The product from the burning of a combination of carbonaceous materials.

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

February 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP (no certificate) study conducted according a proposed guideline. Study is reported as a public company report.

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for testing of chemicals, Draft proposal for a new guideline, Fish Embryo Toxicity (FET) Test (2006)

Deviations:

not specified

GLP compliance:

not specified

Analytical monitoring:

not specified

Details on sampling:

Observed with loupe.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

Preparation of water extract of ashes (WAF):
- Method: SS-EN 14735:2005/AC:2006
- Concentration: L/S 10
- Shaking time: 24 h
- Filtration: 0.45 µm

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish
- Age at study initiation (mean and range, SD): 0

Test type:

static

Water media type:

freshwater

Total exposure duration:

144 h

Post exposure observation period:

Not reported.

pH:

See Table 4.

Salinity:

See Table 4.

Nominal and measured concentrations:

Ash A: 6.25-100%, Ash C: 0.16-25% and Ash F: 6.25-100%

Details on test conditions:

TEST SYSTEM
- Embryo cups (if used, type/material, size, fill volume): 250 µl
- Test vessel: 96 multiplate
- Renewal rate of test solution (frequency/flow rate): None
- No. of fertilized eggs/embryos per vessel: 1
- No. of vessels per concentration (replicates): 16

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO (12890:1999)
- Salinity was adjusted to correspond the salinity in each test concentration
- Chemical parameters: See Table 3

OTHER TEST CONDITIONS
- Adjustment of pH: adjusted to pH 7-8


EFFECT PARAMETERS MEASURED
- Observation intervals: 24, 48 and 144 hours
- Observed effects: deaths, teratogenic and sublethal effects, e.g. malformations in eyes or tails, pigmentation, spontaneous movement, oedema, heart beat, delay in development time

VEHICLE CONTROL PERFORMED: yes

Reference substance (positive control):

yes

Remarks:

Not reported

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

ca. 3.2 other: %

Conc. based on:

other: WAF

Remarks on result:

other: Ash A

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.78 other: %

Conc. based on:

other: WAF

Remarks on result:

other: Ash C

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

ca. 25 other: %

Conc. based on:

other: WAF

Remarks on result:

other: Ash F

Details on results:

Significant delay (p<0.001) in hatching time compared to control was observed for ashes A and C (Figure 1 and 2).

Reported statistics and error estimates:

See Fig. 1 and 2.

Sublethal observations / clinical signs:

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 nitrogen)

Metal		Ash A	Ash C	Ash F
Al	mg/kg TS	53500	39400	30000
Ca	mg/kg TS	69000	210000	144000
Fe	mg/kg TS	34300	11500	11300
K	mg/kg TS	14400	38400	56400
Mg	mg/kg TS	9890	12000	15000
Mn	mg/kg TS	594	677	5440
Na	mg/kg TS	36100	49200	11500
P	mg/kg TS	3420	5020	8420
Si	mg/kg TS	284000	65000	220000
Ti	mg/kg TS	4390	8150	1000
As	mg/kg TS	48,8	98,3	< 3
Ba	mg/kg TS	898	1300	1270
Be	mg/kg TS	1,22	0,712	< 0,6
Cd	mg/kg TS	7,32	66,1	8,14
Co	mg/kg TS	20,1	19,3	6,2
Cr	mg/kg TS	360	402	30,5
Cu	mg/kg TS	1690	2580	59,9
Hg	mg/kg TS	0,341	6,09	0,228
La	mg/kg TS	20,7	24,6	< 6
Mo	mg/kg TS	< 6	15,9	< 6
Nb	mg/kg TS	< 6	9,3	< 6
Ni	mg/kg TS	62,2	75,1	17,7
Pb	mg/kg TS	802	2000	52,5
S	mg/kg TS	5280	30900	24800
Sb	mg/kg TS	118	483	1,29
Sc	mg/kg TS	4,25	3,04	< 1
Se	mg/kg TS	2,07	4,24	1,62
Sn	mg/kg TS	242	452	2,19
Sr	mg/kg TS	222	390	492
V	mg/kg TS	33,6	27,7	21,6
W	mg/kg TS	< 60	< 60	< 60
Y	mg/kg TS	12,8	14	9,71
Zn	mg/kg TS	2690	10100	1270
Zr	mg/kg TS	225	120	158
TS	%	87,4	81,8	59,7
LOI	% TS	3,2	13,9	6,6
TOC	% TS	1	1	< 1
N-tot	% av TS	<0,1	<0,1	< 0,1

Table 3. Amounts of metals, chloride, fluoride, sulfur and nitrogen in the leachates of ashes.

			Ash A	Ash C	Ash F
Parameter	Unit				Column	Shaked
pH		10		12	11,9	12,4
Conductivity	mS/m	292		5540	1108	1489
Metals
Ca	mg/l	436		4330	95,6	373
Fe	mg/l	0,0102		<0,008	<0.004	0,0095
K	mg/l	63,8		3640	2710	2960
Mg	mg/l	0,344		<0,5	<0.5	<0,2
Na	mg/l	193		4240	395	404
S	mg/l	266		131	1170	1020
Si	mg/l	0,832		0,303	17,2	0,752
Al	μg/l	10100		8,81	848	21,1
As	μg/l	5,3		<20	<3	<1
Ba	μg/l	70,5		2710	64,1	142
Cd	μg/l	0,141		0,444	<0.05	<0,07
Co	μg/l	0,18		<0,1	<0.05	0,157
Cr	μg/l	1,46		32,3	55,9	3,96
Cu	μg/l	300		576	<1	1,66
Hg	μg/l	0,0243		0,0775	<0.02	<0,02
Mn	μg/l	1,1		0,569	0,232	4,04
Mo	μg/l	53,6		155	236	221
Ni	μg/l	3,24		30,1	<0,5	0,51
Pb	μg/l	3,66		7350	<0,2	4,91
Sb	μg/l	30,1		0,225	0,129	0,592
Se	μg/l	1,7		10,1	5,63	6,06
V	μg/l	5,83		9,72	25,8	1,5
Zn	μg/l	5,83		3820	<2	10,4
Anions
Chloride	mg/l	470		16000	569	480
Fluoride	mg/l	<0,20		<12,0	<1,20	<6,00
Sulphate	mg/l	570		330	3080	2800
Other
Ammonium	mg/l	1,4		27	0,024	*****
Nitrate	mg/l	<0,50		<28,0	0,75	*****
Nitrite	mg/l	<0,01		1,14	0,31	*****
TOC/DOC	mg/l	15		2,3	<1	4
Phenol Index	mg/l	0,007		0,009	0,011	<0,005
CODCr	mg/l	33		46	18	*****
BOD7	mg/l	*****		6	<1	*****

Table 4. The validity parameters (pH, oxygen in % and salinity in ‰) for the leachates measured before test start.

	Ash A	Ash C	Ash F
pH	9.02	12.55	12.1
Acidity (%)	30	135	93
Salinity (‰ )	3.4	28.8	5.7

Conclusions:

NOEC (144 h) values for three different types of ashes were determined. NOEC for bottom ash formed by burning mainly domestic waste was 3.2 %, NOEC for fresh fly ash formed by burning mainly domestic waste was 0.78 %, and NOEC for fly ash formed by burning mainly biofuels 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.

Executive summary:

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.

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not reported.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP compliant, non-guideline experimental investigation. Study published in scientific, peer reviewed journal.

Qualifier:

no guideline available

Principles of method if other than guideline:

Oxidative stress and enzyme induction study with freshwater fish Channa punctata and fly ash leachate (24 h).

GLP compliance:

no

Analytical monitoring:

not specified

Vehicle:

no

Test organisms (species):

other: Channa punctata

Details on test organisms:

TEST ORGANISM
- Common name: Gerai
- Source: obtained from commersial fish suppliers
- Age at study initiation (mean and range, SD): Not reported
- Length at study initiation (length definition, mean, range and SD): 15-17 cm
- Weight at study initiation (mean and range, SD): 70-75 g
- Feeding during test: No

ACCLIMATION
- Acclimation period: 15 d
- Acclimation conditions (same as test or not): glass aquarium; the tank water was kept oxygen saturated by aeration and temperature was maintained at the ambient laboratory temperature (25±2°C)
- Type and amount of food: commercial fish feed
- Feeding frequency: twice a week

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

24 h

Details on test conditions:

Ten animals were used both in the test water and in the control.

EFFECT PARAMETERS MEASURED: lipid peroxidation, antioxidant enzyme activity and glutathione reduction at the end of the test

TEST CONCENTRATIONS: Fish were exposed to 1 ml/l of fly ash leachate representing 100 mg/mL of fly ash in the original slurry. This concentration was selected on the basis of range finding study.

Reference substance (positive control):

no

Duration:

24 h

Remarks on result:

not measured/tested

Remarks:

Tested parameters included: - induction of lipid peroxidase in liver, kidney and gills - induction of catalase activity in liver and kidney and gills - induction of GST activity in liver and kidney and gills - increase of GSH in liver and kidney and gills. No LC-, EC-, NOEC or LOEC values were determined.

Details on results:

Significant differences in the tested parameters compared to control were observed (see Figures).

Reported statistics and error estimates:

Statistically significant findings compared to control were:
- induction of lipid peroxidase in liver, kidney and gills (p<0.001)
- induction of catalase activity in liver and kidney (p<0.01) and gills (p<0.001)
- induction of GST activity in liver and kidney (p<0.05) and gills (p<0.01)
- increase of GSH in liver and kidney (p<0.05) and gills (p<0.01)

Sublethal observations / clinical signs:

See illustration.

Validity criteria fulfilled:

not specified

Conclusions:

Sub-bituminous coal ash was found to induce oxidative stress and enzyme activity in Channa punctata.

Executive summary:

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.

Reference 3

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Not reported

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Non-GLP compliant, non-guideline experimental investigation. Study published in scientific, peer reviewed journal.

Qualifier:

no guideline available

Principles of method if other than guideline:

Cytological and genetic in-vitro study with hepatocytes from freshwater fish Channa punctata.

GLP compliance:

no

Analytical monitoring:

not specified

Vehicle:

no

Test organisms (species):

other: Hepatocytes from Channa punctata

Test type:

other: in-vitro

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

also 24h

Duration:

48 h

Remarks on result:

not measured/tested

Remarks:

The studied parameters were apoptosis, DNA fragmentation and laddering, caspases, cytochrome-c, lactate dehydrogenase (LDH). No LC-, EC-, NOEC or LOEC values were determined.

Sublethal observations / clinical signs:

Apoptosis, DNA fragmentation and DNA laddering

All the studied parameters indicate a proapoptotic effect of fly ash leachates in fish hepatocytes (See figures 1 -2).

Caspases, cytochrome-c and LDH

A 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.

 

H₂O₂, superoxide ions and LPO

FAL 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).

Validity criteria fulfilled:

not specified

Conclusions:

Sub-bituminous coal fly ash leachate caused significant cytotoxic effects on hepatocytes from Channa punctata. Also, effects in DNA level were obvious.

Executive summary:

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.

Description of key information

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.

Additional information

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.