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Bioaccumulation: aquatic / sediment

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Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Only summaries available
Principles of method if other than guideline:
Bioaccumulation study in fish (field study)
GLP compliance:
no
Test organisms (species):
Lepomis sp.
Details on test organisms:
TEST ORGANISM
- Common name: bluegill sunfish
- Source: collected from 5 sites in the Emory and Clinch Rivers
Route of exposure:
aqueous
Test type:
field study
Water / sediment media type:
natural water: freshwater
Total exposure / uptake duration:
8 - 12 wk

Selenium is elevated in muscle and other tissues of certain fish species sampled downstream of the fly ash spill

Although there were differences between sites in some indicators of exposure and effects in these fish collected 2-3 months after the spill, there were no consistent relationships between metal bioaccumulation and indicator responses.

Female fish collected at and downstream of the spill in spring/summer 2009 had no obvious significant reproductive abnormalities.

Endpoint:
bioaccumulation in sediment species, other
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Only summaries available
Principles of method if other than guideline:
Corbicula fluminea Bioaccumulation Test for Sediments (Provided by USACE Vicksburg; EPA/COE Inland Testing Manual)
GLP compliance:
not specified
Test organisms (species):
other: Corbicula fluminea
Route of exposure:
sediment
Test type:
static
Water / sediment media type:
natural sediment: freshwater
Total exposure / uptake duration:
28 d
Reference substance (positive control):
not specified

Results indicated no appreciable bioaccumulation of metals in Corbicula fluminea exposures (28-d) to whole ash.

The Bioaccumulation Factors (BAFs) are negligible, all BAF values for the measured heavy metals are below 1; the maximum BAF among metals was for zinc (mean = 0.343, range = 0.146 - 0.591 kg sediment (dry wt) / kg body weight (wet wt).

Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to guideline
Guideline:
other: USEPA method 200.8
Version / remarks:
Method was used for Se analysis in water.
Qualifier:
according to guideline
Guideline:
other: USEPA SW-846 method 6010B
Version / remarks:
Method used for Se determination in sediment samples.
Qualifier:
according to guideline
Guideline:
other: US Environmental Protection Agency. 2007. Method SW846-6020A: Inductively coupled plasma-mass spectrometry. Office of Solid Waste, Washington, DC
Version / remarks:
Method was used for Se determination in fish tissue.
Qualifier:
according to guideline
Guideline:
other: US Environmental Protection Agency. 1996. Method SW846-3050B: Acid digestion of sediments, sludges, and soils. Office of Solid Waste, Washington, DC.
Principles of method if other than guideline:
- Principle of test: The purpose of the study was to examine Se bioaccumulation in fish over time (2009 - 2013) at the Kingston ash spill site and potential concerns to human and aquatic life.
- Short description of test conditions: Monitoring was performed after the spill in two affected rivers Emory and Clinch as well as downstream the spill. Reference sites were additionally monitored for comparison. Three fish species with different trophic habits were analyzed.
- Parameters analysed / observed: Water samples, sediment, fish fillets as well as whole body fish were analyzed for Se.
GLP compliance:
no
Remarks:
peer reviewed data - GLP not relevant
Radiolabelling:
no
Details on sampling:
- Sampling intervals/frequency for test organisms: 6 bluegill and largemouth bass respectively were collected from each sampling location twice per year (spring-fall) in the period between 2009 and 2013. Fillets were analyzed for Se. Redear sunfish took place in spring of 2010 to 2013 and fall of 2012.
- Sampling intervals/frequency for test medium and sediment samples: water samples were collected from 2009 to 2013. Sediment samples were collected from the first 15 cm from multiple river locations from 2009 and 2013.
- Sample storage conditions before analysis: Water and sediment samples were shipped on ice (< 6°C) for analysis within 48 h of collection. Fish samples were shipped frozen for homogenization and Se analysis.
Vehicle:
no
Test organisms (species):
other: Lepomis macrochirus, Lepomis microlophus and Micropterus salmoides
Details on test organisms:
TEST ORGANISM
- Common name: Bluegill sunfish, redear sunfish, largemouth bass
- Source: Emory and Clinch rivers.
- Reason for fish selection: Bluegill sunfish and largemouth bass are commonly caught by local fisherman and are relevant for evaluation of human health risk. Largemouth bass represents the upper trophic fish level. Redear sunfish are good representers of local Se conditions and bioaccumulation potential since they are short living and sedentary organisms.
- Weight at study initiation: sunfish: 50–150 g, largemouth bass: 500–2500 g

Route of exposure:
other: multiple depending on fish species. For more details see "Details on test organisms"
Justification for method:
other: monitoring data from the ash contaminated river
Test type:
field study
Water / sediment media type:
other: fish lived in their natural environment (Emory and Clinch River)
Reference substance (positive control):
no
Details on estimation of bioconcentration:
BASIS INFORMATION
- Monitoring data: Se concentrations in water, sediment, fish fillet and whole body fish were determined and compared with protective thresholds from USEPA and other literature.

Remarks on result:
other: not reported
Type:
other: Se concentrations in fish fillet and whole body were determined in this study. For details see field "any other information on results incl. tables" and "overall remarks and attachments".
Remarks on result:
other: Monitoring study
Remarks:
For details see field "any other information on results incl. tables" and "overall remarks and attachments".
Remarks on result:
other: not studied
Reported statistics:
- Statistical analyses were performed with SigmaPlot or SAS/STAT software.
- Nonparametric Holm Sidak method: comparisons between different species, sampling season, and location types (ash-affected vs reference sites).
- Data fitting (log-linear model) was performed to examine the relationship between fillet and whole body Se.
- Covariance analysis was performed to investigate if the relationship between fillet and whole body Se covaried with species or sampling season.
- Durbin Watson test: autocorrelation between residuals in the time series was analyzed

- Aqueous Se concentrations were low within the 5 year investigation period and in most cases below the detection limit. Detectable Se was mostly found in samples close to the spill source. In only one case the concentration was higher (1.34 µg/L) than the USEPA aqueous criterion for Se of 1.3 µg/L in lentic systems. For more details see field "overall remarks, attachments".

- Sediment Se samples were also below the detection limit in multiple cases. Concentrations in sediments were between 1.15 µg/g to 8.25 µg/g in ash affected sites and between 1.01 and 6.17 µg/g in not affected sites. However the difference was statistically not significant. 10% of the sediment samples had elevated Se concentrations exceeding the 4 µg/g which is considered a hazardous concentration with bioaccumulation concerns. Some of these samples were among the reference site samples. For more details see field "overall remarks, attachments".

- Fillet Se concentrations: Se in fish was higher in ash affected sites than reference sites. Fillet concentrations were between 1.2 and 5.5 µg/g at reference sites and between 1.2 and 8 µg/g in contaminated sites. Redear sunfish were affected more than the other species in both affected and non affected sites. This is in line with previous studies and is attributed to their benthic diet. Low Se concentrations were determined in Largemouth bass, which represents the upper trophic level and suggests low biomagnification potential. Se concentrations were found to be higher in fish collected close to the spill site.

- Whole body Se concentrations: the same pattern as for fillet Se was found. Whole body concentrations were between 1.3 and 3.6 µg/g at reference sites and between 1.5 and 4.6 µg/g in contaminated sites.

The authors conclude that fish from the Kingston spill site indeed have elevated Se concentrations in terms of fillet or whole body. However only in few cases the most conservative threshold of Se (4 µg/g) has been exceeded at the most contaminated sites after dredging period while the USEPA proposed whole body Se threshold in fish was not exceeded in any case (8.1 µg/g). The study does not indicate concerns for aquatic life from Se in the spill site. However long-term studies need to follow.

Validity criteria fulfilled:
not applicable
Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to guideline
Guideline:
other: US EPA method 6020
Version / remarks:
guideline used for As and Se analysis in fish tissues.
Principles of method if other than guideline:
- Principle of test: purpose of the study was to investigate the bioaccumulation of As and Se in the liver, ovary and muscle tissues of multiple fish species and the potential reasons of the differences after a coal ash spill in Kingston.
- Short description of test conditions: fish were collected from two affected sites of the Emory River and two reference sites upstreams of the spill of the same river for comparison.
- Parameters analysed / observed: Concentrations of As and Se in liver, ovary and muscle tissues, differences in species stomach and intestine pH as well as different trophic dynamics (that could affect different bioaccumulation patterns).
GLP compliance:
no
Remarks:
peer reviewed data - GLP not relevant
Radiolabelling:
no
Details on sampling:
- Sampling intervals/frequency for test organisms for bioaccumulation study: Fish collection for the bioaccumulation test was conducted between 12.04.2010 and 13.05.2010. Six adult females were collected from each species (4 species) and each sampling location (4 sampling locations).
- Sampling intervals/frequency for test organisms for pH and trophic dynamics study: Based on the results of the bioaccumulation test further fish (only bluegill and redear sunfish) for the pH and trophic dynamics test were collected between 25.04.2011 and 20.05.2011. Between 8 to 16 adult (male and female) bluegill and redear sunfish were collected from all 4 locations. 6 fish collected for the pH study were also used for the trophic dynamics study.
- Sampling intervals/frequency for test organisms for trophic dynamics study:
- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods): For the pH test, stomach and intestine pH of the two fish species were measured. For the trophic dynamics test the investigation was based on stable isotope ratios of δ15N and δ13C.
Vehicle:
no
Test organisms (species):
other: Large mouth bass, white crappie, bluegill and redear sunfish for bioaccumulation study. Bluegill and redear sunfish for pH and trophic dynamics study.
Details on test organisms:
TEST ORGANISM
- Common name: large mouth bass, white crappie, bluegill and redear sunfish
- Source: Emory river
- Age at study initiation: adult females for bioaccumulation study, adult females and males for pH study
- Length and weight at study initiation for bioaccumulation study: large mouth bass(43.3±1.9 cm and 1,256.9± 179.8g), white crappie (31.8± 0.9 cm and 447.5± 40.9 g), bluegill (14.4± 0.4 cm and 52.8 ±4.2g) and redear sunfish (18.6± 0.5 cm and 98.2± 7.0g).
- Length and weight at study initiation for pH and trophic dynamic study: bluegill (14.2±0.2 cm and 50.3±1.9 g) and redear sunfish (18.4±0.4 cm and 103.0±6.2 g)
- Lipid content at test initiation: not reported


Route of exposure:
other: multiple depending on fish species.
Justification for method:
other: monitoring data from the affected river
Test type:
field study
Water / sediment media type:
other: fish lived in their natural environment (Emory River)
Details on test conditions:
TEST SYSTEM
- Monitoring data from natural environment (Emory River - two coal-ash affected sites named EMR 1 and EMR 3 and two reference sites named ERM 6-8 and LERM 2).
Reference substance (positive control):
no
Details on estimation of bioconcentration:
BASIS INFORMATION
- Monitoring data: As and Se concentrations in tissues (ovary, liver, muscle) of 4 fish species, pH analysis of stomach and intestine of 2 fish species, trophic dynamics of 2 fish species.
Remarks on result:
other: not reported
Type:
other: see "any other information on results incl. tables" and "overall remarks, attachments."
Remarks on result:
other: see "any other information on results incl. tables"
Remarks on result:
other: not investigated
Reported statistics:
Fish species and site differences in Se and As concentrations and stable isotopes were studied using two-way ANOVA with Turkeys post hoc test.
pH data were analyzed via one-way ANOVA.
JMP Pro 9 software was used for statistical analysis.
Linear regression modelling was performed to determine the relationship of As and Se bioaccumulation to stable isotopes.

Results of bioaccumulation study:

- For exact concentrations measured see field "overall remarks, attachments"

- As and Se concentrations in fish were generally lower in this study compared to other literature data.

- As: bioaccumulation was higher in ash contaminated sites in multiple fish species (mostly observed in ERM 1 site)

- Se: only sunfish and bluegill fish had higher concentrations in the ash affected sites (these results lead to the further study with pH and trophic dynamics).

- Concentrations of Se in livers and ovaries of redear sunfish in ash affected sites were increased compared to the concentrations in the coal ash itself (approx. 2.2 times). Concentrations of Se were especially elevated in liver of redear sunfish compared to other tissues and species.

Results on trophic dynamics study:

- No significant differences were observed between the two ash sites. The same observation was made for the reference sites. Therefore samples were pooled.

- Differences were however found between ash affected sites and reference sites implying differences of trophic dynamics between ash affected and non affected sites.

- A significant increase of 13C content in fish of the two ash affected sites was observed. Coal ash related carbon could be increased incorporated in carbon compounds of the food web at ash affected sites.

- 15N was enriched in reference sites.

- The relationship of Se and As concentrations to δ13C and δ15N was investigated to examine if there is a relationship between these parameters. A negative relationship was found between

δ15N and Se for bluegill and reader sunfish while a positive relationship was found between δ13C and Se for the two fish species. For As a positive relationship between δ13C and redear sunfish was observed.

Results of pH study:

Although differences in pH was identified between species, no differences were observed between affected and reference sites. Ash affected sites did not have an impact on stomach and intestine pH.

Validity criteria fulfilled:
not applicable
Endpoint:
bioaccumulation in sediment species: invertebrate
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to guideline
Guideline:
other: EPA methods USEPA 6020 and 1631
Version / remarks:
Guidelines were used for metal analysis in mussels.
Principles of method if other than guideline:
- Principle of test: The purpose of the study was to investigate the bioaccumulation in three different mussel species during and after dredging activities in situ in the ash affected sites from the Tennessee Valley Authority Kingston Fossil Plant coal ash spill.
- Short description of test conditions: Mussels were caged for 1 year in ash affected and unaffected river sites for both the dredging period and the post-dredging period. Additionally mussels of each species were kept under laboratory conditions as a reference.
- Parameters analysed / observed: Mussel health (mussel condition index) and concentrations of As, Cd, Cr, Pb, Ni, Se, Hg, U, Fe, Mg, Al, Sb, Ba, Be, Co, Cu, Mn, Mo, Ag, Sr, Tl, V, and Zn in soft tissues were analyzed. Potential differences based on location, dredging activities and species were investigated.
GLP compliance:
no
Remarks:
peer reviewed data - GLP not relevant
Radiolabelling:
no
Details on sampling:
- Sampling intervals/frequency for test organisms: During dredging activities mussels were placed in Emory River at 20th of June 2009 and sampled at 14th of June 2010, in lower Clinch River at 21st of October 2009 and sampled at 31 August 2010. During post dredging activities all mussels were placed at 28th of October 2010 in the rivers and sampled at 3rd of October 2011. Laboratory reference mussels were introduced at 5th of May 2009 and sampled at between July and August of 2010.
- Sample storage conditions before analysis: Tissue was frozen and held at −20 °C until laboratory analysis.
- Details on sampling and analysis of test organisms and test media samples : 75 individuals of each species were collected from the Tennessee River and transported to the Cumberland River Aquatic Center (Gallatin, TN) in aerated coolers (<3 °C). Prior to mussel deployment back in river, mussels were acclimatized in the laboratory and each individual was thoroughly scrubbed, weighed, and affixed with a number. At end of river exposure period mussels were held between 36 and 48 h to ensure purging of gut contents before processing. Mussels were additionally scrubbed before processing. Then mussels were weighed (total and tissue separately). Foot, mantle and gill were removed and remaining tissue was homogenized, dried (106 °C), digested and analyzed for metals. Health condition index was calculated as follows: condition index = tissue wet weight / shell volume.
Vehicle:
no
Test organisms (species):
other: Ligumia recta, Elliptio crassidens, Cyclonaias tuberculata
Details on test organisms:
TEST ORGANISM
- Common name: black sandshell, elephant ear, purple wartyback
- Source: Emory and Clinch Rivers

ACCLIMATION
- Acclimation period: Prior to initiation of field study, organisms were kept for 45 days in the laboratory.
- Acclimation conditions: First submerged in ∼35 cm of water for 45 d then deployed in flow-through raceways (flow rate of 454 L/min). Water source was from the Cumberland River.
- Type and amount of food: Not fed during holding in laboratory.

Route of exposure:
sediment
Justification for method:
other: Monitoring data
Test type:
field study
Water / sediment media type:
natural water / sediment: freshwater
Details on test conditions:
TEST SYSTEM
- Collected and acclimatized mussels were deployed at four sites in the vicinity of the ash spill. During dredging activities mussels were deployed at following locations: Emory River - directly downstream the spill, Emory River - upstream the spill, lower Clinch River - downstream the Emory River cofluence. After the dredging period mussels were placed at following four locations: Emory River - directly downstream the spill, lower Clinch River - downstream the Emory River cofluence, Clinch River - upstream of the Emory River confluence, Tennessee River - downstream of the Clinch River confluence. Additionally, 10 reference mussels of each species were kept in the laboratory in concrete raceways under the same conditions described under "details on test organisms" for the 45-day holding period.
- Test vessel: Custom deployment enclosures constructed of 5-cm PVC pipe in a 1.2×1.2 m square frame design with 2.5 cm squared high-density polyethylene (HDPE) plastic mesh covering the interior of the frame. Anchor supports (1 m long; made of 5-cm PVC) with custom pointed ends (for attachment into the river bottom) were attached to each corner of the frame.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Water source for organisms in laboratory was from the Cumberland River.


Nominal and measured concentrations:
River sites studied had different ash contents.
Reference substance (positive control):
no
Details on estimation of bioconcentration:
BASIS INFORMATION
- Monitoring data: accumulation of 23 metals in mussel tissue of three mussel species of ash-affected and non-ash-affected river sites as well as laboratory organisms was analyzed. Health condition indexes were also calculated and compared to bioaccumulation.

Type:
other: Mussel tissue concentrations for 23 metals were analyzed.
Remarks on result:
other: for results please refer to "any other informaiton on results incl. tables"
Remarks on result:
other: At end of river exposure period mussels were held between 36 and 48 h to ensure purging of gut contents before processing.
Reported statistics:
ANOVAs with Tukey’s post hoc tests: for analyses of metal concentrations and condition index for each species between site and lab reference samples.
JMP Pro 9 software (Cary, NC) was used for calculations.

- In the entire study, only mean lead concentrations were observed in two cases to exceed the respective contaminant advisory level given by the Food and Drug Administration (1.7 mg/kg). This was observed in the post-dredging period for purple wartyback, (3.2 mg/kg, Lower Clinch) and elephant ear (1.9 mg/kg, Upper Clinch).

- Although some mussel tissue accumulation was observed for some of the 23 metals analyzed in the study it was not linked to a specific spacial pattern (reference vs. affected sites or increased distance to dredging activities) for any of the three mussel species.

- In the post-dredging period significantly elevated concentrations of uranium, antimony, beryllium, and thallium were found in purple wartybacks at both the Upper and Lower Clinch sites and elephant ear in the Upper Clinch compared to the respective tissues in lab-reference organisms. Since concentrations were higher in both Upper (non-affected site) and Lower Clinch (affected site) sites it is not concluded that accumulation was linked to ash-related metals.

Comparison of accumulation during dredging and post dredging period:

- Thallium in black sandshell and mercury in purple wartyback were significantly increased in the post dredging period while thallium in purple wartyback and zinc in elephant ear, were significantly decreased in samples of the Emory River.

- Significant increases of eight metals (thallium, cadmium, uranium, antimony, beryllium, molybdenum, and vanadium) in purple wartyback were observed in the Lower Clinch River. Significant decreases of aluminum in black sandshell and purple wartyback as well as copper in purple wartyback were also observed in the same river.

Condition index observations:

Dredging activities seem to have affected the health condition of the mussels. Condition index decreased at Emory River for all mussel species after dredging activities. This was not observed in Clinch River. However lab-reference black sandshell and purple wartback showed in all cases a significant lower condition index compared to field organisms.

In conclusion dredging activities showed effects on mussel health but were not linked to increased accumulation of specific metals.

Validity criteria fulfilled:
not applicable
Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Fish monitoring was performed in the affected Emory and Clinch river. Three affected sites and three reference sites from the same rivers but not affected from the ash spill, were analyzed. Species with different diet preferences were chosen to capture bioaccumulation and biomagnification of metals. Fish were monitored for 5 years. Multiple parameters like body burdens, blood parameters, metrics of fish health and reproduction were examined. Focus was placed on As, Hg, and Se. Bioaccumulation was related through MANCOVA correlation to fish metrics and reproductive health.
GLP compliance:
no
Remarks:
peer reviewed data - GLP not relevant
Radiolabelling:
no
Details on sampling:
- Sampling intervals/frequency for test organisms: Bluegill and largemouth bass were monitored during April–June of 2009–2013 and redear sunfish during April–June 2010–2013.
- Sampling method for fish bioaccumulation: In the laboratory fish were euthanized and dissected in different tissues. Samples were homogenized for analytical investigations.
- Sampling method for fish health condition: 1 mL blood was sampled from each fish directly in the field. Metrics of fish health including measures of bioenergetics, hematology and immune function, carbohydrate- protein metabolism, electrolyte homeostasis, liver condition, and overall fish condition, were assessed. Several metrics of fish condition (CI) were assessed including the liver-somatic, visceral-somatic, and spleno-somatic indices. Fish reproductive condition was quantified by sizing, staging and counting all oocytes above size thresholds for active yolk accumulation (vitellogenesis) that were contained in a weighed subsample of ovary.
- Sample storage conditions before metal analysis for bioaccumulation: fish samples were shipped frozen (< - 10°C)
Vehicle:
no
Test organisms (species):
other: bluegill (Lepomis macrochirus), redear sunfish (L. microlophus), and largemouth bass (Micropterus salmoides)
Details on test organisms:
TEST ORGANISM
- Common name: bluegill, redear sunfish, and largemouth bass
- Reason for selection of these species: selected due to their high abundance in the Emory and Clinch rivers, due to antrhopogenic consumption and because they represent a variety of trophic levels (study of biomagnification). Bluegill and redear sunfish feed on insects, crustaceans, and other zoobenthos. Redear sunfish have a preference for mollusks. Largemouth bass are predators eating other fish species.
- Weight of monitored fish: generally 50–150 g for sunfish, and 500–2500 g for largemouth bass

Route of exposure:
other: multiple depending on fish species. For more details see "Details on test organisms"
Justification for method:
other: monitoring data from the affected river
Test type:
field study
Water / sediment media type:
other: fish lived in their natural environment (Emory and Clinch River)
Details on test conditions:
TEST SYSTEM
- Fish monitoring data from natural environment - Emory and Clinch River

Reference substance (positive control):
no
Details on estimation of bioconcentration:
BASIS INFORMATION
- Monitoring data: concentration of metals in fish tissue (subsequently correlated to health parameters)

BASIS FOR CALCULATION OF BCF
- No calculation of BCF
Remarks on result:
other: not reported
Type:
other: see "any other information on results incl. tables"
Remarks on result:
other: see "any other information on results incl. tables"
Remarks on result:
other: not reported
Details on results:
- Organ specific bioaccumulation: Although fillet, ovary, and liver bioaccumulation data were collected, analyses used data from fillets because that was the only appropriately-large dataset.
Reported statistics:
In order to detect differences among contaminants and fish health, length, site, and year of collection a multivariate analyses of covariance (MANCOVA; proc mixed, SAS) were applied for each species.
Pearson’s correlations was used for data correlation analysis.
Post-hoc Tukey’s honestly significant difference tests were applied to investigate the differences between sites for all MANCOVAs.

Ash spill sites had higher Se concentrations in all fish species compared to Se burdens in reference sites. This was found in some cases to be correlated to short term reproductive disability following the spill. However the authors conclude that due to the small sample size the results need to be treated with caution. Long-term reproductive effects due to the ash spill were negligible in this study.

Mercury tissue concentrations were generally higher at reference sites than ash-affected sites.

The MANCOVA analysis showed in most cases that there was no difference between the reference site and the affected site for most parameters.

The authors conclude from this study that there is only little evidence for severe chronic fish health effects due to bioaccumulation. However further investigations over a longer time period is needed for higher confidence.

Validity criteria fulfilled:
not applicable
Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
- Principle of test: The purpose of the test was to evaluate potential hazards to fish in Watts Bar Reservoir after the Tennessee Valley Authority (TVA) Kingston ash spill in December 2008. Among other observations of the bioaccumulation potential of constituents of potential ecological concern (COPECs) in fish tissues was evaluated. Only these results will be presented here.
- Short description of test conditions: Monitoring data from ash affected sites and reference sites (not affected by the spill) of the three rivers Emory, Clinch and Tennessee were collected.
- Parameters analysed / observed: Fish tissue concentrations of 26 target metals and metalloids in ash were compared with literature-based critical body residues (CBR). Furthermore concentrations of ash-related contaminants in water were compared with US Environmental Protection Agency's Ambient Water Quality Standards for Fish and Aquatic Life. Hazard quotients were calculated.
GLP compliance:
no
Remarks:
peer reviewed data - GLP not relevant
Radiolabelling:
no
Details on sampling:
- Sampling intervals/frequency for test organisms: Fish were collected from the rivers in 2009 (February through April) and 2010 and analyzed for 26 metals and metalloids.
- Sampling intervals/frequency for surface water samples: Samples were collected 8 times from August to October 2010 at 11 monitoring areas in Emory, Clinch and Tennessee rivers. Water samples were collected from the middle of the water column and close to the sediment surface (epi-benthic samples).
- Details on sampling and analysis of test organisms and test media samples: Water samples right above the sediment surface were analyzed unfiltered and 0.45 µm filtered in order to capture total and dissolved fractions of the 26 analytes.
Vehicle:
no
Test organisms (species):
other: Lepomis macrochirus, Ictalurus punctatus, Dorosoma cepedianum, Lepomis microlophus, Pomoxis annularus
Details on test organisms:
TEST ORGANISM
- Common name: bluegill, channel catfish, gizzard shad, largemouth bass, redear sunfish and white crappie
- Source: Emory, Clinch and Tennessee Rivers
Route of exposure:
other: multiple depending on fish species
Justification for method:
other: monitoring data from the ash affected rivers
Test type:
field study
Water / sediment media type:
other: river water from Emory, Clinch and Tennessee rivers
Reference substance (positive control):
no
Details on estimation of bioconcentration:
BASIS INFORMATION
- Monitoring data: Fish tissue concentrations of metals and metalloids (Bioaccumulation of constituents of potential ecological concern (COPEC)) were compared to critical body residues of literature data collected by the US Army Corps of Engineers/USEPA Environmental Residue-Effects Database as a conservative assessment of risks. Hazard quotients (HQs) for fish tissue were calculated by dividing tissue COPEC concentrations by the selected critical body residues. Hazard quotients were calculated for bluegill, channel catfish and largemouth bass fillets; bluegill and gizzard shad whole body samples; whole body gizzard shad without the gut or its contents; bluegill and largemouth bass liver; and bluegill and largemouth bass ovary tissues. Concentrations of analytes in water samples collected right above the sediment were compared to USEPA acute and chronic ambient water quality criteria (AWQC). Hazard quotients were calculated by dividing the measured concentration of each metal with the corresponding water quality criterion.
Remarks on result:
other: not reported
Type:
other: Fish tissue and water concentrations of 26 target metals and metalloids were determined and compared to respective quality criteria. For more details see "any other information on results inlc. tables".
Remarks on result:
other: monitoring study
Remarks:
For details see field "any other information on results incl. tables" and "overall remarks and attachments".
Reported statistics:
1-way analysis of variance (ANOVA) tests: to evaluate potential differences between locations for fillet, whole body, and whole body minus gut and its content, liver, and ovary samples. Analysis was also performed for each species and tissue type.

BIOLOGICAL RESULTS.

In several cases it was observed that COPEC (bioaccumulation of constituents of potential ecological concern) concentrations exceeded NOAEL and LOAEL critical body residues (CBRs). However similar observations were made in samples from reference sites. Furthermore from the 26 metal and metalloids investigated only two (As and Se) showed significantly higher concentrations in fish tissues collected in ash affected sites compared to the reference areas.

WATER CHEMISTRY RESULTS

1. Total concentrations (unfiltered samples)

- Surface water analysis showed that total (unfiltered) concentrations for some metals, mainly Al, and to a lesser extent Pb and Hg, were above the chronic ambient water quality criteria values and exceeded acute criteria in a few cased as well. However Al and Hg were found to exceed chronic criteria also in reference sites.

- Water data from samples collected in the middle of the water column showed that total Hg exceeded the chronic criterion in 3 ash affected sites and one reference site. The acute ambient water quality criteria were not exceeded in any case for total Hg concentrations.

- Benthic water samples: Hg was elevated and exceeded the chronic criterion in 1 ash affected and one reference site. Pb was higher than the chronic criterion in 2 ash affected sites. Acute criteria were not exceeded.

2. Dissolved concentrations (45 µm filtered samples):

- Water data from samples collected in the middle of the water column showed that Al and Hg exceeded chronic criteria for one sampling site. Acute criteria were not exceeded in any case.

- Benthic water samples: all metal concentrations were lower than the chronic and acute criteria.

For more results on water samples see field ""overall remarks, attachments.

In conclusion analysis of tissue concentrations of ash-related metals and metalloids indicated an exceedance of critical body residue values for Al, As, Cd, Cu, Pb, Se and Zn. However these results need to be treated with caution according to the authors due to exceedance of the critical body residues also in reference sites and due to the conservative nature of these values. Elevated values were mostly observed for unfiltered samples (total concentrations). Very little exceedances for dissolved metals were found. Dissolved concentrations might be more relevant for assessment due to better bioavailability. Therefore in general a moderate risk potential by metals incorporated in ash was concluded for fish by the authors from the results of this study.

Validity criteria fulfilled:
not applicable
Endpoint:
bioaccumulation in sediment species: invertebrate
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to guideline
Guideline:
other: US Environmental Protection Agency. 2000. Guidance for assessing chemical contaminant data for use in fish advisories, Vol 1—Fish sampling and analysis. EPA 823-B-00-007. Office of Water, Washington DC.
Version / remarks:
Guideline was used for sample handling procedures.
Qualifier:
according to guideline
Guideline:
other: US Environmental Protection Agency. 2007. Test methods for evaluating solid waste, physical/chemical methods. SW-846, Test methods for evaluating solid waste, physical/chemical methods. Office of Solid Waste, Washington, DC.
Version / remarks:
Guideline used for analysis of mercury in samples.
Qualifier:
according to guideline
Guideline:
other: US Environmental Protection Agency. 1996. Method 3052. Microwave assisted acid digestion of siliceous and organically based matrices. SW- 846, Test methods for evaluating solid waste, physical/chemical methods. Office of Solid Waste, Washington, DC.
Version / remarks:
Guideline was used for nitric acid digestion of samples.
Qualifier:
according to guideline
Guideline:
other: US Environmental Protection Agency. 2007. Method 6020A. Inductively coupled plasma-mass spectrometry. SW-846, Test methods for evaluating solid waste, physical/chemical methods. Office of Solid Waste, Washington, DC.
Version / remarks:
Guideline was used for analysis of remaining 16 metals.
Principles of method if other than guideline:
- Principle of test: Mayfly nymphs (Hexagenia bilineata) were monitored for 17 ash-related metals in the tree ash spill affected rivers Emory, Little Emory, Clinch and Tennessee from 2009 to 2012.
- Short description of test conditions: Hexagenia nymphs from sediment samples were collected from affected and non affected sites for metal analysis.
- Parameters analysed / observed: Spatial and temporal trends for concentrations of metals in nymphs of affected and reference sites were observed and analyzed.
GLP compliance:
no
Remarks:
peer reviewed data - GLP not relevant
Radiolabelling:
no
Remarks:
peer reviewed publication
Details on sampling:
- Sampling intervals/frequency for test organisms: Hexagenia nymphs were collected from the Emory, Clinch and Tennessee rivers at ash affected and non affected sites. In 2009 3 ash affected sites and 3 reference sites were sampled from the Clinch and Emory rivers. After 2009 one site was added to assess the upstream presence of ash-related contaminants, one site approximately 1.0 mile downstream of the confluence with the Emory River, and 2 ash affected and one reference sites of the Tennessee River were added. Mayfly nymphs were collected once per year between early May and mid-June. Four (2009) and three (2010 to 2012) samples were collected from each site (Peterson dredge).
- Sample storage conditions before analysis: Samples were held on ice for transport to the laboratory. In the laboratory samples were stored at < -20°C until further analysis. Collected rinsates were stabilized with nitric acid and stored in a refrigerator until analysis.
- Details on sampling and analysis of test organisms and test media samples: Dredged samples were on site washed with river water and collected nymphs were placed in small glass bottles (60–120 mL). In the laboratory samples were rinsed 3 times with distilled water. Excess external moisture was removed. > 1.5 g nymphs were added in 40 mL glass I-Chem vials. Samples were dried > 90 h in a freeze drier. Dried samples were stored frozen until analysis. Rinsates during the procedure were extra collected. All samples were analyzed for 17 metals (antimony (Sb), arsenic (As), barium (Ba), beryllium (Be), boron (B), cadmium (Cd), chromium (Cr), cobalt (Co), copper (Cu), lead (Pb), mercury (Hg), molybdenum (Mo), nickel (Ni), selenium (Se), strontium (Sr), vanadium (V), and zinc (Zn)).
Vehicle:
no
Test organisms (species):
other: Hexagenia nymphs
Details on test organisms:
TEST ORGANISM
- Common name: mayfly
- Source: Emory, little Emory, Clinch and Tennessee Rivers
Route of exposure:
sediment
Justification for method:
other: Monitoring data from ash affected rivers.
Test type:
field study
Water / sediment media type:
natural water / sediment: freshwater
Reference substance (positive control):
no
Details on estimation of bioconcentration:
BASIS INFORMATION
- Monitoring data: Metal concentrations in Hexagenia nymphs were analyzed.

Type:
other: See "any other information on results incl. tables"
Remarks on result:
other: See "any other information on results incl. tables"
Reported statistics:
Principal components analysis was used to identify spatial and temporal patterns in metal concentrations.

Concentrations of all metals in the rinsates were below the method detection limits.

Principal analysis supports that concentrations of As, Ba, Be, Mo, Sb, Se, Sr, and V were higher concentrated in nymphs at ash-affected sites than at reference sites. From these metals, Se concentrations in nymphs from ash-affected sites were either comparable to or higher than the concentrations in pond ash. This was not the case for the other metals (concentrations were significantly higher in pond ash compared to nymphs). This group of metals showed a decreasing concentration gradient with increasing distance from the spill site.

Concerning B, Cr, Co, Cu, Ni, and Pb, the lattest was comparably concentrated in nymphs and ash. All other metals were higher in ash than in nymphs. These elements did not show a concentration gradient with increasing distance from the spill site suggesting also other sources of contamination.

Hg was not necessarily associated with the ash spill. Hg accumulation was observed but was not associated only to ash sources.

Cd and Zn concentrations in nymphs were much higher than in ash. The accumulation of these elements was not found to be associated with the ash spill, although it cannot be excluded.

Results strongly suggest that the ash associated metals in nymphs started to decline after 2010. This was increasingly observed in 2012 with As, Se, Sr, Sb, Mo, and V bearing the lowest levels in this study. However values were still elevated compared to the reference sites.

Validity criteria fulfilled:
not specified
Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to guideline
Guideline:
other: Palermo MR, Schroeder PR, Estes TJ, Francingues NR. 2008. Technical guidelines for environmental dredging of contaminated sediments. ERDC/EL TR-08-29. U. S. Army Engineer Research and Development Center, Vicksburg, MS.
Deviations:
yes
Remarks:
10-d instead of 1-h mixing time (through aeration) to represent extreme conditions for metal release and dissolution and a conservative assessment of toxicity and bioaccumulation.
Qualifier:
according to guideline
Guideline:
other: Bednar AJ, Chappell MA, Seiter JM, Stanley JK, Averett DE, Jones WT, Pettway BA, Kennedy AJ, Hendrix SH, Steevens JA. 2010. Geochemical investigations of metals release from submerged coal fly ash using extended elutriate tests. Chemosphere 81:1393–1400.
Qualifier:
according to guideline
Guideline:
other: U.S. Environmental Protection Agency. Test methods for evaluating solid waste, physical/chemical methods, 3rd ed. SW-846. Washington, DC.
Principles of method if other than guideline:
- Principle of test: Larval and juvenile fish were exposed for 10 d to Emory River (EMR-EL) and ash recovery ditch elutriates (0, 10, 50 and 100%) as well as controls in order to study the potential effects of dredging activities (metal mobilization) to fish health. A larval and juvenile fish elutriate bioassay as well as a gut metal content experiment were conducted and analysed. 24 metals were measured in fish tissues. However detailed focus was given to 4 metals of higher concern: As, Cr, Hg, Se. Here only the data relevant for bioaccumulation are presented (juvenile fish elutriate bioassay and gut metal experiment).
GLP compliance:
no
Remarks:
peer reviewed data - GLP not relevant
Radiolabelling:
no
Details on sampling:
- Sampling intervals/frequency and method for test organisms in fish toxicity and bioaccumulation experiment: For juvenile fish experiments, survival, whole-fish biomass, length, liver mass (to determine liver somatic index), and bioaccumulation were identified at the end of the test. For fish tissue metal analysis samples were taken at test termination.
- Sampling intervals/frequency and method for test organisms in gut content experiment: digestive tracts were removed (except of liver) and rest of fish carcass was homogenized. The digestive tracts and carcass were dried overnight in a 60° C oven, then ashed in a muffle furnace at 550°C for 3 h. The metal content in the ashed digestive tracts and the homogenized fish carcasses was analyzed.
- Sampling intervals/frequency and method for test medium samples: During the 10-d elutriate preparation period, samples for metal determination were collected at 1, 24, 48, 96, and 240 h. During test water-quality parameters (temperature, pH, conductivity, dissolved oxygen) were recorded for all replicates at test start and end and for one replicate per treatment during test days 1 to 9.
Vehicle:
no
Details on preparation of test solutions, spiked fish food or sediment:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 10 g (wet wt) fly ash was added to 1 L Emory River water or ash recovery ditch water to prepare the elutriate mixture. Solution was mixed for 10 d to represent extreme conditions of metal release. The elutriate sediment load was calculated to be approx. 5.3 g/L.
- Controls: 0% (Emory River water from reference site), procedure control (dechlorinated tap water)
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: fathead minnow
- Source: commercial supplier: Aquatic Biosystems
- Length at study initiation: Juvenile fish: 4.2 ± 0.3 cm (were selected to supply adequate tissue mass for chemical analysis)
- Feeding during test : yes
- Food type: Fish flakes for juvenile P. promelas (Zeigler® AquaTox Feed, Aquatic Eco-Systems).
- Frequency: Every other day to maintain water quality.

ACCLIMATION
- Acclimation period: Juvenile fish - 72 h
- Acclimation conditions: same as test
- Type and amount of food: Fish flakes for juvenile fish.
- Feeding frequency: daily
Route of exposure:
aqueous
Justification for method:
aqueous exposure method used for following reason: in order to assess the potential risk from ash dredging activities in the river to fish
Test type:
semi-static
Water / sediment media type:
other: elutriates prepared with natural river water and ash
Total exposure / uptake duration:
10 d
Total depuration duration:
10 d
Hardness:
20 - 100 mg/L
Test temperature:
19.2 - 21 °C
pH:
7.12 - 8.47
Dissolved oxygen:
6.0 - 9.9
Conductivity:
90 - 290 mS/cm
Details on test conditions:
TEST SYSTEM
- Test vessel: 3.75-L glass jars for juvenile fish
- Aeration: yes
- Renewal rate of test solution: renewal on test days 3 and 7 (fish tissue metal experiment), renewal on day 5 (gut metal content experiment)
- No. of organisms per vessel: 10 (fish tissue metal experiment), 5 (gut metal content experiment)
- No. of vessels per concentration (replicates): 5 (fish tissue experiment), 1 (gut metal content experiment)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 2 elutriates were prepared for tests with fly ash and 1. Emory River water (EMR-EL) and 2. with water from the ash recovery ditch (ARD-EL). Reference water for elutriates and toxicity tests was collected from the Emory river approximately 12 miles upstream of the fly ash affected site. Fly ash was collected from 5 locations of the same river where dredging activities were occurring. Fly ash and water samples were also collected from the ash recovery ditch. All fish were exposed to elutriate water collected on day 10 of the elutriate water preparation. Juvenile fish were exposed only to EMR-EL water.
- Particulate matter: no data
- Alkalinity: 64 - 160 mg/L
- Holding medium different from test medium: yes

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: not mentioned
- Light intensity: not mentioned

RANGE-FINDING / PRELIMINARY STUDY
- Test concentrations: no
Nominal and measured concentrations:
Fish tissue metal experiment: 100% elutriate (the 10-d elutriate preparation), 50% and 10% elutriate treatments (diluted with Emory River reference water), 0% (Emory River reference site water), control (dechlorinated tap water)
Gut metal content experiment: 100% elutriate (the 10-d elutriate preparation), 0% (Emory River reference site water), control (dechlorinated tap water)
Reference substance (positive control):
yes
Remarks:
KCl
Details on estimation of bioconcentration:
BASIS INFORMATION
- Dissolved concentrations of metals in elutriates were compared with screening values (criterion continuous concentration - CCC - US EPA screening values and literature) in order to assess the exposure of fish. Metal concentrations measured in fish were evaluated through comparison with literature-based metal critical body residues (CBRs e.g. NOECs).

BASIS FOR CALCULATION OF BCF
- No BCF calculated
Remarks on result:
other: not reported
Type:
other: See "any other information on results incl. tables"
Remarks on result:
other: See "any other information on results incl. tables"
Results with reference substance (positive control):
48- and 96- h median lethal concentration = 0.69 g KCl/L
Details on results:
- Mortality of test organisms: No mortality of juvenile fish during 10 d test period.
- Behavioural abnormalities: none observed
- Observations on feeding behavior: Feeding was observed in all treatments during the whole test period.
- Observations on body length and weight: No difference of fish length, whole-fish biomass, liver mass and liver somatic index in any of the treatments compared to the reference site water (0% treatment).
- Reproduction during test period: no
- Organ specific bioaccumulation: Metal content was measured in gut. Concentrations of As, Cr and Se were significantly higher in the gut compared to remaining fish carcass. For EMR-EL elutriates these metal concentrations in the gut were 50 - 2800 times higher than in remaining fish carcass.
- Mortality and/or behavioural abnormalities of control: No mortalities.


No fish mortalities or other effects (decreased weight, length etc.) were observed in any treatment compared to the controls.

Whole fish analysis (incl. tissue and gut) showed significant increases in whole-body burdens for following metals: As, Se, Hg, Al, Ba, Cd, Co, Cu, Fe, K, Mg, Mo, Pb, Tl, and V.

Metal content analysis in gut showed As, Cr and Se were substantially higher in gut than rest parts of the fish. In Emory River elutriates the concentration in gut was 50 - 2800 x higher in the gut compared to the remaining fish carcass. This suggests that the exposure dependent increase of metal concentration in the tested juvenile fish were mainly attributed to metal content in gut than accumulated in fish tissues.  

The authors conclude that maximum dissolved As (identified as As(V)), Cr (identified as Cr(III)) and Hg (inorganic), measured in elutriates were still lower than concentrations of concern according to U.S. EPA recommendations and other literature. Se (identified as Se(IV)) exceeded in some time points the U.S. EPA and Tennessees maximum limit of 5 µg/L. However the exposure route to Se is primarily the diet and based on the body-burden values, authors conclude low concern from Se.

All mean measured body burdens of As, Cr, Hg and Se in fish were below critical body residues (CBRs) based on literature search by the authors. In one case measured As was slightly higher compared to literature critical body residue values. However, based on the gut metal content experiments, it is very possible that the elevated concentration was a contribution of the metal in the gut.

Validity criteria fulfilled:
yes
Endpoint:
bioaccumulation in aquatic species: fish
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Remarks:
Summary of available data used for the endpoint assessment of the target substance.
Adequacy of study:
weight of evidence
Justification for type of information:
Refer to analogue justification provided in IUCLID section 13
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Type:
other:
Remarks on result:
other: Based on results from WoE source studies, the potential for bioaccumulation in the aquatic/sediment environment are expected to be low.

Description of key information

The bioaccumulation potential of ashes (residues), including cenospheres, is low.

Key value for chemical safety assessment

Additional information

No bioaccumulation study is available for the cenospheres, but it can be concluded that cenospheres are not bioavailable for aquatic organisms due to its physical and chemicals properties as inert, hollow balls of sand-like material; Thus considering the extremely low recovery rate of ash contents, experienced from toxicological investigations and based on the structure-related properties of the substance, bioavailability and therefore bioconcentration of cenospheres contents is not expected.

Since no studies investigating the bioaccumulation of cenospheres, fly ash (CAS 93924-19-7) are available, in accordance to Regulation (EC) No 1907/2006 Annex XI, 1.5 a read-across to structurally related ashes (CAS 931-322-8) was conducted. This read-across is justified within the analogue justification in IUCLID Section 13.

Ashes, including cenospheres, are inorganic complex compounds consisting of multitude of unknown and variable constituents (UVCB), and it is therefore technically not possible to determine an overall bioaccumulation for this substance. However, most of metals contained in ashes, which are of concern have been reviewed in literature. 

End of 2008 4.1 million m3coal ash were released into the Emory, Tennessee and Clinch rivers due to an accident in the Tennessee Valley Authority (TVA) Kingston Fossil Plant. This unfortunate accidence provided an opportunity to directly study the impact of coal fly ash in a large lotic system since more than one rivers were affected. A variety of studies were initiated by multiple actors to assess ecological risks to different organisms. Since fly ash contains a diversity of metals such as As, Cr, Cu, Pb, Hg, Ni, Se, Tl, V, Zn, it is of a long-term ecological concern. Among multiple toxicological and ecotoxicological studies, bioaccumulation potential of metals associated to fly ash was investigated either as an endpoint solely or in combination with toxicological effects.

Shortly after the spill, 90% of the ash was removed from the river by dredging. Studies concern both the period of dredging (March 2009 – May 2010) and the post-dredging period after the partial removal of the ash. 5 published studies related to the Kingston spill were taken into account as evidence for the bioaccumulation potential of ash related metals in fish. Publications cover both field monitoring data and laboratory experiments. In some studies special attention was given to specific metals or metalloids, such as Se, As and Hg.

In 2009 and 2010 benthic (bottom-dwelling) and pelagic (mid-water column) fish communities were collected from the spill site by the Tennessee Valley Authority in order to examine among others the bioaccumulation potential of ash related constituents (Riggs et al. 2014). Following fish species were chosen for investigation: bluegill (Lepomis macrochirus), channel catfish (Ictalurus punctatus), gizzard shad (Dorosoma cepedianum), largemouth bass (Micropterus salmoides), redear sunfish (Lepomis microlophus), and white crappie (Pomoxis annularus). 26 trace metals and metalloids were measured in order to evaluate if uptake poses a risk for fish health. Fish tissue concentrations were compared with literature-based critical body residues. Concentrations of ash-related contaminants in water were additionally compared to US Environmental Protection Agency's Ambient Water Quality Standards for Fish and Aquatic Life and finally Hazard Quotients were calculated. The outcome of this study revealed little evidence of elevated uptake of ash related metals of potential ecological concern in fish. As and Se were an exception for which tissue concentrations were significantly higher in ash than in reference sites and exceeded consistently critical body burdens, such as NOAEL (NOAEL Se: 0.018 – 0.524 mg/kg ww; NOAEL As: 0.04 – 8.4 mg/kg ww for liver, muscle, ovary and whole body). The authors express a reservation toward the results due to uncertainties in retrieving the critical body residues (US Army Corps of Engineers/USEPA Environmental Residue-Effects Database) considering these values as very conservative. 

Stanley et al. (2013) conducted laboratory studies with juvenile fish in order to study among others the potential bioaccumulation effects of dredging activities and potential metal mobilization to fish health. The experiments were designed in such way to represent extreme conditions of exposure to oxidized and mobilized metal species. Juvenile Pimephales promelas were exposed for 10 d to elutriates prepared by bubbling river water with two different fly ash types for 10 days. Then elutriates were diluted to create different exposure concentrations (0, 10, 50 and 100%). Emory river water and dechlorinated tap water were used as controls. Concerning the study of bioaccumulation potential, a juvenile fish elutriate bioassay and the analysis of metal contents in the gut were performed. 24 metals (As, Cr, Hg, Se, Ag, Al, Ba, Be, Ca, Cd, Co, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, Pb, Sb, Tl, V, Zn) were measured in fish tissues with a special focus on 4 selected metals of higher concern: As, Cr, Hg and Se. No mortality was observed in the study. A dose-response (increased concentrations) relationship was observed between mean concentrations of the four metals in fish (whole body) and elutriates (not significant for Cr). Nevertheless, all mean measured body burdens of As, Cr, Hg and Se in fish were below critical body residues (CBRs) based on literature search by the authors. Only in one case measured As was slightly higher (2.95 (±0.76) mg/kg wet weight) compared to literature critical body residue values. The analysis of gut metal content suggests that above mentioned metals were mostly associated to gut content and not fish tissue. The study results demonstrate a low potential of toxic bioaccumulation from ash occurring at extreme conditions.

Se bioaccumulation and potential health concerns in fish were investigated over time from 2009 through 2013 after the Kingston ash spill (Mathews et al., 2014). A monitoring was performed in two affected rivers i.e. Emory and Clinch as well as downstream of the spill. Reference sites were additionally monitored for comparison. Three fish species (Bluegill sunfish, Redear sunfish, Largemouth bass) with different trophic habits were examined. Water and sediment samples, fish fillets as well as whole body fish were analyzed for Se. Se concentrations in water were low within the 5 year investigation period and in most cases below the detection limit. Detectable Se was mostly found in samples collected close to the spill source. In only one case the Se concentration in water was higher (1.34 µg/L) than the USEPA aqueous criterion for Se of 1.3 µg/L in lentic systems. Concentrations in sediments were between 1.15 µg/g to 8.25 µg/g at ash-affected sites and between 1.01 and 6.17 µg/g in not affected sites, without statistic significant difference. Se concentrations in fish fillet were higher in contaminated (1.2 - 8 µg/g) compared to non-contaminated sites (1.2 - 5.5 µg/g). Similar trends were observed for whole body fish. However, this depends on fish species while the USEPA proposed whole body Se threshold in fish (8.1 µg/g) was not exceeded in any case, the study does not indicate high risks for aquatic life from Se at this spill site. 

Fish health and reproductive metrics were linked to contaminant bioaccumulation at the Tennessee Valley Authority Kingston coal ash spill by Pracheil et al. (2016). Fish monitoring was performed in the affected Emory and Clinch rivers. Three of the affected sites and three reference sites were analyzed. Species with different diet preferences were chosen to capture bioaccumulation and biomagnification of metals. Fish were monitored for 5 years. Multiple parameters like body burdens, blood parameters, metrics of fish health and reproduction were examined, while As, Hg, and Se were investigated intensively. Bioaccumulation was related through MANCOVA correlation to fish metrics and reproductive health. The MANCOVA analysis showed in most cases that there was no difference between the reference site and the affected site for most parameters. Ash spill sites had generally higher Se concentrations in all fish species compared to Se burdens in reference sites and was verified by Tukey’s test. For Hg and As, the trends were different. Hg was significantly elevated only in one of the reference sites compared to the other sites. Elevation of As was observed in bluegill and largemouth bass from spill sites the years directly after the spill (2010-2011). The study did no show consistent patterns in blood chemistry functional response groups over years for any species. A short-term reproductive disability following the spill was observed which however needs to be treated with caution due to the small sample size of the results. In conclusion although some ash associated contaminants were elevated at ash affected sites, there was no clear evidence to affect fish health. Long-term health effects due to the ash spill are considered to be low.

Se and As bioaccumulation were examined by Otter et al. (2012). The bioaccumulation of As and Se in the liver, ovary and muscle tissues of multiple fish species and the potential reasons of the differences after the coal ash spill in Kingston were investigated in this study. Fish were collected from two affected sites of the Emory River and two reference sites upstreams of the spill of the same river for comparison. Concentrations of As and Se in liver, ovary and muscle tissues, differences in species stomach and intestine pH as well as different trophic dynamics (that could affect different bioaccumulation patterns) were explored. As concentrations were elevated in ash contaminated sites in multiple fish species while Se was higher concentrated only in sunfish and bluegill fish in the ash affected sites. However As and Se concentrations in fish were generally low. The trophic dynamic study indicated some significant differences between ash affected and non ash affected sites. The pH in stomach and intestine was not found to have an impact to Se or As bioaccumulation.

Greeley et al. investigated the bioaccumulation and maternal transfer of the often ash-related metals As, Se and Hg in redear sunfish (Lepomis macrolophus), two years after the Kingston spill. Ash affected and non ash affected sites from the Emory and Clinch Rivers were compared. In this study, similar to other studies, it was shown that although whole body (4.9–5.3 mg/kg d.w.) and ovary (6.7–9.0 mg/kg d.w.) concentrations of Se were significantly higher compared to concentrations in fish from reference sites (2.2–3.2 mg/kg d.w.for whole bodies and 3.6–4.8 mg/kg d.w. for ovaries), Se concentrations remained below the proposed U.S. Environmental Protection Agency (USEPA) criteria for the protection of aquatic life.

Further two publications related to the Kingston ash spill are available for the bioaccumulation of ash related metals to two sediment organisms. Sediment organisms had slightly different patterns concerning accumulation of metals that fish.

The first available publication intended to in situ investigate the bioaccumulation of metals in three different mussel species (black sandshell, elephant ear, purple wartyback) during and after dredging activities in the ash affected sites (Otter et al. 2015). Mussels were caged for 1 year in ash affected and unaffected river sites for both the dredging period and the post-dredging period. Additionally mussels of each species were kept under laboratory conditions as a control. Mussel health (mussel condition index) and concentrations of 23 metals (As, Cd, Cr, Pb, Ni, Se, Hg, U, Fe, Mg, Al, Sb, Ba, Be, Co, Cu, Mn, Mo, Ag, Sr, Tl, V, and Zn) in soft tissues were measured. Potential differences based on location, dredging activities and species were investigated. Metal mussel concentrations were compared with contaminant advisory levels given mainly by the Food and Drug Administration in order to identify potential threats. From all metals measured, only the mean of lead concentrations were observed in two cases to exceed the respective contaminant advisory level given by the Food and Drug Administration (1.7 mg/kg). This was observed in the post-dredging period for purple wartyback, (3.2 mg/kg, Lower Clinch) and elephant ear (1.9 mg/kg, Upper Clinch). Dredging activities had in some cases effects on mussel health but were not linked to increased accumulation of specific metals.

In a further study with sediment invertebrates mayfly nymphs (Hexagenia bilineata) were monitored for 17 ash-associated metals in the tree ash spill affected rivers Emory, Little Emory, Clinch and Tennessee from 2009 to 2012 (Smith, 2016). Hexagenia nymphs from sediment samples were collected from affected and reference sites for metal analysis. Spatial and temporal trends for concentrations of metals in nymphs of affected and reference sites were observed and analyzed. Principal analysis support that concentrations of As, Ba, Be, Mo, Sb, Se, Sr, and V were higher in nymphs at ash-affected sites than at reference site and concentrations in animals decreased with increasing distance from the spill. Se concentrations in nymphs were even higher than in pond ash samples. The results of the study strongly suggested that the ash-associated metals in nymphs started to decline after 2010, and this was increasingly observed in 2012 with As, Se, Sr, Sb, Mo, and V bearing the lowest levels in this study. However, the overall values were still elevated compared to the reference sites. The authors conclude that the concentrations found are not high enough to cause adverse effects.

Finally, one study provides information on the bioaccumulation of ash related metals to terrestrial organisms. Site-specific bioaccumulation of ash-associated metals in tetragnathid spiders in ash spill affected sites and the potential transfer from the aquatic to the terrestrial systems was investigated. Spiders and sediment were collected from ash affected and reference sites and analyzed for metal concentrations. Results showed that although concentrations of metals in sediments were higher at ash affect sites compared to reference sites in many cases, this was not reflected in spiders. Most of metals showed no significant site difference in the tested organisms indicating a lack of metal bioavailability for this organism. Se and Ni were found to be elevated in tetragnathid spiders. However, threshold values are scarce for this organisms and therefore it cannot be concluded that the concentrations could be hazardous.

The available studies showed in a weight of evidence approach that bioaccumulation of ash associated metals is possible but did not indicate high concerns towards toxicity to any compartment. In only few cases concentrations in organisms exceeded proposed thresholds (e.g. from US EPA, FDA) for the protection of the environment and humans, even under the extreme conditions of the spill. More than 24 metals (e.g. As, Cr, Hg, Se, Ag, Al, Ba, Be, Ca, Cd, Co, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, Pb, Sb, Tl, V, Zn) were investigated. However, individual metals of concern such as As and Se seem to be longer in flux and therefore of higher concern especially for adult fish. Bioaccumulation was, not surprisingly, found to be species dependent considering diet preferences. Mussels and hexagenia nymphs were representative organisms for the sediment compartment investigated for ash-related metal bioaccumulation. Bioaccumulation of several metals was observed in representative sediment organisms. However, concentrations were in both cases not high enough to cause concerns and were not related to toxicity. Transfer of the ash related metals from the aquatic to the terrestrial compartment as studied for tetragnathid spiders was found to be low except in case of Se, which was found to be elevated in the organisms. However, it needs to be considered that the spill represents an extreme and worst case situation. Bioavailability and consequently bioaccumulation of ash-related metals is complex and dependent on multiple factors like pH, redox potential, geochemical interactions etc. The site monitoring and lab investigations of the presented publications are highly representative for demonstrating the behavior of ash-associated metals in the water, sediment and soil, with indicating the impact of ash contamination on different fish species and potential of accumulations. However, the pool of these studies do not cover the completely possible spectrum on the bioaccumulation potential of ash related metals under the different environmental conditions.

Furthermore, the Tennessee Valley Authority (TVA) initiated two-phase laboratory sediment and surface water toxicity studies in the spring and summer of 2009. The objective of this testing was to determine whether constituents associated with fly ash including cenospheres found in site sediment and surface water are harmful to or are bio-accumulated by benthic invertebrates. The primary focus of the initial studies was on the potential for short-term effects associated with removing ash from the. The toxic effects endpoints measured include survival, growth and reproduction, and metals bioaccumulation elicited by exposure of benthic and aquatic species to whole ash, ash elutriates, dredge plume water, and ash stilling pond effluent. The results from these „A Multi-phased Toxicity Study for Evaluating Potential Risks of Kingston Fossil Plant Fly Ash Exposure to Benthic and Aquatic Biota“ ash composite samples indicated no appreciable bioaccumulation of metals inCorbicula flumineaexposures (28-d) to whole ash (R. Sherrard; poster, SETAC, 2009). The Bioaccumulation Factors (BAFs) are negligible, all BAF values for the measured heavy metals are below 1; the maximum BAF among metals was for zinc (mean = 0.343, range = 0.146 - 0.591 kg sediment (dry wt) / kg body weight (wet wt).

A second 4-dayLumbriculus variegatustoxicity test (pre-bioaccumulation test) shows also no significant difference between resin-treated ash and lab control and criteria for conducting 28-day bioaccumulation test were not met (R. Sherrard; Poster, SETAC, 2009 and Hydrosphere Research, 2009) and also in a field study with fish was no indication for a bioaccumulation potential of the fly ash including cenospheres. There were differences between sites in some indicators of exposure and effects in these fish collected 2-3 months after the spill, there were no consistent relationships between metal bioaccumulation and indicator responses and female fish collected at and downstream of the spill in spring/summer 2009 had no obvious significant reproductive abnormalities.

It can be therefore concluded that a significant bioaccumulation of ash contents including cenospheres is not expected and further bioaccumulation studies are not necessary.

Reference:

Greeley Jr. M.S., Adams S. M., Elmore L.R., McCracken M.K. (2016) Influence of metal(loid) bioaccumulation and maternal transfer onembryo-larval development in fish exposed to a major coal ash spill. Aquatic Toxicology 173, 165–177.

R. Sherrard – Tennessee Valley Authority, Kingston Ash Recovery Project, Kingston, TN and Hydrosphere Research, 11842 Research Circle, Alachua, FL 32615, Ph. 386-462-7889 cited from: SETAC POSTER 2009 and: http://www.tva.gov/)