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EC number: 931-597-4
CAS number: -
Table 2. Total content of the elements in the ashes. (TS= dry substance,
LOI= Loss on ignition, TOC= Total Organic Carbon, Ntot= total amount of
% av TS
Table 3. Amounts of metals, chloride, fluoride, sulfur and nitrogen in
the leachates of ashes.
Table 4. The validity parameters (pH, oxygen in % and salinity in ‰) for
the leachates measured before test start.
A subchronic Fish Embryo Toxicity (FET) test was performed for three
different types of ashes according to an OECD Guideline for testing of
chemicals, Draft proposal for a new guideline (2006). The used species
was Danio rerio. Water Accomodated Fractions (WAFs) of different
types of ashes were prepared according to SS-EN 14735:2005/AC:2006 in a
ratio L/S 10. Due to delayed toxic responses, observation period used
was 144 hours. NOEC for bottom ash formed by burning mainly domestic
waste (Ash A) was 3.2 %, NOEC for fresh fly ash formed by burning mainly
domestic waste (Ash C) was 0.78 %, and NOEC for fly ash formed by
burning mainly biofuels (Ash C) was 25 % of ash WAF in the exposure
mixture. Significant delay (p<0.001) in hatching time was reported for
bottom ash and for fresh fly ash.
Highest ecotoxicological effects were found for Ash C, for which
concentrations of the most toxic heavy metals and salinity were the
highest of the studied ashes. The adjusted high salinity affects the
complex formation, binding in organic carbon, and bioavailability of
metals. The study indicated that the results depend on the preparation
method of the ash WAF and therefore, toxicity may be overestimated.
Oxidative stress induction potential of fly ash leachate (FAL) in Channa
punctata was studied in a non-GLP non-guideline 24 h study. The
sub-bitominous fly ash was obtained from a thermal power plant dumping
site. The leachate was prepared by mixing the ash in water one hour
every day for seven days, after which the leachate was filtrated. Amount
of fly ash in the studied leachate was 100 mg/mL. After the exposure
time, fish were homogenised followed by centrifugation. The supernatant
was used for the biochemical analyses such as rate of lipid
peroxidation, acitivity of catalase and gluthathione S-transferase
enzymes and level of glutathione. Exposure to fly ash leachate induced
lipid peroxidation, caused elevated enzyme activities and increased
levels of glutathione in all the studied organs. All the effects were
statistically significant compared to controls.
DNA fragmentation and DNA laddering
the studied parameters indicate a proapoptotic effect of fly ash
leachates in fish hepatocytes (See figures 1 -2).
cytochrome-c and LDH
concentration-dependent increase in the activity of caspases 3, 7 and 10
was observed in cells exposed to different concentrations of FAL for 48
h. The maximum activity of the caspases was recorded in the cells
exposed to the highest concentration of FAL (10%). The increase was
significant at FAL concentrations (w/v) of 2% (P < 0.05), 5% (P < 0.01)
and 10% (P < 0.001). The increase of caspase-9 was most pronounced of
the caspases (P < 0.001). Compared to control, increase of release of
cytochrome-c was significant (P<0.05) in all concentrations of FAL
except for 1%. LDH activity increased significantly at two highest
concentrations (P<0.05 at 5% and P<0.01 at 10%) with high variation in
the results. See figures 3A-C.
superoxide ions and LPO
exposure also resulted in a significant concentration and time dependent
increase in H2O2 production by hepatocytes (Fig.
4A). The maximum increase in production of H2O2was
observed at 10% FAL concentration. Also at other FAL concentrations (1%,
2% and 5%) there was significant (P<0.01) increase. However, exposure at
low concentration (1% FAL) resulted in a significant increase in 48h
analysis (P<0.05). Instead, higher production of superoxide ions was
observed at 24h than at 48h. At 10% FAL exposure, there was a
significant increase in superoxide ion production at 24 h (P<0.01 and at
48h (P<0.05) compared to control in all concentrations except for 1% at
48h. LPO measurement in C. punctata hepatocytes showed significant
increase at all the concentration of FAL (except 1%) at 24 h as well as
48 h (Fig. 4C).
The pro-apoptotic effects of fly ash leachate (FAL) in hepatocytes of Channa punctata was studied in a non-GLP non-guideline in-vitro study. The sub-bitominous fly ash was obtained from a thermal power plant dumping site. The leachate was prepared by mixing the ash in water one hour every day for seven days, after which the leachate was filtrated.
The hepatocytes were isolated with a double perfusion method. The hepatocyte cell density was 4 x 106cells/mL. The medium used was RPMI-1640 medium with 1% FBS, 25 µL/mL gentamycin sulphate and 2.5 µg/mL amphotericin-B. A slurry containing ash was prepared. Cytologic and genetic effects of 10 % sub-bituminous coal fly ash leachate were studied at concentrations of 0, 1, 2, 5 and 10% of FAL from this 10% w/v FAL solution for 24 and 48 h. The studied parameters were apoptosis, DNA fragmentation and laddering, caspases, cytochrome-c, lactate dehydrogenase (LDH)
H2O2, and superoxide ions and lipid peroxidation (LPO). The used techniques were microscopic observation, plate scanning and electrophoresis.
Apoptotic and DNA fragmentation effects of FLA were evident. Significant effects of FAL were observed on caspase activity (P < 0.001), cytochrome-c release (P < 0.05), lactate dehydrogenase activity (P < 0.01). Also the oxidative stress bimarkers showed significant elevation (P < 0.01): H2O2release, superoxide ion production and lipid peroxidation.
Short-term toxicity to fish was estimated based on three publications from literature. One was a guideline compliant study in which Dania rerio was exposed to water accomodated fractions (WAFs) of three different types of ashes for 144 h. Statistically significant (p<0.001) delay in hatching time was used as the endpoint and for determining NOEC. The other two studies were non-GLP compliant, non guideline investigations in which Channa punctata was exposed to fly ash lechate for 24 h and 48 h. In the first investigation, oxidative stress induction potential was studied using lipid peroxidation, GST activity, levels of GSH and proteins as endpoints. In the second investigation, the pro-apoptotic effects (apoptosis, DNA fragmentation and laddering, caspases, cytochrome-c, lactate dehydrogenase LDH, H2O2, and superoxide ions and lipid peroxidation) were studied. Highest ecotoxicological effects were found for ash, for which concentrations of the most toxic heavy metals and salinity were highest. Significant differences in parameters indicating oxidative stress potential as well significant cytotoxic effects in hepatocytes and effects in DNA level were observed in exposed fish compared to the control. LC50 was not reported in any of the studies.
NOEC was obtained from a guideline compliant study with zebra fish (Dania
rerio) exposed to water accomodated fractions of three different
types of ash. NOEC for bottom ash formed by burning mainly domestic
waste was 3.2 % ash in WAF, NOEC for fresh fly ash formed by burning
domestic waste mainly was 0.78 % ash in WAF, and NOEC for fly ash formed
by burning biofuels mainly was 25 % ash in WAF. For 10 % coal ash
slurry, sublethal effects in fish were significant. It was
suspected that high salinity influenced the outcome of the test by
affecting the complex formation, binding in organic carbon, and
bioavailability of metals. The study indicates overestimation of the
results depending on the preparation method of the ash WAF.
It must be noted that the results represent worst-case scenario
since in typical uses of ash, it is not intended to be released to water
environment directly. Thus, environmental concentrations of ash and
salinity effects in water are lower than in this study.
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