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Ecotoxicological information

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:
2019-08-06 to 2019-09-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The study was conducted for the registration in a non-EU country.
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Qualifier:
according to
Guideline:
other: OECD No 23; Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Duplicate samples from the freshly prepared test media of the only test concentration and the control were taken at the start of the test. For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of the only test concentration and the control were collected at the end of the test (after 96 hours) from the approximate centre of the aquaria. All samples remained undiluted until sample preparation which was performed directly after sampling.
All samples were prepared stand-by immediately after sampling by liquid-liquid extraction with the organic solvent Cyclohexane. An aliquot of the resulting organic solvent extracts of the samples taken at test start and test end were stored in a refrigerator (4 ± 4°C) until analysis which was performed.
Details on test solutions:
The test item is not well soluble in test water. To avoid physical effects of undissolved test item on the daphnids, no concentration above the solubility limit of the test item in test water was tested. Therefore, a supersaturated stock solution of nominal 100 mg test item/L was prepared by suspending 1000.98 mg test item in 10000 mL test water. The stock solution was stirred for 23.5 hours in the dark to dissolve as much test item as possible. Then, after a settling time of 1 h, the undissolved test item was separated by filtration (0.45 µm cellulose acetate filter) and the filtrate was used as test medium.
The test media was prepared just before introduction of the test fish (= start of the test).
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
Species: Rainbow trout (Oncorhynchus mykiss)
Size and Weight: Juveniles; The mean body length of the fish* in the test was 4.32 cm ± 0.2 cm (Mean ± SD), the mean body wet weight 0.94 g ± 0.2 g (Mean ± SD).
* 10 fish from the test fish batch were measured before the start of the test
Origin: The test fish were obtained from Forellenzuchtbetrieb Störk, 88348 Bad Saulgau, Germany Holding Conditions and Acclimatisation:
All fish were obtained and held in the laboratory for at least 12 days 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 under the following conditions:
Light: 12 hours photoperiod daily
Temperature: 10 - 14 °C
Oxygen concentration: at least 80 % of the air saturation value
Feeding: three times per week or daily until 24 hours before the test was started
During the last 7 days prior to the start of the test no fish died in the test fish batch. Therefore the mortalities in the fish batch were below 5 % and the fish batch was accepted.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
no
Hardness:
178.3 mg/L as CaCO3
Test temperature:
12.9 to 13.4 °C
pH:
7.7 to 7.9
Dissolved oxygen:
95 to 99 %
Nominal and measured concentrations:
Due to the low water solubility limit of the test item a filtrate of a supersaturated stock suspension of nominal 100 mg/L and a control were tested corresponding to 0.125 µg test item/L (LOQ/2 value).
Details on test conditions:
Test Environment: Controlled environmental room
Measurement of pH-Values, Dissolved Oxygen and Water Temperature:
The water temperature, pH-values and the dissolved oxygen concentrations were determined daily in the test media of each treatment group.
Water Temperature: 12.9 to 13.4 °C
pH-Values of the Test Media: 7.7 to 7.9
Dissolved Oxygen Concentrations: 95 to 99 % of the air saturation value
Light Regime: 12 h light : 12 h dark; 30 min dawn/dusk period was provided
Light Intensity: 540 to 690 lux Aeration of the Test Media: The test media were slightly aerated during the test.
Recording: Test conditions were recorded with suitable instruments and documented in the raw data.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: The NOEC was determined to be ≥ filtrate of 100 mg test item/L based on nominal concentration (≥ 0.125 µg test item/L based on the arithmetic mean measured concentration). 100 mg test item/L is the nominal loading rate.
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: The 96-hour LC50 was determined to be > filtrate of 100 mg test item/L based on nominal concentration (>0.125 µg test item/L based on the arithmetic mean measured concentration). 100 mg test item/L is the nominal loading rate.
Details on results:
In the control and the only test concentration of the filtrate of 100 mg test item/L, all fish survived until the end of the experiment and showed no sublethal effects during the exposure time.
Reported statistics and error estimates:
No statistical analysis was performed.
The LC50 could not be quantified due to the absence of toxicity of the test item. The NOEC were determined directly from the raw data.
Validity criteria fulfilled:
yes
Remarks:
In the control no fish died during the test. The dissolved oxygen concentration in the test media did not fall below 95 % of air saturation value during the test.
Conclusions:
The toxic effect of the test item to Rainbow Trout (Oncorhynchus mykiss) was assessed in a static limit test. Based on the test results the 96-hour LC50 was determined to be > filtrate of 100 mg test item/L based on nominal concentration (>0.125 µg test item/L based on the arithmetic mean measured concentration). The NOEC was determined to be ≥ filtrate of 100 mg test item/L based on nominal concentration (≥ 0.125 µg test item/L based on the arithmetic mean measured concentration). The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to arithmetic mean measured concentration of the test item since a filtrate was tested as the only test concentration. In addition, the reported results are given based on nominal concentration (loading rate).
This study is classified acceptable and satisfies the guideline requirements for fish acute test studies.
Executive summary:

The purpose of this study was to evaluate the acute toxicity of the test item to fish. For this purpose, juvenile Rainbow Trout were exposed in a static test to the highest concentration which can be dissolved in test water under defined conditions for 96 hours. The recorded effects were the mortality and sublethal effects on the fish. This limit-test was performed in compliance with the test guidelines in order to demonstrate that the test item has no toxic effects on the fish up to this concentration.

The test method of application and the test system are recommended by the test guidelines and Rainbow Trout is one of the recommended test species.

The purpose of the analytical part of this study was to verify the concentration of the test item in the test medium.

This study encompassed two treatment groups (one test item concentration of the filtrate of nominal 100 mg/L and one control) each containing 7 individuals. The acute toxicity to unfed juvenile Rainbow Trout was determined in an aerated, static, 96-hour test. The test fish were observed at test start and after approximately 2, 24, 48, 72 and 96 hours test duration for sublethal effects and mortality.

Due to the low water solubility limit of the test item a filtrate of a supersaturated stock suspension of nominal 100 mg/L and a control were tested corresponding to 0.125 µg test item/L (LOQ/2 value).

Biological test results:

In the control and the only test concentration of the filtrate of 100 mg test item/L, all fish survived until the end of the experiment and showed no sublethal effects during the exposure time.

The 96-hour LC50was determined to be > filtrate of 100 mg test item/L based on nominal concentration (>0.125 µg test item/L based on the arithmetic mean measured concentration). The NOEC was determined to be ≥ filtrate of 100 mg test item/L based on nominal concentration (≥ 0.125 µg test item/L based on the arithmetic mean measured concentration).

Analytical test results:

The quantification of the test item in the test samples was performed using liquid-liquid extraction with Cyclohexane followed by analysis via Gas Chromatography with MS/MS detection. The concentrations of the test item were determined in the duplicate test media samples from the only test concentration (test item stock solution) and the duplicate control samples from all sampling times (0 and 96h hours). The arithmetic mean measured values are:

<LOQ corresponding to 0.125 µg test item/L using LOQ/2 value for calculation (according to OECD23 guidance document specifications) in the filtrate of nominal 100 mg test item/L

The toxic effect of the test item to Rainbow Trout (Oncorhynchus mykiss) was assessed in a static limit test. Based on the test results the 96-hour LC50 was determined to be > filtrate of 100 mg test item/L based on nominal concentration (>0.125 µg test item/L based on the arithmetic mean measured concentration). The NOEC was determined to be ≥ filtrate of 100 mg test item/L based on nominal concentration (≥ 0.125 µg test item/L based on the arithmetic mean measured concentration). The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to arithmetic mean measured concentration of the test item since a filtrate was tested as the only test concentration. In addition, the reported results are given based on nominal concentration (loading rate).

This study is classified acceptable and satisfies the guideline requirements for fish acute test studies.

Description of key information

In the control and the only test concentration of the filtrate of 100 mg test item/L, all fish survived until the end of the experiment and showed no sublethal effects during the exposure time.

GLP OECD 203: LC50 > 100 mg/L (nominal), corresponding to the arithmetic mean measured concentration of >0.125 µg test item/L

Key value for chemical safety assessment

Additional information

The purpose of this study was to evaluate the acute toxicity of the test item to fish. For this purpose, juvenile Rainbow Trout were exposed in a static test to the highest concentration which can be dissolved in test water under defined conditions for 96 hours. The recorded effects were the mortality and sublethal effects on the fish. This limit-test was performed in compliance with the test guidelines in order to demonstrate that the test item has no toxic effects on the fish up to this concentration.

The test method of application and the test system are recommended by the test guidelines and Rainbow Trout is one of the recommended test species.

The purpose of the analytical part of this study was to verify the concentration of the test item in the test medium.

This study encompassed two treatment groups (one test item concentration of the filtrate of nominal 100 mg/L and one control) each containing 7 individuals. The acute toxicity to unfed juvenile Rainbow Trout was determined in an aerated, static, 96-hour test. The test fish were observed at test start and after approximately 2, 24, 48, 72 and 96 hours test duration for sublethal effects and mortality.

Due to the low water solubility limit of the test item a filtrate of a supersaturated stock suspension of nominal 100 mg/L and a control were tested corresponding to 0.125 µg test item/L (LOQ/2 value)

The toxic effect of the test item to Rainbow Trout (Oncorhynchus mykiss) was assessed in a static limit test. Based on the test results the 96-hour LC50 was determined to be > filtrate of 100 mg test item/L based on nominal concentration (>0.125 µg test item/L based on the arithmetic mean measured concentration). The NOEC was determined to be ≥ filtrate of 100 mg test item/L based on nominal concentration (≥ 0.125 µg test item/L based on the arithmetic mean measured concentration). The initial concentrations and the maintenance of the exposure concentrations during the test were verified in the analytical part. All reported results refer to arithmetic mean measured concentration of the test item since a filtrate was tested as the only test concentration. In addition, the reported results are given based on nominal concentration (loading rate).

This study is classified acceptable and satisfies the guideline requirements for fish acute test studies.