- Email: [email protected]
Histopathology of Mercenaria mercenaria as an indicator of pollutant stress
Marine Environmental Research 14 (1984) 521-524
H i s t o p a t h o l o g y of Mercenaria mercenaria as an Indicator o f Pollutant Stress M. R. Tripp, C. R. Fries, M. A. Craven & C. E. Grier School of Life and Health Sciences, University of Delaware, Newark, Delaware 19716, USA
& C. E. Gates Department of Statistics, Texas A & M University, College Station, Texas 77843, USA
M e r c e n a r i a mercenaria is a filter-feeding mollusc and effects on its digestive system might be anticipated if clams are exposed to chemicals dissolved in estuarine water. We evaluated certain morphological and biochemical parameters for cyclic variations and whether they were affected by chemical stressors. The experimental approach consisted of: (1) determining normal digestive tubule and epithelial cell dimensions and whether they change with light/dark or tidal cycles; (2)injecting clams with pollutant chemicals to determine if maximal exposure of tissues to chemicals resulted in changes in tubule size or lysosomal content; and (3) exposing clams to the same chemicals in the water for 6 months to determine if morphological or lysosomal changes occurred. Adult M . mercenaria were collected locally and held in the Broadkill River, Delaware (salinity 28 _+ 2%0; temperature 19 + 1.5 °C). They were sampled at high, mid and low tides for six consecutive tidal cycles. The test chemicals studied were benzo[a]pyrene (BAP), hexachlorobenzene (HCB) and pentachlorophenol (PCP). Because of their limited solubility in water, the chemicals were dissolved in corn oil for injection; each clam received 500/~g in 0.1 ml of corn oil (controls received corn oil only) injected directly into the digestive gland. Injected clams were returned to separate flow-through tanks and sampled twice weekly for 3 521 Marine Environ. Res. 0141-1136/84/$03.00 © Elsevier Applied Science Publishers Ltd, England, 1984. Printed in Great Britain.
522
M. R. Tripp, C. R. Fries, M. A. Craven, C. E. Grief, C. E. Gates INJECI"IOfl EXPERIMENT
67'0
630 I
2 14£
65.0
L
C BC(a~PHOS
1"0
N
C BP(b~HCB
C Br'(C)ICP He8
10"0
C BP(d)IX:P HQS
26"0
N
C BP(e)K:PHC8
C BP(R:::p HCl ) Bf
Fig. 1. Dimensions (in #m) of digestive tubules and absorptive cells of M. mercenaria injected with BAP, HCB or PCP. Data ranked by Duncan's multiple range analysis. Hatched bars differ significantly (P < 0.05) from solid bars. 6 MONTHEXPOSUREEXPERIMENT
I 26C
9"0
°
58"0
111
9"0
25£
52-0i
C BPPCPHCB {a}
C lip R2PHCB (b)
c (C) ~FO=HCB
C BP~CPeCB (d)
c lipPC~eCB (e)
C SptC'pHCS (t)
Fig. 2. Dimensions (in/tm) of digestive tubules and absorptive cells of M. mercenaria held for 6 months in seawater dosed with BAP, HCB or PCP. Data ranked by Duncan's multiple range analysis. Hatched bars differ significantly (P < 0.05) from solid bars.
M. mercenaria as indicator ~?[pollutant stress
523
weeks. Chemicals were added to water by constantly cycling seawater through sand columns impregnated with BAP or HCB; the sodium salt of PCP was dissolved in seawater and pumped into the appropriate tank. 4 or 6 clams were sacrificed per sample. A 1 cm transverse slice was cut through the center of the digestive gland, divided in half, and one piece fixed in Davidson's solution while the other was frozen in hexane-dry ice. Histological sections stained with hematoxylin and eosin were used to measure digestive tubule width and length, lumen width and length, and epithelial cell width and height. Each sample yielded 48 or 72 measurements of each dimension. B-Glucuronidase and acid phosphatase were visualized by standard histochemical techniques (Moore, Cell Tissue Res. 175, 279-87, 1976; Gomori, Stain Tech. 25, 81 -4, 1950, respectively) and quantitation of lysosomes was based on enumeration of colored granules in a standard tissue area. This technique was used to differentiate between lysosomal enzyme (compact granules) and microsomal enzyme (diffuse product in cytoplasm). Statistical significance was determined by use of Anova analysis of variance and ranked by Duncan's multiple range test. Digestive tubule and cell dimensions of normal clams did not change with light/dark cycle or tidal cycle. Considerable variability occurred among individual clams, but these changes were not regular or cyclic. Clams injected with corn oil, with or without chemical, had some immediate inflammatory responses that were resolved in a few days. Tubule dimensions and digestive cell size changed slightly (and were statistically different from controls) in HCB- and PCP-injected clams, but there was no change in BAP-injected clams (Fig. 1). Lysosomal enzyme (acid phosphatase and fl-glucuronidase) content varied irregularly. [3Glucuronidase values were decreased in BAP-injected animals just after injection but returned to normal within a few days; all chemical-injected animals had higher than normal values by day 18. Acid phosphatase values were depressed at day 2, rebounded sharply to above control values by day 4 and gradually decreased towards normal thereafter. Clams held for 6 months in seawater plus test chemicals did not show significant changes in tubule width or length, lumen width or cell height. HCB and BAP both decreased cell width while PCP decreased both lumen length and cell width (Fig. 2). Digestive gland epithelial cells of M. mercenaria do not undergo pronounced cyclic breakdown and reformation as do comparable cells in some other molluscs. Changes in cell and tubule dimensions are not
524
M. R. Tripp, C. R. Fries, M. A. Craven, C. E. Grier, C. E. Gates
strongly influenced by light/dark cycle or tidal cycle. Clams injected with potentially toxic chemicals such as BAP, HCB or PCP have some significant (P < 0.05) changes in tubule or cell dimensions. They also show altered concentrations of lysosomal enzymes but not massive lysis of digestive tubule cells. Our data suggest that BAP (either injected or ingested) has no significant effect on M. mercenaria while HCB and PCP may have mildly toxic effects on the digestive gland. Supported by a grant from the National Science Foundation (OCE-77 24037).
H i s t o p a t h o l o g y of Mercenaria mercenaria as an Indicator o f Pollutant Stress M. R. Tripp, C. R. Fries, M. A. Craven & C. E. Grier School of Life and Health Sciences, University of Delaware, Newark, Delaware 19716, USA
& C. E. Gates Department of Statistics, Texas A & M University, College Station, Texas 77843, USA
M e r c e n a r i a mercenaria is a filter-feeding mollusc and effects on its digestive system might be anticipated if clams are exposed to chemicals dissolved in estuarine water. We evaluated certain morphological and biochemical parameters for cyclic variations and whether they were affected by chemical stressors. The experimental approach consisted of: (1) determining normal digestive tubule and epithelial cell dimensions and whether they change with light/dark or tidal cycles; (2)injecting clams with pollutant chemicals to determine if maximal exposure of tissues to chemicals resulted in changes in tubule size or lysosomal content; and (3) exposing clams to the same chemicals in the water for 6 months to determine if morphological or lysosomal changes occurred. Adult M . mercenaria were collected locally and held in the Broadkill River, Delaware (salinity 28 _+ 2%0; temperature 19 + 1.5 °C). They were sampled at high, mid and low tides for six consecutive tidal cycles. The test chemicals studied were benzo[a]pyrene (BAP), hexachlorobenzene (HCB) and pentachlorophenol (PCP). Because of their limited solubility in water, the chemicals were dissolved in corn oil for injection; each clam received 500/~g in 0.1 ml of corn oil (controls received corn oil only) injected directly into the digestive gland. Injected clams were returned to separate flow-through tanks and sampled twice weekly for 3 521 Marine Environ. Res. 0141-1136/84/$03.00 © Elsevier Applied Science Publishers Ltd, England, 1984. Printed in Great Britain.
522
M. R. Tripp, C. R. Fries, M. A. Craven, C. E. Grief, C. E. Gates INJECI"IOfl EXPERIMENT
67'0
630 I
2 14£
65.0
L
C BC(a~PHOS
1"0
N
C BP(b~HCB
C Br'(C)ICP He8
10"0
C BP(d)IX:P HQS
26"0
N
C BP(e)K:PHC8
C BP(R:::p HCl ) Bf
Fig. 1. Dimensions (in #m) of digestive tubules and absorptive cells of M. mercenaria injected with BAP, HCB or PCP. Data ranked by Duncan's multiple range analysis. Hatched bars differ significantly (P < 0.05) from solid bars. 6 MONTHEXPOSUREEXPERIMENT
I 26C
9"0
°
58"0
111
9"0
25£
52-0i
C BPPCPHCB {a}
C lip R2PHCB (b)
c (C) ~FO=HCB
C BP~CPeCB (d)
c lipPC~eCB (e)
C SptC'pHCS (t)
Fig. 2. Dimensions (in/tm) of digestive tubules and absorptive cells of M. mercenaria held for 6 months in seawater dosed with BAP, HCB or PCP. Data ranked by Duncan's multiple range analysis. Hatched bars differ significantly (P < 0.05) from solid bars.
M. mercenaria as indicator ~?[pollutant stress
523
weeks. Chemicals were added to water by constantly cycling seawater through sand columns impregnated with BAP or HCB; the sodium salt of PCP was dissolved in seawater and pumped into the appropriate tank. 4 or 6 clams were sacrificed per sample. A 1 cm transverse slice was cut through the center of the digestive gland, divided in half, and one piece fixed in Davidson's solution while the other was frozen in hexane-dry ice. Histological sections stained with hematoxylin and eosin were used to measure digestive tubule width and length, lumen width and length, and epithelial cell width and height. Each sample yielded 48 or 72 measurements of each dimension. B-Glucuronidase and acid phosphatase were visualized by standard histochemical techniques (Moore, Cell Tissue Res. 175, 279-87, 1976; Gomori, Stain Tech. 25, 81 -4, 1950, respectively) and quantitation of lysosomes was based on enumeration of colored granules in a standard tissue area. This technique was used to differentiate between lysosomal enzyme (compact granules) and microsomal enzyme (diffuse product in cytoplasm). Statistical significance was determined by use of Anova analysis of variance and ranked by Duncan's multiple range test. Digestive tubule and cell dimensions of normal clams did not change with light/dark cycle or tidal cycle. Considerable variability occurred among individual clams, but these changes were not regular or cyclic. Clams injected with corn oil, with or without chemical, had some immediate inflammatory responses that were resolved in a few days. Tubule dimensions and digestive cell size changed slightly (and were statistically different from controls) in HCB- and PCP-injected clams, but there was no change in BAP-injected clams (Fig. 1). Lysosomal enzyme (acid phosphatase and fl-glucuronidase) content varied irregularly. [3Glucuronidase values were decreased in BAP-injected animals just after injection but returned to normal within a few days; all chemical-injected animals had higher than normal values by day 18. Acid phosphatase values were depressed at day 2, rebounded sharply to above control values by day 4 and gradually decreased towards normal thereafter. Clams held for 6 months in seawater plus test chemicals did not show significant changes in tubule width or length, lumen width or cell height. HCB and BAP both decreased cell width while PCP decreased both lumen length and cell width (Fig. 2). Digestive gland epithelial cells of M. mercenaria do not undergo pronounced cyclic breakdown and reformation as do comparable cells in some other molluscs. Changes in cell and tubule dimensions are not
524
M. R. Tripp, C. R. Fries, M. A. Craven, C. E. Grier, C. E. Gates
strongly influenced by light/dark cycle or tidal cycle. Clams injected with potentially toxic chemicals such as BAP, HCB or PCP have some significant (P < 0.05) changes in tubule or cell dimensions. They also show altered concentrations of lysosomal enzymes but not massive lysis of digestive tubule cells. Our data suggest that BAP (either injected or ingested) has no significant effect on M. mercenaria while HCB and PCP may have mildly toxic effects on the digestive gland. Supported by a grant from the National Science Foundation (OCE-77 24037).