Manganese ores, reduced

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03-03-2010 to 17-03-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP and to a guideline. 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:

equivalent or similar to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

. Species used:Ceriodaphnia dubia

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)

Deviations:

yes

Remarks:

.Species used:Ceriodaphnia dubia

Principles of method if other than guideline:

The study was performed to assess the effect of the test item on the survival and reproduction of Ceriodaphnia dubia over an 8-day period. The method followed that described in the OECD guidelines for Testing of Chemcials No 211 (1998) ''Daphnia magna, Reproduction test'' US EPA short-term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organism (method 1002.0) and OPPTS Ecological effects test guideline, Daphnid chronic toxicity test OPTTS 850.1300.

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The concentration of the test item in the test preparations was verified by chemical analysis on Day 0 (fresh media), 3, 5, 7 (old and fresh media) and 8 (old media).
- Sampling method:The test samples were analysed following addition of nitric acid (2.5 mL per 50 mL of sample).
- Sample storage conditions before analysis: Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low aqueous solubility and complex nature of the test item for the purpose of the definitive test the test item was prepared as a water accommodated fraction (WAF).

Amount of test item (2.5, 8.0, 25, 80 and 250 mg) were each separately added to the surface of 2.5 litres of reconstituted water to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rates respectively. After the addition of the test item, the reconstituted water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a slight dimple at the water surface. The stirring was stopped after 47 hours and the mixtures allowed to stand for 1 hour. Microscopic observations made on the WAFs indicated that a significant amount of dispersed test tiem was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 1.0, 3.2, 10, 32, and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed no undissolved test item or micro-dispersions to be present.

Test organisms (species):

Ceriodaphnia dubia

Details on test organisms:

TEST ORGANISM
- Common name:Water flea
- Justification for species other than prescribed by test guideline: Ceriodapnia dubia is a freshwater invertebrate representative of a wide variety of natural habitats and can therefore be considered as an important non-target organism in freshwater ecosystems.
- Source: In house laboratory cultures
- Age of parental stock (mean and range, SD): 24 hours old
- Feeding during test: Each culture was fed daily
- Food type: Fed a suspension of algae (Pseudokirchneriella subcapitata) and YAT (yeast, alfalfa, trout chow) combination. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.
- Amount: Each daphnid received 1µl of a unicellular algal culture and 100 µl of a YAT combination.
- Frequency:Daily

ACCLIMATION
- Acclimation conditions: same as test
- Type and amount of food: same as test
- Feeding frequency: Fed daily

Test type:

static

Water media type:

freshwater

Total exposure duration:

8 d

Hardness:

240 mg/L as CaCO3

Test temperature:

25°C

pH:

8.0-8.8

Dissolved oxygen:

8.0 - 9.4 mg O2/L
83- 110% - Air saturation value. 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 reported

Nominal and measured concentrations:

1.0, 3.2, 10, 32 and 100 mg/L - Nominal loading rates

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass vessel
- Type (delete if not applicable): closed. Covered to reduce evaporation
- fill volume: 50 mL
- Aeration: No
- Renewal rate of test solution (frequency): The test preparations were renewed 3 times on Day 2, 3, and 7.
- No. of organisms per vessel:1
- No. of vessels per concentration (replicates):10
- No. of vessels per control (replicates):10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The reconstituted water (Elendt M4) used for the definitive test was the same as that used to maintain the stock animals.
- Alkalinity: 39 and 38 mg CaCO3/ L in the control and the 100 mg/L loading rate WAF test group at the start of the test.
- Conductivity: 1250 and 801 µs/cm in the control and the 100 mg/L loading rate WAF test group at the start of the test
- Intervals of water quality measurement: Temperature was recorded, twice in two different locations, and light intensity was recorded daily throughout the test. Dissolved oxygen concentrations and pH were recorded before and after test media renewal.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness with 20 minutes dawn and dusk transition periods for 8 days.
- Light intensity: 526 to 590 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
On a daily basis the number of live and dead of the ''Parental'' (P1) generation, the numbers of live and dead ''Filial'' (F1) Ceriodaphnia and the number of discarded unhatched eggs were counted. An assessment was also made of the general condition and size of the parental Ceriodaphnia as compared with the controls. The number of Ceriodaphnia with eggs or young in the brood pouch was determined daily. Young daphnids were considered to be dead if no sign of movement was apparent during microscopic examination. Adult Ceriodaphnia which were unable to swim for approximately 15 seconds after gentle agitation (ie. immobile), were considered to be dead. An immobilisation criterion for the young daphnids was considered to be inappropriate due to the large numbers of off-spring produced in the flasks.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Results used to determine the conditions for the definitive study: Based on the results of an acute toxicity test to Daphnia magna (Harlan Laboratories Ltd Project Number 2702/0161).

Duration:

8 d

Dose descriptor:

LOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Loading rate WAF

Basis for effect:

reproduction

Remarks on result:

other: (P<0.05)

Duration:

8 d

Dose descriptor:

NOELR

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Loading rate WAF

Basis for effect:

other: Mortalilty(immobilisation)

Remarks on result:

other: Observed in the Parental generation (P1)

Duration:

8 d

Dose descriptor:

other: EL*50

Effect conc.:

27 mg/L

Nominal / measured:

estimated

Conc. based on:

other: Nominal loading rtes

Basis for effect:

immobilisation

Remarks on result:

other: Parental Ceriodaphnia generation (P1). 95% confidence limits of 32-100 mg/L loading rate WAF.

Duration:

8 d

Dose descriptor:

other: EL*50

Effect conc.:

11 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Loading rate WAF

Basis for effect:

reproduction

Remarks on result:

other: With 95% confidence limits of 6.5-20 mg/L loading rate WAF

Details on results:

- Mortality of parent animals: Mortality (immobilisation) occurred predominantly at the highest loading rate of 100 mg/L resulting in 100% mortality by Day 5 indicating a prolonged toxic effect attributable to exposure of Ceriodaphnia dubia of the test item. Mortalities were also observed at the 1.0, and 3.2 mg/L loading rate WAF test groups. However, statistical analysis of the mortality data using the corrected chi-squared statistic (Breslow and Day 1980) showed that the observed mortalities in the 1.0 and 3.2 loading rate WAF test groups were not significantly different (P≥0.05) when compared to the control. No mortality occurred at the 10 and 32 mg/L loading rate WAF test group throughout the test (See table 5
).- No. of offspring produced per day per female:
- Body length and weight of parent animals: There was no significant effect on the size of the daphnids at the 100 mg/L loading rate WAF test group prior to observing 100% mortalities by Day 6. Thedaphnids at the remaining loading rates were observed to be the same size as the control animals.
- Number of males and females (parental): After 8 days there were no statistically significant differences between the control and the 1.0 and 3.2 mg/L loading rate WAF test groups in terms of the number of live young produced per adult. The 10 and 32 mg/L loading rate WAF groups showed a statistically significant difference from the control after 8 days in terms of producing fewer numbers of live young per adult.

Results with reference substance (positive control):

Not reported

Reported statistics and error estimates:

See Any other information on material and methods incl. tables for details.

Table 5.The following EL*50 (immobilisation) values based on nominal loading rates were estimated by inspection of the immobilisation data at 24 an 48 hours and calculated by the trimmed Spearman-Karber method (Hamilton et al 1977) at 96-hours and the geometric mean method at 8 days:

 

Time	EL*50 (mg/L)	95% Confidence Limits (mg/L)
24 hours	>100	-
48 hours	>100	-
96 hours	75	51-110
8 days	57	32-100**

*EL = Effective loading rate

** = Concentrations resulting in 0 and 100% immobilisation respectively. 

 

Table 6. Summary of findings following the exposure of Ceriodaphnia dubia for 8 days.

 

Nominal loading rate (mg/l)	% Survival of P1	No. of live young		No. of dead young		No. of unhatched young
		Total	Per female*(cumulative)	Total	Per female (cumulative)	Total	Per female (cumulative)
Control	80	140	17	0	0	0	0
1.0	80	139	17	1	<1	0	0
3.2	90	128	16	4	<1	0	0
10	100	83	8	2	<1	0	0
32	100	30	3	0	0	0	0
100	0	0	0	0	0	0	0

*The number of live and dead young and unhatched eggs per live female remaining on day 8.

 

Table 7. Summary of observations of the control group

 

Day	Adults Surviving	Number of adults with eggs in brood pounch	Live young	Dead young	Unhatched Eggs
1	10	0	0	0	0
2	10	2	0	0	0
3	10	9	0	0	0
4	9	9	15	0	0
5	9	9	32	0	0
6	9	9	28	0	0
7	9	9	35	0	0
8	8	8	30	0	0
		Total	140	0	0

 

 

Table 8. Summary of observations of the 1.0 mg/L loading rate WAP test group

 

Day	Adults Surviving	Number of adults with eggs in brood pounch	Live young	Dead young	Unhatched Eggs
1	10	0	0	0	0
2	10	3	0	0	0
3	10	7	5	0	0
4	9	9	17	0	0
5	8	8	20	0	0
6	8	8	22	0	0
7	8	8	42	0	0
8	8	8	33	0	0
		Total	132	0	0

 

Table 9. Summary of observations of the 3.2 mg/l loading rate WAP test group

 

Day	Adults Surviving	Number of adults with eggs in brood pounch	Live young	Dead young	Unhatched Eggs
1	10	0	0	0	0
2	10	1	0	0	0
3	10	8	3	0	0
4	10	9	24	0	0
5	9	9	19	0	0
6	9	9	13	0	0
7	8	10	32	0	0
8	8	9	37	0	0
		Total	128	0	0

 

Table 10.Summary of observations of the 10.0 mg/l loading rate WAP test group

 

Day	Adults Surviving	Number of adults with eggs in brood pounch	Live young	Dead young	Unhatched Eggs
1	10	0	0	0	0
2	10	1	0	0	0
3	10	10	0	0	0
4	10	10	12	0	0
5	10	10	11	0	0
6	10	10	21	0	0
7	10	10	13	0	0
8	10	10	26	0	0
		Total	83	0	0

 

Table 11. Summary of observations of the 32 mg/l loading rate WAP test group

 

Day	Adults Surviving	Number of adults with eggs in brood pounch	Live young	Dead young	Unhatched Eggs
1	10	0	0	0	0
2	10	0	0	0	0
3	10	7	0	0	0
4	10	10	14	0	0
5	10	10	7	0	0
6	10	10	1	0	0
7	10	10	6	0	0
8	10	10	2	0	0
		Total	30	0	0

 

Table 12. Summary of observation of the 100 mg/L loading rate WAF test groups. 

 

Day	Adults Surviving	Number of adults with eggs in brood pounch	Live young	Dead young	Unhatched Eggs
1	10	0	0	0	0
2	10	0	0	0	0
3	6	3	0	0	0
4	3	2	0	0	0
5	A/D			0	0
		Total	0	0	0

A/D = all daphnids dead.

 

Table 13. Total cumulative production of live young. 

 

Nominal loading rate (mg/l)	Day
	1	2	3	4	5	6	7	8
Control	0	0	0	2	4	3	4	3
1.0	0	0	1	2	2	3	5	4
3.2	0	0	0	2	2	1	3	4
10	0	0	0	1	1	2	1	3
32	0	0	0	1	1	0	1	0
100	0	0	0	0	A/D

A/D = all daphnids dead.

 

 

Table 14. Number of live young produced per adult (Non-cumulative). 

 

Nominal loading rate (mg/l)	Day
	1	2	3	4	5	6	7	8
Control	0	0	0	15	47	75	110	140
1.0	0	0	5	22	42	64	106	139
3.2	0	0	3	27	46	59	91	128
10	0	0	0	12	23	44	57	83
32	0	0	0	14	21	22	28	30
100	0	0	0	0	A/D

 

 The following validation criteria were achieved during the test:

                                                             

	Required	Actual
a Control mortality	≤ 20%	20%
b Dissolved oxygen	>3 mg O2/L	≥6.5 mg O2/L
c Ph (control group)	Deviation ≤1.5	0.2
d Mean number of live young per surviving adult (control group)	≥ 15 after 8 days	15
e Coefficient of variation for control group	≤ 25%	23%

 

 

 

 

  

 

 

 

 

 

 

 

 

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Ceriodaphnia dubia to the test item resulted in significant mortalities at the loading rate of 100 mg/L resulting in 100% mortality by Day 6.
The 8-day EL*50 (immobilisation) value, based on nominal loading rates, for the parental Ceriodaphnia dubia generation (P1) was calculated to be 27 mg/L loading rate WAF with 95% confidence limits of 32-100 mg/L loading rate WAF.
The 8-Day EL*50(reproduction) based on nominal loading rates was 11 mg/L loading rate WAF with 95% confidence limits of 6.5 -20 mg/L loading rate WAF.
The ''Lowest Observed Effect loading rate'' (LOEL) and the ''No Observed Effect Loading rate'' (NOEL) based on nominal loading rates were 10 and 3.2 mg/L loading rate WAF respectively.

Executive summary:

A study was performed to assess the effect of the test item on the survival and reproduction of Ceriodaphnia dubia over an 8-day period. The study was conducted under GLP conditions and the method followed that described in the OECD guidelines for Testing of Chemcials No 211 (1998) ''Daphnia magna, Reproduction test'' US EPA short-term methods for estimating the chronic toxicity of effluents and receiving waters to freshwater organism (method 1002.0) and OPPTS Ecological effects test guideline, Daphnid chronic toxicity test OPTTS 850.1300.

Exposure of Ceriodaphnia dubia to the test item resulted in significant mortalities at the loading rate of 100 mg/L resulting in 100% mortality by Day 6.

The 8-day EL*50 (immobilisation) value, based on nominal loading rates, for the parental Ceriodaphnia dubia generation (P1) was calculated to be 27 mg/L loading rate WAF with 95% confidence limits of 32-100 mg/L loading rate WAF.

The 8-Day EL*50(reproduction) based on nominal loading rates was 11 mg/L loading rate WAF with 95% confidence limits of 6.5 -20 mg/L loading rate WAF.

The ''Lowest Observed Effect loading rate'' (LOEL) and the ''No Observed Effect Loading rate'' (NOEL) based on nominal loading rates were 10 and 3.2 mg/L loading rate WAF respectively.  

Reference 2

Endpoint:

long-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:

8 d

Dose descriptor:

LOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Loading rate WAF

Basis for effect:

reproduction

Remarks on result:

other: P < 0.05

Duration:

8 d

Dose descriptor:

NOELR

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Loading rate WAF

Basis for effect:

other: Mortalilty (immobilisation)

Remarks on result:

other: Observed in P1 generation

Duration:

8 d

Dose descriptor:

EL50

Effect conc.:

27 mg/L

Nominal / measured:

estimated

Conc. based on:

other: Nominal loading rate

Basis for effect:

immobilisation

Remarks on result:

other: P1 generation, 95 % CL 32 - 100 mg/L loading rate WAF

Duration:

8 d

Dose descriptor:

EL50

Effect conc.:

11 mg/L

Nominal / measured:

nominal

Conc. based on:

other: Loading rate WAF

Basis for effect:

reproduction

Remarks on result:

other: 95 % CL 6.5 - 20 mg/L Loading Rate WAF

Description of key information

Exposure of Ceriodaphnia dubia to the read-across substance, ferromanganese slag, resulted in significant mortalities at the loading rate of 100 mg/L resulting in 100% mortality by Day 6.

The 8-day EL*50 (immobilisation) value, based on nominal loading rates, for the parental Ceriodaphnia dubia generation (P1) was calculated to be 27 mg/L loading rate WAF with 95% confidence limits of 32-100 mg/L loading rate WAF.

The 8-Day EL*50(reproduction) based on nominal loading rates was 11 mg/L loading rate WAF with 95% confidence limits of 6.5 -20 mg/L loading rate WAF.

The ''Lowest Observed Effect loading rate'' (LOEL) and the ''No Observed Effect Loading rate'' (NOEL) based on nominal loading rates were 10 and 3.2 mg/L loading rate WAF respectively. 

Key value for chemical safety assessment

Marine water invertebrates

Marine water invertebrates

Effect concentration:

3.2 mg/L

Additional information

A study was performed to assess the effect of the read-across substance, ferromanganese slag, on the survival and reproduction of Ceriodaphnia dubia over an 8-day period. The study was conducted under GLP conditions and the method followed that described in the standardised guidelines OECD 211 and US EPA OPTTS 850.1300. 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.  

Exposure of Ceriodaphnia dubia to the test item resulted in significant mortalities at the loading rate of 100 mg/L resulting in 100% mortality by Day 6.

The 8-day EL*50 (immobilisation) value, based on nominal loading rates, for the parental Ceriodaphnia dubia generation (P1) was calculated to be 27 mg/L loading rate WAF with 95% confidence limits of 32-100 mg/L loading rate WAF.

The 8-Day EL*50(reproduction) based on nominal loading rates was 11 mg/L loading rate WAF with 95% confidence limits of 6.5 -20 mg/L loading rate WAF.

The ''Lowest Observed Effect loading rate'' (LOEL) and the ''No Observed Effect Loading rate'' (NOEL) based on nominal loading rates were 10 and 3.2 mg/L loading rate WAF respectively.