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Short-term toxicity to fish

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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.

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.

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.

Key value for chemical safety assessment

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.