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Ecotoxicological information

Short-term toxicity to fish

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Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 January 2012 to 10 February 2012
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. The study report was conclusive, done to valid guidelines and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPP 72-1 (Fish Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid (unspecified)
Details on test material:
- Name of test material (as cited in study report): Cobalt Lithium Manganese Nickel oxide
- Appearance: black solid
- Empirical formula: Li (Co 0.33 Mn 0.33 Ni 0.33) O₂
- Homogeneity: homogeneous
- Storage conditions of test material: ambient; under nitrogen
- Storage stability: the stability of the test material under the storage conditions specified was guaranteed by the Sponsor for the duration of the test.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples for analytical determination of the concentration were taken within 30 minutes before insertion of the test organisms (start of exposure), after 48 hours and at the end of the exposure after approximately 96 hours. Any potential undissolved test material was immediately removed from the bulk sample by filtration with a membrane filter (pore width 0.2 µm). The membrane filter used was preconditioned with approximately 80 mL of test solution prior to collecting the samples. Samples not subjected to a separation step were also collected and analysed. Samples were taken from the middle of the test vessel using a glass pipette.
The samples were stored frozen before analysis.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
Since the test material was considered to be poorly soluble in water, the test solutions were prepared following general guidance recommended by OECD 23 and the US EPA OPPTS 850.1000 using a generator system in order to achieve a saturated solution of the test material.
2.0013 g of test material was placed onto a water-moistened cellulose cartridge filter (Micro-Clean type B2, 5 µm nominal size, Cuno Europe). A vessel containing 20 L of test water was connected via tubes with the stainless steel filter housing and a pump. The test water was recirculated through the filter at a rate of approximately 80 litres per minute for a period of 7 days, as recommended by OECD 29. The pump apparatus and test solution were maintained in a cooled room (13 °C) and the temperature of the test solution was measured daily throughout the 7 days as the recirculation process increased the solution temperature. Since the amount of test material used was far in excess of the water solubility of the individual metals, and the solution was re-circulated through the filter for 7 days, the concentration of the saturated solution was not affected by adsorption to the filter material. After 7 days the saturated solution was allowed to equilibrate to the test temperature for up to one day under test conditions.
Any undissolved test material was removed by filtration, after which the water accommodated fraction (WAF) of the test solution appeared colourless and clear.

Test organisms

Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead minnow
- Supplier: the fish were hatched at the testing facility
- Age at study initiation: approximately 9 months
- Length*: 4.0 cm (3.7 - 4.2 cm)
- Weight*: 0.53 g (0.34 - 0.64 g)
- Feeding during test: No. Feeding ceased approximately 48 hours prior to exposure.

* Measurements were performed with all surviving fish from the control and lowest test concentration as a representative sample of the fish batch. Body weight was wet weight and body length was the total length from the top of the snout to the end of the caudal fin.

ACCLIMATION
- Acclimation period: at least 14 days
- Acclimation conditions: same as test, with the exception that the batch of fish was housed in a fibreglass tank (approximately 300 L) receiving a continuous supply of fresh test water.
- Type and amount of food: Commercial fish diet "Teramin" (Tetra-Werke, Melle, Germany) was provided daily on an ad libitum basis. Frozen brine shrimp (Artemia nauplii) was generally provided on week days. Feeding ceased approximately 48 hours prior to exposure.
- Health during acclimation (any mortality observed): 0 % mortality during the week prior to exposure.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h

Test conditions

Hardness:
1.02 mmol/L (approximately 100 mg/L CaCO₃)
Test temperature:
22 - 23 °C
pH:
8.0 - 8.5
Dissolved oxygen:
6.1 - 8.3 mg/L (≥ 60 % ASV)
Nominal and measured concentrations:
0 and 100 mg/L (nominal*)

* As loading rate, based upon test material mass used to prepare the test solution.
Details on test conditions:
TEST SYSTEM
- Test vessel: 10 L glass aquaria (30 x 22 x 24 cm)
- Fill volume: 10 L
- Aeration: none over the first 24 hours, but slight aeration during the subsequent days of exposure as oxygen content after 24 hours was decreased to approximately 69 % saturation.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Test water: charcoal-filtered tap water from the municipal water works of the city of Frankenthal mixed with deionised water. The water is non-chlorinated and aerated.
- Total organic carbon: < 2 mg/L
- Metals: Ca content - ca. 40 mg/L; Mg content - ca. 5 mg/L
- Conductivity: ca 250 µS/cm (at 25 °C)
- Acid capacity: ca. 2.5 mmol/L
- Culture medium different from test medium: no
- Intervals of water quality measurement: the dilution water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal as well as at the test facility. Furthermore the water is also regularly assayed for the presence of microbes by a contract laboratory.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light / 8 hours dark
- Light intensity: Approximately 62 - 415 Lux

EFFECT PARAMETERS MEASURED
The fish were observed for mortality and toxic signs (changes in appearance and abnormal behaviour in comparison to the control group) 1, 6, 24, 48, 72 and 96 hours after the start of exposure. Fish were considered dead if there was no visible movement and no reaction after touching. Dead fish were removed from the test vessels.
At the end of the exposure period, the body weight and total length of a representative sample of fish were determined. The representative sample consisted of all surviving fish from the test solution and control groups.

PHYSICAL PARAMETERS MEASURED
The appearance of the test solutions and dissolution behaviour of the test material was recorded daily. Temperature, oxygen content and pH-value were measured in each test vessel within 1 hour after the start of exposure and after 24, 48, 72 and 96 hours. Temperature was also monitored continuously (hourly) in one control test vessel with an automatic temperature data logger. The TOC, hardness and conductivity of the dilution water was measured at the start of exposure.
Reference substance (positive control):
no

Results and discussion

Effect concentrations
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
None of the fish died during the exposure period and no adverse effects or abnormal behaviour were observed in either the treatment group or control group.
Since the fish were exposed to a saturated solution of all soluble components of the test material at the given loading rate, the effect concentrations are based on the loading rate.
All test treatments were visibly clear over the entire exposure period and no undissolved test material was observed.

VALIDITY CRITERIA
All the following validity criteria were met and the experiment is considered valid.
- ≤ 10 % mortality in the control group
- Constant exposure conditions across the treatment group and control group.
- The pH did not vary by more than one unit.
- Oxygen concentration was ≥ 60 % of the saturation value.
- Measured test concentrations were kept within ± 20 % of the mean measured concentration during the course of the test.

Any other information on results incl. tables

Analytical Determinations

The analytically determined concentrations of Mn and Ni in the test material solutions were:

Mn content: 18.4 g/100 g

Ni content: 19.7 g/100 g

Table 1: Analytically measured concentrations - unfiltered

Nominal loading rate (mg/L)

0 hours

48 hours

96 hours

Mn

(mg/L)

Ni

(mg/L)

Test material

(mg/L)

Mn (mg/L)

Ni (mg/L)

Test material

(mg/L)

Mn (mg/L)

Ni (mg/L)

Test material

(mg/L)

0

< 0.03

< 0.03

< 0.2

< 0.03

< 0.03

< 0.2

< 0.03

< 0.03

< 0.2

100

< 0.03

0.06

< 0.5

< 0.03

0.06

< 0.5

< 0.03

0.06

< 0.5

Table 2: Analytically measured concentrations - filtered (0.2 µm)

Nominal loading rate (mg/L)

0 hours

48 hours

96 hours

Mn

(mg/L)

Ni

(mg/L)

Test material

(mg/L)

Mn

(mg/L)

Ni

(mg/L)

Test material

(mg/L)

Mn

(mg/L)

Ni

(mg/L)

Test material

(mg/L)

0

< 0.03

< 0.03

< 0.2

< 0.03

< 0.03

< 0.2

< 0.03

< 0.03

< 0.2

100

< 0.03

0.05

< 0.5

< 0.03

0.06

< 0.5

< 0.03

0.06

< 0.5

Filtered and unfiltered samples were analysed with nearly identical results, demonstrating the absence of undissolved test material in the test solution. Mn concentrations were below the LOQ at the start and end of exposure. Ni concentrations were stable over the exposure period at 0.06 mg/L. However, the estimated equivalent amount of test material in solution was < 0.5 mg/L. Nevertheless, the test solutions were considered saturated with all soluble components of the test material, in test media and under test conditions.

All reasonable efforts were used to generate a saturated solution of the test material in test media following the guidance in OECD 23 and OECD 29. Since the test material is a mixed metal oxide, the test solution is considered a water accommodated fraction (WAF).

Table 3: Mortality and toxic signs

Nominal loading rate (mg/L)

Number of fish

Time (hours)

0

6

24

48

72

96

Cumulative mortality (number of dead fish)

0

10

0

0

0

0

0

0

100

10

0

0

0

0

0

0

Observed signs of toxicity

0

10

None

None

None

None

None

None

100

10

None

None

None

None

None

None

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, the 96-hour LL50 of the test material was determined to be in excess of 100 mg/L based on the loading rate of the test material in the test media. The results of the study demonstrate that the test material is not acutely toxic to fish up to the limit of solubility.
Executive summary:

The short term toxicity of the test material to freshwater fish was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 203, EU Method C.1, US EPA OPPTS 850.1075 and EPA OPP 72 -1.

During the study, groups of 10 Pimephales promelas were each exposed to test material at nominal concentrations of 0 (dilution water control) and 100 mg/L, under static conditions, for 96 hours. Mortality, adverse effects and any signs of abnormal behaviour were recorded.

The content of manganese and nickel in the test solutions was analysed during the study. Filtered and unfiltered samples were analysed with nearly identical results. Manganese concentrations were below the LOQ at the start and end of exposure. Nickel concentrations were stable over the exposure period at 0.06 mg/L. However, the estimated equivalent amount of test material in solution was < 0.5 mg/L. Nevertheless, the test solutions were considered saturated with all soluble components of the test material, in test media and under test conditions.

Since the fish were exposed to a saturated solution of test material at the given loading rate, the effect concentrations were based on the loading rate (as recommended in OECD 23).

Under the conditions of the study, a saturated solution of the test material prepared at a loading rate of 100 mg/L in test media had no observable acute toxic effect on the fish. The 96-hour LL50 was therefore determined to be in excess of 100 mg/L. The results of the study demonstrate that the test material is not acutely toxic to fish up to the limit of solubility.