Silicomanganese manufacturing fumes dedusting solid residues

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 March 2010 and 12 April 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and to a current guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

. Species used:Ceriodaphnia dubia

Test guideline 2

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 Chemicals 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.
The test item was known to be insoluble in water. 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 saturated solution of the test item in cases where the test item is of high purity and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution was prepared by stirring an excess (100 mg/l) of test item with reconstituted water (Elendt M4) for 48 hours at approximately 25°C, then removing the undissolved test item by filtration through a pre-conditioned filter (0.2 µm) to give a saturated solution.

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994))

Test material

Test material information

Constituent 1

Reference substance name:

Silicomanganese manufacturing fumes dedusting solid residues

EC Number:

930-776-4

IUPAC Name:

Silicomanganese manufacturing fumes dedusting solid residues

Details on test material:

Sponsor's identification : Mn3O4 (Erachem
Description : brown powder
Date received :8 September 2008
Expiry date : Not supplied
Storage conditions : room temperature in the dark
- Lot/batch No.:08100

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not reported

Sampling and analysis

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.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: An amount of test item (250 mg) was dispersed in 2.5 litres of reconstituted water (Elendt M4) with the aid of magnetic stirring at approximately 100 rpm at approximately 25°C. After 48 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 µm Gelman AcroCap filter (first approximate 100 ml discarded in order to pre-condition the filter) to give the 100% v/v saturated solution. Aliquots (50 and 160 ml) of the 100% v/v saturated solution were each separately diluted in a final volume of 500 ml of reconstituted water (Elendt M4) to give the remainder of the test series of 10 and 32% v/v saturated solution.

Test organisms

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 to 5µl of a unicellular algal culture and 100 µl of a YAT combination. The study plan stated that each daphnid would be fed 1 µl of algal suspension daily. During the test each daphnid was fed 1 to 5 µl of algal suspension. This was due to a revised method of feeding whereby the amount of algal suspension fed was based on the cell density of the suspension. This deviation was considered not to have affected the outcome of the test.
- Frequency:Daily

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

Study design

Test type:

static

Water media type:

freshwater

Total exposure duration:

8 d

Test conditions

Hardness:

210 and 250 mg/l as CaCO3 in the control and the 100% v/v saturated solution test group at the start of the test

Test temperature:

25°C

pH:

8.0-8.3

Dissolved oxygen:

7.1 - 9.3 mg O2/L
The oxygen concentration in some of the test vessels was observed to have an air saturation value (ASV) in excess of 100%.

Salinity:

Not reported

Nominal and measured concentrations:

Control, 10, 30 & 100 % saturated solution - Nominal concentration
Control, 0.52, 1.7 & 5.1 mg/L as test item - Mean measured concentration

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass vessel
- Type (delete if not applicable): closed. Covered with a plastic lid 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: 48 and 50 mg CaCO3/l in the control and the 100% v/v saturated solution test group at the start of the test.
- Conductivity: 902 and 906 µs/cmin the control and the 100% v/v saturated solution test group at the start of the test.
- Intervals of water quality measurement: Temperature of the test preparations was recorded twice daily in two different locations and light intensity was recorded daily throughout the test. Dissolved oxygen concentrations, pH and temperature were recorded before and after each test media renewal. The pH and the dissolved oxygen concentration were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter and the temperature was measured using a Hanna Instruments HI 93510 digital thermometer. Measurements were made on an additional sample (approximately 100 ml) of each test concentration run alongside the test due to the small volumes used in the test. The water hardness, conductivity and alkalinity of the control and the highest test concentration were determined at the start of the test.

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours darkness with 20 minutes dawn and dusk transition periods for 8 days.
- Light intensity: 551 to 581 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
On a daily basis the numbers 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).

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

8 d

Dose descriptor:

LOEC

Effect conc.:

100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 100% v/v saturated solution as this test group produced significantly fewer live young per adult (P<0.05) than the control.

Effect concentrations 2

Duration:

8 d

Dose descriptor:

NOEC

Effect conc.:

32 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Mortality(immobilisation)

Remarks on result:

other: There were no significant mortalities (immobilisation) observed in the parental generation (P1) and there were no significant differences (P<0.05) in terms of the number of live young produced per adult when compared to the control after 8 days.

Effect concentrations 3

Duration:

8 d

Dose descriptor:

other: EC50

Effect conc.:

100 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: Parental Ceriodaphnia generation (P1).

Effect concentrations 4

Duration:

8 d

Dose descriptor:

other: EL*50

Effect conc.:

78 other: % v/v saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Mortality of parent animals: Mortality (immobilisation) occurred predominantly at the highest test concentration of 100% v/v saturated solution, resulting in 40% mortality by Day 8, indicating a prolonged toxic effect attributable to exposure of Ceriodaphnia dubia to the test item.
Mortality was also observed at the test concentration of 32% v/v saturated solution. However, statistical analysis of the mortality data using the corrected chi-squared statistic (Breslow and Day 1980) showed that the observed mortality in the 32% v/v saturated solution test group was not significantly different (P > 0.05) when compared to the control group. See table 2.

- No. of offspring produced per day per female: There was a significant effect on size and colour of the daphnids in that 33% of the surviving daphnids on Day 8 at the test concentration of 100% v/v saturated solution were markedly smaller and paler in colour than the control animals.
The daphnids at the remaining test concentrations were observed to be the same size and colour as the control animals.
After 8 days there were no statistically significant differences between the control, 10 and 32% v/v saturated solution test groups in terms of the number of live young produced per adult. The 100% v/v saturated solution test group showed a statistically significant difference from the control and the remaining test groups after 8 days in terms of producing fewer numbers of live young per adult (see Appendix 4).
The EC50 (reproduction) value calculated by the maximum-likelihood probit method (Finney 1971) on Day 8, based on nominal test concentrations was 78% v/v saturated solution.

Information on the effects of the test item on the F1 generation is limited, since, by study design, the young are removed soon after liberation from the brood pouch. However, an assessment made at each media renewal showed the "filial" daphnids produced by all the test groups were in the same general condition as the young produced by the controls over the duration of the test. Young were first produced in the control group on Day 3 of the test. Numbers of unhatched eggs and dead young were zero in all control and treatment groups surviving to maturation.

Results with reference substance (positive control):

Not reported

Reported statistics and error estimates:

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

Any other information on results incl. tables

Table 2: The following EC₅₀(immobilisation) values based on nominal test concentrations as test item were estimated throughout the test:

Time	EC50 (% v/v Saturated Solution)
24 hours	>100
48 hours	>100
96 hours	>100
8 days	>100*

*   It was not possible to calculate an EC₅₀value at this time point as less than 50% immobilisation occurred.

Table 3. Summary of findings following the exposure of Ceriodaphnia dubia for 8 days.

 

Nominal Concentration (% v/v saturated solution)	% 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	90	245	27	0	0	0	0
10	100**	214	24	0	0	0	0
32	90	193	21	0	0	0	0
100	60	62	10	0	0	0	0

*The number of live young per live adult** A single live daphnid failed to produce any young throughout the duration of the test. This was considered to be possibly due to an inferior daphnid given the results from the remaining daphnids in the group. The results from this daphnid were therefore not included in the statistical analysis of the young produced

 

Table 4. 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	5	0	0	0
3	10	9	13	0	0
4	10	9	15	0	0
5	10	9	48	0	0
6	9	9	40	0	0
7	10	9	74	0	0
8	10	9	55	0	0
		Total	245	0	0

 

 

Table 5. Summary of observations of the 10% v/v saturated solution 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	8	11	0	0
4	9	9	13	0	0
5	9	9	45	0	0
6	9	9	55	0	0
7	9	9	59	0	0
8	9	9	31	0	0
		Total	214	0	0

 

Table 6.Summary of observations of the 32% v/v saturated solution 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	4	0	0	0
3	10	8	12	0	0
4	10	10	8	0	0
5	10	10	55	0	0
6	10	10	46	0	0
7	10	9	35	0	0
8	10	9	42	0	0
		Total	198	0	0

 

Table 7.Summary of observations of the 100% v/v saturated solution 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	8	2	0	0	0
3	7	4	0	0	0
4	6	3	4	0	0
5	6	3	11	0	0
6	6	4	15	0	0
7	5	4	1	0	0
8	5	5	31	0	0
		Total	62	0	0

  

Table 8. Total cumulative production of live young. 

 

Nominal Concentration (% v/v saturated solution)	Day
	1	2	3	4	5	6	7	8
Control	0	0	13	28	76	116	190	245
10	0	0	11	24	69	124	183	214
32	0	0	12	20	75	124	156	198
100	0	0	0	4	15	30	31	62

  

Table 9. Number of live young produced per adult (Non-cumulative). 

 

Mean Measured Concentration (mg/l as Test Item)	Day
	1	2	3	4	5	6	7	8
Control	0	0	1	2	5	4	8	6
10	0	0	1	1	5	6	6	3
32	0	0	1	1	6	5	4	5
100	0	0	0	1	2	3	0	6

 

 

 

 

 

  

 

 

 

 

 

 

 

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Ceriodaphnia dubia to the test item resulted in significant mortalities at the test concentration of 100% v/v saturated solution resulting in 40% mortalities by Day 8.The 8-Day EC50 (immobilisation) value, based on nominal test concentrations, for the parental Ceriodaphnia generation (P1) was greater than 100% v/v saturated solution.The 8-Day EC50 (reproduction) based on nominal test concentrations was 78% v/v saturated solution.
The "Lowest Observed Effect Concentration" (LOEC) and the "No Observed Effect Concentration" (NOEC) based on nominal test concentrations were 100% and 32% v/v saturated solution respectively.

Executive summary: