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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:
2012-11-21 to 2013-04-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study without deviations.
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
The dilutions 1:2, 1:4, 1:8 and 1:16 of the undiluted filtrate with the loading rate of 100 mg/L were used as test media. Additionally, a control was tested in parallel.

- Sampling method:
For the determination of the actual test item concentrations in this semi-static test, duplicate samples were taken from each treatment just before the start of the first and of the last renewal periods (Days 0 and 3). At the end of these renewal periods (Days 1 and 4), additional samples were taken for determination of the stability of the test item during the renewal periods of 24 hours.
However, from the undiluted filtrate, the last samples were taken after 24 hours, since all fish were dead at this concentration at the corresponding observation dates.
All samples were taken from the approximate center of each test vessel without mixing the test medium. Immediately after sampling, acetonitrile was added to each sample in the ratio of 9:1 to stabilize the latter during the storage period.

- Sample storage conditions before analysis:
All samples were stored deep-frozen (at about -20 °C) immediately after sampling. The test item was known to be stable in the test water under these storage conditions based on a pre-experiment and prior studies (Harlan D53105).
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Due to the low water solubility of the test item, a dispersion with the loading rate of 100 mg/L was prepared at the start of the test and before each test medium renewal by dispersing 1110 mg of the test item (dosing range: 1100.0–1180.2 mg) in 11 L (range: 11.0–11.1 L) of test water. This preparation was supported by ultrasonic treatment for 15 minutes and intense stirring on a magnetic stirrer over 3 hours in the dark, to dissolve a maximum amount of the test item in the dispersion, in a completely filled and tightly closed stirring vessel. No auxiliary solvent or emulsifier was used.
After the 3-hour stirring period, the dispersion of the test item was filtered through a membrane filter (GF/F, pore size 0.7 μm). The negative pressure of the filtration unit was reduced (900 mbar) to avoid losses of volatile components of the test item during filtration. The filter was pre conditioned with at least 200 mL of the dispersion. The undiluted filtrate was used as a stock solution for the preparation of all test media. For this preparation, aliquots of the filtrate was diluted with test water. The test media were freshly prepared just before introduction of the fish (= start of the exposure) and before each test medium renewal (according to OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000).

- Controls: Test water without test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the entire test duration. However water solubility of test item is ca. 1 mg/L only, whereas the test concentrations where based on 100 mg/L test item.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: zebra fish
- Source: breeding culture at Harlan Laboratories
- Age at study initiation (mean and range, SD): no data
- Length at study initiation (length definition, mean, range and SD): From the acclimated test fish batch, 10 fish were measured at the start of the test. The mean body length of the fish was 2.98 ± 0.12 cm (Mean ± SD). These measured fish were not introduced for the test (same fish were also used for weghing, see below).
- Weight at study initiation (mean and range, SD): Mean body wet weight was 0.19 ± 0.03 g (Mean ± SD).
- Method of breeding: not specified
- Feeding during test: no

ACCLIMATION
- Acclimation period: Prior to test start, the test fish were acclimated for one week to the test water and temperature.
- Acclimation conditions (same as test or not): During holding and acclimatization until one days before the start of the test, the fish were fed with a commercial fish diet
- Type and amount of food: Tetra Min® Hauptfutter, supplied by TETRA-Werke, 49304 Melle / Germany
- Feeding frequency: no data
- Health during acclimation (any mortality observed): During holding and acclimatization, 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
Post exposure observation period:
no
Hardness:
1.25 mmol/L (125 mg/L) as CaCO3
Test temperature:
21-22 °C
pH:
7.2 - 7.4
Dissolved oxygen:
7.8 - 9.8 mg/L
Nominal and measured concentrations:
Undiluted filtrate (loading rate 100 mg/L): measured dissolved test item concentraiton 2.8 mg/L
1:2 dilution: measured dissolved test item concentraiton 1.25 mg/L
1:4, 1:8, and 1:16 dilutions not analysed
Details on test conditions:
TEST SYSTEM
- Test vessel: One 10 liter glass flask was used for each treatment.
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: The glass flasks were nearly completely filled to keep the air-space in the test flasks as small as possible and were tightly sealed with Teflon-lined screw caps.
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): no flow-through design applied
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: 0.13 g fish wet weight per liter test medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water was used
Composition of dilution water (EPA Medium)
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: 0.038mmol/L; 2.9 mg/L

- Total organic carbon: Not applicable as reconstituted water was used
- Particulate matter: Not applicable as reconstituted water was used
- Metals: Toxic metals not applicable as reconstituted water was used. Detailed mineral composition of test medium is given in the study report.
- Pesticides: Not applicable as reconstituted water was used
- Chlorine: Not applicable as reconstituted water was used
- Alkalinity: Not measured; estimated to be 0.4 mmol/L based on hydrogen carbonate concentration of the reconstituted test water
- Ca/Mg ratio: 4:1
- Conductivity: Not measured, however detailed mineral composition is given
- Culture medium different from test medium: yes
- Intervals of water quality measurement: daily

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: A 16 hour light to 8-hour dark photoperiod, with a 30-minute transition period was used
- Light intensity: 140 to 480 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- The recorded effects were mortality and visible abnormalities of the test fish. Dead fish were removed at least once daily and discarded.
- The water temperature, pH values and oxygen concentrations were measured for each treatment with surviving fish at the start of the test and once daily during the test in the freshly prepared and old test media.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study: none conducted
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Basis for effect:
mortality
Remarks on result:
other: 1.3-2.8 mg/L
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
1.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
2.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
1.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
2.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: none observed
- Observations on body length and weight: none observed
- Other biological observations: none observed
- Mortality of control: no mortalities
- Other adverse effects control: none observed
- Abnormal responses: none observed
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none observed
- Effect concentrations exceeding solubility of substance in test medium: yes

Mortality and Visible Abnormalities Observed in the Test Fish

Abbreviations of visible abnormalities for which fish were observed during the study

 

AK:  Strongly extended gills                           :      Fish mainly at the water surface

AP:   Apathy                                                 SA:      Mucus secretion

BA:  Distended abdomen                              SR:      Fish lying on side or back on the bottom

BO:  Fish mainly at the bottom                       SV:      Strong ventilation

        of the test vessel                                   TS:       Tumbling during swimming

GA:  Exophthalmus                                       VF:      Changed body color

KR:  Convulsions

 

Number of fish tested per treatment: 7

 

Treatment / Dilution

Mean measured concentration

[mg/L]

Number of abnormal and dead fish / number of dead fish
Type of visible abnormalities

Observation time

2 hours

24 hours

48 hours

72 hours

96 hours

Control

--

0 / 0

0 / 0

0 / 0

0 / 0

0 / 0

1:16

*

*

*

*

*

*

1:8

n.a.

0 / 0

0 / 0

0 / 0

0 / 0

0 / 0

1:4

n.a.

0 / 0

0 / 0

0 / 0

0 / 0

0 / 0

1:2

1.25

0 / 0

0 / 0

0 / 0

0 / 0

0 / 0

Undiluted filtrate (loading rate 100 mg/L)

2.8

7 / 0
AP

7 / 7

-/-

-/-

-/-

  LC50 #    n.d. 1.9  1.9   1.9  1.9
  95% C.I. #    - 0.94 - 2.1   0.94 - 2.1 0.94 - 2.1   0.94 - 2.1

 

-/-:                  all fish dead

#:                    based on mean measured concentration

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

n.d.:    could not be determined

*:        the dilution 1:16 was lost by accident and not taken into account for the study

Validity criteria fulfilled:
yes
Conclusions:
The fish 96-hour LC50 is 1.9 mg/L.
Executive summary:

The acute toxicity of the test item to zebra fish (Danio rerio) was determined in a 96-hour semi-static test with daily test medium renewal according to the EU Commission Directive 92/69/EEC, Part C.1, the Commission Regulation (EC) No 440/2008, Part C.1 and the OECD Guideline for Testing of Chemicals No. 203 (1992), as well as according to the OPPTS Guideline No. 850.1075 (Public Draft, April 1996). Due to the low water solubility of the test item, a dispersion of the test item with the loading rate of 100 mg/L was continuously stirred at room temperature in the dark over 3 hours supported by 15 minutes ultrasonication. Then, the dispersion was filtered with reduced negative pressure. The dilutions 1:2, 1:4, 1:8 and 1:16 of the undiluted filtrate with the loading rate of 100 mg/L were used as test media. Additionally, a control was tested in parallel. The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000. Since the test item was known to be volatile, the test was performed using 10 L glass flasks completely filled with test medium and tightly sealed with glass stoppers to avoid losses of test item (closed system). In the fresh medium samples from the first test medium renewal period (Day 0), the analytically determined concentrations of (ethoxymethoxy)cyclododecane in the test media (dilution 1:2 and undiluted filtrate) were 1.52 and 2.67 mg/L, respectively. In the fresh medium samples from the last test medium renewal period (Day 3), the analytically determined concentration of (ethoxymethoxy)cyclododecane in the dilution 1:2 was 1.3 mg/L. In the old medium samples from the first test medium renewal period (Day 1) 90 and 113% of the initially measured concentrations were found in the analyzed samples of the dilution 1:2 and the undiluted filtrate, respectively. In the old medium samples from the last test medium renewal period (Day 4) 56% of the initially measured concentration was found in the analyzed sample of the dilution 1:2. The undiluted filtrate was not analyzed in the last medium renewal period, since all fish were dead at that point of the study. The biological results were related to the mean measured test item concentrations calculated as the arithmetic mean of the geometric means of the concentrations measured at the start and end of each test period). Mean measured concentrations (geometric mean) of test dilutions: dilution 1:16 lost by accident; dilution 1:8 and 1:4 not analysed; dilution 1:2 measured 1.3 mg/L; undiluted filtrate (loading rate 100 mg/L) measured 2.8 mg/L. The biological test results (calculated as the arithmetic mean of the geometric means of the concentrations measured at the start and end of each test period) after 96 hours exposure: – 96-hour LC50: 1.9 mg/L (95% confidence interval: 1.3-2.8 mg/L) – 96-hour LC0: 1.3 mg/L – 96-hour LC100: 2.8 mg/L – 96-hour NOEC: 1.3 mg/L – 96-hour LOEC: 2.8 mg/L

Description of key information

The fish 96-hour LC50 is 1.9 mg/L.

Key value for chemical safety assessment

LC50 for freshwater fish:
1.9 mg/L

Additional information

The acute toxicity of the test item to zebra fish (Danio rerio) was determined in a 96-hour semi-static test with daily test medium renewal according to OECD Guideline for Testing of Chemicals No. 203 (1992). Due to the low water solubility of the test item, a dispersion of the test item with the loading rate of 100 mg/L was continuously stirred at room temperature in the dark over 3 hours supported by 15 minutes ultrasonication. Then, the dispersion was filtered with reduced negative pressure. The dilutions 1:2, 1:4, 1:8 and 1:16 of the undiluted filtrate with the loading rate of 100 mg/L were used as test media. Since the test item was known to be volatile, the test was performed using 10 L glass flasks completely filled with test medium and tightly sealed with glass stoppers to avoid losses of test item (closed system). The biological results were related to the mean measured test item concentrations calculated as the arithmetic mean of the geometric means of the concentrations measured at the start and end of each test period). The biological test results (calculated as the arithmetic mean of the geometric means of the concentrations measured at the start and end of each test period) after 96 hours exposure: – 96-hour LC50: 1.9 mg/L (95% confidence interval: 1.3-2.8 mg/L) – 96-hour LC0: 1.3 mg/L – 96-hour LC100: 2.8 mg/L – 96-hour NOEC: 1.3 mg/L – 96-hour LOEC: 2.8 mg/L