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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1.2.2010-19.2.2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
yes
Remarks:
see Overall remarks
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Test organisms (species):
Poecilia reticulata
Details on test organisms:
TEST ORGANISM
- Common name: Poecilia reticulata
- Source: EMPLA, s.r.o., Hradec Králové, Czech republic (certified supplier)
- Age at study initiation (mean and range, SD): 3-4 month
- Length at study initiation (length definition, mean, range and SD): 2.0-3.0 cm
- Method of breeding: all-glass aquaria with aerated drinking water
- Feeding during test
- Food type: Tropica (feed for aquarium fish) ad libitum, sometimes live daphnia


ACCLIMATION
- Acclimation period: 7 days
- Type and amount of food: fish were not fed during 72 hours before test
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
Content of (Ca + Mg) ions: 2.47 mmol/l in preliminary test
Content of (Ca + Mg) ions: 2.47 mmol/l in limit test
Test temperature:
see Tables 3 and 4
pH:
see Tables 3 and 4
Dissolved oxygen:
see Tables 3 and 4
Nominal and measured concentrations:
As the test substance is multi-component poorly soluble substance the water-accommodated fractions (WAFs) were prepared for preliminary and limit test. The WAFs were prepared by adding measured amounts of the test substance to the dilution water and then were stirred for suitable period. After period of stirring the WAFs were filtered through 0.45 µm filter and these solutions were used for testing.
The suitable period for achieving equilibrated concentration of dissolved and dispersed components in aqueous phase was determined in preliminary experiment. For preliminary experiment the WAFs were prepared as follows: the suspension of 100 mg of the test substance in 1000 ml of dilution water was stirred for period 3, 24, 48 and 72 hours on a shaft stirrer, consequently filtered through 0.45 µm filter. During this experiment the concentrations of metals (Al, Ca, Fe, K, Mg, Mn, Na and Zn) in resulted WAFs were determined. The samples of WAFs were prepared in dilution water, which contents metals as Ca, Mg, Na and K. For this reason the analytical determination of the concentrations of metals was performed also in pure dilution water.
The analytical results showed, that the test substance is poorly soluble substance. If we consider the error of determination the analytical results from 3, 24, 48 and 72 hours are comparable. Because of technical performing of the test the 72 hours period of stirring was chosen for preparation the WAFs.
The WAFs for preliminary and limit test were prepared by adding measured amounts of the test substance to the dilution water. The WAFs were stirred for period 72 hours on a shaft stirrer and consequently filtered through 0.45 µm filter. The exact loading rates are given in tables.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open beaker
- Material, size, headspace, fill volume: 1000 ml solution
- Aeration: without
- No. of organisms per vessel: 7 (preliminary test), 10 (limit test)
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Standard dilution water was prepared just before the test by admixing 2.5 ml of stored solutions A, B, C and D in 1000 ml of deionized water.
A 117.6 g CaCl2×2H20 in 1 l deionized water
B 49.3 g MgSO4×7H2O in 1 l deionized water
C 25.9 g NaHCO3 in 1 l deionized water
D 2.3 g KCl in 1 l deionized water


OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: daylight


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.1 (limit test)
- Justification for using less concentrations than requested by guideline: Water-accommodated fractions (WAFs) were prepared for preliminary and limit test because the test material is a mixture of poorly soluble substances. Nominal concentration 100 mg/l was used.
- Range finding study
- Test concentrations: 100, 60, 40, 20 and 10 mg/l
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
24 h
Dose descriptor:
LL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality (fish)
Duration:
48 h
Dose descriptor:
LL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality (fish)
Duration:
72 h
Dose descriptor:
LL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality (fish)
Key result
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality (fish)
Duration:
24 h
Dose descriptor:
NOELR
Effect conc.:
91 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality (fish)
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
91 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality (fish)
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
91 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
91 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality (fish)
Duration:
24 h
Dose descriptor:
LL100
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality (fish)
Duration:
48 h
Dose descriptor:
LL100
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality (fish)
Duration:
72 h
Dose descriptor:
LL100
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LL100
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality (fish)
Results with reference substance (positive control):
Reference test:
The sensitivity of the test species and correctness of test performance is periodically verified in three-month period by testing the reference substance, potassium dichromate. The results of verification test with K2Cr2O7 are the following:
LC50 (48 h) = 126 mg/l (95% confidence limit: 80 - 171 mg/l)
LC50 (96 h) = 117 mg/l (95% confidence limit: 95 - 138 mg/l)
The results of the last interlaboratory test arranged by Centre for Assessment of Laboratories of T.G.Masaryk Water Research Institute Prague in 2008 for potassium dichromate were:
LC50 (96 h) = 81.2 - 190 mg/l
The mortality of test organisms in the reference substance solution meets criteria determined by the interlaboratory test.
Sublethal observations / clinical signs:

Table 1: Fish mortality - preliminary test

Loading rate mg/l 100 60 40 20 10 control
Number of live fish 2 hours 7 7 7 7 7 7
Mortality % 0 0 0 0 0 0
Number of live fish 24 hours 7 7 7 7 7 7
Mortality % 0 0 0 0 0 0
Number of live fish 48 hours 7 7 7 7 7 7
Mortality % 0 0 0 0 0 0
Number of live fish 72 hours 7 7 7 7 7 7
Mortality % 0 0 0 0 0 0
Number of live fish 96 hours 7 7 7 7 7 7
Mortality % 0 0 0 0 0 0

Table 2: Fish mortality - limit test

Loading rate mg/l 100 91 83 75 68 62 control
Number of live fish 2 hours 10 10 10 10 10 10 10
Mortality % 0 0 0 0 0 0 0
Number of live fish 24 hours 9 10 10 10 10 10 10
Mortality % 10 0 0 0 0 0 0
Number of live fish 48 hours 9 10 10 10 10 10 10
Mortality % 10 0 0 0 0 0 0
Number of live fish 72 hours 9 10 10 10 10 10 10
Mortality % 10 0 0 0 0 0 0
Number of live fish 96 hours 9 10 10 10 10 10 10
Mortality % 10 0 0 0 0 0 0

Table 3: Test conditions - preliminary test

Loading rate mg/l 100 60 40 20 10 control t (°C )
O2(mg/l) 0 hour 6.6 6.2 6.2 6.8 6.1 9.6 21.9
pH 8.4 8.3 8.3 8 7.9 7.8
O2(mg/l) 24 hours 7.0 7.6 7.6 7.0 7.0 7.5 22.0
pH 7.7 7.7 7.7 7.6 7.6 7.6
O2(mg/l) 48 hours 6.3 6.6 6.6 6.6 6.7 7.2 22.0
pH 7.5 7.5 7.5 7.5 7.5 7.6
O2(mg/l) 72 hours 6.1 6.2 6.0 6.3 6.2 6.5 21.6
pH 7.5 7.6 7.6 7.6 7.5 7.5
O2(mg/l) 96 hours 7.2 6.8 6.8 7.1 7.1 7.3 22.1
pH 7.5 7.5 7.5 7.5 7.5 7.6

Table 4: Test conditions - limit test

Loading rate mg/l 100 91 83 75 68 62 control t (°C)
O2(mg/l) 0 hour 6.2 6.1 5.5 6.1 6.3 5.4 8.9 22.0
pH 8.1 8.4 8.3 8.2 8.2 8.2 7.9
O2(mg/l) 24 hours 5.8 6.0 5.8 6.0 6.1 6.0 8.6 22.1
pH 7.6 7.7 7.7 7.6 7.6 7.6 7.7
O2(mg/l) 48 hours 6.6 7.1 6.9 6.8 6.4 6.1 7.1 21.8
pH 7.4 7.6 7.5 7.4 7.4 7.5 7.6
O2(mg/l) 72 hours 6.5 6.1 6.2 5.9 5.9 5.7 6.8 21.9
pH 7.4 7.4 7.4 7.4 7.4 7.4 7.4
O2(mg/l) 96 hours 7.0 5.9 5.9 5.7 6.0 5.9 6.3 21.6
pH 7.5 7.4 7.4 7.4 7.4 7.5 7.5
Validity criteria fulfilled:
yes
Remarks:
The evaluation according to point 4 (the test substance concentration in test mixtures should not decrease under 80 % of the initial concentration) is impossible in this study for low solubility of the test material. The study was performed without the an
Conclusions:
The highest observed mortality was 10 % in the limit test. Therefore exact values of LL50 could not be calculated and the values of LL are given in the form of a range. The LL100 and NOELR values are identified by direct observation. The loading rates of the test substance were used for all evaluations and results.
According to the classification criteria given in quoted Directive the test substance, Slimes and Sludges, blast furnace and steelmaking, is not assigned to any of quoted toxicity categories.

LL50 (24 h) > 100 mg/l (loading rate)
LL50 (48 h) > 100 mg/l (loading rate)
LL50 (72 h) > 100 mg/l (loading rate)
LL50 (96 h) > 100 mg/l (loading rate)

NOELR (24 h) = 91 mg/l (loading rate)
NOELR (48 h) = 91 mg/l (loading rate)
NOELR (72 h) = 91 mg/l (loading rate)
NOELR (96 h) = 91 mg/l (loading rate)

LL100 (24 h) > 100 mg/l (loading rate)
LL100 (48 h) > 100 mg/l (loading rate)
LL100 (72 h) > 100 mg/l (loading rate)
LL100 (96 h) > 100 mg/l (loading rate)
Executive summary:

The test substance, Slimes and Sludges, blast furnace and steelmaking, was tested for acute toxicity on fish Poecilia reticulata. The test was performed according to method C.1 - Acute toxicity for fish, Council Regulation (EC) No. 440/2008, published in O.J.L 142, 2008.

Since the test substance is multi-component poorly soluble substance the water-accommodated fractions (WAFs) were prepared by adding measured amounts of the test substance to the dilution water and then were stirred for a period of 72 hours. After period of stirring the WAFs were filtered through 0.45 µm filter and these solutions were used for testing. The WAFs were prepared for preliminary and limit test.

The preliminary test was performed in range of test substance loading rates 10 - 100 mg/l. Based on no toxicity of the test substance found in preliminary test, the limit test was performed. The limit test was performed in range of test substance loading rates 62 - 100 mg/l. The test was performed as static.

The study was performed without the analytical determination of the test substance concentrations. Only the concentrations of metals in the highest loading rate 100 mg/l were analysed in the limit test. For all evaluations and results the loading rates of the test substance were used.

Test results:                                                        

LL50 (24 h) > 100 mg/l    (loading rate)

LL50 (48 h) > 100 mg/l    (loading rate)

LL50 (72 h) > 100 mg/l    (loading rate)

LL50 (96 h) > 100 mg/l    (loading rate)

 

NOELR (24 h) = 91 mg/l   (loading rate)

NOELR (48 h) = 91 mg/l   (loading rate)

NOELR (72 h) = 91 mg/l   (loading rate)

NOELR (96 h) = 91 mg/l   (loading rate)

 

LL100 (24 h) > 100 mg/l  (loading rate)

LL100 (48 h) > 100 mg/l  (loading rate)

LL100 (72 h) > 100 mg/l  (loading rate)

LL100 (96 h) > 100 mg/l  (loading rate)

 

Classification of the test substance on the basis of hazard to aquatic environment was performed according to the Directive 93/21/EEC, Annex IV (article 5.2.1.).

According to the classification criteria given in quoted Directive the test substance, Slimes and Sludges, blast furnace and steelmaking, is not assigned to any of quoted toxicity categories.

Description of key information

The test was performed according to EU Method C.1 (Acute Toxicity for Fish) on Poecilia reticulata species.
The study was performed without the analytical determination of the test substance concentrations. As the test substance is multi-component poorly soluble substance the water-accommodated fractions (WAFs) were prepared for preliminary and full test.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
100 mg/L

Additional information

As the test substance was tested by using the water-accommodated fractions (WAFs) and nominal concentrations (loading rates) were used, LL parameters were used instead of LC values.

LL50 (96 h) > 100 mg/l.