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Tissue and cellular alterations in Asian clam (Potamocorbula amurensis) from San Francisco Bay: toxicological indicators of exposure and effect?
Marine Environmental Research 50 (2000) 301±305 www.elsevier.com/locate/marenvrev
Tissue and cellular alterations in Asian clam (Potamocorbula amurensis) from San Francisco Bay: toxicological indicators of exposure and eect? S.L. Clark *, S.J. Teh, D.E. Hinton Aquatic Toxicology Laboratory, Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616, USA
Abstract The US Geological Survey has reported the presence of a metal contamination gradient in clam tissues, decreased condition indices, and irregular reproductive patterns have been reported in the Asian clam, Potamocorbula amurensis, from San Francisco Bay. If metals are driving the observed patterns in the ®eld, then biomarkers of exposure, and possibly deleterious eect, should show a corresponding gradient. In this study, biomarkers from sub-cellular to tissue levels of biological organization were assessed in P. amurensis collected from the Bay or exposed to cadmium in the laboratory. Cellular and tissue alterations were assessed using histopathology and enzyme histochemistry (EH). Alterations in the ovary, testis, kidney, and gill tissues were most common at the most contaminated station when data were averaged over a 12-month sampling period. EH analysis indicated decreased active transport, energy status, and glucose oxidation in kidney and digestive gland at the most contaminated site which may indicate a decreased potential for growth. Ovarian lesions observed in feral Asian clams were experimentally induced in healthy clams by cadmium exposure in laboratory exposures. Our results suggest a contaminant etiology for tissue alterations. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Asian clam; San Francisco Bay; Cadmium; Exposure; Eect
The Asian clam (Potamocorbula amurensis) is an euryhaline species of bivalve mollusc introduced to San Francisco Bay in the mid-1980s. The exotic molluscan species spread proli®cally, accounting for 95% of the benthic biomass in some areas of the estuary (Carlton, Thompson, Schemel & Nichols, 1990). The US Geological * Corresponding author. 0141-1136/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0141-1136(00)00100-8
302
S.L. Clark et al. / Marine Environmental Research 50 (2000) 301±305
Survey (USGS) examined metal bioaccumulation in P. amurensis in San Francisco Bay between 1990 and 1999 and observed a contamination gradient (mg/g) in tissues of clams collected from four contaminated sites (Station 4.1>6.1>8.1>12.5), low condition indices, and asynchronous spawning at stations with elevated metal body burden (Brown & Luoma, 1995; Parchaso, Brown, Thompson & Luoma, 1997; Thompson, Parchaso, Brown & Luoma, 1996). Among the metals present in the ®eld, the most consistent correlation with these endpoints was found with cadmium (C. Brown, pers. comm.). The primary goal of the present study was to determine whether enzymatic and histopathological biomarkers were correlated with contaminant exposure in Asian clams. Cellular and tissue alterations were assessed in clams collected from Stations 4.1, 6.1, 8.1, and 12.5 using histopathology and enzyme histochemistry. Further, we conducted laboratory exposures to determine the role of cadmium in the production of lesions. Asian clam were collected from four stations in northern San Francisco Bay from July 1996 through June 1997 on a monthly basis (weather permitting) with a Van Veen grab aboard the USGS RV Polaris. Ten clams from each station were processed for histopathology and ®ve for enzyme histochemistry. Clams were also collected from the Martinez mud¯ats (Martinez, CA) for a static renewal laboratory exposure to cadmium (i.e. 0, 5, 10, 20, and 40 ppb nominal concentration for 14days in aqueous media at 10%/14 C). Clams for histopathological assessments were ®xed in buered formalin, cut along the sagital plane, and embedded in paran with the cut surface down (e.g. with the internal organs exposed) while the other half was embedded in the same block with the gill surface down. A semi-quantitative scoring method was used to examine histopathology tissues with scores ranging from 0 to 3 based on percent of tissue aected or presence/absence of lesions (Table 1). Enzyme histochemical analyses for intracellular digestion (acid phosphatase, ACP), active transport (alkaline phosphatase, ALKP), energy status (adenosine triphosphatase, ATPase), and cell proliferation (glucose-6-phosphate dehydrogenase, G6PDH) were performed on freeze-dried and glycomethacrylate embedded clams following standard methods (Teh & Hinton, 1993; Teh, Clark, Brown, Luoma & Hinton, in press).
Table 1 Histopathological scoring system for lesions in Asian clam tissues Histopatholoical Focal score necrosis in ovary
Chronic in¯ammation and necrois in testis
Mucus cell swelling in gill
0
Not present
Not present
1 2 3
Not present
Edema in kidney
Scored as percentage of clams with lesion <5% of tissue <10%/®eld of view (40) <10%of lamella aected N/A 5±10% of tissue 10±20%/®eld of view (40) 10±25% lamella aected N/A >10% of tissue >20%/®eld of view (40) >25%lamella aected N/A
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A histopathological assessment of Asian clam collected from four stations in northern San Francisco Bay revealed chronic in¯ammation and necrosis of the testis, regions of focal necrosis in ovary, edema in the kidney, and mucus cell swelling in the gill (Fig. 1). Although lesion prevalence varied during 12 months of sampling, averaging histological scores within stations over this period revealed increased lesion severity at the station with the highest metal body burden (Station 4.1) when compared to the least contaminated station (Station 12.5; Fig. 1). A comparison of enzyme histochemistry revealed dierences between Stations 4.1 and 8.1 during May and June 1997, but could not be used to distinguish dierences between stations 6.1 and 8.1. Clams from Station 8.1 had increased cell proliferation, increased energy (ATPase) localization, and increased intracellular digestion in the digestive diverticulum and kidney when compared to Station 4.1, which is similar to results reported for April 1997 (Teh et al., in press). Laboratory exposures of clams to cadmium resulted in ovarian (Fig. 2B) and gill lesions, indicating these two lesion types are appropriate biomarkers of exposure to cadmium. Furthermore, the presence of ovarian lesions in cadmium-exposed clams provides some evidence of the potential role of this metal in inducing similar lesions in the ®eld. ATPase localization was greatly diminished in the gill of clams exposed to cadmium when compared to controls, although no other enzyme alterations were
Fig. 1. Comparison of mean histopathologic lesions for clams collected monthly from July 1996 through June 1997 from four stations along an established metals contamination gradient (Stations. 4.1>6.1>8.1>12.5) in northern San Francisco Bay. Note the highest average histopathological score for each lesion occurred at the most contaminated station and decreased along the gradient. Histopathological score methods are described in Table 1. Lesions are: focal necrosis in the ovary (FNO), chronic in¯ammation and necrosis in the testis (CINT), mucus cell swelling in the gill (MCSG), and edema in the kidney (EK). Error bars represent S.E.
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Fig. 2. (A) Normal ovary in Asian clam exposed to 0 ppb cadmium with clearly discernable ovarian follicles (OF), mature oocytes (O), and immature oocytes (arrowhead). (B) Ovary of clam exposed to 40 ppb cadmium for 14 days exhibiting break down of follicular structure, few mature (O) and immature oocytes (arrowhead), pyknotic nuclei (PN), and large regions of focal necrosis. Bar represents 275 mm. Similar lesions were found in ®eld collected clams (Fig. 1).
detected in other clam tissues. The severity and prevalence of histopathological biomarkers and diminished enzyme activities at the station with consistently elevated metal body burdens provides additional correlative evidence that these biomarkers are useful indicators of exposure. Furthermore, these histopathologic biomarkers (e.g. reproductive tissue lesions) appear to indicate deleterious eects when USGS ®ndings of altered reproductive patterns are considered. Both histological damage in the reproductive organs and enzyme alterations in the digestive diverticulum and kidney, which play important roles in the conversion of food to energy and removal of waste products, also provide evidence re¯ecting an overall decreased potential for growth in the individuals at the Delta Station. Clearly, further investigation of other confounding anthropogenic (e.g. organic contaminants) and environmental stressors (e.g. salinity) is warranted and could provide the additional evidence needed to more clearly link these biomarkers to deleterious eects in situ. Acknowledgements We thank USGS sta Cindy Brown, Sam Luoma, Francis Parcheso, and Jan Thompson of the US Geological Survey in Menlo Park and the crew of the RV Polaris. Funding for this research was provided, in part, by the US EPA-UC Davis Center for Ecological Health Research (No. 819658), by grant No. 823297 from the US EPA, and by a training grant from the UC Systemwide Toxic Substances Research and Teaching Program's Lead Campus Program in Ecotoxicology.
S.L. Clark et al. / Marine Environmental Research 50 (2000) 301±305
305
References Brown, C. L., & Luoma, S. N. (1995). Use of the euryhaline bivalve Potamocorbula amurensis as a biosentinal species to assess trace metal contamination in San Francisco Bay. Mar. Ecol. Prog. Ser., 124, 129±142. Carlton, J. T., Thompson, T. K., Schemel, L. E., & Nichols, F. H. (1990). Remarkable invasion of San Francisco Bay (California, USA) by Asian clam Potamocorbula amurensis. I. Introduction and dispersal. Mar. Ecol. Prog. Ser., 66(1±2), 81±94. Parchaso, F., Brown, C. L., Thompson, J., & Luoma, S. N. (1997). In situ eects of trace contaminants on the ecosystem in the San Francisco Bay Estuary, 1995: the necessary link to establishing water quality standards II, Open-File Report 97-240. Teh, S. J., & Hinton, D. E. (1993). Detection of enzyme histochemical markers of hepatic preneoplasia and neoplasia in medaka (Oryzias latipes). Aquatic Tox., 24, 163±182. Teh, S. J., Clark, S. L., Brown, C. L., Luoma, S. N., & Hinton, D. E., (in press). Enzymatic and histopathologic biomarkers as indicators of contaminant exposure and eect in Asian clam (Potamocorbula amurensis). Biomarkers (in press). Thompson, J. K., Parchaso, F., Brown, C. L., & Luoma, S. N. (1996). In situ eect of trace contaminants on the ecosystem in the San Francisco Bay Estuary: the necessary link to establishing water quality standards II, Open-File Report 96±437.
Tissue and cellular alterations in Asian clam (Potamocorbula amurensis) from San Francisco Bay: toxicological indicators of exposure and eect? S.L. Clark *, S.J. Teh, D.E. Hinton Aquatic Toxicology Laboratory, Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California-Davis, Davis, CA 95616, USA
Abstract The US Geological Survey has reported the presence of a metal contamination gradient in clam tissues, decreased condition indices, and irregular reproductive patterns have been reported in the Asian clam, Potamocorbula amurensis, from San Francisco Bay. If metals are driving the observed patterns in the ®eld, then biomarkers of exposure, and possibly deleterious eect, should show a corresponding gradient. In this study, biomarkers from sub-cellular to tissue levels of biological organization were assessed in P. amurensis collected from the Bay or exposed to cadmium in the laboratory. Cellular and tissue alterations were assessed using histopathology and enzyme histochemistry (EH). Alterations in the ovary, testis, kidney, and gill tissues were most common at the most contaminated station when data were averaged over a 12-month sampling period. EH analysis indicated decreased active transport, energy status, and glucose oxidation in kidney and digestive gland at the most contaminated site which may indicate a decreased potential for growth. Ovarian lesions observed in feral Asian clams were experimentally induced in healthy clams by cadmium exposure in laboratory exposures. Our results suggest a contaminant etiology for tissue alterations. # 2000 Elsevier Science Ltd. All rights reserved. Keywords: Asian clam; San Francisco Bay; Cadmium; Exposure; Eect
The Asian clam (Potamocorbula amurensis) is an euryhaline species of bivalve mollusc introduced to San Francisco Bay in the mid-1980s. The exotic molluscan species spread proli®cally, accounting for 95% of the benthic biomass in some areas of the estuary (Carlton, Thompson, Schemel & Nichols, 1990). The US Geological * Corresponding author. 0141-1136/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0141-1136(00)00100-8
302
S.L. Clark et al. / Marine Environmental Research 50 (2000) 301±305
Survey (USGS) examined metal bioaccumulation in P. amurensis in San Francisco Bay between 1990 and 1999 and observed a contamination gradient (mg/g) in tissues of clams collected from four contaminated sites (Station 4.1>6.1>8.1>12.5), low condition indices, and asynchronous spawning at stations with elevated metal body burden (Brown & Luoma, 1995; Parchaso, Brown, Thompson & Luoma, 1997; Thompson, Parchaso, Brown & Luoma, 1996). Among the metals present in the ®eld, the most consistent correlation with these endpoints was found with cadmium (C. Brown, pers. comm.). The primary goal of the present study was to determine whether enzymatic and histopathological biomarkers were correlated with contaminant exposure in Asian clams. Cellular and tissue alterations were assessed in clams collected from Stations 4.1, 6.1, 8.1, and 12.5 using histopathology and enzyme histochemistry. Further, we conducted laboratory exposures to determine the role of cadmium in the production of lesions. Asian clam were collected from four stations in northern San Francisco Bay from July 1996 through June 1997 on a monthly basis (weather permitting) with a Van Veen grab aboard the USGS RV Polaris. Ten clams from each station were processed for histopathology and ®ve for enzyme histochemistry. Clams were also collected from the Martinez mud¯ats (Martinez, CA) for a static renewal laboratory exposure to cadmium (i.e. 0, 5, 10, 20, and 40 ppb nominal concentration for 14days in aqueous media at 10%/14 C). Clams for histopathological assessments were ®xed in buered formalin, cut along the sagital plane, and embedded in paran with the cut surface down (e.g. with the internal organs exposed) while the other half was embedded in the same block with the gill surface down. A semi-quantitative scoring method was used to examine histopathology tissues with scores ranging from 0 to 3 based on percent of tissue aected or presence/absence of lesions (Table 1). Enzyme histochemical analyses for intracellular digestion (acid phosphatase, ACP), active transport (alkaline phosphatase, ALKP), energy status (adenosine triphosphatase, ATPase), and cell proliferation (glucose-6-phosphate dehydrogenase, G6PDH) were performed on freeze-dried and glycomethacrylate embedded clams following standard methods (Teh & Hinton, 1993; Teh, Clark, Brown, Luoma & Hinton, in press).
Table 1 Histopathological scoring system for lesions in Asian clam tissues Histopatholoical Focal score necrosis in ovary
Chronic in¯ammation and necrois in testis
Mucus cell swelling in gill
0
Not present
Not present
1 2 3
Not present
Edema in kidney
Scored as percentage of clams with lesion <5% of tissue <10%/®eld of view (40) <10%of lamella aected N/A 5±10% of tissue 10±20%/®eld of view (40) 10±25% lamella aected N/A >10% of tissue >20%/®eld of view (40) >25%lamella aected N/A
S.L. Clark et al. / Marine Environmental Research 50 (2000) 301±305
303
A histopathological assessment of Asian clam collected from four stations in northern San Francisco Bay revealed chronic in¯ammation and necrosis of the testis, regions of focal necrosis in ovary, edema in the kidney, and mucus cell swelling in the gill (Fig. 1). Although lesion prevalence varied during 12 months of sampling, averaging histological scores within stations over this period revealed increased lesion severity at the station with the highest metal body burden (Station 4.1) when compared to the least contaminated station (Station 12.5; Fig. 1). A comparison of enzyme histochemistry revealed dierences between Stations 4.1 and 8.1 during May and June 1997, but could not be used to distinguish dierences between stations 6.1 and 8.1. Clams from Station 8.1 had increased cell proliferation, increased energy (ATPase) localization, and increased intracellular digestion in the digestive diverticulum and kidney when compared to Station 4.1, which is similar to results reported for April 1997 (Teh et al., in press). Laboratory exposures of clams to cadmium resulted in ovarian (Fig. 2B) and gill lesions, indicating these two lesion types are appropriate biomarkers of exposure to cadmium. Furthermore, the presence of ovarian lesions in cadmium-exposed clams provides some evidence of the potential role of this metal in inducing similar lesions in the ®eld. ATPase localization was greatly diminished in the gill of clams exposed to cadmium when compared to controls, although no other enzyme alterations were
Fig. 1. Comparison of mean histopathologic lesions for clams collected monthly from July 1996 through June 1997 from four stations along an established metals contamination gradient (Stations. 4.1>6.1>8.1>12.5) in northern San Francisco Bay. Note the highest average histopathological score for each lesion occurred at the most contaminated station and decreased along the gradient. Histopathological score methods are described in Table 1. Lesions are: focal necrosis in the ovary (FNO), chronic in¯ammation and necrosis in the testis (CINT), mucus cell swelling in the gill (MCSG), and edema in the kidney (EK). Error bars represent S.E.
304
S.L. Clark et al. / Marine Environmental Research 50 (2000) 301±305
Fig. 2. (A) Normal ovary in Asian clam exposed to 0 ppb cadmium with clearly discernable ovarian follicles (OF), mature oocytes (O), and immature oocytes (arrowhead). (B) Ovary of clam exposed to 40 ppb cadmium for 14 days exhibiting break down of follicular structure, few mature (O) and immature oocytes (arrowhead), pyknotic nuclei (PN), and large regions of focal necrosis. Bar represents 275 mm. Similar lesions were found in ®eld collected clams (Fig. 1).
detected in other clam tissues. The severity and prevalence of histopathological biomarkers and diminished enzyme activities at the station with consistently elevated metal body burdens provides additional correlative evidence that these biomarkers are useful indicators of exposure. Furthermore, these histopathologic biomarkers (e.g. reproductive tissue lesions) appear to indicate deleterious eects when USGS ®ndings of altered reproductive patterns are considered. Both histological damage in the reproductive organs and enzyme alterations in the digestive diverticulum and kidney, which play important roles in the conversion of food to energy and removal of waste products, also provide evidence re¯ecting an overall decreased potential for growth in the individuals at the Delta Station. Clearly, further investigation of other confounding anthropogenic (e.g. organic contaminants) and environmental stressors (e.g. salinity) is warranted and could provide the additional evidence needed to more clearly link these biomarkers to deleterious eects in situ. Acknowledgements We thank USGS sta Cindy Brown, Sam Luoma, Francis Parcheso, and Jan Thompson of the US Geological Survey in Menlo Park and the crew of the RV Polaris. Funding for this research was provided, in part, by the US EPA-UC Davis Center for Ecological Health Research (No. 819658), by grant No. 823297 from the US EPA, and by a training grant from the UC Systemwide Toxic Substances Research and Teaching Program's Lead Campus Program in Ecotoxicology.
S.L. Clark et al. / Marine Environmental Research 50 (2000) 301±305
305
References Brown, C. L., & Luoma, S. N. (1995). Use of the euryhaline bivalve Potamocorbula amurensis as a biosentinal species to assess trace metal contamination in San Francisco Bay. Mar. Ecol. Prog. Ser., 124, 129±142. Carlton, J. T., Thompson, T. K., Schemel, L. E., & Nichols, F. H. (1990). Remarkable invasion of San Francisco Bay (California, USA) by Asian clam Potamocorbula amurensis. I. Introduction and dispersal. Mar. Ecol. Prog. Ser., 66(1±2), 81±94. Parchaso, F., Brown, C. L., Thompson, J., & Luoma, S. N. (1997). In situ eects of trace contaminants on the ecosystem in the San Francisco Bay Estuary, 1995: the necessary link to establishing water quality standards II, Open-File Report 97-240. Teh, S. J., & Hinton, D. E. (1993). Detection of enzyme histochemical markers of hepatic preneoplasia and neoplasia in medaka (Oryzias latipes). Aquatic Tox., 24, 163±182. Teh, S. J., Clark, S. L., Brown, C. L., Luoma, S. N., & Hinton, D. E., (in press). Enzymatic and histopathologic biomarkers as indicators of contaminant exposure and eect in Asian clam (Potamocorbula amurensis). Biomarkers (in press). Thompson, J. K., Parchaso, F., Brown, C. L., & Luoma, S. N. (1996). In situ eect of trace contaminants on the ecosystem in the San Francisco Bay Estuary: the necessary link to establishing water quality standards II, Open-File Report 96±437.