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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:
06 Oct - 05 Dec 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
June 18, 2019
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Samples were taken from all test item concentrations and from the control
- Sampling method: Duplicate samples were taken from the freshly prepared test media at test start. At test end after 96 h the contents of test beakers of each treatment were poured together and duplicate samples were taken from the appr. centre of test aquaria. Samples were taken from the two highest test item concentrations (45.5 and 100 mg/L) after 24 h since all fish were dead at this timepoint.
- Sample storage conditions before analysis: All samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Differential loading:
Defined amounts of the test item were added directly to the test water for each test concentration and were carefully stirred for 24 h in the dark to dissolve as much of the test item as possible. After cessation of mixing and a following period (1 h) of settling to allow phase separation, the aqueous phase, i.e. the water accommodated fraction, was drawn off carefully and used as the test medium of the corresponding nominal test concentration.
The highest loading rate of 100 mg test item/L was prepared by mixing 1100.0 mg test item into 11000 mL test water, for the loading rate of 45.5 mg test item/L, 500.3 mg test item were mixed into 10995.6 mL test water, for the loading rate of 20.7 mg test item/L, 227.6 mg were mixed into 10995.2 mL test water. The loading rate of 9.39 mg test item/L was prepared by mixing 103.3 mg into 11000 mL test water and for the loading rate of 4.27 mg test item/L was prepared by mixing 47.1 mg into 11023.4 mL test water. For the loading rate of 1.94 mg test item/L two stock solutions were prepared by mixing 10.7 mg were mixed into 5500 mL test water.
- Controls: Yes, test water without test item
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow trout
- Source: Forellenzuchtbetrieb Störk, Bad Saulgau, Germany
- Length at study initiation: 4.63 cm ± 0.3 cm (mean ± SD)
- Weight at study initiation: 0.94 g ± 0.1 g (mean ± SD)
- Method of breeding: All fish were obtained and held in the laboratory before the start of the test. They were held in water of the quality to be used in the test for at least seven days immediately before testing.

ACCLIMATION
- Acclimation period: At least 9 days
- Acclimation conditions: same as for the test
- Type and amount of food during acclimation: SKRETTING Pro Aqua Brut 0.7 GR (02140 Fontaine les Vervins, France) three times per week or daily until 24 hours before the test was started
- Feeding frequency during acclimation: Three times per week or daily until 24 hours before the test was started.
- Health during acclimation: During the last 7 days prior to the start of the test no fish died in the test fish batch.

FEEDING DURING TEST : No feed during the test
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
13.2 - 13.7 °C (Control)
12.9 - 13.8 °C (Test item concentrations)
pH:
7.1 - 7.6 (Control)
6.8 - 7.4 (Test item concentrations)
Dissolved oxygen:
93 - 99% (Control)
91 - 98% (Test item concentrations)
Conductivity:
≤ 10 µScm-1
Nominal and measured concentrations:
0 (Control), 1.94, 4.27, 9.39, 20.7, 45.5 and 100 mg/L (nominal loading rate)
For the measured concentrations please see table 2 in section “Any other information on results incl. tables”.
Details on test conditions:
TEST SYSTEM
Test vessel:
- Type: open
- Material, size, headspace, fill volume: 12 litre glass aquaria with 10 litre test medium and 2 L headspace
- Aeration: Test media were slightly aerated during the test
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.731 g fish/L test water

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO Medium (analytical grade salts were added in deionised water)
- Water hardness: 1.78 mmol/L (= 178.4 mg/L) as CaCO3
- Alkalinity: 0.55 mmol/L
- Ca/Mg ratio: 4 : 1 (based on molarity)
- Na/K ratio: 10 : 1 (based on molarity)
- Culture medium different from test medium: No
- Intervals of water quality measurement: Water temperature, pH-values and the dissolved oxygen concentrations were determined daily in the test media of each treatment group.

OTHER TEST CONDITIONS
- Photoperiod: 12 h light : 12 h dark; 30 min dawn/dusk period was provided
- Light intensity: 730 to 820 lux

EFFECT PARAMETERS MEASURED: The test fish were observed at test start and after approximately 2, 5, 24, 28, 48, 51.5, 70, 75 and 96 h test duration for sublethal effects and mortality.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
Range finding study:
- Test concentrations: 0 (Control) and 15.9 mg/L
- Results used to determine the conditions for the definitive study: The results of the limit test were considered to be inappropriate since the analytical recovery was low.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: Behavioural abnormalities as loss of equilibrium and abnormal skin pigmentation were observed only up to an exposure period of 48 h. At the loading rate of nominal 20.7 mg test item/L six fish were dead after 96 h and the surviving fish showed loss of equilibrium and abnormal skin pigmentation. At the loading rates of nominal 45.5 and 100 mg test item/L all fish were dead after 96 h.
- Other biological observations: At the loading rate of nominal 9.39 mg test item/L, all fish survived until the end of the experiment.
- Mortality of control: No mortality was observed in the control
Reported statistics and error estimates:
The LC50, LC20 and LC10 at the observation times were calculated by Probit analysis. The NOEC and LOEC after 96 h were calculated by Step-down Cochran Armitage test procedure, whereas the LC0 and the LC100 were determined directly from the raw data. The software used to perform the statistical analysis was ToxRat Professional, Version 3.3.0, ToxRat Solutions GmbH.

Table 1: Observed Mortality of unfed Rainbow Trout (Oncorhynchus mykiss) exposed to test item for 96 h

Nominal Loading Rate

Mortality

mg test item/L

0 h

2 h

24 h

48 h

72 h

96 h

# mort

# mort

# mort

# mort

# mort

# mort

Control

0

0

0

0

0

0

1.94

0

0

0

0

0

0

4.27

0

0

0

0

0

0

9.39

0

0

0

0

0

0

20.7

0

0

1

4

6

6

45.5

0

0

7

7

7

7

100

0

0

7

7

7

7

EL50 [mg test item/L]

n.d.

n.d.

23.6

20.1

18.0

18.0

95% CI.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

# mort: Cumulative number of dead fish

n.d.: not determinable

Values refer to nominal loading rates

Table 2 Summary of Analytical Results

Sample Description

% of nominal 1

[mg test item/L]

fresh (0 h) [%]

Aged [%]

Control

n.a.

n.a.

1.94 mg/L Loading Rate

71

27 (96 h)

4.27

92

37 (96 h)

9.39

82

35 (96 h)

20.7

57

35 (96 h)

45.5

47

44 (24h)

100

23

22 (24 h)

1 mean value of all measured samples per treatment group; RSD: relative standard deviation per treatment group; n: number of analysed samples; n.a.: not applicable;

Analytical Results:

The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to loading rate because the test was dosed via the water accommodated fraction (WAF) approach.

Validity criteria

Criterion from the guideline

Outcome

Validity criterion fulfilled

The mortality in the control(s) should not exceed 10% (or one fish if less than ten are used) at the end of the test

No mortality

Yes

The dissolved oxygen concentration must have been at least 60 per cent of the air

saturation value throughout the test

≥ 91%

Yes

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.

Description of key information

LL50 (96 h): 18 mg/L (nominal, OECD 203, O. mykiss)

Key value for chemical safety assessment

Additional information

There is one experimental study available, in which the toxicity of the test substance (EC 944-336-4) to fish was assessed according to OECD guideline 203 and GLP.In a static test, Oncorhynchus mykiss was exposed to nominal loading rates of 1.94, 4.27, 9.39, 20.7, 45.5 and 100 mg test item/L for 96 h. Since the substance is a UVCB consisting of poorly soluble compounds, the test item was applied as water accommodated fraction (WAF). The actual test item concentrations were analytically verified via quantification of one component of the UVCB for fresh and aged samples by HPLC- UV detection. The initial measured concentration was between 23 and 92% of nominal for each test item concentration. Based on the quantified UVCB component the test concentrations were found to decrease over the course of the 96 h test in the test concentrations. The measured test concentration in the aged samples ranged from 22 to 44% of nominal. The test concentrations 100 mg/L and 45.5 mg/ L were analysed after 24 h because all fish were dead. Both test concentrations remained stable over the 24 hour test period. Based on the test results the 96-hour LL50 was determined to be 18.0 mg test item/L based on nominal loading rates. The NOEL was determined to be 9.39 mg test item/L also based on nominal loading rates.