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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 2012-03-22 to 2012-03-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
Samples were collected from one test chamber of each of the 37 and 73 ng/L treatment groups and the control group one day prior to the start of the test after conditioning the diluter for approximately 2 days. Samples were also collected from alternating replicate test chambers of the 37 and 73 ng/L treatment groups and the control group at the start of the test and after 48 and 96 hours to measure concentrations of the test substance.

The samples were collected, placed in glass volumetric flasks, and processed immediately for analysis. Samples of all stock solutions were also collected prior to the start of the test and at the end of the test to confirm delivery of the test substance to the test system.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Individual stock solutions (3.65, 1.85, 0.90, 0.46, 0.23 μg/mL) were prepared in DMF for each of the test concentrations by serial dilution of a master stock solution prepared at a concentration of 3.65 μg/mL. The solutions were mixed by inversion and were clear and colourless. Aliquots of each stock, sufficient for the duration of the test, were placed in the syringe pumps at the initial of the test solution flows. Any remaining stock solution was stored in amber glass bottles.

The five test substance stock solutions were injected into the diluter mixing chambers at a rate of 4.0 μL/min where they were mixed with dilution water that was being delivered at a rate of 200 mL/min, to achieve the desired test concentrations. The negative control received dilution water only. The solvent control was prepared by delivering HPLC-grade DMF to the mixing chamber at a rate sufficient to achieve a final concentration of 0.02 mL/L.

- Controls: Dilution water and Dilution water + 0.02 mL/L DMF.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: rainbow trout
- Source: Thomas Fish Company, Andersen, California 96007
- Age at study initiation: juveniles
- Length at study initiation (mean, range): 5.0 cm, 4.6-5.3 cm
- Weight at study initiation (mean and range): 1.23 g, 0.98-1.60 g
- Loading at start of test: 0.086 g fish/L/day (0.82 g fish/L instantaneous loading)
- Feeding during test: none

ACCLIMATION
- Acclimation period: at least 14 days
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: Commercially prepared diet (Ziegler Brothers Inc., PA).
- Feeding frequency: Daily but no feed in the two days preceding the test
- Health during acclimation (any mortality observed): no adverse symptoms
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
136 mg/L as CaCO3
Test temperature:
12.1 - 12.6°C
pH:
range 8.0 to 8.2
Dissolved oxygen:
≥7.6 mg/L (≥70% ASV)
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: negative control, solvent control, 4.6 ng/l, 9.1 ng/l, 18 ng/l, 37 ng/l, 73 ng/l
Mean measured concentrations: * The analytical methodology could not measure the test substance at this concentration.
The results are expressed relative to the mean measured concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 25 L Teflon-lined stainless steel aquaria filled with 15 L of test water. Water depth was 18 cm. The aquaria were placed in a temperature-controlled water bath.
- Type: open
- Aeration: Dilution water aerated prior to use
- Type of flow-through (e.g. peristaltic or proportional diluter): Continuous flow diluter. Syringe pumps were used to deliver the test substance stock solutions or solvent to the mixing chambers. The flow of dilution water into each mixing chamber was controlled by rotameters.
- Renewal rate of test solution (frequency/flow rate): 10 volume replacements each day
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2
- Biomass loading rate: 0.086 g fish/L/day (0.82 g fish/L instantaneous loading)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Moderately hard well water. Filtered (0.45 μm)and UV-sterilised prior to use
- Alkalinity: 174 mg/L as CaCO3
- Conductivity: 370 μS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: Temperature was monitored continuously. Dissolved oxygen and pH were measured at the start of the test, at 24-hour intervals through the test and at the end of the test. Hardness, alkalinity and conductance were measured at the start of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Photoperiod: 16 h light, 8 h darkness
- Light intensity: 350 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : observations of mortality and other signs of toxicity were made at 2.5, 24, 48, 72 and 96 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 14.2 ng/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 14.2 ng/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: none reported, no overt signs of toxicity were observed
- Other biological observations: none
- Mortality of control: none
- Other adverse effects control: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: the LC50 and NOEC are around the solubility limit of the substance
Reported statistics and error estimates:
None required - no effects were observed in any treatment

Table 1. Test results

Nominal concentration (ng/L)

Mean measured concentration (ng/L)

Percentage mortality after 96 hours

0 (Control)

Not determined*

0

0 (Solvent control)

Not determined*

0

4.6

Not determined*

0

9.1

Not determined*

0

18

Not determined*

0

37

5.1

0

73

14.2

0

Validity criteria fulfilled:
yes
Conclusions:
A 96-hour LC50 value of >14.2 ng/L has been determined for the effects of the registered substance on mortality of Oncorhynchus mykiss based on mean measured concentrations of the substance. The result was obtained under flow-through test conditions.

Description of key information

LC50 (96-h): >14.2 ng/l, mortality Oncorhynchus mykiss, reliability 1, Wildlife International Ltd., 2012

Key value for chemical safety assessment

Additional information

A 96 hour LC50 value of >14.2 ng/l has been determined for the effects of the registered substance on mortality of Oncorhynchus mykiss based on mean measured concentrations of the substance in accordance with OECD TG 203 and in compliance with GLP (Wildlife International 2012). The result was obtained under flow-through test conditions.

Due to the slow hydrolysis of the test substance and the test media preparation and exposure method, it is likely that the test organisms were predominantly exposed to the parent substance.

The test substance has a water solubility of <10 ng/l and therefore, the results indicate that it is not toxic at its solubility limit.