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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:
From September 3rd to November 16th, 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
July 1992
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Representative samples for analytical determination of the concentration from the first and the third test interval were taken at start and end of use of the test solution: 30 min before insertion of the test organisms (at start of exposure), after 24 h (old test solution), after 48 h (new test solution) and after 72 h (old test solution).
Water samples were taken from the middle of the test vessel using a glass pipette and were transported to the analytical laboratory in glass ampoules, which were rinsed with test solution before they were filled.
The transport to the analytical laboratory was done on the day of sampling.
Vehicle:
no
Details on test solutions:
The test concentrations were prepared separately for each test vessel. Fresh test solutions were made on each day and the fish were transferred to new test solutions in 24 hour intervals. The test substance was weighed into a beaker. The defined volume of test water was filled into the test vessel. An aliquot from the test water in the test vessel was taken and mixed with the test substance using an ultra turrax and the mixture was poured into the test vessel and the whole test batch was homogenized again with an ultra turrax.The test solutions were blue and clear. In the higher concentrations, foam could be observed on the water surface.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Source: Zierfisch-Center Klöckner, Ludwigshafen, Germany
- Age at study initiation: approx. 6 months
- Body length: from the top of the snout to the end of the caudal fin, 4.2 cm (4.0 - 4.3 cm)
- Body weight: 0.74 g (0.58 - 0.90 g), wet weight.
- Method of breeding: all animals used for the study were derived from the same batch of fish.
- Diet: commercial fish diet "Tetramin", supplier Tetra-Werke, Melle, Germany, ad libitum, additionally generally on workdays live brine shrimp (artemia).
- Withdrawal of feed: during the last day before start of exposure.
- Feeding during test: none.
- Health check: all test animals were observed for their health state during insertion into the test vessels. No signs for sickness, injuries or abnormalities were observed.
- Hatching date: 03 May 2007
- Arrival at the testing facility: 13 Aug 2007

ACCLIMATION
- Acclimation period: acclimatization to testing conditions including test water and light regime for 14 days.
- Photoperiod: 16 hours light, 8 hours dark.
- Water quality: non chlorinated charcoal filtered tap water (Frankenthal, Germany), mixed with deionized water.
Total hardness: approx. 1 mmol/l = 100 mg/l CaCO3
Acid capacity: approx. 2.5 mmol/l
Oxygen content greater than 80 % saturation
pH value: generally 7.5 – 8.5
Temperature: approx. 23 °C
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
approx. 1 mmol/l = 100 mg/l CaCO3
Test temperature:
23 ± 1 °C
pH:
7.8 – 8.2
Dissolved oxygen:
8.1 - 8.6 mg/l; the concentration of dissolved oxygen were maintained above 60 % of the maximum saturation.
Nominal and measured concentrations:
Nominal concentrations: 0, 5.0, 10, 22, 50 and 100 mg/l
Details on test conditions:
TEST SYSTEM
- Test vessel: glass aquaria (30 cm long, 22 cm wide, 24 cm high).
- Material, size, headspace, fill volume: 10 litres.
- Aeration: slight.
- Renewal rate of test solution: water renewal was done every 24 hours because the test substance precipitates in the presence of impurities in water.
- No. of organisms per vessel: 7
- Loading: 0.5 g fish/l water.
- No. of vessels per concentration: one replicate.
- No. of vessels per control: one replicate.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: non chlorinated charcoal filtered tap water (Frankenthal, Germany) mixed with deionized water, aerated. contaminants.
- Ca content: approx. 40 mg/l
- Mg content: approx. 5 mg/l
- Acid capacity: approx. 2.5 mmol/l
- Conductivity: approx. 250 μS/cm (at 25 °C)

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light, 8 hours darkness.
- Light intensity: approx. 82 - 293 Lux; the light intensity is determined in regular intervals at the surface of the aquaria.

EFFECT PARAMETERS MEASURED
The fish were observed for survival and toxic signs (changes appearance, swimming behaviour and behaviour in comparison to the control group) within 1 h after start of exposure and 6, 24, 48, 72 and 96 h after start of exposure. For this, the fish were directed towards the front pane with a net, since the observation was disturbed by the coloration of the test solution. Fish were considered dead if there was no visible movement and no reaction after touching. Dead fish were removed from the test vessels.

TEST CONCENTRATIONS
The test concentrations were selected on the basis of a range finding part (experimental conduct in accordance with GLP without a GLP status) of the study. LC50 range derived from 96 hour range finding: 10 - 100 mg/l.
The results from experiments 1 and 2 revealed inconclusive results with toxicity at concentrations equal/higher than 10 mg/l, which was, however, very likely not caused by the test substance only.
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
18.2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
MORTALITY AND SYMPTOMS
No symptoms were observed during the entire exposure period in the control and in surviving fish in all test groups. The No Observed Effect Concentration at 96 hours was stated at 8.2 mg/l (meas. arithm. mean).

ANALYTICAL CONCENTRATION
The analytically determined concentrations of the test substance in the freshly prepared test solutions were in the range of 77 – 89 % of the nominal concentration and in the 24 h old test solutions in the range of 65 – 90 % of the nominal concentration (in both cases no background correction was considered). However, all measured values were within a range of ± 20 % of the mean measured concentration, indicating that the test concentrations were kept sufficiently constant over the exposure period. In conclusion, the test results could be based on the mean analytically determined concentrations.
A slight adsorption was measured also in the control group. This is considered to be caused by components released to the test water by the fish rather than from any contamination by the test substance in the control group. Therefore a background correction was carried out for the mean values. A background correction for the individual determinations was considered to be not appropriate, since the variability between the measurements was relatively high.
Sublethal observations / clinical signs:

MORTALITY

Nominal conc.
(mg/l)
Number of fish 1 h 6 h 24 h* 48 h* 72 h* 96 h
0.0 7 0 0 0 0 0 0
5.0 7 0 0 0 0 0 0
10.0 7 0 0 0 0 0 0
22.0 7 0 0 0 0 3 3
50.0 7 0 0 0 0 7 7
100.0 7 0 0 7 7 7 7

* old test solution

ANALYTICAL CONCENTRATION

Dissolution behaviour

Dissolution behaviour was monitored in the control for 96 h in old and new test solutions. In each test group, dissolution behaviour was monitored up to the time point where some fish remained alive. In the control, no remarkable observations were made. The test medium was clear. At 5.0 mg/l, blue colouration caused by the test substance was observed. At higher concentrations (10, 22, 50 and 100 mg/l), blue colouration caused by the test substance and/or signs of inhomogeneous dispersion of the test articles i.e. suspended particles, was observed.

The following observations were recorded for the dissolution behaviour of the test substance in the test water

nominal concentration (mg/l) 1 h 24 hrs* 24 hrs** 48 hrs* 48 hrs** 72 hrs* 72 hrs** 96 hrs
0 0 0 0 0 0 0 0 0
5 3 3 3 3 3 3 3 3
10 3, 7 3 3 3 3 3 3 3
22 3, 7 3 3, 7 3 3 3 3, 7 3, 7
50 3, 7 3, 7 3, 7 3, 7 3, 7 3, 7 - -
100 3, 7 3, 7 - - - - - -

* old test solution ** new test solution

Explanation of abreviations:

0: no remarkable observations, clear test medium

1: homogeneous dispersion of the test substance in water, turbidity

2: strong turbidity caused by the test substance

3: colouration caused by the test substance (blue)

4: inhomogeneous dispersion of the test article, suspended particles

5: precipitation, undissolved test substance at the bottom of the test vessel

6: undissolved test substance on the water surface

7: foam on the water surface

-: not determined

Test concentrations

Nominal conc. (mg/l) Mean over all measurements
(mg/l)
Mean values after background correction:
-0.34 mg/l
0.0 0.34 0
5.0 4.08 3.74
10.0 8.53 8.19
22.0 18.20 17.86
50.0 38.13 37.79
100.0 81.14 80.80

Nominal conc. (mg/l)

0.0 5.0 10.0 22.0 50.0 100.0

At start (0h), fresh test solution

330/331 nm 0.09 4.33 8.93 18.81 38.47 88.13
602-/603 nm 0.37 4.48 8.89 18.53 38.41 87.11
Mean 0.23 4.41 8.91 18.67 38.44 87.62
% of nominal - 88 89 85 77 88

At the end of interval, 24 h old test solution

330/331 nm 0.02 3.12 9.1 16.88 36.52 75.05
602-/603 nm 0.32 3.40 8.8 16.8 36.31 74.25
Mean 0.17 3.26 8.95 16.84 36.42 74.65
% of nominal - 65 90 77 73 75

At the start of interval (48 h), fresh test solution

330/331 nm 0.48 4.28 8.46 19.16 38.37 -
602-/603 nm 0.53 4.47 8.42 19.41 39.69 -
Mean 0.51 4.48 8.44 19.29 39.03 -
% of nominal - 88 84 88 78 -

At the end of interval (72h), 24 h old test solution

330/331 nm 0.41 4.26 7.83 18.31 38.9 -
602-/603 nm 0.50 4.26 7.77 17.66 38.38 -
Mean 0.46 4.26 7.8 17.99 38.64 -
% of nominal - 85 78 82 77 -

-: not determined

Validity criteria fulfilled:
no
Remarks:
the mortality in the control does not exceed 10 % and the dissolved oxygen concentration had been at least 60 %. Nevertheless, the concentration of the substance tested was in the rage of 65 - 90 % of the nominal
Conclusions:
LC50 (96 h): 18.2 mg/l (meas. arithm. mean)
Executive summary:

The acute toxic effect of the test substance to Danio rerio was assessed over 96 h according to OECD guideline 203. The test was carried out in a semistatic system with water renewal was done every 24 hours because the test substance precipitates in the presence of impurities in water, which can lead to a decrease in the analytically determined concentrations over the exposure period.

Each test concentration was prepared separately. Nominal test concentrations were 5.0, 10, 22, 50 and 100 mg/l. The test solutions were blue and clear. In the higher concentrations foam could be observed on the water surface. The analytically determined concentrations of the test substance in the freshly prepared test solutions were in the range of 77 – 89 % of the nominal concentration and in the 24 h old test solutions in the range of 65 – 90 % of the nominal concentration (in both cases no background correction was considered). However, all measured values were within a range of ± 20 % of the mean measured concentration, indicating that the test concentrations were kept sufficiently constant over the exposure period. In conclusion, the test results could be based on the mean analytically determined concentrations.

No symptoms were observed during the entire exposure period in the control and in surviving fish in all test groups. No mortality was observed in control fish. The No Observed Effect Concentration at 96 hours was stated at 8.2 mg/l (meas. arithm. mean); the LC50 was recorded at 18.2 mg/l (meas. arithm. mean).

Conclusion

LC50 (96 h): 18.2 mg/l (meas. arithm. mean)

Description of key information

LC50 (96 h): 18.2 mg/l (meas. arithm. mean)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
18.2 mg/L

Additional information

The acute toxic effect of the test substance to Danio rerio was assessed over 96 h, according to OECD guideline 203. The test was carried out in a semistatic system with water renewal was done every 24 hours because the test substance precipitates in the presence of impurities in water, which can lead to a decrease in the analytically determined concentrations over the exposure period. Nominal test concentrations were 5.0, 10, 22, 50 and 100 mg/l. In the higher concentrations foam could be observed on the water surface.

The analytically determined concentrations of the test substance in the freshly prepared test solutions were in the range of 77 – 89 % of the nominal concentration and in the 24 h old test solutions in the range of 65 – 90 % of the nominal concentration (in both cases no background correction was considered). No symptoms were observed during the entire exposure period in the control and in surviving fish in all test groups. No mortality was observed in control fish. The No Observed Effect Concentration at 96 hours was stated at 8.2 mg/l (meas. arithm. mean); the LC50 was recorded at 18.2 mg/l (meas. arithm. mean).