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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 2018-04-30 to 2018-06-14
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:
1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
other: OECD Guidance Document No. 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 20141015
- Analytical purity: 99.35% w/w
- Appearance: clear liquid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature. Keep container tightly closed in a dry and well-ventilated place.
- Stability under test conditions: stable
Analytical monitoring:
yes
Details on sampling:
- Sampling method: Duplicate samples were taken from all test media with surviving fish and the control at the start and end of all test medium renewal periods.
- Sample storage conditions before analysis: Immediately after sampling, all samples were frozen (at -20 ±5 °C). Based on pre-experiments for investigation of the storage stability (IES Study 20170461), the test item was found to be stable in the test water under these storage conditions.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
At the start of the test (Day 0) and at the test medium renewals at Days 1, 2, and 3, the test media were prepared as follows:

First, an emulsion of the test item was prepared by dosing 198 µL of the test item in 2000 mL test water. In this way, the loading rate of the emulsion was 100 mg/L, considering the relative density of the test item of 1.01. The test item was mixed into test water by intensive stirring for three hours in the dark in a closed vessel to dissolve a maximum of test item in test water. The stirring time was based on the stirring pre-experiment (performed under IES Study 20170459) which showed, that the equilibrium between dissolved and undissolved test item was reached after this stirring time.

After stirring, the emulsions were filtered through a 0.45 µm membrane filter (Whatman, NC45). As a pre-caution, the filter was pre-conditioned with 200 mL filtrate to avoid losses of dissolved test item due to adsorption on the filter material. The negative pressure of the filtration unit was reduced to a minimum to avoid losses of the volatile test item by evaporation during the filtration process.

This equilibrated filtrate was diluted with test water to prepare the test media, i.e. the dilutions 1:8, 1:16, 1:32, 1:64 and 1:128.

The preparation of the test media was based on the OECD Guidance Document No. 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: zebrafish
- Source: IES Laboratories
- Length at study initiation (length definition, mean, range and SD): 2.7 ± 0.09 cm
- Weight at study initiation (mean and range, SD): 0.14 ± 0.02 g
- Feeding during test: no

ACCLIMATION
- Acclimation period: at least 1 week after delivery
- Acclimation conditions (same as test or not): yes
- Type and amount of food during acclimation: Until one day before the start of the test, the fish were fed with a commercial fish diet (Tetra Min® Hauptfutter, supplied by TETRA-Werke, 49324 Melle / Germany).
- Feeding frequency during acclimation: daily
- Health during acclimation (any mortality observed): During the last three weeks prior to the test, no fish died in the test fish batch and all fish were healthy.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
1.25 mmol/L or 125 mg/L as CaCO3
Test temperature:
21°C
pH:
7.3 - 7.6
Dissolved oxygen:
7.8 - 8.6 mg/L
Salinity:
not relevant
Conductivity:
not indicated
Nominal and measured concentrations:
Range-finder
Nominal concentrations: Control, 1:100, 1:20 dilutions of a filtrate with a loading rate of 100 mg/L
Measured concentrations: not determined

First main test (limit test):
Nominal concentrations: control, 1:8 dilutions of a filtrate with a loading rate of 100 mg/L (treshold concentration was the EC50 value determined in an algae study (OECD 201) and in an acute daphnia study (OECD 202))
Measured concentrations: not determined

Second main test
Nominal concentrations: Control, 1:128, 1:64, 1:32, 1:16, 1:8 l dilutions of a filtrate with a loading rate of 100 mg/L
Measured concentrations (range at t=0): Measured concentrations (range at t=96): n.a. : not analysed as below the detection limit
Details on test conditions:
TEST SYSTEM
- Test vessel: glass bottle
- Type: closed
- Material, size, headspace, fill volume: One full glass bottle with approximately five liters test medium was used per treatment. As the test item was considered to be volatile a closed exposure system was used, i.e. the test vessels were completely filled with test medium (without headspace) and were tightly sealed with a screw cap to avoid losses of the test item by evaporation.
- Aeration: The test water was aerated prior to the preparation of the test media until oxygen saturation was reached.
- Renewal rate of test solution (frequency/flow rate): test medium renewal every 24 hours
- No. of organisms per vessel: 7 fish per vessel
- Biomass loading rate: 0.2 g fish wet weight per liter medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water was used in the study. It consisted of analytical grade salts dissolved in purified water to obtain the following nominal concentrations:
CaCl2 × 2H2O 1.0 mmol/L (147 mg/L)
MgSO4 × 7H2O 0.25 mmol/L (61.5 mg/L)
NaHCO3 0.38 mmol/L (32.5 mg/L)
KCl 0.038 mmol/L (2.9 mg:L)
The ratio of Ca:Mg and Na:K was 4:1 and 10:1, respectively, based on molarity.

- Intervals of water quality measurement: The water temperature, pH values and oxygen concentrations were measured for each treatment with surviving fish at the start and the end of all test medium renewal periods. At the same dates the appearance of the test media was recorded.

OTHER TEST CONDITIONS
- Photoperiod: A 16 hour light to 8-hour dark photoperiod, with a 30-minute transition period was used
- Light intensity: The light intensity during the light period was approximately 15-16 µE s-1 m-2.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The test fish were observed for mortality and visible abnormalities after approximately 3, 24, 48, 72 and 96 hours test duration.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Test concentrations: 1:8, 1:16, 1:32, 1:64 and 1:128
- Results used to determine the conditions for the definitive study: yes
- Analysis of the test item concentrations: For measurement of the actual concentrations of the test item, duplicate samples were taken from all test media with surviving fish and the control at the start and end of all test medium renewal periods. Immediately after sampling, all samples were frozen (at -20 ±5 °C). Based on pre-experiments for investigation of the storage stability (IES Study 20170461), the test item was found to be stable in the test water under these storage conditions. The concentrations of the test item in the test media and the control were analyzed in one of the duplicate samples from the dilutions 1:8, 1:16, and 1:32 from all sampling times. The samples of the lower test concentrations (dilution 1:64, and 1:128) were not analyzed since these concentrations were below the 96-hour NOEC determined in this test and were therefore not relevant for the interpretation of the biological results.


PRE-EXPERIMENTS FOR SELECTION OF THE STUDY DESIGN OF THE MAIN TEST:
In the first main test, a semi-static limit test in a closed system was performed following the threshold approach as developed for chemical substances at the European Commission’s joint Research Centre. Thus, only one test concentration and a control (test water without test item) were tested. The test concentration corresponded approximately to the lower EC50 value determined in an algae study (OECD 201) and in an acute daphnia study (OECD 202). The threshold concentration was the dilution 1:8 of a filtrate of an equilibrated test item emulsion with a test item loading rate of 100 mg/L. This limit test should demonstrate that the test organism fish is not the most sensitive test organism compared to algae or daphnia. However, a 100 % mortality of the test fish was observed at the threshold concentration at Day 2 of the test.

Therefore a range finding test had to be performed and the main test had to be repeated as a full test with five test concentrations.

For determination of the appropriate test concentrations for the second main test, a range-finding test was performed. The dilutions 1:100 and 1:20 of a filtrate of an equilibrated test item emulsion with a loading rate of 100 mg/L were tested. The test was performed in a closed system. For each treatment, three fish were used. The results of the range finding test were as follows:
- Control: 0 of 3 dead fish (96-hour mortality)
- 1:100 dilution: 0 of 3 dead fish (96-hour mortality); no symptoms observed in the surviving fish
- 1:20 dilution: 2 of 3 dead fish (96-hour mortality); no symptoms observed in the surviving fish

Based on the results of the first main test and on the range finding test the following test concentrations for the second main test were chosen: The dilutions 1:8, 1:16, 1:32, 1:64 and 1:128 of the filtrate of an equilibrated test item emulsion with a loading rate of 100 mg/L. Additionally, a control (test water without test item) was tested in parallel.

As the test item was volatile, a closed exposure system was applied. The test was performed semi-static with daily test medium renewals to keep the test item concentrations as constant as possible during the test period of 96 hours and to keep the oxygen concentration of the test medium sufficiently high in the closed system.

At the start of the test, seven fish were introduced into each test vessel in a random order. The loading rate of the fish was 0.2 g fish wet weight per liter medium. At the renewal dates, the fish of the test item treatments and the control were transferred into new test vessels with freshly prepared test media and test water, respectively.

Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% C.I.: 1.03-2.1 mg/L
Details on results:
- Mortality of control: none
- Other adverse effects control: no
- Abnormal responses: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No remarkable observations were made concerning the appearance of the test media. They were clear solutions throughout the test medium renewal periods of 24 hours.
- 96-h NOEC = 1.03 mg/L
- 96-h LOEC* = 2.1 mg/L
*: Based on visible abnormalities and mortality observed in the fish


Results with reference substance (positive control):
no
Reported statistics and error estimates:
Statistical analysis was performed using ToxRat Professional

Table    Mortality and Visible Abnormalities Observed in the Test Fish

Treatment / Dilution*



[mg/L]

Mean Measured
Test Item Concentration
[mg/L]

Number of Abnormal Fish / Number of Dead Fish

Observation Time [Hours]

3

24

48

72

96

Control

< LOQ

0 / 0

0 / 0

0 / 0

0 / 0

0 / 0

1:128

n.a.

0 / 0

0 / 0

0 / 0

0 / 0

0 / 0

1:64

n.a.

0 / 0

0 / 0

0 / 0

0 / 0

0 / 0

1:32

1.03

0 / 0

0 / 0

0 / 0

0 / 0

0 / 0

1:16

2.1

0 / 0

0 / 0

0 / 1

2 / 5

0 / 7

1:8

4.3

0 / 0

0 / 7

--

--

--

LC50

> 4.3

3.0

2.7

1.8

1.5

95 % C.I.

n.d.

n.d.

1.8–4.2

1.0–3.2

1.03–2.1

*:                  Dilutions of a filtrate of an equilibrated test item emulsion with a loading rate of 100 mg/L.

95% C.I.:    95% confidence interval of the LC50

n.a.:             Not analyzed since below the NOEC determined in this test

n.d.:             Could not be determined

--:                 All fish dead

LOQ:          Limit of Qantification =0.572 mg test item /L

Validity criteria fulfilled:
yes
Remarks:
The test was considered valid as no fish in the control died during the 96-hour test period. The oxygen concentration in the test media and the control did not drop below 60 % of the air saturation during the test.
Conclusions:
The acute toxicity of the test article to zebrafish (Danio rerio) was determined in a 96-hour semi-static test according to OECD guideline 203. The 96-hour LC50 was calculated to be 1.5 mg/L with 95 %-confidence limits of 1.03 and 2.1 mg/L (based on the geometric mean measured concentrations). The results of the test can be considered reliable without restriction.

Description of key information

In the results obtained during the guideline study (Peither, 2019) on the test item the 96-h LC50 of zebrafish (Danio rerio) was calculated to be 1.5 mg/L with 95%-confidence limits of 1.03 and 2.1 mg/L (based on the geometric mean measured concentrations). The results of the test can be considered reliable without restriction. Based on the results it can be considered that the test item is harmful to fish.

Key value for chemical safety assessment

LC50 for freshwater fish:
1.5 mg/L

Additional information