Manganese ores, reduced

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 10 February 2010 and 19 March 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

yes

Remarks:

See below.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

See below.

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. Previous experience gained from studies conducted on poorly water soluble test items has shown that a mixing period of 24 - 48 hours is sufficient to ensure equilibration between the test item and water phase. At the completion of mixing, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Inspection: 15/09/09 Date of signature: 26/11/09

Analytical monitoring:

yes

Details on sampling:

Chemical analysis of test loading rates:

Water samples were taken from the control (replicates R1 – R2 pooled) and the 100 mg/l.

Loading rate WAF test group (replicates R1 – R2 and R3 – R4 pooled) at 0 and 48 hours for quantitative analysis.

Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

The concentration of the test item in the test preparations was verified by chemical analysis at 0 and 48 hours (see appendix 2 in the attachment section).

Vehicle:

yes

Details on test solutions:

Reconstituted Water
i) Stock Solutions
a) CaCl2.2H2O 11.76 g/l
b) MgSO4.7H2O 4.93 g/l
c) NaHCO3 2.59 g/l
d) KCl 0.23 g/l

Preparation
An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain: 1st instar Daphnia magna derived from in-house laboratory cultures.
- Source: 1st instar Daphnia magna derived from in-house laboratory cultures.
- Age at study initiation (mean and range, SD): less than 24 hours old
- Weight at study initiation (mean and range, SD): not stated
- Length at study initiation (length definition, mean, range and SD): not stated
- Valve height at study initiation, for shell deposition study (mean and range, SD): not stated
- Peripheral shell growth removed prior to test initiation: not applicable
- Method of breeding: parthenogenesis
- Feeding during test
The daphnids received no food during the test

ACCLIMATION
- Acclimation period: not stated

QUARANTINE (wild caught)
not applicable

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

Immobilisation and any adverse reactions to exposure were observed 24 and 48 hours after exposure.

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.

Test temperature:

Water temperature was recorded daily throughout the test. The temperature was measured using a Hanna Instruments HI 93510. The test temperature was maintained at 21 to 23 °C throughout the study.

Some of the temperatures during the range-finding and definitive tests were measured to be slightly in excess of the 20 ± 1°C given in the protocol. This was considered not to affect the results of the test as no adverse effects of exposure were observed throughout the duration of the test and that the temperatures were within the test guideline specification.

pH:

The pH values were recorded at the start and termination of the test. The pH concentrations were measured using a WTW pH/Oxi 340I pH meter.

Dissolved oxygen:

Dissolved oxygen concentrations were recorded at the start and termination of the test. The dissolved oxygen concentrations were measured using a
dissolved oxygen meter.

The oxygen concentration in the test vessels was observed to have an air saturation value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed.

Salinity:

Not measured as freshwater was used.

Nominal and measured concentrations:

Nominal concentrations: 10, 18, 32, 56 and 100 mg/l.
Measured concentrations:

Details on test conditions:

TEST SYSTEM
- Test vessel: As in the range-finding test 250 ml glass jars containing approximately 200 ml of test preparation were used.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: fill volume: 200mL
- Aeration: the test vessels were not aerated.
- Type of flow-through (e.g. peristaltic or proportional diluter): not reported
- Renewal rate of test solution (frequency/flow rate): not reported
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): not reported
- Biomass loading rate: not reported

TEST MEDIUM / WATER PARAMETERS
Reconstituted Water
i) Stock Solutions
a) CaCl2.2H2O 11.76 g/l
b) MgSO4.7H2O 4.93 g/l
c) NaHCO3 2.59 g/l
d) KCl 0.23 g/l

Preparation
An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.


OTHER TEST CONDITIONS
- Adjustment of pH: yes the reconstituted water was adjusted if necessary
- Photoperiod: lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods.
- Light intensity:

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline: not stated
- Range finding study
- Test concentrations: In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/l.
- Results used to determine the conditions for the definitive study: Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 10, 18, 32, 56 and 100 mg/l.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.78 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL 0.58 - 0.72mg/l.

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Range-finding Test
Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1 (in the any other information on results section)

Immobilisation was observed predominately at the 100 mg/l loading rate WAF.
A single immobilised daphnid was observed at the 1.0 and 10 mg/l loading rate WAFs after 48 hours exposure. These were considered to be due to natural causes rather than a toxic effect given that no further immobilisation was observed.

Analysis of the 100 mg/l loading rate WAF (see Appendix 2) showed a measured concentration of manganese of 2.79 mg/l.
Based on this information loading rates of 10, 18, 32, 56 and 100 mg/l, using a stirring period of 47 hours followed by a 1-Hour standing period, were selected for the definitive test.

Results with reference substance (positive control):

- Results with reference substance valid?

Analysis of the immobilisation data by the probit method (Finney 1971) at 24 hours and
the trimmed Spearman-Karber method (Hamilton et al 1977) at 48 hours based on the
nominal test concentrations gave the following results:

Time (h) EC50 (mg/l) 95% confidence limits (mg/l)
24 0.84 0.72 - 0.97
48 0.65 0.58 - 0.72

The No Observed Effect Concentration was 0.32 mg/l. The No Observed Effect Concentration is based upon zero immobilisation rates.

 Immobilisation data:

Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2. The relationship between percentage immobilisation and loading rate at 48 hours is given in Figure 1 (see attachment).

Inspection of the immobilisation data at 24 hours and analysis of the immobilisation data by the trimmed Spearman-Karber method (Hamilton et al1977) at 48 hours based on the nominal loading rates gave the following results:

Time (h)	EL*50(mg/l)	95% Confidence limits (mg/l)
24	>100**	-
48	43	41 - 46

*EL = Effective Loading rate

** An EL₅₀value could not be calculated as less than 50% immobilisation occurred at this time point

The No Observed Effect Loading rate after 24 and 48 hours exposure was 32 mg/l loading rate WAF. The No Observed Effect Loading rate is based upon zero immobilisation at this loading rate.

A single immobilised daphnid was observed at the 10 mg/l loading rate WAF after 24 hours exposure. This was considered to be due to natural causes rather than a toxic effect given that no further immobilisation was observed and no immobilisation was observed at the 18 and 32 mg/l loading rate WAFs.

Table 1              Cumulative Immobilisation Data in the Range-findingTest

Nominal Loading Rate (mg/l)	Cumulative ImmobilisedDaphnia (Initial Population: 10 Per Replicate)
	24 Hours	48 Hours
Control	0	0
1.0	0	1[1]
10	0	1*
100	5	10

 

[1]Immobilisation considered to be due to natural causes rather than a toxic effect given that no further immobilisation was observed

Table2              Cumulative Immobilisation Data in the DefinitiveTest

Nominal Loading Rate (mg/l)	Cumulative ImmobilisedDaphnia (Initial Population: 10 Per Replicate)
	24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0
10	1[1]	0	1*	5	1*	0	1*	5
18	0	0	0	0	0	0	0	0
32	0	0	0	0	0	0	0	0
56	2	2	4	20	9	10	19	95
100	1	1	2	10	10	10	20	100

R₁– R₂= Replicates 1 and 2

[1]Immobilisation considered to be due to natural causes rather than a toxic effect given that no further immobilisation was observed and no immobilisation was observed at the 18 and 32 mg/l loading rate WAFs

Table 3              Vortex Depth Measurements at the Start and End of the Mixing Period

	Nominal Loading Rate (mg/l)
	Control		10		18
	*	+	*	+	*	+
Height of Water Column (cm)	~17	~17	~17	~17	~17	~17
Depth of Vortex (cm)	~0.2	~0.2	~0.2	~0.2	~0.2	~0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present

 

	Nominal Loading Rate (mg/l)
	32		56		100
	*	+	*	+	*	+
Height of Water Column (cm)	~17	~17	~17	~17	~17	~17
Depth of Vortex (cm)	~0.2	~0.2	~0.2	~0.2	~0.2	~0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present

*= Start of mixing period

+= End of mixing period

Table 4              Cumulative Immobilisation Data in the Positive Control

Nominal Concentration (mg/l)	Cumulative ImmobilisedDaphnia (Initial Population: 10 Per Replicate)
	24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0
0.32	0	0	0	0	0	0	0	0
0.56	1	1	2	10	3	2	5	25
1.0	7	7	14	70	10	10	20	100
1.8	10	9	19	95	10	10	20	100
3.2	10	10	20	100	10	10	20	100

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL*50 value of greater than 100 mg/l loading rate WAF. Correspondingly the No Observed Effect Loading rate was 100 mg/l loading rate WAF.

Executive summary:

Introduction:

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed that described in the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp, Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods:

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/l for 48 hours at a temperature of 21°C to 23ºC under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

A positive control conducted approximately every six months used potassium dichromate as the reference item. Daphnia magna was exposed to an aqueous solution of the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l for 48 hours at a temperature of approximately 20°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results:

The 48-Hour EL*50 for the test item to Daphnia magna based on nominal loading rates was greater than 100 mg/l loading rate WAF and correspondingly the No Observed Effect Loading rate was 100 mg/l loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/l. The test preparations were analysed for manganese content. Analysis of the test preparations at 0 hours showed measured concentrations of manganese to be 0.330 and 0.326 mg/l. Analysis of the old media at 48 hours showed measured test concentrations of manganese to be 0.290 and 0.298 mg/l.

The dissolved test item may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

The 48-Hour EC50 for the reference item to Daphnia magna based on nominal concentrations was 0.78 mg/l with 95% confidence limits of 0.58 – 0.72 mg/l. The No Observed Effect Concentration was 0.32 mg/l.

Reference 2

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

The study was conducted between 10 February 2010 and 9 April 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. Read-across from FeMn slag to sinter ore is justified on the basis of similar constituents, very poor water solubility and leaching in the TDp study. Sinter ore has a lower solubility and leaching compared to FeMn slag.

Reason / purpose for cross-reference:

other: read-across target

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

yes

Remarks:

See principles of method if other than guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

yes

Remarks:

See principles of method if other than guideline.

Principles of method if other than guideline:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996 and OECD 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. Previous experience gained from studies conducted on poorly water soluble test items has shown that a mixing period of
24 - 48 hours is sufficient to ensure equilibration between the test item and water phase. At the completion of mixing, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Inspection: 15/09/09 Date of signature: 26/11/09

Analytical monitoring:

yes

Details on sampling:

- Concentrations:
- Sampling method: Water samples were taken from the control and each loading rate WAF test group (replicates R1 – R2 pooled) at 0 and 48 hours for quantitative analysis.
- Sample storage conditions before analysis: Duplicate samples were taken and stored at approximately -20C for further analysis if necessary.

Details on test solutions:

Reconstituted water:

i) Stock Solutions
a) CaCl2.2H2O 11.76 g/l
b) MgSO4.7H2O 4.93 g/l
c) NaHCO3 2.59 g/l
d) KCl 0.23 g/l

ii) Preparation

An aliquot (25 ml) of each of solutions a) to d) was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.

The reconstituted water used for both the range-finding and definitive tests was the same as that used to maintain the stock animals.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water flea
- Strain: 1st instar Daphnia magna derived from in-house laboratory cultures.
- Source: 1st instar Daphnia magna derived from in-house laboratory cultures.
- Age at study initiation (mean and range, SD): less than 24 hours old
- Weight at study initiation (mean and range, SD): not stated
- Length at study initiation (length definition, mean, range and SD): not stated
- Valve height at study initiation, for shell deposition study (mean and range, SD): not stated
- Peripheral shell growth removed prior to test initiation: not applicable
- Method of breeding: parthenogenesis
- Feeding during test
The daphnids received no food during the test

ACCLIMATION
- Acclimation period: overnight

QUARANTINE (wild caught)
not applicable

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

The number of immobilised Daphnia were recorded after 24 and 48 hours.

Hardness:

The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3. (see appendix 3 in the attachments for detailed results).

Test temperature:

Water temperature was recorded daily throughout the test. The temperature was measured using a Hanna Instruments HI 93510. The test temperature was maintained at 21 °C throughout the study. (see appendix 3 in the attachments for detailed results).

pH:

The pH values were recorded at the start and termination of the test. The pH concentrations were measured using a WTW pH/Oxi 340I pH meter. Differences in pH were observed at 0 hours between the control and the 10, 18, 32, 56 and 100 mg/l loading rate WAFs. (see appendix 3 in the attachments for detailed results).

Dissolved oxygen:

Dissolved oxygen concentrations were recorded at the start and termination of the test. The dissolved oxygen concentrations were measured using a
dissolved oxygen meter. There were no treatment related differences for oxygen concentration. (see appendix 3 in the attachments for detailed results).

The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100%. This was considered to be due to the presence of microscopic air bubbles in the media super-saturating the diluent and was considered not to have had an impact on the outcome or integrity of the test as no adverse effects were observed in the control group.

Salinity:

Not measured as freshwater was used.

Nominal and measured concentrations:

Nominal concentrations:
In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/l.

Measured concentrations:
Analysis of the fresh media at 0 hours showed measured concentrations of manganese to range from 1.13 to 3.79 mg/l. Analysis of the old media at 48 hours showed measured concentrations of manganese to range from less than the limit of 1.16 to 3.62 mg/l.

Analysis of the control samples at 0 and 48 hours showed measured concentrations of 0.0155 and 0.0159 mg manganese/l.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250ml glass jars
- Type (delete if not applicable): closed top reduce evaporation.
- Material, size, headspace, fill volume: 200ml
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): not reported
- Renewal rate of test solution (frequency/flow rate): no test solution renewable
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.

Stock Solutions:
a) CaCl2.2H2O 11.76 g/l
b) MgSO4.7H2O 4.93 g/l
c) NaHCO3 2.59 g/l
d) KCl 0.23 g/l

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod: The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. - Light intensity: not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Not reported
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study
- Test concentrations: In the range-finding test Daphnia magna were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/l.
- Results used to determine the conditions for the definitive study: Based on the results from the range finding study, loading rates of 10, 18, 32, 56 and 100 mg/l, using a stirring period of 47 hours followed by a 1-Hour standing period, were selected for the definitive test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

43 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: 95% CL 41 - 46

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.78 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Remarks on result:

other: 95% CL

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: immobilisation

Details on results:

Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1 (please see the any other information on results section for all tables). Immobilisation was observed predominately at the 100 mg/l loading rate WAF.

A single immobilised daphnid was observed at the 1.0 and 10 mg/l loading rate WAFs after 48 hours exposure. These were considered to be due to natural causes rather than a toxic effect given that no further immobilisation was observed.

Analysis of the 100 mg/l loading rate WAF (see appendix 2) showed a measured concentration of manganese of 2.79 mg/l.

Based on this information loading rates of 10, 18, 32, 56 and 100 mg/l, using a stirring period of 47 hours followed by a 1-Hour standing period, were selected for the definitive test.

Results with reference substance (positive control):

Cumulative immobilisation data from the exposure of Daphnia magna to the reference item (Harlan Laboratories Ltd Project No: 0039/1133) during the positive control are given in Table 4. The relationship between percentage immobilisation and concentration at 24 and 48 hours is given in Figures 2 and 3.
Analysis of the immobilisation data by the probit method (Finney 1971) at 24 hours and the trimmed Spearman-Karber method (Hamilton et al 1977) at 48 hours based on the nominal test concentrations gave the following results:

Time (h) EC50 (mg/l) 95% Confidence limits (mg/l)
24 0.84 0.72 - 0.97
48 0.65 0.58 - 0.72

The No Observed Effect Concentrations after 24 and 48 hours was 0.32 mg/l. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

The slope and its standard error of the response curve at 24 hours was 7.7 (SE = 1.6). Due to the unsuitable nature of the data it was not possible to calculate the slope and error of response curve at 48 hours.

The results from the positive control with potassium dichromate were within the normal range for this reference item. The mean 48-Hour EC50 value calculated from all positive controls was 0.77 mg/l (sd = 0.20).

Reported statistics and error estimates:

An estimate of the EL*50 value at 24 hours was given by inspection of the immobilisation data.

The EL*50 value and associated confidence limits at 48 hours were calculated by the trimmed Spearman-Karber method (Hamilton et al 1977) using the ToxCalc computer software package (ToxCalc 1999).

When only one partial response is shown the trimmed Spearman-Karber method is appropriate.

DefinitiveTest:

Immobilisation data:

Cumulative immobilisation data from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2. The relationship between percentage immobilisation and loading rate at 48 hours is given in Figure 1.

Inspection of the immobilisation data at 24 hours and analysis of the immobilisation data by the trimmed Spearman-Karber method (Hamilton et al 1977) at 48 hours based on the nominal loading rates gave the following results:

Time (h)	EL*50(mg/l)	95% Confidence limits (mg/l)
24	>100**	-
48	43	41 - 46

The No Observed Effect Loading rate after 24 and 48 hours exposure was 32 mg/l loading rate WAF. The No Observed Effect Loading rate is based upon zero immobilisation at this loading rate.

A single immobilised daphnid was observed at the 10 mg/l loading rate WAF after 24 hours exposure. This was considered to be due to natural causes rather than a toxic effect given that no further immobilisation was observed and no immobilisation was observed at the 18 and 32 mg/l loading rate WAFs.

*EL = Effective Loading rate

** An EL₅₀value could not be calculated as less than 50% immobilisation occurred at this time point

Table1              Cumulative Immobilisation Data in the Range-finding Test

Nominal Loading Rate (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	24 Hours	48 Hours
Control	0	0
1.0	0	1[1]
10	0	1*
100	5	10

[1]Immobilisation considered to be due to natural causes rather than a toxic effect given that no further immobilisation was observed

Table 2              Cumulative Immobilisation Data in the Definitive Test

Nominal Loading Rate (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0
10	1[1]	0	1*	5	1*	0	1*	5
18	0	0	0	0	0	0	0	0
32	0	0	0	0	0	0	0	0
56	2	2	4	20	9	10	19	95
100	1	1	2	10	10	10	20	100

R₁– R₂= Replicates 1 and 2

[1]Immobilisation considered to be due to natural causes rather than a toxic effect given that no further immobilisation was observed and no immobilisation was observed at the 18 and 32 mg/l loading rate WAFs

Table 3              Vortex Depth Measurements at the Start and End of the Mixing Period

	Nominal Loading Rate (mg/l)
	Control		10		18
	*	+	*	+	*	+
Height of Water Column (cm)	~17	~17	~17	~17	~17	~17
Depth of Vortex (cm)	~0.2	~0.2	~0.2	~0.2	~0.2	~0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present

 

	Nominal Loading Rate (mg/l)
	32		56		100
	*	+	*	+	*	+
Height of Water Column (cm)	~17	~17	~17	~17	~17	~17
Depth of Vortex (cm)	~0.2	~0.2	~0.2	~0.2	~0.2	~0.2
Observation of Vortex	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present	Dimple present

*= Start of mixing period

+= End of mixing period

Table 4              Cumulative Immobilisation Data in the Positive Control

Nominal Concentration (mg/l)	Cumulative Immobilised Daphnia (Initial Population: 10 Per Replicate)
	24 Hours				48 Hours
	R1	R2	Total	%	R1	R2	Total	%
Control	0	0	0	0	0	0	0	0
0.32	0	0	0	0	0	0	0	0
0.56	1	1	2	10	3	2	5	25
1.0	7	7	14	70	10	10	20	100
1.8	10	9	19	95	10	10	20	100
3.2	10	10	20	100	10	10	20	100

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL*50 value of 43 mg/l loading rate WAF with 95% confidence limits of 41 - 46 mg/l loading rate WAF. The No Observed Effect Loading rate at 48 hours was 32 mg/l loading rate WAF.

Executive summary:

Introduction:

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed that described in the OECD Guidelines for Testing of Chemicals (April 2004) No 202, "Daphnia sp, Acute Immobilisation Test" referenced as Method C.2 of Commission Regulation (EC) No. 440/2008.

Methods:

Following a preliminary range-finding test, twenty daphnids (2 replicates of 5 animals) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 10, 18, 32, 56 and 100 mg/l for 48 hours under at a temperature of 21°C static test conditions. The number of immobilised Daphnia were recorded after 24 and 48 hours.

A positive control conducted approximately every six months used potassium dichromate as the reference item. Daphnia magna was exposed to an aqueous solution of the reference item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/l for 48 hours at a temperature of approximately 20°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results:

The 48-Hour EL*₅₀ for the test item to Daphnia magna based on nominal loading rates was 43 mg/l loading rate WAF with 95% confidence limits of 41 - 46 mg/l loading rate WAF. The No Observed Effect Loading rate was 32 mg/l loading rate WAF.

The test item contains manganese. The test concentrations were monitored by analysis of the test samples for manganese only.

Analysis of the fresh media at 0 hours showed measured concentrations of manganese to range from 1.13 to 3.79 mg/l. Analysis of the old media at 48 hours showed measured concentrations of manganese to range from less than the limit of 1.16 to 3.62 mg/l. 

The dissolved test item may have been one or several components of the test item. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

The 48-Hour EC₅₀ for the reference item to Daphnia magna based on nominal concentrations was 0.78 mg/l with 95% confidence limits of 0.58 – 0.72 mg/l. The No Observed Effect Concentration was 0.32 mg/l.

 

Reference 3

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material.

Reason / purpose for cross-reference:

read-across source

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

43 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95 % CL 41 - 46 mg/L

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

0.78 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% CL

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Description of key information

The acute toxicity of the registered substance to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL50 value of greater than 100 mg/l loading rate WAF. Correspondingly the No Observed Effect Loading rate was 100 mg/l loading rate WAF.

The acute toxicity of the read-across substance, ferromanganese slags, to the freshwater invertebrate Daphnia magna has also been investigated and gave a 48-Hour EL50 value of 43 mg/l loading rate WAF with 95% confidence limits of 41 - 46 mg/l loading rate WAF.  The No Observed Effect Loading rate at 48 hours was 32 mg/l loading rate WAF.

Additional information

Key Study: Goodbland & Mullee (2010)

A study was performed to assess the acute toxicity of the registered substance to Daphnia magna. The method followed that described in the standardised guidelines OECD 202 and EU Method C.2 and was performed under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test material, at a single nominal loading rate of 100 mg/L for 48 hours at a temperature of 21 °C to 23 ºC under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

A positive control conducted approximately every six months used potassium dichromate as the reference material. Daphnia magna was exposed to an aqueous solution of the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L for 48 hours at a temperature of approximately 20 °C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

The 48-Hour EL*50 for the test material to Daphnia magna based on nominal loading rates was greater than 100 mg/L loading rate WAF and correspondingly the No Observed Effect Loading rate was 100 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L. The test preparations were analysed for manganese content. Analysis of the test preparations at 0 hours showed measured concentrations of manganese to be 0.330 and 0.326 mg/L. Analysis of the old media at 48 hours showed measured test concentrations of manganese to be 0.290 and 0.298 mg/L.

The dissolved test material may have been one or several components of the test material. Given that toxicity cannot be attributed to a single component or mixture of components but to the test material as a whole, the results were based on nominal loading rates only.

Under the conditions of the test the acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EL*50 value of greater than 100 mg/L loading rate WAF. Correspondingly the No Observed Effect Loading rate was 100 mg/L loading rate WAF.

Supporting Study: Priestly & Mullee (2010) - Read-Across

A study was performed on the read-across substance, ferromanganese slags, to assess the acute toxicity of the test material to Daphnia magna. The method followed that described in the standardised guidelines OECD 202 and EU Method C.2 and was performed under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997). Read-across from FeMn slag to sinter ore is justified on the basis of similar constituents, very poor water solubility and leaching in the TDp study. Sinter ore has a lower solubility and leaching compared to FeMn slag.  

Following a preliminary range-finding test, twenty daphnids (2 replicates of 5 animals) were exposed to Water Accommodated Fractions (WAFs) of the test material over a range of nominal loading rates of 10, 18, 32, 56 and 100 mg/L for 48 hours under at a temperature of 21°C static test conditions. The number of immobilised Daphnia was recorded after 24 and 48 hours.

A positive control conducted approximately every six months used potassium dichromate as the reference material. Daphnia magna was exposed to an aqueous solution of the reference material at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L for 48 hours at a temperature of approximately 20°C under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

The 48-Hour EL*50 for the test material to Daphnia magna based on nominal loading rates was 43 mg/L loading rate WAF with 95 % confidence limits of 41 - 46 mg/L loading rate WAF. The No Observed Effect Loading rate was 32 mg/L loading rate WAF.

The test material contains manganese. The test concentrations were monitored by analysis of the test samples for manganese only.

Analysis of the fresh media at 0 hours showed measured concentrations of manganese to range from 1.13 to 3.79 mg/L. Analysis of the old media at 48 hours showed measured concentrations of manganese to range from less than the limit of 1.16 to 3.62 mg/L. 

The dissolved test material may have been one or several components of the test material. Given that toxicity cannot be attributed to a single component or mixture of components but to the test material as a whole, the results were based on nominal loading rates only.

The 48-Hour EC50 for the reference material to Daphnia magna based on nominal concentrations was 0.78 mg/L with 95 % confidence limits of 0.58 – 0.72 mg/L. The No Observed Effect Concentration was 0.32 mg/L.