EFFECTS OF DDT IN FUNDULUS: STUDIES ON TOXICITY, FATE, AND REPRODUCTION 1 RICHARD B. CRAWFORD~ Trinity College, HaroCord, Connecticut 06106 and ANTHONY M. GUARINO Laboratory of Toxicology, National Cancer Institute, Bethesda. Maryland 20014
The toxicity, absorption, distribution, metabolism, and effects on reproduction of DDT was studied using the killifish (Fundulus heteroclitus), a species of economic importance because of its widespread abundance and its presence toward the lower e n d of the food chain, x4C-DDT was administered by exposure from the ambient water. There was a rapid removal of the radioactive pesticide from the water accompanied by uptake of radioactivity primarily by carcass (primarily muscle tissue) and eggs of the fish. Most (>92%) of the radioactivity in the carcass was shown by TLC methods to be the parent pesticide. One day after a single 24-hr exposure to x4C-DDT, approximately 70% of the administered radioactivity was found in the carcass and the levels of this tissue decayed with a ttA, of three days. One day after a single 24-hr exposure to 0. I ppm of 14C-DDT, the organs that contained the highest concentration of the pesticide (ca. 5 ppm) were intestine and liver. When the pesticide was administered by two 24-hr exposures from water, the intestine, liver and ovaries contained the major concentration of radioactivity (7 to 14 ppm). Untreated Fundulus contained less than 0.2 ppm of total DDT-like compounds. A variety of doses and schedules were tested in an effort to maximize the absorption of DDT, while minimizing the mortality to the fish. An intermittent schedule of 24 hr in 0.1 ppm DDT followed by 24 hr in DDT-free sea water, repeated two times, was found to be optimal. At the levels examined, DDT delayed the rate of normal development of fertilized eggs from Fundulus, but did not appear to cause any observable alterations in the hatched fry. Fertilization of Fundulus eggs was significantly diminished when insemination was carried out in DDTcontaining sea water. Of the vast quantities of persistent organochlorine c o m p o u n d s that are dispersed into our e n v i r o n m e n t , most ultimately enter the oceans of the world (Risebrough 1968, Woodwell et al. 1971). It has been estimated that 25% of the DDT produced has already entered the ocean ( C o m m o n e r 1972). Thus, even if widespread use of this pesticide is terminated, aquatic species will continue to be exposed to these agents for m a n y more years. Such expbsures in water have been extensively
:Studies were conducted at Mount Desert Island Biological Laboratory, Salisbury Cove, ME 04672 under the partial support of a grant from the National Science Foundation (6B-28139). 2To whom requests for reprints should be directed. Archives of Environmental Contamination and Toxicology, Vol. 4, 334-348 (1976) 9 1976 by Springer-Verlag New York Inc.
334
Effects of DDT in Fundulus
335
documented to result in rapid absorption by the bottom sediment, plankton, algae, aquatic invertebrates, aquatic vegetations, and fish (Edwards 1970; Whitacre et al. 1972). Some of these organisms take up xenobiotics directly from the water flowing through their bodies, or accumulation may occur through their food. Small fish can absorb pesticides via both these routes, and furthermore these animals represent a food source for many aquatic and other species at higher trophic levels. The studies presented here document the uptake, metabolic fate, and distribution of DDT in the small teleost, Fundulus heteroclitus. These investigations were conducted on the fish in its natural habitat and on fish exposed to very high levels of the pesticide for short term analysis of lethal dose and distribution. Very little is known about the effects of pesticides on reproduction and development in fish. Therefore, we have initiated studies on fertilization and embryo development patterns in DDT-treated gametes from Fundulus.
Materials and methods Biological materials. Fundulus heteroclitus (Linnaeus) were obtained from estuarine waters of Frenchman Bay and kept in floating live cars in sea water. Gametes were obtained, fertilized, and embryos reared according to methods described previously (Crawford et al. 1973). Eggs, stripped from females, were mixed with a sperm suspension obtained by mincing dissected testes. Filtered 50% sea water (sea water:distilled water, 1:1) kept at 16~ was the incubation medium. Developmental stage references are according to Oppenheimer (1937). Fish were kept in 1000-ml Erlenmeyer flasks containing 750 ml of sea water at 13~ Under standard conditions each flask held four fish. All sea water was filtered through membrane filters (0.45/x pore size) to eliminate the influence of other marine organisms and particles on DDT uptake by Fundulus. Flask contents were mixed and aerated from a central pump via an aquarium stone. The water, whether it contained DDT or not, was changed daily. DDT solutions were prepared by adding 2.0 ml of ethanolic solutions containing the appropriate amount of the pesticide to 1000 ml of filtered sea water immediately prior to use. Control fish were kept in 0.2% ethanol-sea water. The lethal dosage of DDT for F u n d u l u s was determined under a variety of exposure conditions. First, fish were maintained in DDT continuously for up to 24 hr at concentrations from 0.01 to 1.0 ppm. In another type of study, fish were subjected to DDT (0.05 or 0.10 ppm) for 24-hr periods interspersed with 24 hr in sea water containing no DDT. Uptake of 14C-DDT (Amersham/Searle, ring-labelled, 32.3 mCi/mmole) was measured both by direct measurements in the fish and indirectly by determination of the loss of DDT from the media. Aliquots from the 14C-DDT-containing media were extracted with 18 ml of toluene scintillation solution (4 g PPO and 50 mg POPOP/L). Fifteen ml of the extract was placed in a vial and counted in the Nuclear Chicago Mark I liquid scintillation spectrometer.
336
R. B. Crawford and A. M. Guarino
The distribution of DDT in various tissues of Fundulus was determined as follows: Four fish were placed in each flask containing 750 ml of medium (containing 0.1 ppm of 14C-DDT) and they were removed at the appropriate time for assay. Tissues were removed, weighed, and aliquots placed in two ml of NCS Tissue Solubilizer at 50~ When the tissue dissolved, 18 ml of toluene scintillation solution, indicated above, was added and the samples were counted. The remaining carcass was homogenized and aliquots representing approximately 100 mg were prepared with NCS and the scintillation solution in the usual manner. Additional samples of whole Fundulus which had been treated with 0.1 ppm of '14C-DDT for 24 hr, as described above, were extracted for thin layer chromatographic (TLC) analysis of DDT and metabolites. Fish were homogenized for two minutes with three ml of acet-onitrile per g of fish and sufficient perchloric acid to bring the mixture to pH 2, as described previously (Pritchard et al. 1973). The homogenate was centrifuged at 2000 x g for five min and the resulting supernatant was concentrated under a stream of nitrogen.
Thin layer chromatography. Extracts were chromatographed in the following solvent systems: (I) heptane; and (2) heptane:ethanol (5:1). Silica gel glass plates (250/z) from Analtech, Inc., Wilmington, DE 19801, were used. Extracts were co-chromatographed with authentic samples of DDT, 1 DDD, DDE and DDA. The positions of the pesticide analogs were determined by spraying with alcoholic AgNO3 (0.5%) and exposure to ultraviolet light. The darkened regions of the plates containing the known standards were scraped and counted employing the same scintillation fluid as above. Ambient pesticide analysis. Two pools of Fundulus, obtained from the same source as the experimental fish, were sacrificed, immediately frozen, and less than one month later analyzed for DDT and metabolites by gas chromatography using standardized procedures (Pesticide Manual 1968). Whole Fundulus samples were ground in a blender to obtain a homogenous sample and fat was extraced from a 20-ml aliquot. Following acetonitrile partition and Florisil chromatography, the extract was concentrated and analyzed on a Glowall Chromalab instrument equipped with a ~2~radium electron capture detector. The column was packed with a 1:1 mixture of 7% QF-1 and 9% OV-17 (both on 80/100 mesh Gas-Chrom Q) and run at 226~ Identification and quantitation of sample peaks were performed by comparison of retention times and calibration curves obtained from standard solutions of reference compounds. Results Ambient pesticide level. Gas chromatographic analysis of two pools of fish provided an estimate of background " D D T residues" in the local Fundulus population of Mt. Desert Island, Maine (Table I). On a wet weight basis, individual values for both pools of fish were virtually identical. Total " D D T residues" were 0.17 ppm in the whole body. Both isomers of DDT itself (0.085 ppm) accounted for 50%
Effects of DDT in Fundulus
337
of the pesticide, while DDD (0.02 ppm), DDE (0.02 ppm) and DDMU (0.045 ppm) isomers made up the remainder. It is noted that while values in the two fish pools were quite similar on a wet weight basis, they were rather different on a lipid weight basis. The profile of DDT analogs on this basis is the same as for the wet weight assays and the total DDT residues were about nine ppm in terms of fat weight.
Identification of 14C-DDT metabolites in the fundulus. Since preliminary results had shown that the dominant site for uptake of DDT was the carcass, homogenates of whole Fundulus were subjected to thin layer chromatographic analyses of the metabolites. Table II shows that the dominant radioactive material is the parent pesticide. Approximately 93% occurred as DDT whether analyses were conducted at one day or eight days after administration of the 14C-DDT. The most abundant metabolite (4%) seen is DDD at the earliest time and DDE at the later time. Only about i% of the radioactivity occurred as the further metabolite DDA. Overall toxicity studies. Maintaining fish in DDT-containing sea water continuously gave the results seen in Table III. One ppm was 100% lethal for the three fish tested within 24 hr while 0.5 and 0.1 ppm gave approximately 82% and 9% mortality, respectively, in the same time period. From these data the graphically calculated 24 hr-LCso was about 0.25 ppm for Fundulus. Emphasis in these studies was placed at the 0.1 ppm dosage which appears to he the maximum dosage tolerated with minimal mortality. When Fundulus are exposed to DDT on an intermittent schedule, it is apparent (Table IV) that the fish cannot long tolerate additional doses at the 0.1 ppm level. However, several fish survived four treatments at 0.05 ppm interspersed with 24 hr free of the pesticide. At 0. I ppm, 44% of the animals died after two exposures. From Day 5 on, about 40% more of the fish died, whereas by Day 15 of exposure, 94% had died. At the lower exposure level of 0.05 ppm, very little mortality occurred up to Day 5 and no more than about one-third of the animals were dead even up to Day 13. The overall mortality from the two doses differed by about two-fold and suggested analysis of these data in terms of c x t (Wilbur 1969) as is shown in Table V. For these calculations, c was taken as the initial concentration of DDT added to the flask in ppm and t was one day for each day of exposure. The validity of the c x t constant appeared to hold up for values up to 0.2 for both exposure levels. At higher c x t values there was about two-fold increased lethality at the higher (0.1 ppm) concentrations of DDT.
Uptake of DDT and distribution in tissues. The results of indirect analyses of DDT-uptake are seen in Figure 1. The flasks which contained no fish showed a large 1Abbreviations: DDT = l,l,l-trichloro-2,2-bis (p-chlorophenyl) ethane; DDD = 1,1-dichloro-2,2-bis (p-ehlorophenyl) ethane; DDE = l,l-dichloro-2,2,-bis (p-chlorophynyl) ethylene; DDA = 2,2-bis (pchlorophenyl) acetic acid and DDMU = l-chloro-2,2-bis (p-chlorophenyl) ethylene.
338
R. B. Crawford and A. M. Guarino
Background levels of DDT analogs in Fundulus a
T a b l e I.
Parts per million Analog
Wet weight
Fat weight
DDMU
0.045 (0.04-0.05)
2.55 (2.26-2.84)
p,p'DDE
0.02
1.00 (0.87-1.12)
o,p'DDD
0.01
0.28 (0.26-0.29)
o,p'DDT
0.03
1.65 (0.36-1.94)
p,p'DDD
0.01
0.36 (0.34-0.38)
p,p'DDT
0.055 (0.05-0.06)
2.88 (2.48-3.27)
Sum
0.17
8.72
aValues are mean of two pools of five fish each. Numbers in parentheses are ranges of values. Where no ranges are given, replicate assays were equal. Fish were acquired, maintained and assayed as described in Methods. Table II.
TLC identification of 14C-DDT and metabolites in whole Fundulus a % 0f-radioactivity
Days after initial exposure
DDT
DDE
DDD
DDA
1
92.9 • 1.3
2.2 __. 0.6
4.1 + 0.8
0.9 _ 0.7
8
92.6 _ 1.6
4.1 _+ 1.0
2.3 +__0.7
0.9 -+- 0.6
aFish were exposed to 14C-DDT, 0. l ppm as indicated in Methods. Values are mean percent -+ S.D. of total radioactivity residing in indicated spot for four to six fish at each time point. Identity of radioactive spots was determined by co-chromatography with authentic samples of DDT, DDE, DDD, and DDA. Typical Rf.s expressed as RooT, where the Rr of DDT is normalized to 1.00, were as follows: Solvent I, heptane, 0.00, 0.63, 1.00 and 1.34; Solvent II, heptane-ethanol (5:1), 0.15, 0.92, 1.00, and 1.03, for DDA, DDD, DDT, and DDE. respectively. Table III.
Percentage of deaths in Fundulus maintained in DDT-containing media Hours in DDT Solution
DDT (ppm)
6
10
19
22
24
1.0
0
0
33
67
100
(3) a
0.5
0
0
0
55
82
(ll)
0.1
0
0
0
0
9
(56)
0.01
0
0
0
0
0
(8)
aFigure in parentheses refers to number of fish studied.
Effects of DDT in Fundulus
339
Percentage of deaths in Fundulus given long term dosages of DDT
Table IV.
Days from first DDT dose a DDT (ppm)
1
3
5
7
9
0.i
25
44
75
81
88
0.05
0
6
13
38
38
11
13
15
88
94
94
(16) b
38
38
56
(16)
aEach 24-hr dose was followed by 24 hr free of DDT. Doses were initiated on Day 0 and then continued on even numbered days. bFigure in parentheses refers to number of fish studied.
T a b l e V.
C x T effects of D D T exposure on Fundulus a Cumulative toxicity, % dead
c • t
0.05 ppm DDT
0.10 p p m DDT
0.05
0
--
0.I0
6
25
0.15
13
--
0.20
38
44
0.25
38
--
0.30
38
75
0.35
38
--
0.40
56
81
0.05 -0.80
--
88 -94
aData are derived from Table IV as explained in text.
decrease in l a C - D D T within 24 hr due to volatilization and adsorption onto the glass, as had been reported by Acree et al. (1963) and by Gakstatter and Weiss (1967). H o w e v e r , the flasks containing fish showed a p p r o x i m a t e l y 80% diminution of r a d i o a c t i v i t y within four hr. E x a m i n a t i o n o f these curves suggests that at least 50% o f the dose was taken up by the fish in four hr, thus offering e v i d e n c e o f the ability o f the Fundulus to r e m o v e this pesticide from water. S i m i l a r results had been observed in studies on the b r o w n trout ( H o l d e n 1962). Tables V I and V I I show the distribution results in two types o f experiments differing in the time o f exposure o f the fish to the DDT. These data indicate the concentration o f D D T in tissues and the percent o f a b s o r b e d dose in each tissue both one and eight d a y s post dose. The fish o f Table VI were e x p o s e d to 0.1 p p m D D T for 24 hr while the fish o f Table VII were in the D D T - c o n t a i n i n g sea water 24 hr, in sea water alone 24 hr, and then returned to the D D T solution for another 24-hr exposure. A f t e r the one or two intervals o f exposure to D D T , the fish were returned
R. B. Crawford and A. M. Guarino
340
to DDT-free water for either one or eight days. It is clear that over half of the absorbed dose was found in the carcass (primarily within muscle), regardless of which exposure technique was used. The concentration ratio of radioactivity in the fish (carcass) to the initial radioactivity in the media was about 15. In terms of concentration (Table VI) the actual values for eggs were quite low (ca-0.5 ppm) compared with all other tissues sampled. One or eight days after exposure, hepatic and intestinal levels were three to five ppm. The remaining tissues had one or two ppm of the pesticide. It is of special interest to note that a major quantity of DDT accumulates in the ovaries and that significant quantities appear in the eggs, indicating a degree of transport of substances across egg membranes. Experiments with male fish showed incorporation of DDT into testes at levels much higher than for eggs.
100
90
80
70 E .E ._~
60
~
50
o 0 I ~
40;
r
t'\\\ \
\,~ NO fish in flask
\\ \\
\\ \
4 fish in flask
\ \
\
\ ~
~,.....
20
10
0
i 10
I 20
I I ~1 30 40 9 Hours
I
50
60
Fig. 1, Effect of fish on DDT loss from incubation media. At time zero, each flask contained 750 ml of 0.1 ppm ~4C-DDT. 4 fish were added to each flask except in the no-fish controls. 1.0 ml aliquots were removed and analyzed at the indicated time intervals.
Effects o f DDT in Fundulus
341
T a b l e V I . DDT incorporation and distribution in Fundulus a (single 24-hr DDT dose) 1 day post DDT dose Tissue
ppm DDT
% of the absorbed dose in tissue
8 days post DDT dose ppm DDT
Intestine
5.11 • 0.88 b
7.83 +-- 1.80
Liver
4.91 •
8.70 --+ 1.33
2.92 • 0.33
8.06 • 0.95
Eggs e
0.25 • 0.09
3.02 •
0.57 • 0.12
10.92 • 2.60
9.33 •
42.85 • 2.28
Ovaries e
1.01
1.12
3.08 • 0.44
% of the absorbed dose in tissue
1.86
11.80 •
1.71
--
--
Testes
1.55 • 0.53
2.32 • 0.59
--
Brain
2.63 • 0.66
0.88 +-- 0 . I 0
1.74 • 0.21
1.00 • 0.13
Heart
2.07 • 0.55
0.23 • 0.04
0.97 • 0.12
0.24 • 0.04
Spleen
2.37 • 0.50
0.14 • 0.04
0.56 • 0.08
0.15 • 0.03
Gills
2.17 • 0.64
0.97 • 0.24
1.59 • 0.17
1.69 • 0.38
--
Muscle
1.50 • 0.36
--
0.83 • 0.06
--
Carcass
1.54 • 0.20
72.83 • 3.65
0.79 • 0.08
52.50 • 5.38
aDosage and assay performed as described in the text. bValues are mean • standard error for seven fish used in each study. eTissue assays on each fish were performed on either eggs or ovaries, never both from the same animal.
T a b l e V I I . D D T incorporation and distribution in Fundulus a (two 24-hr D D T doses) 8 days post DDT dose
1 day post DDT dose Tissue Intestine
ppm DDT
% of the absorbed dose in tissue
ppm DDT
14.24 -~ 4.42 b
18.07 • 3.80
% of the absorbed dose in tissue
2.57 - 0.30
5.69 __. 1.51
Liver
8.18 -4- 1.03
7.43 •
1.17
5.19 • 0.31
6.67 __. 0.33
Ovaries
6.80 • 0.82
15.20 •
1.12
10.40 • 3.00
25.16 • 6.29
Brain
3.59 • 0.60
0.77 • 0.10
3.07 • 0.53
0.78 • 0.14
Heart
2.39 • 0.18
0.17 • 0.03
1.82 - 0.42
0.20 • 0.04
Spleen
1.84 • 0.38
0.15 • 0.02
0.94 • 0.20
0.17 ___ 0.07
Gills
2.50 • 0.29
0.98 • 0,16
2.32 • 0.40
1.41 __. 0.33
Muscle
1.76 • 0.33
--
1.23 • 0.16
--
Carcass
1.73 • 0.09
55.58 • 2.90
1.35 • O. 15
56.17 __ 7.58
aDosage and assay performed as described in the text. bValues are mean -- standard error for five fish used in each study.
342
R. B. Crawford and A. M. Guarino
The major effect obtained from the double-exposure technique (Table VII), is that more DDT is absorbed. Note, for example, that five ppm was present in the intestine one day after exposure to one dose, whereas this same tissue had 14 ppm after two doses. It should be noted that a major portion of the labeled material is still present in the fish eight days following removal from the DDT-containing medium. The txl= (calculated graphically by extrapolation back to to) for carcass was three days after the single dose of DDT, and eight days after the double dosing (Table VIII). After single-day doses, these carcass values were about the same as are seen in this table for muscle, spleen and heart. The rather long tt/2 (nine days) for gills is interesting in that is suggests that this organ is a site of high binding and/or excretion of the pesticide. The h/z's for intestine, liver and brain also were pro'longed 9 to 12 days. The usual effect of the two-day dosing, was to prolong the tissue halftime. Thus, when compared with the double exposure, brain, heart, and gill t~/2's were three to four-fold longer than after the single exposure (see Table VIII). Liver, muscle and carcass values were approximately doubled after two exposures. The only tl/2 which decreased after the two 24-hour treatment _periods, was that of intestine, and it diminished by one-half. Thus it is obvious that this is a primary site of entry for DDT and that absorption from the intestine is very rapid, leading to a comparatively low concentration by Day 8 post-treatment. It is also of interest that in the relatively large organ, the intestine (ca. 250 mg) there is a decrease by a factor of one-half while the brain, heart, liver and gills (total weight of all four organs equals 290 rag) increased in half-time by a factor of two to three. The gill tz/~ of 43 days is best explained in terms of binding rather than excretion of the pesticide via this tissue. The prolonged tz12of brain (30 days) is of particular importance in light of the notorious CNS effects of chlorinated hydrocarbon pesticides (Wooley 1970).
Effects on reproduction and development. Having established the distribution of DDT and finding large quantities in the gonads, we became interested in the possible effects of this dosage on fertilization and embryogenesis. At least a range of concentration vs. effect of these functions might be established. Female Fundulus were given DDT dosage in the usual manner. Each flask contained four fish in 750 ml of sea water. The exposure was for 24 hr in 0.1 ppm 14C-DDT, followed by a 24-hr pesticide-free period and then another 24-hr period of 0.I ppm 14C-DDT. Two days after the second dose, eggs were collected from each fish and fertilized. The mass of eggs from each fish was incubated separately. Fertilization of all eggs was accomplished using aliquots from a common pool of minced testes. The quantity of DDT in the eggs was measured by determining the radioactivity of aliquots of eggs removed from the incubation medium. DDT content of eggs from pre-loaded .females ranged from approximately 0.1 to 0.3 ppm as seen in the footnote to Table IX. Development of these eggs was compared with controls from the time of fertilization to hatching (approximately 40 days). The fertilization percentage was unaffected by the DDT at the levels used in this experiment. However, the developmental rate was markedly reduced (Table IX). The DDT-containing embryos remained one or two stages behind the controls
343
Effects of DDT in Fundulus
Tissue half-times (t%) for 14C-DDT treated Fundulus a
Table V I I I .
t V2(Days)
Tissue
Intestine Liver Brain Heart Spleen Gills Muscle Carcass
Single 24-hr exposure
Double 24-hr exposure
12 I1 9 4 3 9 3 3
6 16 30 12 4 43 6 8
aValues calculated, to the nearest whole day, as described in text, from data in Tables VI and VII.
throughout the processes of blastulation, gastrulation, and neurulation. Only during the late stages of organogenesis and growth did the treated embryos " c a t c h - u p " with the controls. Development from that time on was apparently normal, even through hatching. In another series of experiments, eggs were fertilized in sea water containing DDT. Eggs were place in DDT suspensions (1.0 ppm and 10 ppm) for 30 min, sperm were added, and then the eggs were washed free of sperm with the appropriate DDT-containing sea water five min later. Under these conditions, the most dramatic effect was a major reduction in the fertilization rate (Table X). It should be noted that even in experiment No. 3, where fertilization was poor in the controls, the percentage was markedly reduced in the presence of DDT. Aliquots of eggs removed from the incubation media and washed four times with 100 ml of water were analyzed for 14C-DDT content. As seen in Table XI, the eggs 'were able to take up and concentrate the pesticide with both a time and dose dependency. The 24-hr tissue:media ratio was 12 when one ppm was used, while the ten ppm dose gave a ratio of about five suggesting saturation at the higher concentration. Those eggs which did fertilize showed a morphogenetic lag similar to that seen in the previous experiment (Table X). Discussion
In terms of the wet weight of Funduius tissue, the anlogs of DDT " n a t u r a l l y " occuring in the greatest concentrations were p , p ' - D D T and DDMU (Table I). The total DDT-like compounds present in captured Fundulus were 0.17 ppm and thus the quantities to which these fish were exposed in most of these experiments (0.1 ppm) were less than the background levels. These residue levels are approximately the
344
R. B. Crawford and A. M. Guarino
same (0.11 ppm) as were reported by Pritchard et al. (1973) in flounder. When expressed in terms of fat weight, the Fundulus had 8.72 ppm of total DDT analogs in fat. This represents a concentration factor of more than 50-fold and compares favorably with the factor reported for ambient levels in the lobster carcass (Guarino et al. 1974). In studying DDT and its metabolites in extracts of whole Fundulus, it was found that DDT itself is the dominant material, present at more than 93% (Table II) whether analyses were done one or eight days after exposure to ~4C-DDT. The most abundant metabolite occuring at the earlier time was DDD, as has been reported previously for other aquatic species (Guarino et al. 1974, Menzie 1972). After eight days, more DDE than any other metabolite is present, and this was shown previously in longer term experiments employing Gambusia affinis fish in a model ecosystem (Kapoor et al. 1970). Less of the polar metabolites (e.g., DDA) occurred in Fundulus ( < 1 % ) than has been reported to occur in flounder tissues after intravenous treatment (Pritchard et al. 1973). After completion of the dose range-finding experiment summarized in Table III, a variety of techniques were designed to increase the absorption of DDT without excessively increasing the mortality. The intermittent schedule (Table I V and V)
T a b l e IX.
Effect o f D D T on Fundulus development rate a Developmental stage b
Days postfertilization
Controls
DDT-containing eggs
1
8
7
4
17-18
16-17
6
20
19
8
24
23
11
25
25
18
28
28
38
hatching
hatching
aObtaining eggs, fertilization, and staging were carried out as described in the text. In this experiment, eggs from 30 DDT-treated females were used. Egg masses from each female were reared separately. Numbers of eggs per fish ranged from 34 to 250. DDT cbntent in eggs ranged from 0.051 ppm to 0.37 ppm with a mean of 0.16 ppm. DDTcontaining embryos were all at the same stage regardless of their position in the range of DDT content. Fertilization rate was over 95% for all eggs. bDevelopmental stage according to Oppenheimer (1937).
345
Effects of DDT in Fundulus
appeared to increase the survival over continuous exposure, while providing increased levels of absorption (Tables VI and VII). Looking at the data in further detail, it is noted that at 0.1 ppm the more susceptible animals died after the first two doses. Furthermore, it is observed that from Day 5 on, there was not much additional increase in mortality. A similar trend was seen at the 0.05 ppm dose except that by Day 15, there seemed to be about 50% less mortality than there was at the 0.1 level. These results suggested further evaluation of these data in terms of c x t. Since this technique is an expression of both concentration and duration of exposure, similar toxicity is expected at similar c x t values. In Table V, up to c x t = 0.2, the concept seems generally valid since up to this point there is a similar degree of toxicity. The only exception to this appears to be that early deaths occur in the more susceptible individuals. At the c x t values of 0.3 and 0.4, there is only half as much toxicity at the lower exposure
Table X.
Effects of DDT-media on fertilization of Fundulus eggs a % Fertilization
Experiment No.
Controls
1 ppm DDT
1
92
66
2
95
76
3
22
4
aAnalysis performed as described in the text. Eggs were considered fertilized when they developed to a two-cell stage. Fertilization percentage was determined by examination of 100 eggs per experimental group.
Table XI.
Uptake of 14C-DDT by Fundulus eggs a DDT in eggs (ppm)
Time postfertilization
0
1 ppm media
10 ppm media
0.85
3.49
15 min
1.41
8.78
2 hr
3.28
50.53
14.37
48.63
1 day
aAnalyses performed as described in the text. ~4Ccontent was determined on aliquots containing 25 eggs.
346
R. B. Crawford and A. M. Guarino
level as at the higher level. This deviance from the concept suggests the occurrence of a concentration plateau and/or that at the lower concentration of DDT, lifesparing redistribution is occurring. Some evidence for the latter can be cited from Table VI where the concentration of DDT in carcass decreased about 50% from 1.5 to 0.79 ppm after eight days while egg values doubled from 0.25 tO 0.57 ppm. Although this may be a mechanism for decreasing maternal mortality, the present studies show that delayed development (Table IX) and diminished fertility (Table X) can occur in the animals. It is certainly clear from the data in Figure 1 that Fundulus take up DDT from the media and do so at a very rapid rate. We recognize the problem in this type of study of adsorption onto glass and volatilization of the pesticide. However, the incorporation by the fish is rapid enough to minimize the adsorption-volatilization problem in pre-loading experiments. Observation of the rapid loss of DDT f r o m solutions containing no fish does lead to speculation regarding the concentration of DDT on solid surfaces in streams and open bodies of water. That is, in addition to the levels known to occur in water sediment and aquatic life, a long term storage shed for pesticides may reside on the rocks and other solid surfaces of the ocean bottoms and if accounted for, may increase the total quantities of pesticides potentially available in these waters. Although this observation has been made before, it remains unsubstantiated by direct and detailed analysis (Woodwell et al. 1971). Effects of DDT on developmental patterns in Fundulus are reassuring. At least at levels far in excess of those found in free swimming fish the only observed effect on development was. that of a slight delay. The hatched fry appeared to be normal. However, in the total picture of development, " n o r m a l c y " would have to include such things as sexual maturation and fertilizability of the gametes of successive generations. Such study is beyond the scope of the present investigation. It also should be noted that the delayed but otherwise normal development occured in eggs whose DDT concentration was higher than that which is compatible with life in the adult fish. Thus, the developing embryo appeared to be more resistant to DDT than the adult. The more dramatic observation Was that in DDT-containing media, successful fertilization of Fundulus eggs by sperm was markedly reduced. At this juncture we have no way of suggesting whether the effect was on the sperm or egg or both. Clearly, the eggs were able to concentrate DDT; we have no data yet to indicate the pesticide levels in the sperm, but the testicular levels were 1.6 ppm (Table VI). At least qualitatively one might speculate that a reduction of fertilizability in a population, where under normal circumstances few will survive to adulthood, could have a marked effect on the adult population in a few generations.
Effects of DDT in Fundulus
347
Acknowledgement The authors wish to acknowledge the technical assistance of Jacqueline B. Anderson, Patrick B. Briley and Marion A. Kinter.
References Acree, F., M. Beroza, and M. C. Bowman: Codistillation of DDT with water. Agr. Food Chem. 11, 278 (1963). Commoner, B.: The closing circle. A. A. Knopf, NY, p. 227 (1972). Crawford, R. B., C. E. Wilde, M. H. Heinemann, and F. J. Hendler: Morphogenetic disturbances from timed inhibitions of protein synthesis in Fundulus. J. Embryol. Exp. Morph. 29, 363 (1973). Edwards, C. A.: Persistent Pe.sticides in the Environment. Cleveland, CRC Press, p. 27 (1970). Gakstatter, J. H., and C. M. Weiss: The elimination of DDT-C 14, dieldrin-C 14, and lindane-C ~4 from a single sublethal exposure in aquaria. Trans. Amer. Fish. Soe. 96, 301 (1967). Guarino, A. M., J. B. Pritchard, J. B. Anderson, and D. P. Rail: Tissue distribution of ~4C-DDT inthe lobster after administration via intravascular or oral routes or after exposure from ambient sea water. Tox. Appl. Pharmacol. 28, 277 (1974). Holden, A. N.: A study of the absorption of ~4C-labelled DDT from water by fish. Ann. Appl. Biol. 50, 467 (1962). Kapoor, I. P., R. L. Metcalf, R. F. Nystrom, and G. K. Sangha: Comparative metabolism of methoxychlor, methichlor, and DDT in mouse, insects, and in a model ecosystem. Agr. Food Chem. 18, 1145 (1970). Menzie, C. M.: Fate of pesticides in the environment. Ann. Rev. Entomol. 17, 199 (1972). Oppenheimer, J. M.: The normal stages ofFundulus heteroclitus. Anat. Rec. 68, 1 (1937). Pesticide Analytical Manual. Dept. of Health, Education and Welfare, Food and Drug Administration, Washington, D.C. Revised ed. 2 (1968). Pritchard, J. B., A. M. Guarino, and W. B. Kinter: Distribution metabolism and excretion of DDT and mirex by a marine teleost, the winter flouder, Environ. Health Persp. 4, 45 (1973). Risebrough, R. W.: Chlorinated hydrocarbons in marine ecosystems. In: Chemical Fallout, M. W. Miller and G. G. Berg, Eds. Springfield: C. C. Thomas, pp. 5-23 (1968). Whitacre, D. M., C. C. Roan, and G. W. Ware: Pesticides and aquatic microorganisms. Search 3, 150 (1972).
348
R. B. Crawford and A. M. Guarino
Wilber, C. G.: The Biological Aspects of Water Pollution. Springfield: C. C. Thomas, pp. 31-32 (1969). Woodwell, G. M., P. P. Craig, and H. A. Johnson: DDT in the biosphere: where does it go? Science 174, 1101 (1971). Wooley, D. Effect of DDT on the nervous system of the rat. In: Biological Impact of Pesticides in the Environment. J. W. Gillette, Ed. Oregon State Press. p. 114 (1970).
Manuscript received November 11, 1974; accepted June 20, 1975
The toxicity, absorption, distribution, metabolism, and effects on reproduction of DDT was studied using the killifish (Fundulus heteroclitus), a species of economic importance because of its widespread abundance and its presence toward the lower e n d of the food chain, x4C-DDT was administered by exposure from the ambient water. There was a rapid removal of the radioactive pesticide from the water accompanied by uptake of radioactivity primarily by carcass (primarily muscle tissue) and eggs of the fish. Most (>92%) of the radioactivity in the carcass was shown by TLC methods to be the parent pesticide. One day after a single 24-hr exposure to x4C-DDT, approximately 70% of the administered radioactivity was found in the carcass and the levels of this tissue decayed with a ttA, of three days. One day after a single 24-hr exposure to 0. I ppm of 14C-DDT, the organs that contained the highest concentration of the pesticide (ca. 5 ppm) were intestine and liver. When the pesticide was administered by two 24-hr exposures from water, the intestine, liver and ovaries contained the major concentration of radioactivity (7 to 14 ppm). Untreated Fundulus contained less than 0.2 ppm of total DDT-like compounds. A variety of doses and schedules were tested in an effort to maximize the absorption of DDT, while minimizing the mortality to the fish. An intermittent schedule of 24 hr in 0.1 ppm DDT followed by 24 hr in DDT-free sea water, repeated two times, was found to be optimal. At the levels examined, DDT delayed the rate of normal development of fertilized eggs from Fundulus, but did not appear to cause any observable alterations in the hatched fry. Fertilization of Fundulus eggs was significantly diminished when insemination was carried out in DDTcontaining sea water. Of the vast quantities of persistent organochlorine c o m p o u n d s that are dispersed into our e n v i r o n m e n t , most ultimately enter the oceans of the world (Risebrough 1968, Woodwell et al. 1971). It has been estimated that 25% of the DDT produced has already entered the ocean ( C o m m o n e r 1972). Thus, even if widespread use of this pesticide is terminated, aquatic species will continue to be exposed to these agents for m a n y more years. Such expbsures in water have been extensively
:Studies were conducted at Mount Desert Island Biological Laboratory, Salisbury Cove, ME 04672 under the partial support of a grant from the National Science Foundation (6B-28139). 2To whom requests for reprints should be directed. Archives of Environmental Contamination and Toxicology, Vol. 4, 334-348 (1976) 9 1976 by Springer-Verlag New York Inc.
334
Effects of DDT in Fundulus
335
documented to result in rapid absorption by the bottom sediment, plankton, algae, aquatic invertebrates, aquatic vegetations, and fish (Edwards 1970; Whitacre et al. 1972). Some of these organisms take up xenobiotics directly from the water flowing through their bodies, or accumulation may occur through their food. Small fish can absorb pesticides via both these routes, and furthermore these animals represent a food source for many aquatic and other species at higher trophic levels. The studies presented here document the uptake, metabolic fate, and distribution of DDT in the small teleost, Fundulus heteroclitus. These investigations were conducted on the fish in its natural habitat and on fish exposed to very high levels of the pesticide for short term analysis of lethal dose and distribution. Very little is known about the effects of pesticides on reproduction and development in fish. Therefore, we have initiated studies on fertilization and embryo development patterns in DDT-treated gametes from Fundulus.
Materials and methods Biological materials. Fundulus heteroclitus (Linnaeus) were obtained from estuarine waters of Frenchman Bay and kept in floating live cars in sea water. Gametes were obtained, fertilized, and embryos reared according to methods described previously (Crawford et al. 1973). Eggs, stripped from females, were mixed with a sperm suspension obtained by mincing dissected testes. Filtered 50% sea water (sea water:distilled water, 1:1) kept at 16~ was the incubation medium. Developmental stage references are according to Oppenheimer (1937). Fish were kept in 1000-ml Erlenmeyer flasks containing 750 ml of sea water at 13~ Under standard conditions each flask held four fish. All sea water was filtered through membrane filters (0.45/x pore size) to eliminate the influence of other marine organisms and particles on DDT uptake by Fundulus. Flask contents were mixed and aerated from a central pump via an aquarium stone. The water, whether it contained DDT or not, was changed daily. DDT solutions were prepared by adding 2.0 ml of ethanolic solutions containing the appropriate amount of the pesticide to 1000 ml of filtered sea water immediately prior to use. Control fish were kept in 0.2% ethanol-sea water. The lethal dosage of DDT for F u n d u l u s was determined under a variety of exposure conditions. First, fish were maintained in DDT continuously for up to 24 hr at concentrations from 0.01 to 1.0 ppm. In another type of study, fish were subjected to DDT (0.05 or 0.10 ppm) for 24-hr periods interspersed with 24 hr in sea water containing no DDT. Uptake of 14C-DDT (Amersham/Searle, ring-labelled, 32.3 mCi/mmole) was measured both by direct measurements in the fish and indirectly by determination of the loss of DDT from the media. Aliquots from the 14C-DDT-containing media were extracted with 18 ml of toluene scintillation solution (4 g PPO and 50 mg POPOP/L). Fifteen ml of the extract was placed in a vial and counted in the Nuclear Chicago Mark I liquid scintillation spectrometer.
336
R. B. Crawford and A. M. Guarino
The distribution of DDT in various tissues of Fundulus was determined as follows: Four fish were placed in each flask containing 750 ml of medium (containing 0.1 ppm of 14C-DDT) and they were removed at the appropriate time for assay. Tissues were removed, weighed, and aliquots placed in two ml of NCS Tissue Solubilizer at 50~ When the tissue dissolved, 18 ml of toluene scintillation solution, indicated above, was added and the samples were counted. The remaining carcass was homogenized and aliquots representing approximately 100 mg were prepared with NCS and the scintillation solution in the usual manner. Additional samples of whole Fundulus which had been treated with 0.1 ppm of '14C-DDT for 24 hr, as described above, were extracted for thin layer chromatographic (TLC) analysis of DDT and metabolites. Fish were homogenized for two minutes with three ml of acet-onitrile per g of fish and sufficient perchloric acid to bring the mixture to pH 2, as described previously (Pritchard et al. 1973). The homogenate was centrifuged at 2000 x g for five min and the resulting supernatant was concentrated under a stream of nitrogen.
Thin layer chromatography. Extracts were chromatographed in the following solvent systems: (I) heptane; and (2) heptane:ethanol (5:1). Silica gel glass plates (250/z) from Analtech, Inc., Wilmington, DE 19801, were used. Extracts were co-chromatographed with authentic samples of DDT, 1 DDD, DDE and DDA. The positions of the pesticide analogs were determined by spraying with alcoholic AgNO3 (0.5%) and exposure to ultraviolet light. The darkened regions of the plates containing the known standards were scraped and counted employing the same scintillation fluid as above. Ambient pesticide analysis. Two pools of Fundulus, obtained from the same source as the experimental fish, were sacrificed, immediately frozen, and less than one month later analyzed for DDT and metabolites by gas chromatography using standardized procedures (Pesticide Manual 1968). Whole Fundulus samples were ground in a blender to obtain a homogenous sample and fat was extraced from a 20-ml aliquot. Following acetonitrile partition and Florisil chromatography, the extract was concentrated and analyzed on a Glowall Chromalab instrument equipped with a ~2~radium electron capture detector. The column was packed with a 1:1 mixture of 7% QF-1 and 9% OV-17 (both on 80/100 mesh Gas-Chrom Q) and run at 226~ Identification and quantitation of sample peaks were performed by comparison of retention times and calibration curves obtained from standard solutions of reference compounds. Results Ambient pesticide level. Gas chromatographic analysis of two pools of fish provided an estimate of background " D D T residues" in the local Fundulus population of Mt. Desert Island, Maine (Table I). On a wet weight basis, individual values for both pools of fish were virtually identical. Total " D D T residues" were 0.17 ppm in the whole body. Both isomers of DDT itself (0.085 ppm) accounted for 50%
Effects of DDT in Fundulus
337
of the pesticide, while DDD (0.02 ppm), DDE (0.02 ppm) and DDMU (0.045 ppm) isomers made up the remainder. It is noted that while values in the two fish pools were quite similar on a wet weight basis, they were rather different on a lipid weight basis. The profile of DDT analogs on this basis is the same as for the wet weight assays and the total DDT residues were about nine ppm in terms of fat weight.
Identification of 14C-DDT metabolites in the fundulus. Since preliminary results had shown that the dominant site for uptake of DDT was the carcass, homogenates of whole Fundulus were subjected to thin layer chromatographic analyses of the metabolites. Table II shows that the dominant radioactive material is the parent pesticide. Approximately 93% occurred as DDT whether analyses were conducted at one day or eight days after administration of the 14C-DDT. The most abundant metabolite (4%) seen is DDD at the earliest time and DDE at the later time. Only about i% of the radioactivity occurred as the further metabolite DDA. Overall toxicity studies. Maintaining fish in DDT-containing sea water continuously gave the results seen in Table III. One ppm was 100% lethal for the three fish tested within 24 hr while 0.5 and 0.1 ppm gave approximately 82% and 9% mortality, respectively, in the same time period. From these data the graphically calculated 24 hr-LCso was about 0.25 ppm for Fundulus. Emphasis in these studies was placed at the 0.1 ppm dosage which appears to he the maximum dosage tolerated with minimal mortality. When Fundulus are exposed to DDT on an intermittent schedule, it is apparent (Table IV) that the fish cannot long tolerate additional doses at the 0.1 ppm level. However, several fish survived four treatments at 0.05 ppm interspersed with 24 hr free of the pesticide. At 0. I ppm, 44% of the animals died after two exposures. From Day 5 on, about 40% more of the fish died, whereas by Day 15 of exposure, 94% had died. At the lower exposure level of 0.05 ppm, very little mortality occurred up to Day 5 and no more than about one-third of the animals were dead even up to Day 13. The overall mortality from the two doses differed by about two-fold and suggested analysis of these data in terms of c x t (Wilbur 1969) as is shown in Table V. For these calculations, c was taken as the initial concentration of DDT added to the flask in ppm and t was one day for each day of exposure. The validity of the c x t constant appeared to hold up for values up to 0.2 for both exposure levels. At higher c x t values there was about two-fold increased lethality at the higher (0.1 ppm) concentrations of DDT.
Uptake of DDT and distribution in tissues. The results of indirect analyses of DDT-uptake are seen in Figure 1. The flasks which contained no fish showed a large 1Abbreviations: DDT = l,l,l-trichloro-2,2-bis (p-chlorophenyl) ethane; DDD = 1,1-dichloro-2,2-bis (p-ehlorophenyl) ethane; DDE = l,l-dichloro-2,2,-bis (p-chlorophynyl) ethylene; DDA = 2,2-bis (pchlorophenyl) acetic acid and DDMU = l-chloro-2,2-bis (p-chlorophenyl) ethylene.
338
R. B. Crawford and A. M. Guarino
Background levels of DDT analogs in Fundulus a
T a b l e I.
Parts per million Analog
Wet weight
Fat weight
DDMU
0.045 (0.04-0.05)
2.55 (2.26-2.84)
p,p'DDE
0.02
1.00 (0.87-1.12)
o,p'DDD
0.01
0.28 (0.26-0.29)
o,p'DDT
0.03
1.65 (0.36-1.94)
p,p'DDD
0.01
0.36 (0.34-0.38)
p,p'DDT
0.055 (0.05-0.06)
2.88 (2.48-3.27)
Sum
0.17
8.72
aValues are mean of two pools of five fish each. Numbers in parentheses are ranges of values. Where no ranges are given, replicate assays were equal. Fish were acquired, maintained and assayed as described in Methods. Table II.
TLC identification of 14C-DDT and metabolites in whole Fundulus a % 0f-radioactivity
Days after initial exposure
DDT
DDE
DDD
DDA
1
92.9 • 1.3
2.2 __. 0.6
4.1 + 0.8
0.9 _ 0.7
8
92.6 _ 1.6
4.1 _+ 1.0
2.3 +__0.7
0.9 -+- 0.6
aFish were exposed to 14C-DDT, 0. l ppm as indicated in Methods. Values are mean percent -+ S.D. of total radioactivity residing in indicated spot for four to six fish at each time point. Identity of radioactive spots was determined by co-chromatography with authentic samples of DDT, DDE, DDD, and DDA. Typical Rf.s expressed as RooT, where the Rr of DDT is normalized to 1.00, were as follows: Solvent I, heptane, 0.00, 0.63, 1.00 and 1.34; Solvent II, heptane-ethanol (5:1), 0.15, 0.92, 1.00, and 1.03, for DDA, DDD, DDT, and DDE. respectively. Table III.
Percentage of deaths in Fundulus maintained in DDT-containing media Hours in DDT Solution
DDT (ppm)
6
10
19
22
24
1.0
0
0
33
67
100
(3) a
0.5
0
0
0
55
82
(ll)
0.1
0
0
0
0
9
(56)
0.01
0
0
0
0
0
(8)
aFigure in parentheses refers to number of fish studied.
Effects of DDT in Fundulus
339
Percentage of deaths in Fundulus given long term dosages of DDT
Table IV.
Days from first DDT dose a DDT (ppm)
1
3
5
7
9
0.i
25
44
75
81
88
0.05
0
6
13
38
38
11
13
15
88
94
94
(16) b
38
38
56
(16)
aEach 24-hr dose was followed by 24 hr free of DDT. Doses were initiated on Day 0 and then continued on even numbered days. bFigure in parentheses refers to number of fish studied.
T a b l e V.
C x T effects of D D T exposure on Fundulus a Cumulative toxicity, % dead
c • t
0.05 ppm DDT
0.10 p p m DDT
0.05
0
--
0.I0
6
25
0.15
13
--
0.20
38
44
0.25
38
--
0.30
38
75
0.35
38
--
0.40
56
81
0.05 -0.80
--
88 -94
aData are derived from Table IV as explained in text.
decrease in l a C - D D T within 24 hr due to volatilization and adsorption onto the glass, as had been reported by Acree et al. (1963) and by Gakstatter and Weiss (1967). H o w e v e r , the flasks containing fish showed a p p r o x i m a t e l y 80% diminution of r a d i o a c t i v i t y within four hr. E x a m i n a t i o n o f these curves suggests that at least 50% o f the dose was taken up by the fish in four hr, thus offering e v i d e n c e o f the ability o f the Fundulus to r e m o v e this pesticide from water. S i m i l a r results had been observed in studies on the b r o w n trout ( H o l d e n 1962). Tables V I and V I I show the distribution results in two types o f experiments differing in the time o f exposure o f the fish to the DDT. These data indicate the concentration o f D D T in tissues and the percent o f a b s o r b e d dose in each tissue both one and eight d a y s post dose. The fish o f Table VI were e x p o s e d to 0.1 p p m D D T for 24 hr while the fish o f Table VII were in the D D T - c o n t a i n i n g sea water 24 hr, in sea water alone 24 hr, and then returned to the D D T solution for another 24-hr exposure. A f t e r the one or two intervals o f exposure to D D T , the fish were returned
R. B. Crawford and A. M. Guarino
340
to DDT-free water for either one or eight days. It is clear that over half of the absorbed dose was found in the carcass (primarily within muscle), regardless of which exposure technique was used. The concentration ratio of radioactivity in the fish (carcass) to the initial radioactivity in the media was about 15. In terms of concentration (Table VI) the actual values for eggs were quite low (ca-0.5 ppm) compared with all other tissues sampled. One or eight days after exposure, hepatic and intestinal levels were three to five ppm. The remaining tissues had one or two ppm of the pesticide. It is of special interest to note that a major quantity of DDT accumulates in the ovaries and that significant quantities appear in the eggs, indicating a degree of transport of substances across egg membranes. Experiments with male fish showed incorporation of DDT into testes at levels much higher than for eggs.
100
90
80
70 E .E ._~
60
~
50
o 0 I ~
40;
r
t'\\\ \
\,~ NO fish in flask
\\ \\
\\ \
4 fish in flask
\ \
\
\ ~
~,.....
20
10
0
i 10
I 20
I I ~1 30 40 9 Hours
I
50
60
Fig. 1, Effect of fish on DDT loss from incubation media. At time zero, each flask contained 750 ml of 0.1 ppm ~4C-DDT. 4 fish were added to each flask except in the no-fish controls. 1.0 ml aliquots were removed and analyzed at the indicated time intervals.
Effects o f DDT in Fundulus
341
T a b l e V I . DDT incorporation and distribution in Fundulus a (single 24-hr DDT dose) 1 day post DDT dose Tissue
ppm DDT
% of the absorbed dose in tissue
8 days post DDT dose ppm DDT
Intestine
5.11 • 0.88 b
7.83 +-- 1.80
Liver
4.91 •
8.70 --+ 1.33
2.92 • 0.33
8.06 • 0.95
Eggs e
0.25 • 0.09
3.02 •
0.57 • 0.12
10.92 • 2.60
9.33 •
42.85 • 2.28
Ovaries e
1.01
1.12
3.08 • 0.44
% of the absorbed dose in tissue
1.86
11.80 •
1.71
--
--
Testes
1.55 • 0.53
2.32 • 0.59
--
Brain
2.63 • 0.66
0.88 +-- 0 . I 0
1.74 • 0.21
1.00 • 0.13
Heart
2.07 • 0.55
0.23 • 0.04
0.97 • 0.12
0.24 • 0.04
Spleen
2.37 • 0.50
0.14 • 0.04
0.56 • 0.08
0.15 • 0.03
Gills
2.17 • 0.64
0.97 • 0.24
1.59 • 0.17
1.69 • 0.38
--
Muscle
1.50 • 0.36
--
0.83 • 0.06
--
Carcass
1.54 • 0.20
72.83 • 3.65
0.79 • 0.08
52.50 • 5.38
aDosage and assay performed as described in the text. bValues are mean • standard error for seven fish used in each study. eTissue assays on each fish were performed on either eggs or ovaries, never both from the same animal.
T a b l e V I I . D D T incorporation and distribution in Fundulus a (two 24-hr D D T doses) 8 days post DDT dose
1 day post DDT dose Tissue Intestine
ppm DDT
% of the absorbed dose in tissue
ppm DDT
14.24 -~ 4.42 b
18.07 • 3.80
% of the absorbed dose in tissue
2.57 - 0.30
5.69 __. 1.51
Liver
8.18 -4- 1.03
7.43 •
1.17
5.19 • 0.31
6.67 __. 0.33
Ovaries
6.80 • 0.82
15.20 •
1.12
10.40 • 3.00
25.16 • 6.29
Brain
3.59 • 0.60
0.77 • 0.10
3.07 • 0.53
0.78 • 0.14
Heart
2.39 • 0.18
0.17 • 0.03
1.82 - 0.42
0.20 • 0.04
Spleen
1.84 • 0.38
0.15 • 0.02
0.94 • 0.20
0.17 ___ 0.07
Gills
2.50 • 0.29
0.98 • 0,16
2.32 • 0.40
1.41 __. 0.33
Muscle
1.76 • 0.33
--
1.23 • 0.16
--
Carcass
1.73 • 0.09
55.58 • 2.90
1.35 • O. 15
56.17 __ 7.58
aDosage and assay performed as described in the text. bValues are mean -- standard error for five fish used in each study.
342
R. B. Crawford and A. M. Guarino
The major effect obtained from the double-exposure technique (Table VII), is that more DDT is absorbed. Note, for example, that five ppm was present in the intestine one day after exposure to one dose, whereas this same tissue had 14 ppm after two doses. It should be noted that a major portion of the labeled material is still present in the fish eight days following removal from the DDT-containing medium. The txl= (calculated graphically by extrapolation back to to) for carcass was three days after the single dose of DDT, and eight days after the double dosing (Table VIII). After single-day doses, these carcass values were about the same as are seen in this table for muscle, spleen and heart. The rather long tt/2 (nine days) for gills is interesting in that is suggests that this organ is a site of high binding and/or excretion of the pesticide. The h/z's for intestine, liver and brain also were pro'longed 9 to 12 days. The usual effect of the two-day dosing, was to prolong the tissue halftime. Thus, when compared with the double exposure, brain, heart, and gill t~/2's were three to four-fold longer than after the single exposure (see Table VIII). Liver, muscle and carcass values were approximately doubled after two exposures. The only tl/2 which decreased after the two 24-hour treatment _periods, was that of intestine, and it diminished by one-half. Thus it is obvious that this is a primary site of entry for DDT and that absorption from the intestine is very rapid, leading to a comparatively low concentration by Day 8 post-treatment. It is also of interest that in the relatively large organ, the intestine (ca. 250 mg) there is a decrease by a factor of one-half while the brain, heart, liver and gills (total weight of all four organs equals 290 rag) increased in half-time by a factor of two to three. The gill tz/~ of 43 days is best explained in terms of binding rather than excretion of the pesticide via this tissue. The prolonged tz12of brain (30 days) is of particular importance in light of the notorious CNS effects of chlorinated hydrocarbon pesticides (Wooley 1970).
Effects on reproduction and development. Having established the distribution of DDT and finding large quantities in the gonads, we became interested in the possible effects of this dosage on fertilization and embryogenesis. At least a range of concentration vs. effect of these functions might be established. Female Fundulus were given DDT dosage in the usual manner. Each flask contained four fish in 750 ml of sea water. The exposure was for 24 hr in 0.1 ppm 14C-DDT, followed by a 24-hr pesticide-free period and then another 24-hr period of 0.I ppm 14C-DDT. Two days after the second dose, eggs were collected from each fish and fertilized. The mass of eggs from each fish was incubated separately. Fertilization of all eggs was accomplished using aliquots from a common pool of minced testes. The quantity of DDT in the eggs was measured by determining the radioactivity of aliquots of eggs removed from the incubation medium. DDT content of eggs from pre-loaded .females ranged from approximately 0.1 to 0.3 ppm as seen in the footnote to Table IX. Development of these eggs was compared with controls from the time of fertilization to hatching (approximately 40 days). The fertilization percentage was unaffected by the DDT at the levels used in this experiment. However, the developmental rate was markedly reduced (Table IX). The DDT-containing embryos remained one or two stages behind the controls
343
Effects of DDT in Fundulus
Tissue half-times (t%) for 14C-DDT treated Fundulus a
Table V I I I .
t V2(Days)
Tissue
Intestine Liver Brain Heart Spleen Gills Muscle Carcass
Single 24-hr exposure
Double 24-hr exposure
12 I1 9 4 3 9 3 3
6 16 30 12 4 43 6 8
aValues calculated, to the nearest whole day, as described in text, from data in Tables VI and VII.
throughout the processes of blastulation, gastrulation, and neurulation. Only during the late stages of organogenesis and growth did the treated embryos " c a t c h - u p " with the controls. Development from that time on was apparently normal, even through hatching. In another series of experiments, eggs were fertilized in sea water containing DDT. Eggs were place in DDT suspensions (1.0 ppm and 10 ppm) for 30 min, sperm were added, and then the eggs were washed free of sperm with the appropriate DDT-containing sea water five min later. Under these conditions, the most dramatic effect was a major reduction in the fertilization rate (Table X). It should be noted that even in experiment No. 3, where fertilization was poor in the controls, the percentage was markedly reduced in the presence of DDT. Aliquots of eggs removed from the incubation media and washed four times with 100 ml of water were analyzed for 14C-DDT content. As seen in Table XI, the eggs 'were able to take up and concentrate the pesticide with both a time and dose dependency. The 24-hr tissue:media ratio was 12 when one ppm was used, while the ten ppm dose gave a ratio of about five suggesting saturation at the higher concentration. Those eggs which did fertilize showed a morphogenetic lag similar to that seen in the previous experiment (Table X). Discussion
In terms of the wet weight of Funduius tissue, the anlogs of DDT " n a t u r a l l y " occuring in the greatest concentrations were p , p ' - D D T and DDMU (Table I). The total DDT-like compounds present in captured Fundulus were 0.17 ppm and thus the quantities to which these fish were exposed in most of these experiments (0.1 ppm) were less than the background levels. These residue levels are approximately the
344
R. B. Crawford and A. M. Guarino
same (0.11 ppm) as were reported by Pritchard et al. (1973) in flounder. When expressed in terms of fat weight, the Fundulus had 8.72 ppm of total DDT analogs in fat. This represents a concentration factor of more than 50-fold and compares favorably with the factor reported for ambient levels in the lobster carcass (Guarino et al. 1974). In studying DDT and its metabolites in extracts of whole Fundulus, it was found that DDT itself is the dominant material, present at more than 93% (Table II) whether analyses were done one or eight days after exposure to ~4C-DDT. The most abundant metabolite occuring at the earlier time was DDD, as has been reported previously for other aquatic species (Guarino et al. 1974, Menzie 1972). After eight days, more DDE than any other metabolite is present, and this was shown previously in longer term experiments employing Gambusia affinis fish in a model ecosystem (Kapoor et al. 1970). Less of the polar metabolites (e.g., DDA) occurred in Fundulus ( < 1 % ) than has been reported to occur in flounder tissues after intravenous treatment (Pritchard et al. 1973). After completion of the dose range-finding experiment summarized in Table III, a variety of techniques were designed to increase the absorption of DDT without excessively increasing the mortality. The intermittent schedule (Table I V and V)
T a b l e IX.
Effect o f D D T on Fundulus development rate a Developmental stage b
Days postfertilization
Controls
DDT-containing eggs
1
8
7
4
17-18
16-17
6
20
19
8
24
23
11
25
25
18
28
28
38
hatching
hatching
aObtaining eggs, fertilization, and staging were carried out as described in the text. In this experiment, eggs from 30 DDT-treated females were used. Egg masses from each female were reared separately. Numbers of eggs per fish ranged from 34 to 250. DDT cbntent in eggs ranged from 0.051 ppm to 0.37 ppm with a mean of 0.16 ppm. DDTcontaining embryos were all at the same stage regardless of their position in the range of DDT content. Fertilization rate was over 95% for all eggs. bDevelopmental stage according to Oppenheimer (1937).
345
Effects of DDT in Fundulus
appeared to increase the survival over continuous exposure, while providing increased levels of absorption (Tables VI and VII). Looking at the data in further detail, it is noted that at 0.1 ppm the more susceptible animals died after the first two doses. Furthermore, it is observed that from Day 5 on, there was not much additional increase in mortality. A similar trend was seen at the 0.05 ppm dose except that by Day 15, there seemed to be about 50% less mortality than there was at the 0.1 level. These results suggested further evaluation of these data in terms of c x t. Since this technique is an expression of both concentration and duration of exposure, similar toxicity is expected at similar c x t values. In Table V, up to c x t = 0.2, the concept seems generally valid since up to this point there is a similar degree of toxicity. The only exception to this appears to be that early deaths occur in the more susceptible individuals. At the c x t values of 0.3 and 0.4, there is only half as much toxicity at the lower exposure
Table X.
Effects of DDT-media on fertilization of Fundulus eggs a % Fertilization
Experiment No.
Controls
1 ppm DDT
1
92
66
2
95
76
3
22
4
aAnalysis performed as described in the text. Eggs were considered fertilized when they developed to a two-cell stage. Fertilization percentage was determined by examination of 100 eggs per experimental group.
Table XI.
Uptake of 14C-DDT by Fundulus eggs a DDT in eggs (ppm)
Time postfertilization
0
1 ppm media
10 ppm media
0.85
3.49
15 min
1.41
8.78
2 hr
3.28
50.53
14.37
48.63
1 day
aAnalyses performed as described in the text. ~4Ccontent was determined on aliquots containing 25 eggs.
346
R. B. Crawford and A. M. Guarino
level as at the higher level. This deviance from the concept suggests the occurrence of a concentration plateau and/or that at the lower concentration of DDT, lifesparing redistribution is occurring. Some evidence for the latter can be cited from Table VI where the concentration of DDT in carcass decreased about 50% from 1.5 to 0.79 ppm after eight days while egg values doubled from 0.25 tO 0.57 ppm. Although this may be a mechanism for decreasing maternal mortality, the present studies show that delayed development (Table IX) and diminished fertility (Table X) can occur in the animals. It is certainly clear from the data in Figure 1 that Fundulus take up DDT from the media and do so at a very rapid rate. We recognize the problem in this type of study of adsorption onto glass and volatilization of the pesticide. However, the incorporation by the fish is rapid enough to minimize the adsorption-volatilization problem in pre-loading experiments. Observation of the rapid loss of DDT f r o m solutions containing no fish does lead to speculation regarding the concentration of DDT on solid surfaces in streams and open bodies of water. That is, in addition to the levels known to occur in water sediment and aquatic life, a long term storage shed for pesticides may reside on the rocks and other solid surfaces of the ocean bottoms and if accounted for, may increase the total quantities of pesticides potentially available in these waters. Although this observation has been made before, it remains unsubstantiated by direct and detailed analysis (Woodwell et al. 1971). Effects of DDT on developmental patterns in Fundulus are reassuring. At least at levels far in excess of those found in free swimming fish the only observed effect on development was. that of a slight delay. The hatched fry appeared to be normal. However, in the total picture of development, " n o r m a l c y " would have to include such things as sexual maturation and fertilizability of the gametes of successive generations. Such study is beyond the scope of the present investigation. It also should be noted that the delayed but otherwise normal development occured in eggs whose DDT concentration was higher than that which is compatible with life in the adult fish. Thus, the developing embryo appeared to be more resistant to DDT than the adult. The more dramatic observation Was that in DDT-containing media, successful fertilization of Fundulus eggs by sperm was markedly reduced. At this juncture we have no way of suggesting whether the effect was on the sperm or egg or both. Clearly, the eggs were able to concentrate DDT; we have no data yet to indicate the pesticide levels in the sperm, but the testicular levels were 1.6 ppm (Table VI). At least qualitatively one might speculate that a reduction of fertilizability in a population, where under normal circumstances few will survive to adulthood, could have a marked effect on the adult population in a few generations.
Effects of DDT in Fundulus
347
Acknowledgement The authors wish to acknowledge the technical assistance of Jacqueline B. Anderson, Patrick B. Briley and Marion A. Kinter.
References Acree, F., M. Beroza, and M. C. Bowman: Codistillation of DDT with water. Agr. Food Chem. 11, 278 (1963). Commoner, B.: The closing circle. A. A. Knopf, NY, p. 227 (1972). Crawford, R. B., C. E. Wilde, M. H. Heinemann, and F. J. Hendler: Morphogenetic disturbances from timed inhibitions of protein synthesis in Fundulus. J. Embryol. Exp. Morph. 29, 363 (1973). Edwards, C. A.: Persistent Pe.sticides in the Environment. Cleveland, CRC Press, p. 27 (1970). Gakstatter, J. H., and C. M. Weiss: The elimination of DDT-C 14, dieldrin-C 14, and lindane-C ~4 from a single sublethal exposure in aquaria. Trans. Amer. Fish. Soe. 96, 301 (1967). Guarino, A. M., J. B. Pritchard, J. B. Anderson, and D. P. Rail: Tissue distribution of ~4C-DDT inthe lobster after administration via intravascular or oral routes or after exposure from ambient sea water. Tox. Appl. Pharmacol. 28, 277 (1974). Holden, A. N.: A study of the absorption of ~4C-labelled DDT from water by fish. Ann. Appl. Biol. 50, 467 (1962). Kapoor, I. P., R. L. Metcalf, R. F. Nystrom, and G. K. Sangha: Comparative metabolism of methoxychlor, methichlor, and DDT in mouse, insects, and in a model ecosystem. Agr. Food Chem. 18, 1145 (1970). Menzie, C. M.: Fate of pesticides in the environment. Ann. Rev. Entomol. 17, 199 (1972). Oppenheimer, J. M.: The normal stages ofFundulus heteroclitus. Anat. Rec. 68, 1 (1937). Pesticide Analytical Manual. Dept. of Health, Education and Welfare, Food and Drug Administration, Washington, D.C. Revised ed. 2 (1968). Pritchard, J. B., A. M. Guarino, and W. B. Kinter: Distribution metabolism and excretion of DDT and mirex by a marine teleost, the winter flouder, Environ. Health Persp. 4, 45 (1973). Risebrough, R. W.: Chlorinated hydrocarbons in marine ecosystems. In: Chemical Fallout, M. W. Miller and G. G. Berg, Eds. Springfield: C. C. Thomas, pp. 5-23 (1968). Whitacre, D. M., C. C. Roan, and G. W. Ware: Pesticides and aquatic microorganisms. Search 3, 150 (1972).
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Wilber, C. G.: The Biological Aspects of Water Pollution. Springfield: C. C. Thomas, pp. 31-32 (1969). Woodwell, G. M., P. P. Craig, and H. A. Johnson: DDT in the biosphere: where does it go? Science 174, 1101 (1971). Wooley, D. Effect of DDT on the nervous system of the rat. In: Biological Impact of Pesticides in the Environment. J. W. Gillette, Ed. Oregon State Press. p. 114 (1970).
Manuscript received November 11, 1974; accepted June 20, 1975
