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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study was carried out between 28 June and 1 July 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of signature 08.10.2010

Analytical monitoring:

yes

Details on sampling:

- Test water was sampled and pH, hardness, conductivity and alkalinity were measured by the study laboratory. Water quality analysis including DOC, COD, suspended solids, N, pesticides, PCBs and metals was conducted in a separate non-GLP laboratory. Metal analysis of four test solutions (3.2 mg/l, 56 mg/l and two controls) were conducted in another non-GLP laboratory.
- Sample storage conditions before analysis: Corresponding to the storage condition of test material.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test solutions were prepared by the addition of a known quantity of mixed ash directly to 1 litre volumes of culture medium. These solutions were stirred overnight using magnetic followers and then allowed to stand for 15 minutes. After this time the solutions were filtered through 0.45 µm cellulose nitrate filters, which had been previously washed with 100 mL Reverse Osmosis (RO) water. The pH of each solution was measured and adjusted, if necessary, with 1 M hydrochloric acid to 8.0 ± 0.25. The resulting solutions were clear and colourless.
- Controls: The control consisted of culture medium only.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Source: continuous laboratory cultures held at Brixham Environmental Laboratory
- Age at study initiation (mean and range, SD): <24 hours
- Method of breeding: Diploid parthenogenesis
- Feeding during test: No
- No evidence of disease in the organisms and the culture before test

Test type:

static

Water media type:

freshwater

Total exposure duration:

48 h

Hardness:

181 mg/L CaCO3.

Test temperature:

20 ± 1°C (nominal)

pH:

7.1 - 8.2

Dissolved oxygen:

6.2 - 9.2 mg/L (68 - 101% of the air saturation value)

Salinity:

0.2 to 0.3‰

Nominal and measured concentrations:

Nominal loading concentrations: 10, 18, 32, 56, 100, 180 and 320 mg/L.
Measured concentration of trace elements in test waters were following (in µg/L):
320 mg/L: Al: 557, As: 2.54, Cd: 0.07, Cr 3.0, Pb: 0.46, Hg: 4.37, Ni: 0.30
56 mg/L: Al: 348, As: 3.99, Cd: 0.08, Cr: 0.58, Pb: 1.17, Hg: 3.02, Ni: 0.05
10 mg/L: Al: 63.6, As: 1.93, Cd: 0.05, Cr: 0.16, Pb: 0.17, Hg: 1.79, Ni: 0.16
3.2 mg/L: Al 3.16, As 5.28, Cd: 0.02, Cr: 0.07, Pb: 0.51, Hg: 1.64, Ni: 0.10
control: Al: 2.03, As: 7.07, Cd: 0.42, Cr: 0.47, Pb: 0.87, Hg: 2.99, Ni: 0.38
control 2: Al: 2.94, As: 1.83, Cd: 0.05, Cr: 0.09, Pb: 0.16, Hg: 2.24, Ni 0.26

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 ml glass beakers
- Type (delete if not applicable): closed, covered with loose fitting glass lid
- Material, size, headspace, fill volume: test soluition depth 60 mm, fill vol. 200 ml
- Aeration: No
- No. of organisms per vessel: five (25 D. magna/L)
- No. of vessels per concentration (replicates): four replicates per test concentration
- No. of vessels per control (replicates): four

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Elendt's M4 D. magna medium
- Conductivity: @25°C 679 µS/cm
- Other water parameters: see Table 6.
- Intervals of water quality measurement: For pH, salinity and DOC 0 and 48h. Salinity was measured in the dilution water control and the nominal 10, 56 and 320 mg L-1 loading concentrations only. Temperature values were determined daily, in an additional test vessel, using a mercury-in-glass thermometer calibrated to BS593.


OTHER TEST CONDITIONS
- Adjustment of pH: Adjusted, if necessary, with 1 M hydrochloric acid to 8.0 ± 0.25.
- Photoperiod: 16 hours light: 8 hours dark, with 20 minute dusk and dawn transition periods
- Nominal test solution temperature: 20 ± 1°C

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobilisation

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Test concentrations: 10, 18, 32, 56, 100, 180 and 320 mg/L (nominal)

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

95.9 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal loading concentrations

Basis for effect:

mobility

Remarks on result:

other: 95% CL 79.0-119 mg/L

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

57.1 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal loading concentrations

Basis for effect:

mobility

Remarks on result:

other: 95% CL 46.0-71.0 mg/L

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

180 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal loading concentrations

Basis for effect:

mobility

Remarks on result:

other: The EC100 is defined as the lowest nominal loading concentration that caused 100% immobilisation

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

other: nominal loading concentrations

Basis for effect:

mobility

Remarks on result:

other: The NOEC is defined as the highest nominal loading concentration in which there was no immobility observed

Details on results:

- Behavioural abnormalities: None
- Mortality of control: No, also no immobilisation
- Other adverse effects control: No other symptoms of toxicity were observed
- Abnormal responses: None

Table 4 Daphnia magna response

Time	Nominal conc of mixed ash	Number immobilised per replicate				Total number tested	Total number immobilised	Percentage immobilised
hour	mg/L	A	B	C	D
24	Dilution water control	0	0	0	0	20	0	0
	10	0	0	0	0	20	0	0
	18	0	0	0	0	20	0	0
	32	0	0	0	0	20	0	0
	56	0	0	0	1	20	1	5
	100	1	1	4	3	20	9	45
	180	5	5	5	5	20	20	100
	320	5	5	5	5	20	20	100
48	Dilution water control	0	0	0	0	20	0	0
	10	0	0	0	0	20	0	0
	18	0	0	0	0	20	0	0
	32	0	0	0	0	20	0	0
	56	4	2	4	3	20	13	65
	100	5	5	5	4	20	19	95
	180	5	5	5	5	20	20	100
	320	5	5	5	5	20	20	100

Physical and chemical data

Dissolved oxygen concentrations ranged from 6.2 to 9.2 mg/L (68 to 101% of the air saturation value), pH values (after time 0 adjustments) ranged from 7.1 to 8.2, and the salinity ranged from 0.2 to 0.3‰. Individual data are shown in Table 5.

Table 5 Dissolved oxygen concentrations and pH values

Nominal loading concentration of mixed ash	Dissolved oxygen concentration mg/L		pH			Salinity (‰)
mg/L	0 hours	48 hours	0 hours		48 hours	0 hours	48 hours
			Before adjustment	After adjustment
Dilution water control	7.2	9.0	8.0	-	7.9	0.2	0.2
10	6.8	9.0	8.4	8.1	8.0	0.2	0.2
18	6.8	9.0	8.6	8.2	8.0	-	-
32	6.8	9.0	8.7	8.1	8.0	-	-
56	6.6	9.0	8.7	7.9	8.0	0.2	0.2
100	6.6	9.2	9.0	8.0	7.9	-	-
180	6.2	8.6^a	9.9	8.0	7.3^a	-	-
320	6.2	8.8^a	10.8	8.1	7.1^a	0.2	0.3^a

a Measurements taken at 24 hours, as 100% mortalities at that time

Temperature values taken at 0, 24 and 48 hours were 21.6, 21.4 and 21.1°C.

The hardness of the D. magna media was 181 mg/L CaCO3, and the conductivity was 679 µS/cm. Water quality parameters of a recent batch of D. magna media are shown in Table 6.

Table 6 Dilution water parameters

Parameter	Result
*pH	7.9
*Conductivity @ 25ºC	679 µS cm^-1
*Total hardness as CaCO3	181 mg L^-1
*Alkalinity as CaCO3	34 mg L^-1
Solids suspended @105ºC	<3 mg L^-1
Dissolved organic carbon	0.210 mg L^-1
Chemical oxygen demand	<12mg L^-1
Ammoniacal nitrogen as N	<0.002 mg L^-1
Highest organophosphorous pesticides	<0.01 µg L^-1
Highest organochlorine pesticides	0.1 µg L^-1
Highest PCB	<0.001 µg L^-1
Aluminium	<10 µg L^-1
Arsenic	<1 µg L^-1
Boron	527 µg L^-1
Cadmium	<0.1 µg L^-1
Chromium	<0.5 µg L^-1
Cobalt	2.30 µg L^-1
Copper	5.32 µg L^-1
Iron	74.0 µg L^-1
Lead	<2 µg L^-1
Manganese	<10 µg L^-1
Mercury	<0.01 µg L^-1
Nickel	<1 µg L^-1
Silver	<1 µg L^-1
Zinc	<5 µg L^-1

*Analyses conducted at Brixham Environmental Laboratory (BEL) on the batch of media used in the study.Other analyses were performed on a recent batch of media by The Environment Agency (EA), National Laboratory Service (NLS), Olympia House, Gelderd Lane, Gelderd Road, Leeds, LS12 6DD, for analysis at NLS laboratories. The EA is a non-GLP compliant facility.

Validity criteria fulfilled:

yes

Conclusions:

The EC50 (48h) value of Daphnia magna for Ash was 57.1 mg/L.

Executive summary:

Short-term toxicity of Ash was determined in a GLP compliant study according to OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). In the test, young, less than 24 h old D. magna were exposed to the test substance for 48 hours in a static test. Due to low water solubility of Ash, test solutions were filtrated to form Water Accommondable Fractions (WAF) of Ash. Immobilisation was observed 24 and 48 hours after initiation of the test.The EC50 value was determined to be 57.1 mg/L.

Description of key information

Toxicity of Ash to Daphnia magna was determined in a GLP compliant laboratory study according to OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test). The EC50 (48h) was found to be 57.1 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

57.1 mg/L

Additional information

Due to low water solubility of Ash, Water Accommondated Fractions of the test solutions were prepared.