies (Table IV). The greatest difference in mean percentage increase in size per
Table III. Percentage Survival of Animals Exposed to Mirex and Chi-Square Values After Each of Four 28-Day Seasonal Experiments. Data on Treated Tanks and on Control Tanks were Combined Percentage survival and ehi-square Animals
Number
Spring
Summer
Fall
Winter
Sheepshead minnows Control
75
63
96
91
99
Treated
75
55
99
92
100
N.S. a
N.S.
N.S.
N.S.
100
98
95
95
81
95
Chi-square
Blue crabs Control
42
Treated
42
Chi-square
98
71
N.S.
11.01 **
4.09*
N.S.
Pink shrimp Control
75
86
87
91
91
Treated
75
76
0
19
49
78.44**
30.51 **
96
97
59
89
Chi-square
N.S.
114.70"*
Grass shrimp Control
150b
95
Treated
150b
86
Chi-square
-c
79 10 145.87"*
41.69"*
6.20*
aN.S. = non-significant, ** significant at I% level (X 2, 1 d.f. = 6.63), * significant at 5% level (X2, 1 d.f. = 3.84). b 105 used in the fall experiment. eSome small grass shrimp escaped through the mesh of the cages, and all those free in the tanks may not have been captured (values in table include those captured).
Seasonal Effects of Mirex
379
molt between treated and control groups was less than 3%; not decisive in a species normally characterized by variable growth among individuals (Tagatz 1968). Taking into account deaths that occurred among treated crabs, frequency of molting did not differ greatly between treated and control groups. Animals exposed to leached mirex concentrated the chemical in body tissues (Table V). During 28 days' exposure, depending on the experiment, surviving sheepshead minnows accumulated from 10,500 to 40,800X the average concentration of mirex in the test water. Residues in young fish (
Four 28-day seasonal experiments were conducted using selected estuarine animals in outdoor tanks that received continuous flow of mirex-laden water. Mirex (dodecachlorooctahydro-l,3,4-metheno-2H-cyclobuta [cd] pentalene) leached from fire ant bait (0.3% mirex) by fresh water and then mixed with salt water was toxic to blue crabs (Callinectes sapidus), pink shrimp (Penaeus duorarum), and grass shrimp (Palaemonetes pugio) but not to sheepshead minnows (Cyprinodo~ variegatus), at concentrations less than 0.53 /zg/L in water. The amount of leachin~ was greatest in summer and least in spring. Greatest mortality occurred in summer at the highest water temperature and concentration of mirex; least mortality occurred in spring at next to the lowest temperature and at the lowest concentration. Earliest deaths of blue crabs occurred after six days of exposure and shrimps after two days. Small juvenile crabs were more sensitive to leached mirex than were large juveniles. Mirex did not appear to affect growth or frequency of molting in crabs. All exposed animals concentrated mirex. Among animals that survived for 28 days, sheepshead minnows concentrated mirex 40,800X above the concentration in the water, blue crabs 2,300X, pink shrimp 10,000X, and grass shrimp 10,800X. Sand substrata contained mirex up to 770X that in the water. Most control and exposed animals in samples examined histologically had normal tissues, but alteration in gills of some exposed fish and natural pathogens in some exposed and control crabs and shrimp were observed. The experiments demonstrated that mirex can be leached from bait by fresh water, concentrated by estuarine organisms, and can be toxic .to crabs and shrimps. This study was conducted to determine the seasonal effects, on various estuarine organisms, of mirex 1 leached from mirex fire and bait (84.7% corn cob grits, 15.0% goybean oil, and 0.3% mirex) by fresh water. Mirex is a chlorinated hydrocarbon insecticide applied in bait form (1.4 kg per ha) to control the imported fire ant, Solenopsis richteri Forel, in the southeastern United States. Field studies have shown translocation of mirex from treated land and high marsh areas to estuarine biota (Borthwick et al. 1973). Possible routes of entry into the estuarine environment include, but may not be limited to, biological transport, tidal action, or fresh water runoff containing .the bait or mirex leached from bait. Low concentrations of mirex (ng/L or ppt range) have been detected in natural waters. In three cases during periods of heavy runoff, a residue of 0.03 ~g/L (part 1Dodecachlorooctahydro-l,3,4,-metheno-2H cyclobuta [cd] pentalene. Archives of Environmental Contamination and ToxicologyVol. 3,371-383 (1975) 9 1975 by Springer-Verlag New York Inc.
371
372
M.E. Tagatz et al.
per billion) was found in samples of water from various streams of Mississippi after application of mirex bait in the watershed (Alley, personal communication2). In almost all studies on the effects of mirex on non-target aquatic organisms, experimental animals were exposed directly to the bait or to the technical compound dissolved in a water-miscible solvent (Butler 1963, Muncy and Oliver 1963, Van Valin et al. 1968, McKenzie 1970, Lowe et al. 1971, Ludke et al. 1971, Bookhout et al. 1972, Cooley et al. 1972, Collins et al. 1973, Redmann 1973). However, the study by Ludke et al. (1971) included exposing crayfish in small aquaria to mirex leached from bait enclosed in filter paper and screen wire. Our study considers the possibility that in field applications of mirex, estuarine organisms may not come into direct contact with the bait, but could be exposed to mirex leached from the bait and carried from treated areas by rainwater runoff into estuarine drainage systems. Because solvents (other than the constituent soybean oil) are not used during application of mirex bait in the field, they were not used in the experimental design of the present work.
Materials and methods Four 28-day replicate experiments were conducted, using caged animals in outdoor tanks that received a continuous flow of mirex-laden water from gravity-flow columns that contained mirex ant bait. Our experimental design (mixing of mirexladen tap water from the columns with salt water) was chosen to simulate conditions in an estuary, where fresh water runoff from the watershed may contain mirex leached from bait and the mirex could be introduced into the estuary when fresh water mixes with salt water. The experiments were conducted seasonally: spring (April 25 - May 23, 1973), summer (July 10 - August 7, 1973), fall (October 6 - November 13, 1973), and winter (January 15 - February 12, 1974). Six 2.4-m diameter fiberglass tanks and six 10-cm diameter glass columns were used (Figure 1). Three treated tanks received filtered (cartridges of one-/zpore size) tap water that had trickle-filtered through columns of mirex bait before being mixed with unfiltered sea water. Similarly, three control tanks received water from columns containing all components of the bait except mirex. Tap water contained no detectable chlorine (< 0.1 rag/L). Tap water (0.5 L/min) and natural seawater (I .0 L/min) siphoned from constant-head boxes were mixed in glass troughs, positioned below the columns, before entering the tanks. The water level in each tank was maintained at 30.5 cm by a standpipe opposite the site where water entered from the mixing trough. The tank area was partially enclosed by a fiberglass roof and back wall for protection from severe weather. Each column consisted of an outer cylinder containing three 15-cm-high and 9-cm-diameter cylinder inserts (Figure 2). Each insert contained a 50-g layer of bait 2E. G. Alley, Mississippi State Chemical Laboratory, State College, Miss. 39762.
Seasonal Effects of Mirex
373
(total of 150 g per column) which was soaked for 24 hr in fresh water to allow swelling before being placed in the columns. The bottoms of the inserts were covered by nylon screen (0.84 mm mesh) to retain the bait. A funnel attached to the bottom of the column concentrated the flow of tap water leaving the column. Because exploratory tests indicated relatively high concentrations (> 2 ~g/L) of mirex in column runoff during the first few days, we passed tap water through the columns (0.5 L/rain.) for 4 to 8 days (spring, 8 days; summer, 4 days; fall and winter, 6 days) before the tanks were filled. Tanks were filled a day prior to the start of animal exposure. The amount of bait used in the column and the water flow were chosen because they produced low but detectable residues of mirex in tank water (desired range, 0.01-0.50 ~g/L). Temperatures (12:00 Noon, mercury thermometer) and salinities (8:00 AM, temperature-compensated refractometer) of the tank water were measured at the start of each test and four times weekly thereafter. Temperature and salinity changes were gradual. Temperature responded to natural changes in air and sea water temperatures; salinity, to natural fluctuations in salinity of the sea water source (Santa Rosa Sound, Florida).
Fig. 1. Experimental system showing outdoor tanks and gravity-flowcolumns.
374
M.E. Tagatz et al.
Caged animals were placed on two cm of beach sand covering the bottom of the tanks. Each tank held four cages that contained, respectively, 25 adult sheepshead minnows, Cyprinodon variegatus; 14 juvenile blue crabs, Callinectes sapidus; 25 juvenile pink shrimp, Penaeus duorarum; and 50 (35 in the fall experiment) adult grass shrimp, Palaemonetes pugio. Fish ranged from 32 to 59 mm total length; crabs, 21 to 75 mm carapace width; pink shrimp, 42 to 92 mm rostrum-telson length; and grass shrimp, 22 to 32 mm rostrum-telson in the spring study and 33 to 36 mm in the summer, fall and winter studies. All animals were captured in local waters and acclimated for 3 to 16 hr to the initial salinity and temperature of each experiment by gradual addition of tap water to the salt water stock-aquaria. Cages for crabs (115.5 cm x 33.0 cm x 23.0 cm deep) were made of stainlesssteel screening (7.1 mm mesh) over wooden frames, and cages for fish and shrimps (76.0 cm • 76.0 cm x 30.5 cm deep) were made of nylon screening (3.3 mm mesh) over wooden frames. To prevent cannibalism, crabs were confined in indi-
Freshwater siphon (301/hr)
)
Constant-hea( (Filtered fres!
Constant-head I (Unfiltered seat
c bait)
Flow into tank (901/hr)
Fig. 2. Gravity-flow column
Seasonal Effects of Mirex
375
vidual compartments (16.5 cm x 16.5 cm x 23.0 cm deep). Pink shrimp were provided 7.5 cm of sand on the cage bottom for burrowing. Crabs were fed small fish, and pink shrimp were fed cubes of fish meat, weekly. Fish and grass shrimp were not given food but could consume plankton. To determine growth of crabs, carapace width (in mm between the tips of the lateral spines) was measured at the beginning, middle and end of the experiment. Percentage survival of animals was determined at the end of the experiment. The chi-square test was used to determine significant differences in numbers of dead and living animals between treated and control tanks. Samples of water, sand, or animals were analyzed by electron-capture gas chromatography to determine mirex concentrations. Sensitivity was 0.01/zg/g (ppm) for whole animals (wet-weight basis) and for sediment samples (air-dried weight basis), and 0.01 /zg/L (ppb) for water samples. Samples to which known amounts of mirex were added gave recovery rates greater than 85%, but concentrations were not corrected for percentage recovery. Techniques for most residue analyses were those of Lowe et al. (1971) and for tissue samples that weighed less than five g, those of Hansen et al. (in press). Samples of water from each tank were obtained at the start of the experiment (when the animals were placed in the tank) and twice a week thereafter. A water sample consisted of a composite collection from two sites in each tank. At 14 and 28 days, a composite sample of sand from four sites was obtained from each tank. Surviving animals (composite samples) were analyzed for mirex at the end of the experiment, and dead animals (individual or composite samples) were analyzed as deaths occurred. No residues of mirex were detected in pretest samples of sand and animals or in fish used as food. Samples of sheepshead minnows, blue crabs, or pink shrimp surviving at the end of the experiments, and samples of dead pink shrimp, were taken for histological examination. Surviving pink shrimp were sampled in all experiments; fish and crab, in all experiments except winter. Samples of survivors consisted of 10 or 15 individuals of a species from treated tanks and 10 or 15 individuals from control tanks. Samples of dead pink shrimp consisted of seven treated shrimp in fall and nine treated and three control shrimp in winter.
Results and discussion Measured concentrations of mirex (~g/L) in the three treated tanks averaged approximately 0.04 in spring, 0.12 in summer, 0.06 in fall, and 0.09 in winter (Table I). Analysis of variance showed" no significant differences ( o : = 0.05) among the three treated tanks in each test. Also, mirex residues did not increase or decrease statistically ( o: = 0.05) with time within individual tanks. Seasonal temperatures and salinities of tank water and average residues of mirex are summarized in Table II. Temperatures averaged 23.1~ in spring, 29.8~ in
0.07 0.03
0.03 0.09
0.03
Week 3
Week 4
Average
0.04
0.04 0.11
0.07 0.06
0.03 < 0.01 a
0.02 0.03
0.03
0.11
0.12
0.01-
0.04
0.03 0.08
0.05 0.02
0.04 0.01
0.12 0.02
0.02
a0.005 used for calculating average.
0.09
< 0.01-< 0.01-
0.02 0.01
Week 2
Range
0.02 0.02
< 0.01 a
Week 1
Start
Spring
Tank 1 Tank 2 Tank 3
Time
elapsed
.0.36
0.03"
0.09
0.05 0.09
0.03 0.05
0.04 0.04
0.14 0.05
0.36
0.23
0.04-
0.10
0.10 0.23
0.09 0.10
0.04 0.08
0.08 0.04
0.17
0.52
0.05-
0.16
0.11 0.11
0.10 0.19
0.05 0.07
0.20 0.06
0.52
Tank 1 Tank 2 Tank 3
Summer
0.20
0.03-
0.07
0.04 0.05
0.05 0.04
0.03 0.04
0.14 0.07
0.20
0.23
0.03-
0.06
0.03 0.04
0.03 0.04
0.03 0.03
0.09 0.04
0.23
0.12
0.01-
0.04
0.02 0.02
0.02 0.02
0.01 0.02
0.07 0.04
0.12
Tank 1 Tank 2 Tank 3
Fall
0.25
0.03-
0.08
0.04 0.05
0.05 0.03
0.03 0.05
0.11 0.07'
0.25
0.24
0.02-
0.07
0.05 0.04
0.04 0.03
0.03 0.02
0.09 0.05
0.24
0.52
0.03-
0.11
0.03 0.04
0.05 0.03
0.04 0.08
0.12 0.04
0.52
Tank 1 Tank 2 Tank 3
Winter
Table I. Concentrations of Mirex (gg/L ) in Treated Tank Water During Four 28-Day Seasonal Experiment~
S
0~
t-t1 ,-q
Seasonal Effects of Mirex summer, 23.4~ in fall, and 19. I~ 19 parts per thousand.
377
in winter. Overall salinity range was from 7 to
In mirex-contaminated tanks, survival of crabs and shrimps usually was significantly reduced; survival of fish was not affected (Table III). The number of deaths was greatest in the summer, followed by fall, winter and spring: Observed deaths among exposed shrimps occurred after 2 to 15 days in summer (all pink shrimp died in 15 days), after 8 to 28 days in fall, and after 17 to 28 days in winter. During summer and fall, significantly more exposed blue crabs died than did control crabs, deaths occurring after 6 to 28 days of exposure. At the end of the summer experiment, one-third of the crabs surviving in treated tanks were paralyzed or had lost equilibrium. Most deaths occurred among the smaller crabs. Of 12 deaths among 42 crabs exposed in summer, I I were among 19 crabs 28 to 40 mm carapace width, but only one died among 23 crabs 41 to 64 mm. Survival of sheepshead minnows was unaffected in all experiments. This species can spawn in the presence of mirex since each tank contained 50 to 100 young sheepshead minnows (up to 14 mm total length) at the end of the spring studies and 75 to 200 young (up to 26 mm) at the end of the summer studies. Lowe et al. (1971) also found that fish were relatively unaffected, juvenile pinfish (Lagodon rhomboides) living for five months on a diet that contained approximately 20 /zg/g of technical mirex. Mortality and delayed toxicity to crabs and shrimps exposed to mirex (technical grade) occurred in other studies. Lowe et al. (1971) found that a significant number of juvenile Penaeus duorarum died during a seven-day exposure to 1.0 /zg/L of mirex in sea water averaging 17~ but few died during a 21-day exposure to 0.1 /~g/L in sea water averaging 14~ All juvenile Callinectes sapidus died within three weeks after a 96-hr exposure to 0.1 mg/L (Lowe et al. 1970). Redmann (1973) reported a 40% mortality ofPalaemonetes pugio in 12 days from a 48-hr exposure to 0.01 ~g/g of mirex in sea water at 20~ McKenzie (1970) and Lowe et al. (1971) demonstrated that smaller blue crabs were more sensitive to mirex bait than were larger crabs. Table II. Temperature, Salinity and Average Concentration of Mirex in Tank Water During Four 28-Day Seasonal Experiments
Temperature(~
Salinity (Parts per thousand) Av. Range
Mirex /~g/L
Av.
Range
Spring
23.1
19.3-25.2
13.2
10-18
0.04
Summer
29.8
28.0-30.8
15.7
14-18
0.12
Fall
23.4
17.0-27.0
17.5
15-19
0.06
Winter
19.1
13.8-22.9
13.6
7-16
0.09
M . E . Tagatz et al.
378
The greater mortality observed in summer could be caused by increased leaching and by increased toxicity of mirex in warmer water. Analysis of variance showed significant differences ( cc = 0.05) in mirex residues in water between summer and spring and between summer and fall; other paired treatments were not significant. Most deaths of affected animals occurred in summer at the highest water temperature and concentration of mirex; fewest in spring at next to the lowest temperature and at the lowest concentration. McKenzie (1970) reported that survival time and rate in juvenile blue crabs exposed to mirex bait decreased as temperatures increased from 20 to 27~ Mirex had no marked affect on growth of juvenile blue crabs exposed in these studies (Table IV). The greatest difference in mean percentage increase in size per
Table III. Percentage Survival of Animals Exposed to Mirex and Chi-Square Values After Each of Four 28-Day Seasonal Experiments. Data on Treated Tanks and on Control Tanks were Combined Percentage survival and ehi-square Animals
Number
Spring
Summer
Fall
Winter
Sheepshead minnows Control
75
63
96
91
99
Treated
75
55
99
92
100
N.S. a
N.S.
N.S.
N.S.
100
98
95
95
81
95
Chi-square
Blue crabs Control
42
Treated
42
Chi-square
98
71
N.S.
11.01 **
4.09*
N.S.
Pink shrimp Control
75
86
87
91
91
Treated
75
76
0
19
49
78.44**
30.51 **
96
97
59
89
Chi-square
N.S.
114.70"*
Grass shrimp Control
150b
95
Treated
150b
86
Chi-square
-c
79 10 145.87"*
41.69"*
6.20*
aN.S. = non-significant, ** significant at I% level (X 2, 1 d.f. = 6.63), * significant at 5% level (X2, 1 d.f. = 3.84). b 105 used in the fall experiment. eSome small grass shrimp escaped through the mesh of the cages, and all those free in the tanks may not have been captured (values in table include those captured).
Seasonal Effects of Mirex
379
molt between treated and control groups was less than 3%; not decisive in a species normally characterized by variable growth among individuals (Tagatz 1968). Taking into account deaths that occurred among treated crabs, frequency of molting did not differ greatly between treated and control groups. Animals exposed to leached mirex concentrated the chemical in body tissues (Table V). During 28 days' exposure, depending on the experiment, surviving sheepshead minnows accumulated from 10,500 to 40,800X the average concentration of mirex in the test water. Residues in young fish (
