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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:
2015-04-21 to 2016-07-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
(1992)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
(2008)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: saturated solutiuon with a nominal loading rate of 1 mL test item/L
- Sampling method: Analytical evaluation of the saturated solution of the test substance and the control was carried out from freshly prepared media after 0 and 72 h and from corresponding aged test media after 24 and 96 h.
- Sample storage conditions before analysis: All samples were stored at 6 ± 2 °C before and at room temperature after preparation, if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Saturated solutions with a nominal loading of 1 ml test item / L were prepared with dilution water three days prior to the start of the exposure and the following three consecutive days. The
test item was placed by pipetting on the surface of the dilution water. To ensure that the solubility limit of the test item in the test medium was obtained, a slow stirring procedure for 72 ± 2 h was applied. Gentle stirring (to avoid a formation of an emulsion) was carried out with a magnetic stirrer over the bottom of the flask at approximately 25 °C. After a separation of the phases after at least 1 hour of standing, the saturated solution was taken from the homogeneous phase and was used for testing without further dilution.
- Test concentration: The saturated solution was tested in a limit test.
- Controls: 7 fish in dilution water (without test item) under the same test conditions
- Evidence of undissolved material: Since the formation of a (micro-) emulsion had to be excluded, the stock solution was checked after stirring via a laser beam (Tyndall effect) for any signs of (micro-) emulsions. No Tyndall effect was observed for any of the solutions.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Danio rerio (zebrafish)
- Strain: Danio rerio (zebrafish), Gnathostoma, Pisces, Osteichthyes, Teleostei, Cypriniformes, Cyprinidae
- Source: gained at Noack Laboratorien GmbH from a single brood stock
- Length at study initiation (length definition, mean, range and SD): Average and overall range for body length at the test end: 2.83 (2.7 to 2.9) cm
- Weight at study initiation (mean and range, SD): Average body weight at the test end: 0.204 g (no individual measurements).

ACCLIMATION
- Acclimation period: Zebrafish with at least 12 days of acclimatization and mortality < 5 %within these days prior to the start of exposure, were used for testing.
- Type and amount of food during acclimation: Sera vipan (Sera). The amount of food was 2 % of the fish body weight per feeding day.
- Feeding frequency during acclimation: Food was provided 3 times per week. The test fish were not fed 24 h before test start.
- Health during acclimation: Zebrafish with at least 12 days of acclimatization and mortality < 5 %within these days prior to the start of exposure, were used for testing. No disease treatments were administered throughout holding and testing.

FEEDING DURING TEST
The fish were not fed during the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Remarks on exposure duration:
daily renewal of the test media
Hardness:
63 mg CaC03/L
Test temperature:
23.6 ±0.2 °C
pH:
6.8 - 7.8
Dissolved oxygen:
> 60 % of the air saturation value, 90-100 % oxygen saturation
Nominal and measured concentrations:
Nominal concentration: 100 % saturated solution. The measured concentrations of the test item at the start of the exposure intervals were 103 and 85.9 µg/L (0 and 72 hours). At the end of the exposure intervals (24 and 96 hours), the measured concentrations were 5 and 40 % of the initially measured values (4.81 and 34.3 µg test item/L). Therefore, all effect values given are based on the geometric mean measured test item concentrations (38.3 µg/L).
Details on test conditions:
TEST SYSTEM
- Test vessel: glass vessel
- Material, size, headspace, fill volume: glass, 3L volume, dimensions 11.5/13/20 em, Test volume 2 L/ vessel
- Aeration: Gentle aeration was provided during the test
- Renewal rate of test solution: daily renewal
- No. of organisms per vessel: 7 (in total 20 fish)
- No. of vessels per concentration (replicates): One replicate
- No. of vessels per control (replicates): One replicate
- Biomass loading rate: Fish density was 0.714 g fish per liter test solution.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water, dechlorinated, charcoal treated, aerated for 24 h
- pH-value: 6.0- 8.5
- Ca/mg ratio: 10- 250 mg CaC03/L
- Culture medium different from test medium: Same as used for holding

OTHER TEST CONDITIONS
- Photoperiod: corresponds to the natural daylight hours
- Light intensity: 0.1 - 10 µmol · m^-2 · s^-1 (corresponding to approximately 7-750 Lux).

EFFECT PARAMETERS MEASURED : Mortality after 2, 24, 48, 72 and 96 h. Visilble abnormalities were also observed.

TEST CONCENTRATIONS
- Range finding study: A range finding test (non-GLP) with a saturated solution has been performed
- Test concentrations: 100 % saturated solution,nominal loading rate of 1 mL test item/L
- Results used to determine the conditions for the definitive study: The saturation solution as limit concentration is based on the results of a non GLP preliminary range finding test carried under semi-static conditions with a saturated solution. No mortality has been observed in the range finding study and all fish showed normal behaviour.
Reference substance (positive control):
no
Remarks:
No reference item is recommended for this test according to the guideline.
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 38.3 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
> 38.3 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
>= 38.3 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other:
Remarks:
The saturated solution caused no effects on vitality in Danio rerio.
Details on results:
- Behavioural abnormalities: No abnormalities observed. All fish showed normal behaviour.
- Mortality of control: No mortality observed.
- Other observations: The test medium was clear throughout the study.

All effect levels were given based on the geometric mean of the measured concentration.- Behavioural abnormalities:
- Observations on body length and weight:
- Other biological observations:
- Mortality of control:
- Other adverse effects control:
- Abnormal responses:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:

The concentrations of the test substance were analytically verified via LC-MS/MS at the start of the exposure intervals (0 and 72 hours) and at the end of the exposure intervals (24 and 96 hours) in the limit concentration and the control. Details of the analytical method are presented in section 12.1. The measured concentrations of the test item at the start of the exposure intervals were 103 and 85.9 µg/L (0 and 72 hours). At the end of the exposure intervals (24 and 96 hours), the measured concentrations were 5 and 40 % of the initially measured values.
Sublethal observations / clinical signs:

Observations of sub lethal effects in the Test Vessels

Nominal test item loading

 

Effect*

Number of fish effected at observation time [hours]

2

24

48

72

96

Saturated solution

(1)

7/7

7/7

7/7

7/7

7/7

Control

(1)

7/7

7/7

7/7

7/7

7/7

*)The numbers in brackets correspond to the following observations: (1)=Normal behavior

Cumulative Mortality [%] in the Test Vessels

Nominal test item loading

 

Cumulative mortality at observation time [hours]

2

24

48

72

96

Saturated solution

0

0

0

0

0

Control

0

0

0

0

0

Measured Concentrations of the Test Item during the Definitive Test

Sapmling

 

Cumulative mortality at observation time [hours]

0 h

24b h

72 h

96 h

Geometric mean measured test item conc. [µg/L]2)

 

Meas.

conc.

[µg/L]

Meas.

conc.

[µg/L]

%

Meas.

conc.

[µg/L]

Meas.

conc.

[µg/L]

%

Saturated solution

103

4.811)

5

85.9

34.3

40

38.3

Control

< LOQ

< LOQ

< LOQ

< LOQ

 

 

Meas. conc. = measured concentration of the test item

% = percent of the initially measured concentration of the test item at the start of the respective exposure interval

LOQ =limit of quantification of theanalytical method(15.00 µg/L of the test item)

1) =<LOQ but > systemquantification limit(2 µg/L), mean value of two replicates

2) = value < LOQ (24 h) taken into account with % LOQ

Validity criteria fulfilled:
yes
Remarks:
02-saturation >= 60 %; Mortality in the control <= 1/7 fish; pH value in the control group did not deviate by more than one unit over the test period; All effect values given are based on the geometric mean of the measured test substance concentrations.
Conclusions:
All effect levels are given based on the geometric mean measured concentration of the test substance Di-(iso)-pentyl terephthalate (DPT). The water soluble fraction of the test item was found to have no effect on the vitality of zebrafish. The LC0 corresponds to the geometric mean measured concentration of >=38.3 µg/L, the LC50 and the LC100 of > 38.3 µg/L.

Description of key information

The saturated solution with a geometric mean measured concentration of 38.3 µg/L caused no effects on vitality in Danio rerio. Therefore, the LC50 (96 h) is > 38.3 µg/L (geom. mean).

Key value for chemical safety assessment

Additional information

A short-term toxicity test with danio rerio has been conducted according to OECD Guideline for Testing of Chemicals No. 203 (1992) and the EU Council Regulation (EC) No. 440/2008/Method C.1 (2008). The test was performed in GLP compliance and with an analytical determination of the test substance concentration. A semi-static test with daily renewal of the test media was performed as a limit test using the saturated solution with a nominal loading of 1 mL test item /L. All effect values given are based on the geometric mean measured test substance concentrations. Exposure to the saturated solution of the test substance with a geometric mean measured concentration of 38.3 µg/L caused no effects on vitality in Danio rerio. The LC0 corresponds to a geometric mean measured concentration of >= 38.3 µg/L. The LC50 and the LC100 correspond to a geometric mean measured concentration of > 38.3 µg/L.