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Long-term toxicity to fish

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Reference
Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Remarks:
This information is submitted based on ECHA decision number TPE-D-2114343940-51-01.
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The RSS is based on the draft report.
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Version / remarks:
July 2013
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance: stable for up to 72 hours in test water
Analytical monitoring:
yes
Details on sampling:
Duplicate samples were taken from all test concentrations at the start and the end of seven test medium renewal periods distributed over the test period. Four renewal periods of two days and three renewal periods of three days were sampled.
From the control, samples were taken at the start and the end of five renewal periods.
At the start of the renewal periods, the samples were taken in the test medium preparation beakers before distributing the test media to the replicate test vessels. At the end of the renewal periods, the samples were taken alternately from the four different replicates of the respective test concentration.
All samples were frozen (at about –20 °C) immediately after sampling and stored until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Although the water solubility of the test material is 1.85 g/L some precipitation was observed in test water. Thus, at the start of the test and before each test medium renewal a preparation with nominal 10 mg test material/L was prepared. For this adequate amounts of the test item were dispersed in test water by ultrasonic treatment for 15 minutes and intensive stirring for 3 hours. Depending on the different volumes of test media needed during the different periods of the test, 30, 120, and 500 mg of the test item were weighed into 3, 12, and 50 L of test water (effective ranges: 29.9–30.2 mg; 119.9–120.5 mg and 499.9–500.3 mg). After stirring, the suspension of the test item was filtered through a membrane filter (Whatman, Type NC45, pore size 0.45 µm). As a precaution, the filter was pre-conditioned with 200 mL filtrate to avoid possible losses of dissolved test item due to adsorption on the filter material.
The undiluted filtrate was used as highest test concentration and was diluted with adequate volumes of test water for preparation of the test media with the lower test concentrations i.e. nominal 3.2, 1.0, 0.32, and 0.10 mg/L (dilutions 1:3.2, 1:10, 1:32, and 1:100)
- Controls: test water only
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: zebrafish
- Source: The origin of the strain of zebrafish is IME, Fraunhofer Institute, 57392 Schmallenberg, Germany. A brood batch of this strain is held at the testing laboratory

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Method of collection of fertilised eggs: Glass dishes covered with a stainless steel mesh were placed on the bottom of the aquarium one day prior to test start (Day –1) for collecting the newly fertilised eggs for the test. The mesh separated fertilised eggs (which sank to the bottom into the glass dishes) from the parent fish. Spawning started in the morning on Day 0 (start of the test = introduction of the eggs into the test media) after turning on the light. The eggs were collected from the glass dishes and were randomly exposed to the test media.

POST-HATCH FEEDING
- Start date: Day 5 (end of the hatching period, i.e. all larvae hatched in the control)
- Type/source of feed: see below
- Amount given: ad libitum while minimizing the surplus
- Frequency of feeding: at least three times each working day. On weekends, food was given twice per day. Fish were not fed 24 hours prior to the end of the test to allow clearance of the digestive tract before weighing the fish.

The following quality of food was provided:
Hatching period: No food for embryos and newly hatched larvae
From Day 5 to Day 20: Living ciliates (raised at IES Laboratories)
Starting from Day 10: Additionally freshly hatched larvae of Artemia salina (raised at IES Laboratories)
Starting from Day 16: Additionally commercial fish dry-food (TETRA-Werke, 49324 Melle / Germany)

The test vessels were replaced by clean ones on each test medium renewal. Dead eggs, dead larvae or fish were removed at the day of observation. Faeces and surplus food were removed regularly from the test vessels.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
35 d
Remarks on exposure duration:
Day 0 to Day 5: hatching period; Day 5 to Day 35: post hatch period (Day 5 post fertilization equals Day 0 post hatch).
Hardness:
1.25 mmol/L (125 mg CaCO3/L)
Test temperature:
26.0-26.8°C
pH:
7.1-7.8
Dissolved oxygen:
≥ 5.1 mg/L (oxygen saturation ≥ 63%)
Nominal and measured concentrations:
See "Attached background material" - Table 1 Test Material Concentrations.pdf.
As the test item was stable during the test medium renewal periods of 48 and 72 hours, the mean measured test item concentrations over the test period were calculated for each test concentration as the arithmetic mean over all measurements in the freshly prepared and aged test media of the seven analyzed renewal periods. For the aged test media, the analytical measurements from samples with food were taken into account.
Details on test conditions:
TEST SYSTEM
- Emybro cups (if used, type/material, size, fill volume): not used
- Test vessel: Glass test vessels were used. With increasing fish size, the volumes of the test vessels were gradually increased as follows: Day 0-12: 400 mL glass dishes (diameter 11 cm); Day 12-24: Glass beakers with approximately 2 liter test medium volume; Day 24-35: 10 liter full-glass aquaria with 7 liter test medium volume. The test vessels were placed into a temperature regulated water bath to keep the water temperature constant during the test period.
- Type (delete if not applicable): open
- Aeration: slight aeration during the test
- Renewal rate of test solution (frequency/flow rate): three times per week (every two and three days)
- No. of fertilized eggs/embryos per vessel: 20
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- Biomass loading rate: maximum 0.12 g fish wet weight/liter (calculated at the end of the test period, based on the volume of the 7-liter aquaria).

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water as described in OECD Guideline for Testing of Chemicals, No. 203, "Fish, Acute Toxicity Test" (July 17, 1992), however at half of the concentration:
1.0 mM CaCl2 × 2H2O (147 mg/L)
0.25 mM MgSO4 × 7H2O (61.5 mg/L)
0.38 mM NaHCO3 (32.5 mg/L)
0.038 mM KCl (2.9 mg/L)
Alkalinity 0.4 mM
- 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:
Oxygen concentration and pH were measured in all test concentrations and in the control at the start of the test and at least two times per week thereafter in the freshly prepared and aged test media.
The water temperature was measured at the start and at the test medium renewals in all replicates of each treatment in the freshly prepared, temperature equilibrated media before introduction/transfer of the fish. Additionally, the water temperature was monitored continuously by a data logger in the control.
The appearance of the test media was checked each working day in the freshly prepared and aged test media. Observations were recorded at least once a week.
OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hour light to 8-hour dark with a 30-minute transition period
- Light intensity: between 5 and 20 µE s-1 m-2

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Egg development and hatching rate:
The embryonic development and hatching of larvae were recorded daily for all replicate test vessels. Eggs showing a change in colour (white non-transparent colour caused by coagulation and/or precipitation of protein) and embryos, which showed no body movement, were considered dead. Dead eggs and embryos were removed from the test vessels.

Development and survival of larvae and juvenile fish:
The survival of the larvae and juvenile fish was observed each day. Mortality criterion for larvae and juvenile fish was lack of reaction to mechanical stimulus.
The larvae and juvenile fish were also observed for visible abnormalities as abnormal appearance (body form) and abnormal behaviour (e.g. uncoordinated swimming or atypical quiescence). Eggs, larvae and fish, which were missing at the observation dates, were considered dead. Observations were recorded at least 3 times per week or at the date of the finding.

Fish length and fish weight:
At the end of the test the fish length (total length from the mouth to the end of the tail fin) and the body wet weight were determined for each individual fish. The dry weight of the fish (drying for 24 hours at 60 °C) was determined from the sum of all fish per replicate, since the fish were very light after drying and individual weight measurement was not feasible.

RANGE-FINDING STUDY
14-day semi-static range finder
- Test concentrations: nominal 10, 3.2, 1.0, and 0.32 mg/L
- Results used to determine the conditions for the definitive study: reduced growth at nominal 3.2 and 10 mg/L.
Reference substance (positive control):
not required
Key result
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
0.33 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
length
Key result
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
0.33 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Remarks:
wet and dry weight
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
9.9 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
number hatched
Duration:
35 d
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
survival of juvenile fish
Duration:
35 d
Dose descriptor:
LC10
Effect conc.:
> 9.9 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
number hatched
Remarks on result:
other: 95% confidence interval: not determinable
Duration:
35 d
Dose descriptor:
LC10
Effect conc.:
8.9 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
survival of juvenile fish
Remarks on result:
other: 95% confidence interval: 5.8-14.3
Duration:
35 d
Dose descriptor:
EC10
Effect conc.:
2.3 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
length
Remarks on result:
other: 95% confidence interval: 1.3-3.1
Duration:
35 d
Dose descriptor:
EC10
Effect conc.:
0.9 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Remarks:
wet weight
Remarks on result:
other: 95% confidence interval: 0.56-1.2
Duration:
35 d
Dose descriptor:
EC10
Effect conc.:
1.3 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Remarks:
dry weight
Remarks on result:
other: 95% confidence interval: 0.79-1.8
Details on results:
see attachment "Tables_fish early life stage.pdf".
Reported statistics and error estimates:
The parameters assessed were evaluated for significant differences to the control. Statistical analysis was performed using ToxRat Professional®, Version 3.2.1. The following statistical tests were applied:
The mortality of fish was tested for significant differences to the control by a Fisher’s Exact Binominal Test (one-sided greater, α = 0.05).
The fish body length, fish wet weight and fish dry weight were compared to the control by a Williams t-test, one-sided smaller, α = 0.05.
The 21-days LC10 value for mortality and the 95% confidence interval were calculated by Logit Analysis.
The 21-days LC10 value for body length, wet weight and dry weight and their 95% confidence interval were calculated by Probit Analysis.
For the hatching rate the mean values calculated for the test concentrations did not indicate an adverse effect (all values equal or higher than the control values) and therefore no statistical evaluation of the data was performed.
Validity criteria fulfilled:
yes
Remarks:
Hatching success in the controls was >70% and post-hatch success was >75%; the water temperature did not differ by more than ±1.5 °C between the aquaria or between successive days at any one time during the test; oxygen concentration was >60% ASV.

Description of key information

overall 35-d NOEC 0.33 mg/L for Danio rerio (OECD TG 210)

Key value for chemical safety assessment

EC10, LC10 or NOEC for freshwater fish:
0.33 mg/L

Additional information