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Uptake and release of p-dichlorobenzene in early life stages of Salmo gairdneri
ECOTOXICOLOGY
AND
ENVIRONMENTAL
SAFETY
6, 439-447
(1982)
Uptake and Release of pDichlorobenzene Early Life Stages of Salmo gairdneri S. GALASSI,* *Istituto
di Ricerca
D. CALAMARI,*
sulle Acque. CNR, Ecologia, Universitd
AND F. SETTI~
Brugherio, Milan, and tlstituto degli Studi di Milano. Milan,
Received
February
in
di Zoologia, Italy
Cattedra
di
1, 1982
Early life stages of Salmo gairdneri from eggs to alevins were exposed to p-dichlorobenzene (p-DCB) in a series of short-term uptake and release studies and in a long-term continuous test at three different concentrations from 3 to 79 fig/liter. Water concentration was frequently checked and the concentration of p-DCB in eggs and alevins was determined by gas chromatographic analysis of hexane extracts. Total and neutral lipids in the different stages were also determined. Neither macroscopic malformations nor histological changes were observed at hatching. The highest concentration (about l/lOth of the incipient lethal level for alevins of S. gairdneri) did not show any significant difference in mortality compared to the control. Bioco’ncentration factors (BCF) experimentally determined on alevins agreed with the theoretical ones, calculated on the basis of water solubility, whereas higher contents of p-DCB up to one order of magnitude were observed in some stages before the hatching. The hypothesis relating accumulation to lipid content was tested; higher concentration factors (CF) of p-DCB were found in stages with higher lipid levels, particularly eggs. Metabolic modifications, occurring during the hatching, on the other hand, increase the rate of release in spite of the still high lipid content at this stage. Kinetic constants of uptake and release were determined for eggs and alevins. It is concluded that more attention has to be paid to compounds with medium theoretical BCF to evaluate the effective accumulation potential of early life stages as in general compounds with very high BCF have a slow rate of uptake.
INTRODUCTION The rlelevance of investigations into developmental and young stages of fish to the evaluation of the deleterious consequences of pollution has been recognized and the importance of these studies in establishing water quality criteria for aquatic life has been stressed by several authors (McKim, 1977; Calamari and Marchetti, 1978). Nevertheless studies on bioaccumulation of organic substances have been carried out mainly on adult fish, with few exceptions (Korn and Stanley, 1981). Bioaccumulation of organic substances in fish can be predicted on the basis of their physical and chemical characteristics (Neely et al., 1974). This has been verified in a number of cases and the margin of error is relatively limited (Veith et al., 1979). Organic substances are stored mainly in the deposited fat of the organisms and significant correlations between pesticide residues and lipid content were found in some species of fish (Earnest and Benville, 197 1; Keck and Raffenot, 1979). As it is well known that lipid content in developmental stages of fish is higher than in alevins or adult fish it can be expected that early life stages accumulate higher quantities of chemicals with a potential for chronic effects. In order to test this hypothesis and explore the possible differences in the manner of uptake and release during various stages of development a series of tests was set up to study accumulation of para-dichlorobenzene (p-DCB) in Salmo guirdneri early 439
Ol47-6513/82/050439-09$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of roprcduction in any form reserved.
440
GALAS%,
CALAMARI,
AND
SETTI
life stages. p-DCB, on which an ample report was recently published (Jori et al., 1982) was selected as it ( 1) has a very high production volume, (2) has been frequently detected in aquatic environment, (3) has been included in several lists of substances to be studied with priority, and (4) is part of a wider research project on chlorobenzenes carried out at the Water Research Institute in Italy. Some results on toxicity and biodegradability of p-DCB in an aquatic environment have already been published (Calamari et al., 1982). MATERIALS
Toxicological
AND
METHODS
Tests
Long-term tests and short-term uptake and release tests were performed in continuous-flow tanks (600 ml/min) at 10-l 2°C. The toxicant was added to the water before it entered the tanks, by means of a multichannel peristaltic pump with Tygon tubes of different diameters dipping in a big reservoir of water saturated with pDCB. By this system almost stable concentrations of p-DCB in the treatment water were kept. They were 12.4 and 3.2 pg/liter in the first continuous series; 79.5, 16.4, and 3.6 pg/liter in the second continuous series; and 65.6, 13.4, and 4.5 pg/liter in the short-term series with standard errors from 7 to 15%. At each stage lo-20 specimens from each concentration were removed from the long-term series for the analysis of p-DCB. At hatching 25 embryos from each concentration were fixed in Bouin’s fluid for histological examination, according to the procedure described by Calamari et al. ( 198 1). Short-term uptake studies were performed exposing about 100 untreated specimens at different developmental stages for 48 hr and then leaving them in clean water for a 24-hr release phase. The p-DCB contents were measured at 24 and 48 hr of exposure and after a 24-hr release. A last experiment was carried out exposing alevins (2-3 cm) to concentrations very similar to those employed in the previous tests for the same period of uptake and release but with a more frequent sampling.
Analytical
Methods
The p-DCB concentration in water was determined at the beginning of the experiments and was frequently checked during and at the end of the tests. Data always refer to observed mean concentrations. Quantitative analysis of p-DCB was carried out on gas chromatographic equipment: a Hewlett-Packard 5750 with a flame ionization detector (FID) and a Fractovap C. Erba 4200 with a 63Ni electron-capture detector (ECD). Aqueous solution (4 ~1) containing more than 0.5 mg/liter of p-DCB was injected directly into the FID-equipped gas chromatograph on a glass column of 2 m X 3 mm i.d. at 110°C packed with 3% SE30 on Gas Chrom W, loo-120 mesh. More diluted solutions were extracted with pesticide-grade n-hexane (1:25) and the extracts were analyzed by the ECD-equipped gas chromatograph with the same stationary phase at the same temperature. The detection limit was 0.002 mg/liter of p-DCB in the initial aqueous solution. Cleaned-up tissue extracts were analyzed by the same technique (ECD detector) and the detection limit in this case was 100 pg of p-DCB/kg of wet tissue. The following method was adopted to determine the levels of p-DCB in eggs and
p-DICHLOROBENZENE
IN Salmo
441
guirdneri
embryos: l-2 g of wet tissue was homogenized with 5 g of anhydrous Na2S04 and 5 ml pesticide-grade n-hexane. Extraction was repeated two or three times with smaller volumes of solvent. Pooled extracts were filtered onto filter paper and diluted to 10 ml. A 2-ml portion of this extract was passed through a Florisil column (2 cm X 0.7 cm i.d.) and eluted with n-hexane. The first 4 ml of eluate were collected for gas-chromatographic analyses. A single extraction procedure gave a 90% recovery for concentrations from 100 to 25,000 pg/kg wet tissue. Higher concentrations required further extractions. To determine neutral lipids 100 specimens were collected in duplicate from the control tank and weighed, and anhydrous Na$O., was added and then blended for 3-4 min. The homogenate was transferred into a Soxhlet apparatus and extracted with n-hexane for 8 hr. Neutral lipids were determined weighing the residue after complete hexane removal. Total lipids were extracted with a similar procedure using chloroform:methanol (2:1, v/v) as solvent. Before evaporating the solvents the extracts were cleaned up from nonlipid substances, according to Folck et al. (1956).
Kinetic
Model
The model utilized was that currently described in the literature (Rescigno and Segre, 1966) and based on the assumption that uptake and release of a substance in fish from and to water can be described by a two-compartment model: KI G-2g cr. Assuming first-order system is
kinetics
the closed form of the initial
differential
equation
cf = $ c,( 1 - epK2’), 2
where K, /K2 - c, = c, is the asymptotic factorily (describes the phenomenon
concentration
cs K, -=-= CW
in fish. If the model satis-
BCF.
K2
This model was applied to the results of the continuous-exposure test before the hatching, expressing data on the basis of lipid content. Not only changes in lipids but also deep metabolic modifications occur after the hatching stage, not permitting an extention of the same model. A second computation was made on the result of short-term exposure of alevins. The model was not applied to short-term uptake and release tests on different developmental stages due to the limited number of experimental points. Uptake, in this case, was expressed as CF (not equilibrium concentration factors) and release was expressed as percentage of p-DCB disappearance. RESULTS The lipid contents in early life stages of S. gairdneri are shown in Table 1. The correlation between neutral and total lipids is significant (P > 95%) and the straight-
442
GALAS%,
CALAMARI, TABLE
WET
WEIGHTS
AND
LIPID
CONTENTS OF
Days after fertilization 2 8 20 32 41 50 56
SETTI
1
OF DIFFERENT AT 10°C
DEVELOPMENTAL
STAGES
S. gairdneri
Neutral lipids (So)
Total lipids (%)
73.9 76.3 75.1 65.5 86.2
3.4 2.8 4.0 4.7 3.8
4.3 8.1 7.6 -
108.0
2.4
3.6
109.6
1.7
2.3
Wet weight per specimen (mid
Developmental stage Egg Egg Eyed
AND
egg
Hatching Adsorbed half-yolk Not completely adsorbed yolk Alevin
line equation isy = - 1.0687 + 1.9989x; therefore computations involving lipid content were made on p-DCB concentrations in neutral lipids. The relationship between developmental stagesand p-DCB content in total body weight for continuous exposure is shown in Fig. 1. The test was repeated twice in different years. The highest concentration was tested only once. A peak of uptake is evident during the hatching phase; the maximum was clearly identified in the first experiment. The accumulation pathway is very similar for comparable concentrations. Eating stages accumulated the compound at very low levels in comparison to eggs. Mortality lower than 30% was identical in all the treatments as in the control. Histological examination of embryos at the hatching did not reveal any abnormality.
0
0
‘-\iP-T ‘\
,T !i ! 1 I i
,/ .:
\ i
.\ u?,
.!
..I/ ._--.H 1s 23 1 0-o (A) (0)
40 5055 e
I
_. --L.-
‘\ ,
20
32 41 5056
093
-
days
FIG. 1. (I) First series of long-term uptake tests of p-DCB from eggs to alevins at two concentrations: 12.4 and(m) 3.2 pg/liter at 12’C. (II) Second series of long-term uptake tests at three concentrations: 79.5, (A) 16.4, and (m) 3.6 *g/liter at 10°C.
p-DICHLOROBENZENE
2
(0)
6
FIG. 2. Short-term uptake and release 73.2. (A) 14.6, and (u) 3.0 rg/liter.
12
IN Salmo
443
gairdneri
f
I 24
tests on alevins
4324 hours
at three
different
concentrations
of p-DCB:
Uptake and release of p-DCB in alevins 2-3 cm long are shown in Fig. 2. It is to be noted that the release curves are particulary sharp and that the levels of uptake are not high. Concentration factors after 24- and 48-hr exposures at three different levels for various developmental stages are reported in Table 2, together with the percentage of release after 24 hr in clean water. The right part of the table refers to the results of the experiment as in Fig. 2, since the alevin stage was tested twice. Eggs until the hatching stage have a particular inability to release the p-DCB. Immediately after the hatching, uptake and release are notably more rapid than in the previous stages. A comparison between different toxicokinetic parameters is given in Table 3 for alevins and eggs. Data for eggs are from the continuous-exposure experiment; data for alevins are from the 48-hr exposure test. Wide differences are evident for all the parameters considered: cf, K,, K2, and t,,>. However, the referred theoretical asymptotic concentration can never be reached by eggs as the time required is too long. At the hatching only 50% of the possible maximum was attained. Concentration factors in the different developmental stages, expressed on the basis of wet weight, for continuous, 24- and 48-hr uptake tests are reported in Fig. 3. Only values calculated for the highest concentration of exposure are shown to avoid visual confusion. However, the trend for analogous experiments at lower concentrations was very similar. DISCUSSION Theoretical calculations indicate a low level of BCF for p-DCB, according to the equations proposed by Chiou et al. (1977) and Kenaga (1980). They are 105 and 52, respectively. Experimental values for adult fish are 60 (Barrow et al., 1980) and 212 (Neely et al., 197,4). BCFs found in the present work for alevins are around 50 and therefore approximately in agreement with both calculated and experimental values.
a Days after fertilization. b From a second test.
134.0 168.7 4.2
69.4 83.9 0
24.8 0
24 hr CF 48 hr CF 24 hr (% release)
4.5
0.5
43.4
23.5
24 hr (% release)
12.1 122.3 152.3
29.4 67.5 138.1
13.4
24 hr (% release) 24 hr CF 48
16.6 30.1 55.1
65.6
81.7 106.4
Hatching (32 days)
39.3 98.5
Eyed egg (20 days)
DISAPPEARANCE
2
12.0 32.1
Egg (8 days)
FACTORS AND PERCENTAGE OF p-DCB
24 hr CF 48 hr CF
CONCENTRATION
TABLE
130.8 133.9 100
70.2 146.1 72.0
49.2 49.2 100
95.2 63.4 100
155.0 150.3 100
48.0 40.1 100
Alevin (56 days) 138.4 96.6 86.7
Not completely adsorbed yolk (SO days)
37.3 48.0 100
42.8 44.9 100
56.5 48.4 100
Alevinb
UPTAKE AND RELEASE TESTS AT 10°C
79.3
81.8 220.0 111.9
118.1 188.0
Adsorbed half-yolk (41 days)
Stage
FROM SHORT-TERM
5 Lo 2 =!
E -;
2 y
% E .-
Q
p-DICHLOROBENZENE
IN Salmo
TABLE KINETIC
CONSTANTS AND AND RELEASE
445
guirdneri
3 FOR p-DCB
ASYMPTOTIC CONCENTRATIONS IN EGGS AND ALEVINS OF
FROM CONTINUOUS-EXPOSURE
TESTS AT 10°C Alevins
Eggs (rgf;iter) 19.5 16.4
(pg/k~lipicl) 1355 x 10’ 303x10’
UPTAKE
S. gairdneri,
K, (hr-I) 22 26
K2 (hr-I)
:A:)
0.0013 0.0014
531 493
(rg$er) 13.2 14.6
(pg/k;lipid)
K, (hr-I)
K2 (hr-‘)
(rl?)
460 390
0.17 0.11
4 6
IO3
200 x 52 x
lo3
The kinetic constants for alevins are very similar to those found in the literature for adult rainbow trout. If the K, for alevins in Table 3 are transformed into values for wet weight instead of lipid content, they result in 7.76 and 6.58 hr~’ whereas Neely et (21.(1974) report 5.76 hr-‘. The K2, which does not need any transformation, being identical for both fresh and fat weights, are 0.17 and 0.11 compared to 0.026 found by Neely et al., (1974). This limited difference also influences the half-time of release which is 4-6 hr for alevins and 15 hr for adults. Kinetic constants and bioaccumulation factors for alevins are also very similar to those found by Konemann and Van Leeuwen ( 1980) for Poecilia reticulata. Comparisons cannot be: made for bioaccumulation of p-DCB in developmental stages as no research was carried out on this subject. The potential of bioaccumulation is quite
2
I 0
I 20
32
Cl
41
50
1 56
r=D
77
days
FIG. 3. Concentration factors of p-DCB in different developmental stages of S. tinuous pg/liter.
long-term
test at 79.5 @g/liter,
(A)
4%hr
uptake
at 65.6 @g/liter,
gairdneri:
and (m) 24-hr
uptake
(0)
con-
at 65.6
446
GALAS%
CALAMARI,
AND
SETTI
high, particularly at the hatching stage. In one case a CF as high as 1000 was observed (Fig. 1, first long-term exposure). For most of the cases the ratios of accumulation in critical developmental stages in regard to alevins range from 6 to 20. Moreover the ability to release is very poor in all the prehatching stages. On the contrary uptake and release are quite fast in the posthatching stages (Table 2). A greater potential for accumulation in eggs in regard to posthatching stages is still evident even if the concentrations of p-DCB are expressed on the basis of lipid content. A theoretical BCF of 17,000 can be calculated for eggs, compared to 3000 for alevins. Despite the high levels of accumulation of p-DCB in eggs and other early life stages neither teratogenic effects nor mortality, higher than in the control tanks, was observed. It is to be noted that the highest concentration employed for the 60day treatment in eggs and alevins is around 0.1 of the incipient lethal level (ILL) found in the 14-day test with alevins by Calamari et al., (1982). A substance that does not cause any evident damage on the very sensitive developmental stages at 1/lOth of the threshold could not be considered as high risk even if it must be classified as “toxic” according to GESAMP (1969), being the acute LCsO in the range l-10 mg/liter for several aquatic organisms (Calamari et al., 1982). These findings confirm that p-DCB is a molecule that apparently would not cause great concern in aquatic environments, notwithstanding the fact that it is widely diffused in biotic and abiotic compartments as already stated by Jori et al. (1982). Considering the result of the tests from a methodological point of view several considerations can be drawn: -Developmental stages of fish are very useful for bioaccumulation studies as well as for mortality and teratogenicity. -Certain stages accumulate more than others having a low release capacity. -The particular substance tested here reached CF up to 1000 in developmental stages when theoretical BCF is around 100; BCFs calculated from the n-octanol/ water partition coefficient, reliable for adult fish, are therefore not applicable to early life stages. -Even if data refer to lipid content, a higher possibility of assumption has been demonstrated in eggs by means of a toxicokinetic model. -There is a need to explore the possibility of prediction of accumulation also for developmental stages of fish; on the basis of the results obtained in the present study it seems more advantageous to test a limited number of key stages (eyed eggs, hatching) with short-term exposures and frequent sampling; kinetic constants and steady-state concentrations can be calculated applying the toxicokinetic models. -It is necessary in this kind of study to separate the thermodynamic point of view (maximum bioaccumulation potential at the steady state) from the kinetic aspects because of the deep biological modifications involved in the developmental stages. Linear inverse relationships between log K,,, and K2 were found for homologous series of organic molecules (Zitko, 1980; Kijnemann and Van Leeuwen, 1980) indicating a very slow accumulation rate for molecules with very high BCF. These kinds of molecules are expected to accumulate to a limited extent in early fife stages owing to the limited exposure time. Considering the possibility of extending these studies to other organic molecules
p-DICHLOROBENZENE
IN
Salmo
gairdneri
447
and assuming that compounds with BCF more than 200-300 are potentially suspicious for bioaccumulation problems (Zitko, 1980), attention should be given to molecules with medium BCF due to their quick uptake. REFERENCES BARROW, M. E., PETROCELLI, S. R., MACEK, K. J., AND CARROLL, J. J. (1980). Bioconcentration and elimination of selected water pollutants by bluegill sunfish (Lepormis machrochirus). In Dynamics, Exposure and Hazard Assessment of Toxic Chemicals (R. Hague, ed.). Ann Arbor Science, Ann Arbor, Mich. CALAMARI, D., GALASSI, S., AND SETTI, F. (1982). Evaluating the hazard of organic substances on aquatic lil‘e: The paradichlorobenzene example. Ecotoxicol. Environ. Saf: 6, 369-378. CALAMARI, D., AND MARCHETTI, R. (I 978). Relevance of studies on developmental and young stages of Salmo gairdneri in establishing water quality criteria for fisheries. Ber. Umwelfbundesamt (Ger) IO, 201-210. CALAMARI, D., MARCHETTI, R., AND VAILATI, G. (1981). Effects of long term exposure to ammonia on developmental stages of rainbow trout (Salmo gairdneri Rich). Rapp. P.-v. R&n. Cons. inc. Explor. Mer. 178, 81-86. CHIOU, C. T., FREED, V. H., SCHMEDDING, D. W.. AND KOHNERT, R. L. (1977). Partition coefficient and bioaccumulation of selected organic chemicals. Environ. Sri. Technol. 5, 475-478. EARNEST. R. D., AND BENVILLE, P. E. (1971). Correlation of DDT and lipid levels for certain San Francisco bay fish. Pestic. Monif. J. 5, 235. FOLCK, J.. LEES, M., AND SLOONE STANLEY, G. H. (1956). A simple method for the isolation and purification of total lipids from animal tissue. J. Biol. Chem. 226, 497-509. GESAMP (I 969). Abstract of the report of the first session. Wafer Res. 3, 995. JORI, A., CALI\MARI. D., CATTABENI, F.. DI DOMENICO, A., GALLI, C. L., GALLI, E., RAMUNDO, A., AND SILANO, V. (1982). Ecotoxicological profile of p-dichlorobenzene (p-DCB). EcoroxicoL Environ. So/: 6, 413-432. KECK, G., AND RAFFENOT, J. ( 1979). Etude &co-toxicologique de la contamination chimique par les PCB dam la riviere du Furaus (Aiu). Rev. Med. Vet. 130, 339-358. KENAGA, E. E. (1980). Predicted bioconcentration factors and soil sorption coefficients of pesticides and other chemicals. Ecotoxicol Environ. Saf: 4, 26-38. K~NEMANI\, H.. AND VAN LEEUWEN. K. (1980). Toxicokinetics in fish: Accumulation and elimination of six chlorobenzenes by guppies. Chemosphere 9, 3319. KORN, S.. AND STANLEY, R. (1981). Sensitivity to, and accumulation and depuration of, aromatic petroleum components by early life stages of coho salmo Oncorhynchus kisutch. Rapp. P.-v. R&n. Cons. int. E.uplor. Mu. 178, 65571. MCKIM. J. M. (1977). Evaluation of tests with early life stages of fish for predicting long-term toxicity. J. Fish. Res. Board Canad. 34, I I48- 1 154. NEELY, W. B., BRANSON, D. R.. AND BLAU, G. E. (1974). Partition coefficient to measure bioconcentration potential of organic chemicals in fish. Environ. Sci. Technol. 13, 1 I 13-l 1 15. RESCIGNO. A., AND SEGRE, G. ( 1966). Drug and Tracer Kinetics. Blaiswell Publishing Co.. Waltham, Mass. VMTH, G. D.. DE FEO, D. L.. AND BERGSTEDT, B. V. (1979). Measuring and estimating the bioconcentration factor of chemicals in fish. J. Fish. Res. Board Cunad. 36, 1040-1047. ZITKO, V. (1980). Relationships governing the behavior of pollutants in aquatic ecosystems and their use in risk assessment. Canad. Tech. Rep. Aquar. Sci. 975, 243-265.
AND
ENVIRONMENTAL
SAFETY
6, 439-447
(1982)
Uptake and Release of pDichlorobenzene Early Life Stages of Salmo gairdneri S. GALASSI,* *Istituto
di Ricerca
D. CALAMARI,*
sulle Acque. CNR, Ecologia, Universitd
AND F. SETTI~
Brugherio, Milan, and tlstituto degli Studi di Milano. Milan,
Received
February
in
di Zoologia, Italy
Cattedra
di
1, 1982
Early life stages of Salmo gairdneri from eggs to alevins were exposed to p-dichlorobenzene (p-DCB) in a series of short-term uptake and release studies and in a long-term continuous test at three different concentrations from 3 to 79 fig/liter. Water concentration was frequently checked and the concentration of p-DCB in eggs and alevins was determined by gas chromatographic analysis of hexane extracts. Total and neutral lipids in the different stages were also determined. Neither macroscopic malformations nor histological changes were observed at hatching. The highest concentration (about l/lOth of the incipient lethal level for alevins of S. gairdneri) did not show any significant difference in mortality compared to the control. Bioco’ncentration factors (BCF) experimentally determined on alevins agreed with the theoretical ones, calculated on the basis of water solubility, whereas higher contents of p-DCB up to one order of magnitude were observed in some stages before the hatching. The hypothesis relating accumulation to lipid content was tested; higher concentration factors (CF) of p-DCB were found in stages with higher lipid levels, particularly eggs. Metabolic modifications, occurring during the hatching, on the other hand, increase the rate of release in spite of the still high lipid content at this stage. Kinetic constants of uptake and release were determined for eggs and alevins. It is concluded that more attention has to be paid to compounds with medium theoretical BCF to evaluate the effective accumulation potential of early life stages as in general compounds with very high BCF have a slow rate of uptake.
INTRODUCTION The rlelevance of investigations into developmental and young stages of fish to the evaluation of the deleterious consequences of pollution has been recognized and the importance of these studies in establishing water quality criteria for aquatic life has been stressed by several authors (McKim, 1977; Calamari and Marchetti, 1978). Nevertheless studies on bioaccumulation of organic substances have been carried out mainly on adult fish, with few exceptions (Korn and Stanley, 1981). Bioaccumulation of organic substances in fish can be predicted on the basis of their physical and chemical characteristics (Neely et al., 1974). This has been verified in a number of cases and the margin of error is relatively limited (Veith et al., 1979). Organic substances are stored mainly in the deposited fat of the organisms and significant correlations between pesticide residues and lipid content were found in some species of fish (Earnest and Benville, 197 1; Keck and Raffenot, 1979). As it is well known that lipid content in developmental stages of fish is higher than in alevins or adult fish it can be expected that early life stages accumulate higher quantities of chemicals with a potential for chronic effects. In order to test this hypothesis and explore the possible differences in the manner of uptake and release during various stages of development a series of tests was set up to study accumulation of para-dichlorobenzene (p-DCB) in Salmo guirdneri early 439
Ol47-6513/82/050439-09$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of roprcduction in any form reserved.
440
GALAS%,
CALAMARI,
AND
SETTI
life stages. p-DCB, on which an ample report was recently published (Jori et al., 1982) was selected as it ( 1) has a very high production volume, (2) has been frequently detected in aquatic environment, (3) has been included in several lists of substances to be studied with priority, and (4) is part of a wider research project on chlorobenzenes carried out at the Water Research Institute in Italy. Some results on toxicity and biodegradability of p-DCB in an aquatic environment have already been published (Calamari et al., 1982). MATERIALS
Toxicological
AND
METHODS
Tests
Long-term tests and short-term uptake and release tests were performed in continuous-flow tanks (600 ml/min) at 10-l 2°C. The toxicant was added to the water before it entered the tanks, by means of a multichannel peristaltic pump with Tygon tubes of different diameters dipping in a big reservoir of water saturated with pDCB. By this system almost stable concentrations of p-DCB in the treatment water were kept. They were 12.4 and 3.2 pg/liter in the first continuous series; 79.5, 16.4, and 3.6 pg/liter in the second continuous series; and 65.6, 13.4, and 4.5 pg/liter in the short-term series with standard errors from 7 to 15%. At each stage lo-20 specimens from each concentration were removed from the long-term series for the analysis of p-DCB. At hatching 25 embryos from each concentration were fixed in Bouin’s fluid for histological examination, according to the procedure described by Calamari et al. ( 198 1). Short-term uptake studies were performed exposing about 100 untreated specimens at different developmental stages for 48 hr and then leaving them in clean water for a 24-hr release phase. The p-DCB contents were measured at 24 and 48 hr of exposure and after a 24-hr release. A last experiment was carried out exposing alevins (2-3 cm) to concentrations very similar to those employed in the previous tests for the same period of uptake and release but with a more frequent sampling.
Analytical
Methods
The p-DCB concentration in water was determined at the beginning of the experiments and was frequently checked during and at the end of the tests. Data always refer to observed mean concentrations. Quantitative analysis of p-DCB was carried out on gas chromatographic equipment: a Hewlett-Packard 5750 with a flame ionization detector (FID) and a Fractovap C. Erba 4200 with a 63Ni electron-capture detector (ECD). Aqueous solution (4 ~1) containing more than 0.5 mg/liter of p-DCB was injected directly into the FID-equipped gas chromatograph on a glass column of 2 m X 3 mm i.d. at 110°C packed with 3% SE30 on Gas Chrom W, loo-120 mesh. More diluted solutions were extracted with pesticide-grade n-hexane (1:25) and the extracts were analyzed by the ECD-equipped gas chromatograph with the same stationary phase at the same temperature. The detection limit was 0.002 mg/liter of p-DCB in the initial aqueous solution. Cleaned-up tissue extracts were analyzed by the same technique (ECD detector) and the detection limit in this case was 100 pg of p-DCB/kg of wet tissue. The following method was adopted to determine the levels of p-DCB in eggs and
p-DICHLOROBENZENE
IN Salmo
441
guirdneri
embryos: l-2 g of wet tissue was homogenized with 5 g of anhydrous Na2S04 and 5 ml pesticide-grade n-hexane. Extraction was repeated two or three times with smaller volumes of solvent. Pooled extracts were filtered onto filter paper and diluted to 10 ml. A 2-ml portion of this extract was passed through a Florisil column (2 cm X 0.7 cm i.d.) and eluted with n-hexane. The first 4 ml of eluate were collected for gas-chromatographic analyses. A single extraction procedure gave a 90% recovery for concentrations from 100 to 25,000 pg/kg wet tissue. Higher concentrations required further extractions. To determine neutral lipids 100 specimens were collected in duplicate from the control tank and weighed, and anhydrous Na$O., was added and then blended for 3-4 min. The homogenate was transferred into a Soxhlet apparatus and extracted with n-hexane for 8 hr. Neutral lipids were determined weighing the residue after complete hexane removal. Total lipids were extracted with a similar procedure using chloroform:methanol (2:1, v/v) as solvent. Before evaporating the solvents the extracts were cleaned up from nonlipid substances, according to Folck et al. (1956).
Kinetic
Model
The model utilized was that currently described in the literature (Rescigno and Segre, 1966) and based on the assumption that uptake and release of a substance in fish from and to water can be described by a two-compartment model: KI G-2g cr. Assuming first-order system is
kinetics
the closed form of the initial
differential
equation
cf = $ c,( 1 - epK2’), 2
where K, /K2 - c, = c, is the asymptotic factorily (describes the phenomenon
concentration
cs K, -=-= CW
in fish. If the model satis-
BCF.
K2
This model was applied to the results of the continuous-exposure test before the hatching, expressing data on the basis of lipid content. Not only changes in lipids but also deep metabolic modifications occur after the hatching stage, not permitting an extention of the same model. A second computation was made on the result of short-term exposure of alevins. The model was not applied to short-term uptake and release tests on different developmental stages due to the limited number of experimental points. Uptake, in this case, was expressed as CF (not equilibrium concentration factors) and release was expressed as percentage of p-DCB disappearance. RESULTS The lipid contents in early life stages of S. gairdneri are shown in Table 1. The correlation between neutral and total lipids is significant (P > 95%) and the straight-
442
GALAS%,
CALAMARI, TABLE
WET
WEIGHTS
AND
LIPID
CONTENTS OF
Days after fertilization 2 8 20 32 41 50 56
SETTI
1
OF DIFFERENT AT 10°C
DEVELOPMENTAL
STAGES
S. gairdneri
Neutral lipids (So)
Total lipids (%)
73.9 76.3 75.1 65.5 86.2
3.4 2.8 4.0 4.7 3.8
4.3 8.1 7.6 -
108.0
2.4
3.6
109.6
1.7
2.3
Wet weight per specimen (mid
Developmental stage Egg Egg Eyed
AND
egg
Hatching Adsorbed half-yolk Not completely adsorbed yolk Alevin
line equation isy = - 1.0687 + 1.9989x; therefore computations involving lipid content were made on p-DCB concentrations in neutral lipids. The relationship between developmental stagesand p-DCB content in total body weight for continuous exposure is shown in Fig. 1. The test was repeated twice in different years. The highest concentration was tested only once. A peak of uptake is evident during the hatching phase; the maximum was clearly identified in the first experiment. The accumulation pathway is very similar for comparable concentrations. Eating stages accumulated the compound at very low levels in comparison to eggs. Mortality lower than 30% was identical in all the treatments as in the control. Histological examination of embryos at the hatching did not reveal any abnormality.
0
0
‘-\iP-T ‘\
,T !i ! 1 I i
,/ .:
\ i
.\ u?,
.!
..I/ ._--.H 1s 23 1 0-o (A) (0)
40 5055 e
I
_. --L.-
‘\ ,
20
32 41 5056
093
-
days
FIG. 1. (I) First series of long-term uptake tests of p-DCB from eggs to alevins at two concentrations: 12.4 and(m) 3.2 pg/liter at 12’C. (II) Second series of long-term uptake tests at three concentrations: 79.5, (A) 16.4, and (m) 3.6 *g/liter at 10°C.
p-DICHLOROBENZENE
2
(0)
6
FIG. 2. Short-term uptake and release 73.2. (A) 14.6, and (u) 3.0 rg/liter.
12
IN Salmo
443
gairdneri
f
I 24
tests on alevins
4324 hours
at three
different
concentrations
of p-DCB:
Uptake and release of p-DCB in alevins 2-3 cm long are shown in Fig. 2. It is to be noted that the release curves are particulary sharp and that the levels of uptake are not high. Concentration factors after 24- and 48-hr exposures at three different levels for various developmental stages are reported in Table 2, together with the percentage of release after 24 hr in clean water. The right part of the table refers to the results of the experiment as in Fig. 2, since the alevin stage was tested twice. Eggs until the hatching stage have a particular inability to release the p-DCB. Immediately after the hatching, uptake and release are notably more rapid than in the previous stages. A comparison between different toxicokinetic parameters is given in Table 3 for alevins and eggs. Data for eggs are from the continuous-exposure experiment; data for alevins are from the 48-hr exposure test. Wide differences are evident for all the parameters considered: cf, K,, K2, and t,,>. However, the referred theoretical asymptotic concentration can never be reached by eggs as the time required is too long. At the hatching only 50% of the possible maximum was attained. Concentration factors in the different developmental stages, expressed on the basis of wet weight, for continuous, 24- and 48-hr uptake tests are reported in Fig. 3. Only values calculated for the highest concentration of exposure are shown to avoid visual confusion. However, the trend for analogous experiments at lower concentrations was very similar. DISCUSSION Theoretical calculations indicate a low level of BCF for p-DCB, according to the equations proposed by Chiou et al. (1977) and Kenaga (1980). They are 105 and 52, respectively. Experimental values for adult fish are 60 (Barrow et al., 1980) and 212 (Neely et al., 197,4). BCFs found in the present work for alevins are around 50 and therefore approximately in agreement with both calculated and experimental values.
a Days after fertilization. b From a second test.
134.0 168.7 4.2
69.4 83.9 0
24.8 0
24 hr CF 48 hr CF 24 hr (% release)
4.5
0.5
43.4
23.5
24 hr (% release)
12.1 122.3 152.3
29.4 67.5 138.1
13.4
24 hr (% release) 24 hr CF 48
16.6 30.1 55.1
65.6
81.7 106.4
Hatching (32 days)
39.3 98.5
Eyed egg (20 days)
DISAPPEARANCE
2
12.0 32.1
Egg (8 days)
FACTORS AND PERCENTAGE OF p-DCB
24 hr CF 48 hr CF
CONCENTRATION
TABLE
130.8 133.9 100
70.2 146.1 72.0
49.2 49.2 100
95.2 63.4 100
155.0 150.3 100
48.0 40.1 100
Alevin (56 days) 138.4 96.6 86.7
Not completely adsorbed yolk (SO days)
37.3 48.0 100
42.8 44.9 100
56.5 48.4 100
Alevinb
UPTAKE AND RELEASE TESTS AT 10°C
79.3
81.8 220.0 111.9
118.1 188.0
Adsorbed half-yolk (41 days)
Stage
FROM SHORT-TERM
5 Lo 2 =!
E -;
2 y
% E .-
Q
p-DICHLOROBENZENE
IN Salmo
TABLE KINETIC
CONSTANTS AND AND RELEASE
445
guirdneri
3 FOR p-DCB
ASYMPTOTIC CONCENTRATIONS IN EGGS AND ALEVINS OF
FROM CONTINUOUS-EXPOSURE
TESTS AT 10°C Alevins
Eggs (rgf;iter) 19.5 16.4
(pg/k~lipicl) 1355 x 10’ 303x10’
UPTAKE
S. gairdneri,
K, (hr-I) 22 26
K2 (hr-I)
:A:)
0.0013 0.0014
531 493
(rg$er) 13.2 14.6
(pg/k;lipid)
K, (hr-I)
K2 (hr-‘)
(rl?)
460 390
0.17 0.11
4 6
IO3
200 x 52 x
lo3
The kinetic constants for alevins are very similar to those found in the literature for adult rainbow trout. If the K, for alevins in Table 3 are transformed into values for wet weight instead of lipid content, they result in 7.76 and 6.58 hr~’ whereas Neely et (21.(1974) report 5.76 hr-‘. The K2, which does not need any transformation, being identical for both fresh and fat weights, are 0.17 and 0.11 compared to 0.026 found by Neely et al., (1974). This limited difference also influences the half-time of release which is 4-6 hr for alevins and 15 hr for adults. Kinetic constants and bioaccumulation factors for alevins are also very similar to those found by Konemann and Van Leeuwen ( 1980) for Poecilia reticulata. Comparisons cannot be: made for bioaccumulation of p-DCB in developmental stages as no research was carried out on this subject. The potential of bioaccumulation is quite
2
I 0
I 20
32
Cl
41
50
1 56
r=D
77
days
FIG. 3. Concentration factors of p-DCB in different developmental stages of S. tinuous pg/liter.
long-term
test at 79.5 @g/liter,
(A)
4%hr
uptake
at 65.6 @g/liter,
gairdneri:
and (m) 24-hr
uptake
(0)
con-
at 65.6
446
GALAS%
CALAMARI,
AND
SETTI
high, particularly at the hatching stage. In one case a CF as high as 1000 was observed (Fig. 1, first long-term exposure). For most of the cases the ratios of accumulation in critical developmental stages in regard to alevins range from 6 to 20. Moreover the ability to release is very poor in all the prehatching stages. On the contrary uptake and release are quite fast in the posthatching stages (Table 2). A greater potential for accumulation in eggs in regard to posthatching stages is still evident even if the concentrations of p-DCB are expressed on the basis of lipid content. A theoretical BCF of 17,000 can be calculated for eggs, compared to 3000 for alevins. Despite the high levels of accumulation of p-DCB in eggs and other early life stages neither teratogenic effects nor mortality, higher than in the control tanks, was observed. It is to be noted that the highest concentration employed for the 60day treatment in eggs and alevins is around 0.1 of the incipient lethal level (ILL) found in the 14-day test with alevins by Calamari et al., (1982). A substance that does not cause any evident damage on the very sensitive developmental stages at 1/lOth of the threshold could not be considered as high risk even if it must be classified as “toxic” according to GESAMP (1969), being the acute LCsO in the range l-10 mg/liter for several aquatic organisms (Calamari et al., 1982). These findings confirm that p-DCB is a molecule that apparently would not cause great concern in aquatic environments, notwithstanding the fact that it is widely diffused in biotic and abiotic compartments as already stated by Jori et al. (1982). Considering the result of the tests from a methodological point of view several considerations can be drawn: -Developmental stages of fish are very useful for bioaccumulation studies as well as for mortality and teratogenicity. -Certain stages accumulate more than others having a low release capacity. -The particular substance tested here reached CF up to 1000 in developmental stages when theoretical BCF is around 100; BCFs calculated from the n-octanol/ water partition coefficient, reliable for adult fish, are therefore not applicable to early life stages. -Even if data refer to lipid content, a higher possibility of assumption has been demonstrated in eggs by means of a toxicokinetic model. -There is a need to explore the possibility of prediction of accumulation also for developmental stages of fish; on the basis of the results obtained in the present study it seems more advantageous to test a limited number of key stages (eyed eggs, hatching) with short-term exposures and frequent sampling; kinetic constants and steady-state concentrations can be calculated applying the toxicokinetic models. -It is necessary in this kind of study to separate the thermodynamic point of view (maximum bioaccumulation potential at the steady state) from the kinetic aspects because of the deep biological modifications involved in the developmental stages. Linear inverse relationships between log K,,, and K2 were found for homologous series of organic molecules (Zitko, 1980; Kijnemann and Van Leeuwen, 1980) indicating a very slow accumulation rate for molecules with very high BCF. These kinds of molecules are expected to accumulate to a limited extent in early fife stages owing to the limited exposure time. Considering the possibility of extending these studies to other organic molecules
p-DICHLOROBENZENE
IN
Salmo
gairdneri
447
and assuming that compounds with BCF more than 200-300 are potentially suspicious for bioaccumulation problems (Zitko, 1980), attention should be given to molecules with medium BCF due to their quick uptake. REFERENCES BARROW, M. E., PETROCELLI, S. R., MACEK, K. J., AND CARROLL, J. J. (1980). Bioconcentration and elimination of selected water pollutants by bluegill sunfish (Lepormis machrochirus). In Dynamics, Exposure and Hazard Assessment of Toxic Chemicals (R. Hague, ed.). Ann Arbor Science, Ann Arbor, Mich. CALAMARI, D., GALASSI, S., AND SETTI, F. (1982). Evaluating the hazard of organic substances on aquatic lil‘e: The paradichlorobenzene example. Ecotoxicol. Environ. Saf: 6, 369-378. CALAMARI, D., AND MARCHETTI, R. (I 978). Relevance of studies on developmental and young stages of Salmo gairdneri in establishing water quality criteria for fisheries. Ber. Umwelfbundesamt (Ger) IO, 201-210. CALAMARI, D., MARCHETTI, R., AND VAILATI, G. (1981). Effects of long term exposure to ammonia on developmental stages of rainbow trout (Salmo gairdneri Rich). Rapp. P.-v. R&n. Cons. inc. Explor. Mer. 178, 81-86. CHIOU, C. T., FREED, V. H., SCHMEDDING, D. W.. AND KOHNERT, R. L. (1977). Partition coefficient and bioaccumulation of selected organic chemicals. Environ. Sri. Technol. 5, 475-478. EARNEST. R. D., AND BENVILLE, P. E. (1971). Correlation of DDT and lipid levels for certain San Francisco bay fish. Pestic. Monif. J. 5, 235. FOLCK, J.. LEES, M., AND SLOONE STANLEY, G. H. (1956). A simple method for the isolation and purification of total lipids from animal tissue. J. Biol. Chem. 226, 497-509. GESAMP (I 969). Abstract of the report of the first session. Wafer Res. 3, 995. JORI, A., CALI\MARI. D., CATTABENI, F.. DI DOMENICO, A., GALLI, C. L., GALLI, E., RAMUNDO, A., AND SILANO, V. (1982). Ecotoxicological profile of p-dichlorobenzene (p-DCB). EcoroxicoL Environ. So/: 6, 413-432. KECK, G., AND RAFFENOT, J. ( 1979). Etude &co-toxicologique de la contamination chimique par les PCB dam la riviere du Furaus (Aiu). Rev. Med. Vet. 130, 339-358. KENAGA, E. E. (1980). Predicted bioconcentration factors and soil sorption coefficients of pesticides and other chemicals. Ecotoxicol Environ. Saf: 4, 26-38. K~NEMANI\, H.. AND VAN LEEUWEN. K. (1980). Toxicokinetics in fish: Accumulation and elimination of six chlorobenzenes by guppies. Chemosphere 9, 3319. KORN, S.. AND STANLEY, R. (1981). Sensitivity to, and accumulation and depuration of, aromatic petroleum components by early life stages of coho salmo Oncorhynchus kisutch. Rapp. P.-v. R&n. Cons. int. E.uplor. Mu. 178, 65571. MCKIM. J. M. (1977). Evaluation of tests with early life stages of fish for predicting long-term toxicity. J. Fish. Res. Board Canad. 34, I I48- 1 154. NEELY, W. B., BRANSON, D. R.. AND BLAU, G. E. (1974). Partition coefficient to measure bioconcentration potential of organic chemicals in fish. Environ. Sci. Technol. 13, 1 I 13-l 1 15. RESCIGNO. A., AND SEGRE, G. ( 1966). Drug and Tracer Kinetics. Blaiswell Publishing Co.. Waltham, Mass. VMTH, G. D.. DE FEO, D. L.. AND BERGSTEDT, B. V. (1979). Measuring and estimating the bioconcentration factor of chemicals in fish. J. Fish. Res. Board Cunad. 36, 1040-1047. ZITKO, V. (1980). Relationships governing the behavior of pollutants in aquatic ecosystems and their use in risk assessment. Canad. Tech. Rep. Aquar. Sci. 975, 243-265.