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Fish-embryo toxicity associated with exposure to soils and sediments contaminated with varying concentrations of dioxins and furans
Marine Environmental Research 35 (1993) 177-180
Fish-Embryo Toxicity Associated with Exposure to Soils and Sediments Contaminated with Varying Concentrations of Dioxins and Furans K. R. Cooper, J. Schell, T. Umbreit & M. Gallo Joint Graduate Program in Toxicology, Ruters University and Robert Wood Johnson Medical School, Piscataway, New Jersey, USA
A BSTRA CT Studies were carried out to evaluate the toxicity to Japanese medaka (Oryzias latipes) embryos after exposure to soils and sediments from various sites contaminated with dioxins and furans. Eggs were exposed to 1-10 mg of material in 1 ml of rearing solution held in a glass vial The eggs were monitored on a daily basis for death, stage development, and lesions. The soils that were evaluated included: two sites from Newark, N J (2200 and 180 #g/kg 2,3,7,8-TCDD); Times Beach, Missouri (9501~g/kg 2,3,7,8-TCDD); and Seveso, Italy (128 #g/kg 2,3,7,8-TCDD). Exposure to these soils resulted in 100% lethality. Based on the Newark, N J, soils, the LC50 to post-hatch survival was 0"09 (0"01 0"93) Ilg/kg of TCDD soil-bound equivalents. Exposure to estuarine core sediments collected near the Newark, N J, site that contained 0"73-21 ~tg/kg were also toxic to the embryo, but the toxicity was less in the sediments than in the soil. The estimated bioavailability from the Newark soil was less than 0.2%. This type of biological-based toxicity can be useful in evaluating the extent of clean-up that may be required for any specific site.
Tetrachloroop-dioxins (TCDD) and tetrachlorodibenzofurans (TCDF) are highly lipophilic and very environmentally persistent compounds that occur as unwanted contaminants from a number of industrial processes. 1 They are found as trace contaminants throughout the world and at very high concentrations in a number o f industrialized countries. 2 Their potential to bioaccumulate in the aquatic food web and their extreme toxicity to a number of developing animals raises concern when heavily contaminated 177
178
K. R. Cooper, J. Schell, T. Umbreit, M. Gallo
sites are identified. 3 The 2,3,7,8-TCDD and 2,3,7,8-TCDF are the most toxic of the isomers and readily bind to soil or sediment. Although very high levels of sediment- or soil-bound dioxins and furans can be chemically stripped from a sample, there is the question of the bioavailability of these compounds. 4 The amount bound onto a particle may not pose a hazard to an organism if it is not able to be adsorbed as a free compound by that organism and reach the sensitive tissue. 5 The development of a sensitive bioassay that can evaluate this parameter would allow for setting acceptable levels that could protect the most sensitive life stages. The developing organism (more precisely, the Japanese medaka) has been shown to be a very sensitive animal for the detection of these compounds. 3'6"7 The results that are reported in this paper examine the toxicity associated with soils and sediments contaminated with varying concentrations of dioxins and furans. The soils that were tested included: a chlorinated phenol-manufacturing site (2,3,7,8-TCDD 2280 pg/kg and 2,3,7,8-TCDF 168/~g/kg), a salvage yard (2,3,7,8-TCDD 180/~g/kg) in Newark, N J; Seveso, Italy (2,3,7,8-TCDD 176 #g/kg); and Times Beach, Missouri (2,3,7,8-TCDD 950 #g/kg). The control soil was from the manufacturing site but stripped of all organic contaminants. The sediments that were tested were dated sections (supplied by Dr Richard Bopp, Columbia University) from cores collected from Dundee Lake (< 58 ng/kg control), the Passaic River (730-21 000 ng/kg), and Newark Bay (430-2900 ng/kg), New Jersey. 9 The pure 2,3,7,8-TCDD (98% pure) was purchased from Cambridge Isotope Laboratories (Woburn, MA). The Japanese medaka (Oryzias latipes) embryo-larval assay (ELA) as previously described was used in these studies. 6 The individual eggs (N= 10-20) were exposed to 1-10mg of particulate material or varying concentrations of pure 2,3,7,8-TCDD in glass vials. The occurrence of lesions, stage development, death, and survival post-hatch were monitored. Testing with either the pure c o m p o u n d or the particulate material resulted in the following sequence of events: no lesions until the appearance of the liver, hemorrhage in the caudal vein and vitelline veins, hepatomegaly, edema and hydropericardium, death prior to or during hatch, and premature hatching. The LC50 and 95% confidence interval for hatch, survival, and lesions following exposure to the salvage yard soil are summarized in Table 1. Exposure of the eggs to the soils from Newark, Times Beach, and Seveso caused 100% death with similar lesions and appearance of lesions to the pure compound. Mixing of control soil with the scrapmetal-yard soil resulted in a dose response with an LC50 to hatch equal to 5"0 (0.56-44.8)/~g/kg of 2,3,7,8-TCDD (Tables 1 and 2). The LC50 to posthatch was 0-09 (0-01-0-93) #g/kg. Even at 1"8 ng/kg, the animals that died were severely deformed. On the basis of response curves between the pure c o m p o u n d and the soils, the amount ofdioxin-free was less than 0.2% from
179
Fish-embryo toxicity associated with exposure to soils TABLE 1
Dose Response with Scrap-Metal-Yard Soil on the Development and Survival of the Japanese Medaka (Oryzias latipes) Treatment "'b
N
Hatched
Lesions
Hatched dead
Survived 3 days
None Control soil Contaminated (pptr) 180000 18 000 1800 180 18 1"8
20 10
95 90
0 10
0 0
95 90
9 9 10 11 10 9
0 44 100 81c 90c 100
100 100 50 36 40 22
0 11 20 9 0 0
a The hThe ' The aThe
0 11 80 63 60 77~
soil was mixed with control soil. table values are percentage response. animal(s) that died had severe abnormal development. two animals that survived less than 3 days were severely deformed.
the N e w a r k soils. T h e D u n d e e L a k e sediments ( < 5 8 n g / k g ) c a u s e d n o toxicity. Sediments t h a t c o n t a i n e d m o r e t h a n l l 0 0 n g / k g o f 2,3,7,8-TCDD resulted in s o m e toxicity, b u t this was less severe t h a n that f r o m c o m p a r a b l e soil c o n c e n t r a t i o n s a n d the p u r e c o m p o u n d tested (Table 2). It also a p p e a r e d that the greater the d e p t h o f the m a t e r i a l in the core, the less toxic it was, even with c o n c e n t r a t i o n s ten times as high. This w o u l d suggest t h a t the 2,3,7,8T C D D , w h e n p r e s e n t in the sediment, is even less available t h a n the 0.2% e s t i m a t e d for the surface soils. TABLE 2
Comparison of Toxicity between Acetone, Nonane, Soil, and Sediment Contaminated with Dioxins and Furans a Parameter
Acetone
Nonane
Scrap-metal soil
Sediment cores
Hatch
19 (10-25)
32 (25-40)
5 000 (560-44 800)
2 900-21 000 b
Survival
13 (10-17)
21 (18-24)
95 (9-930)
2 900-21 000
Lesions
15 (12-19)
!9 (15-24)
400 (30-4 850)
870-6 000
"The values in the table represent ng/kg (soil) or ng/liter (solvent) with LC50 and in ( ) the 95% confidence limits. The acetone and nonane are only 2,3,7,8-TCDD. This is the range within which the LC50 would fall.
180
K. R. Cooper, J. Schell, T. Umbreit, M. Gallo
C o m p a r i s o n of the pure c o m p o u n d s and soil- and sediment-toxicity data illustrates the point that the presence of a concentration of a chemical (determined by chemical extraction and detection) does not mean that it poses an equal hazard to similar concentrations associated with a different matrix. There is only a hazard if the c o m p o u n d is in a free form in a highenough concentration to cause toxicity. The Japanese medaka E L A is a very rapid and sensitive bioassay for evaluating toxicity from different media, s The lesions are not p a t h o n o m o n i c for these compounds, but the low concentrations needed to cause toxicity and the stage-specific toxicity appear to be unique to toxic dioxine and furans. 7 The sensitivity of the developing organism to these c o m p o u n d s is probably due to the alteration of gene regulation, which is involved in both cell proliferation and cell differentiation. 5 The inappropriate expression of genes in the developing embryo (fish and chicken) is magnified because of the importance of both temporal and spatial cellular interactions. It would appear from the sequence of events in the pathogenesis of the lesions involving the blood vessels, blood cells, and heart that the lateral-plate mesoderm is the target for the effects seen in the developing embryo} ° The de-novo formation of blood vessels and the heart from the mesoderm (vasculogenesis) is not affected, but the proliferation of pre-existing blood vessels (angiogenesis) are the target sites. Studies need to be carried out to examine the effects of these c o m p o u n d s on the angiogenesis factors released by proliferating tissues that p r o m o t e the mitosis and migration of endothelial cells.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
Poland, A. & Knutson, J. C. Annu. Rev. PharmacoL, 22, 517-24 (1982). Rappe, C. et al. Chemosphere, 16, 1603-18 (1987). Cooper, K. R. Aquat. Sci., i, 227~,2 (1989). Umbriet, T. H., Hesse, E. J. & Gallo, M. A. Science, 232, 497-9 (1986). Whitlock, J. P. Annu. Rev. Pharmacol. ToxicoL, 30, 251-77 (1990). Wisk, J. D. & Cooper, K. R. Chemosphere, 20, 361-77 (1990). Wisk, J. D. & Cooper, K. R. Environ. Toxicol. & Chem., 9, 1159-69 (1990). Cooper, K. R. & McGeorge, L. In Aquatic Toxicology and Risk Assessment: Fourteenth Volume (ASTM STP 1124). American Society for Testing and Materials, Philadelphia, PA, USA, 1991. 9. Bopp, R. E et al. Environ. Sci. Technol., 25, 951-6 (1992). 10. Pardanaud, L., Yassine, E & Dieterlen-Liervre, E Develop., 105, 473-85 (1989).
Fish-Embryo Toxicity Associated with Exposure to Soils and Sediments Contaminated with Varying Concentrations of Dioxins and Furans K. R. Cooper, J. Schell, T. Umbreit & M. Gallo Joint Graduate Program in Toxicology, Ruters University and Robert Wood Johnson Medical School, Piscataway, New Jersey, USA
A BSTRA CT Studies were carried out to evaluate the toxicity to Japanese medaka (Oryzias latipes) embryos after exposure to soils and sediments from various sites contaminated with dioxins and furans. Eggs were exposed to 1-10 mg of material in 1 ml of rearing solution held in a glass vial The eggs were monitored on a daily basis for death, stage development, and lesions. The soils that were evaluated included: two sites from Newark, N J (2200 and 180 #g/kg 2,3,7,8-TCDD); Times Beach, Missouri (9501~g/kg 2,3,7,8-TCDD); and Seveso, Italy (128 #g/kg 2,3,7,8-TCDD). Exposure to these soils resulted in 100% lethality. Based on the Newark, N J, soils, the LC50 to post-hatch survival was 0"09 (0"01 0"93) Ilg/kg of TCDD soil-bound equivalents. Exposure to estuarine core sediments collected near the Newark, N J, site that contained 0"73-21 ~tg/kg were also toxic to the embryo, but the toxicity was less in the sediments than in the soil. The estimated bioavailability from the Newark soil was less than 0.2%. This type of biological-based toxicity can be useful in evaluating the extent of clean-up that may be required for any specific site.
Tetrachloroop-dioxins (TCDD) and tetrachlorodibenzofurans (TCDF) are highly lipophilic and very environmentally persistent compounds that occur as unwanted contaminants from a number of industrial processes. 1 They are found as trace contaminants throughout the world and at very high concentrations in a number o f industrialized countries. 2 Their potential to bioaccumulate in the aquatic food web and their extreme toxicity to a number of developing animals raises concern when heavily contaminated 177
178
K. R. Cooper, J. Schell, T. Umbreit, M. Gallo
sites are identified. 3 The 2,3,7,8-TCDD and 2,3,7,8-TCDF are the most toxic of the isomers and readily bind to soil or sediment. Although very high levels of sediment- or soil-bound dioxins and furans can be chemically stripped from a sample, there is the question of the bioavailability of these compounds. 4 The amount bound onto a particle may not pose a hazard to an organism if it is not able to be adsorbed as a free compound by that organism and reach the sensitive tissue. 5 The development of a sensitive bioassay that can evaluate this parameter would allow for setting acceptable levels that could protect the most sensitive life stages. The developing organism (more precisely, the Japanese medaka) has been shown to be a very sensitive animal for the detection of these compounds. 3'6"7 The results that are reported in this paper examine the toxicity associated with soils and sediments contaminated with varying concentrations of dioxins and furans. The soils that were tested included: a chlorinated phenol-manufacturing site (2,3,7,8-TCDD 2280 pg/kg and 2,3,7,8-TCDF 168/~g/kg), a salvage yard (2,3,7,8-TCDD 180/~g/kg) in Newark, N J; Seveso, Italy (2,3,7,8-TCDD 176 #g/kg); and Times Beach, Missouri (2,3,7,8-TCDD 950 #g/kg). The control soil was from the manufacturing site but stripped of all organic contaminants. The sediments that were tested were dated sections (supplied by Dr Richard Bopp, Columbia University) from cores collected from Dundee Lake (< 58 ng/kg control), the Passaic River (730-21 000 ng/kg), and Newark Bay (430-2900 ng/kg), New Jersey. 9 The pure 2,3,7,8-TCDD (98% pure) was purchased from Cambridge Isotope Laboratories (Woburn, MA). The Japanese medaka (Oryzias latipes) embryo-larval assay (ELA) as previously described was used in these studies. 6 The individual eggs (N= 10-20) were exposed to 1-10mg of particulate material or varying concentrations of pure 2,3,7,8-TCDD in glass vials. The occurrence of lesions, stage development, death, and survival post-hatch were monitored. Testing with either the pure c o m p o u n d or the particulate material resulted in the following sequence of events: no lesions until the appearance of the liver, hemorrhage in the caudal vein and vitelline veins, hepatomegaly, edema and hydropericardium, death prior to or during hatch, and premature hatching. The LC50 and 95% confidence interval for hatch, survival, and lesions following exposure to the salvage yard soil are summarized in Table 1. Exposure of the eggs to the soils from Newark, Times Beach, and Seveso caused 100% death with similar lesions and appearance of lesions to the pure compound. Mixing of control soil with the scrapmetal-yard soil resulted in a dose response with an LC50 to hatch equal to 5"0 (0.56-44.8)/~g/kg of 2,3,7,8-TCDD (Tables 1 and 2). The LC50 to posthatch was 0-09 (0-01-0-93) #g/kg. Even at 1"8 ng/kg, the animals that died were severely deformed. On the basis of response curves between the pure c o m p o u n d and the soils, the amount ofdioxin-free was less than 0.2% from
179
Fish-embryo toxicity associated with exposure to soils TABLE 1
Dose Response with Scrap-Metal-Yard Soil on the Development and Survival of the Japanese Medaka (Oryzias latipes) Treatment "'b
N
Hatched
Lesions
Hatched dead
Survived 3 days
None Control soil Contaminated (pptr) 180000 18 000 1800 180 18 1"8
20 10
95 90
0 10
0 0
95 90
9 9 10 11 10 9
0 44 100 81c 90c 100
100 100 50 36 40 22
0 11 20 9 0 0
a The hThe ' The aThe
0 11 80 63 60 77~
soil was mixed with control soil. table values are percentage response. animal(s) that died had severe abnormal development. two animals that survived less than 3 days were severely deformed.
the N e w a r k soils. T h e D u n d e e L a k e sediments ( < 5 8 n g / k g ) c a u s e d n o toxicity. Sediments t h a t c o n t a i n e d m o r e t h a n l l 0 0 n g / k g o f 2,3,7,8-TCDD resulted in s o m e toxicity, b u t this was less severe t h a n that f r o m c o m p a r a b l e soil c o n c e n t r a t i o n s a n d the p u r e c o m p o u n d tested (Table 2). It also a p p e a r e d that the greater the d e p t h o f the m a t e r i a l in the core, the less toxic it was, even with c o n c e n t r a t i o n s ten times as high. This w o u l d suggest t h a t the 2,3,7,8T C D D , w h e n p r e s e n t in the sediment, is even less available t h a n the 0.2% e s t i m a t e d for the surface soils. TABLE 2
Comparison of Toxicity between Acetone, Nonane, Soil, and Sediment Contaminated with Dioxins and Furans a Parameter
Acetone
Nonane
Scrap-metal soil
Sediment cores
Hatch
19 (10-25)
32 (25-40)
5 000 (560-44 800)
2 900-21 000 b
Survival
13 (10-17)
21 (18-24)
95 (9-930)
2 900-21 000
Lesions
15 (12-19)
!9 (15-24)
400 (30-4 850)
870-6 000
"The values in the table represent ng/kg (soil) or ng/liter (solvent) with LC50 and in ( ) the 95% confidence limits. The acetone and nonane are only 2,3,7,8-TCDD. This is the range within which the LC50 would fall.
180
K. R. Cooper, J. Schell, T. Umbreit, M. Gallo
C o m p a r i s o n of the pure c o m p o u n d s and soil- and sediment-toxicity data illustrates the point that the presence of a concentration of a chemical (determined by chemical extraction and detection) does not mean that it poses an equal hazard to similar concentrations associated with a different matrix. There is only a hazard if the c o m p o u n d is in a free form in a highenough concentration to cause toxicity. The Japanese medaka E L A is a very rapid and sensitive bioassay for evaluating toxicity from different media, s The lesions are not p a t h o n o m o n i c for these compounds, but the low concentrations needed to cause toxicity and the stage-specific toxicity appear to be unique to toxic dioxine and furans. 7 The sensitivity of the developing organism to these c o m p o u n d s is probably due to the alteration of gene regulation, which is involved in both cell proliferation and cell differentiation. 5 The inappropriate expression of genes in the developing embryo (fish and chicken) is magnified because of the importance of both temporal and spatial cellular interactions. It would appear from the sequence of events in the pathogenesis of the lesions involving the blood vessels, blood cells, and heart that the lateral-plate mesoderm is the target for the effects seen in the developing embryo} ° The de-novo formation of blood vessels and the heart from the mesoderm (vasculogenesis) is not affected, but the proliferation of pre-existing blood vessels (angiogenesis) are the target sites. Studies need to be carried out to examine the effects of these c o m p o u n d s on the angiogenesis factors released by proliferating tissues that p r o m o t e the mitosis and migration of endothelial cells.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
Poland, A. & Knutson, J. C. Annu. Rev. PharmacoL, 22, 517-24 (1982). Rappe, C. et al. Chemosphere, 16, 1603-18 (1987). Cooper, K. R. Aquat. Sci., i, 227~,2 (1989). Umbriet, T. H., Hesse, E. J. & Gallo, M. A. Science, 232, 497-9 (1986). Whitlock, J. P. Annu. Rev. Pharmacol. ToxicoL, 30, 251-77 (1990). Wisk, J. D. & Cooper, K. R. Chemosphere, 20, 361-77 (1990). Wisk, J. D. & Cooper, K. R. Environ. Toxicol. & Chem., 9, 1159-69 (1990). Cooper, K. R. & McGeorge, L. In Aquatic Toxicology and Risk Assessment: Fourteenth Volume (ASTM STP 1124). American Society for Testing and Materials, Philadelphia, PA, USA, 1991. 9. Bopp, R. E et al. Environ. Sci. Technol., 25, 951-6 (1992). 10. Pardanaud, L., Yassine, E & Dieterlen-Liervre, E Develop., 105, 473-85 (1989).