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Ecotoxicological information

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 2008-08-04 to 2008-09-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to international guidelines including a GLP statement. The relevant information is reported and all validity criteria are fulfilled. The mean measured test concentrations were between 38 and 64% of the nominal values therefore the biological results are related to the mean measured test item concentrations.
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)
Version / remarks:
Commission Directive 92/69/ECC
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
2008-11-12
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Nominal test item concentration = 0.46, 1.0, 2.2, 4.6 and 10 mg/L ; mean measured test item concentration = n.a., 0.72, 1.5, 2.7, 2.1 mg/L.
- Sampling method: For the determination of the actual test item concentrations in this semi-static test, duplicate samples were taken from the freshly prepared test media of all test concentrations with surviving fish and from the control at the start of the test (Day 0) and on Day 3. The concentrations of the test item tert-Butyl-peroxybenzoate were analyzed in all test medium samples of the nominal test concentrations of 1.0-10 mg/L from all sampling times (or from the sampling time at the end of the respective exposure period when all fish were dead). The samples taken from the lowest nominal test concentration of 0.46 mg/L were not analyzed, since this test concentration was below the 96-hour NOEC, determined in this test. From the control samples, only one of the duplicate samples was analyzed from each sampling time.
- Sample storage conditions before analysis: Samples were stored at about -20°C immediately after sampling (protected from light until analysis)
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A concentrated stock solution of nominal 50mg/L was freshly prepared by mixing 113mg of the test item (effective weights: 112.9-113.1 mg) into 2.26 liters of test water in a completely filled and tightly closed stirring vessel. This stock solution was stirred for 3 hours to dissolve the test item completely in the test water. Adequate volumes of this intensively stirred stock solution were added to the test water in the test vessels and were intensively mixed to prepare the test media with the test concentrations as stated above. The test media were freshly prepared just before introduction of the fish (= start of the test) and before each test medium renewal.
- Eluate: no data
- Differential loading: no data
- Controls: test water without test item.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable
- Concentration of vehicle in test medium (stock solution and final test solution): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no data
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: zebra fish
- Strain: Brachydanio rerio
- Source: RCC's laboratories
- Age at study initiation (mean and range, SD): no data
- Length at study initiation (length definition, mean, range and SD): The mean body length of the fish was 3.2 +/- 0.12 cm (Mean +/- SD)
- Weight at study initiation (mean and range, SD): The mean body wet weight was 0.28 +/- 0.01 g (Mean +/- SD)
- Method of breeding: according to the guideline.
- Feeding during test: The test duration was 96 hours and the fish were not fed during the test.
- Food type: not applicable
- Amount: At the start of the test, 7 fish were introduced into each test vessel in a random order.
- Frequency: no data

ACCLIMATION
- Acclimation period: prior to test start, the test fish were acclimated for one week
- Acclimation conditions (same as test or not): same test water and temperature.
- Type and amount of food: Tetra Min Hauptfutter, supplier by TETRA-Werke, 49304 Melle / Germany.
- Feeding frequency: During holding and acclimatization until one day before the start of the test, the fish were fed with a commercial fish diet
- Health during acclimation (any mortality observed): no medication was applied during holding and acclimatization.

QUARANTINE (wild caught)
- Duration: not applicable
- Health/mortality: not applicable
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Remarks on exposure duration:
no remarks
Post exposure observation period:
none
Hardness:
Water hardness = 1.25 mmol/L (= 125 mg/L as CaCO3).
Test temperature:
The water temperature in the test vessels was measured daily and maintained at 22-23°C.
pH:
The pH values in the test media and the control was measured daily and ranged from 7.3 to 7.5.
Dissolved oxygen:
The oxygen concentration was measured daily and ranged from 8.1 to 8.2 mg/L at the start of the renewal periods. At the end of the renewal periods the oxygen concentration was in minimum 5.1 mg/L (62% saturation), and thus higher than 60% oxygen saturation as required by the validity criteria.
Salinity:
not applicable
Nominal and measured concentrations:
See table 1 in "Any other information on materials and methods incl. tables" field.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flask
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 5 liter
- Aeration: The Erlenmeyer flasks were nearly completely filled to keep the air-space in the test flasks as small as possible and were tightly sealed. Since a close system was used, the air from the small air-space above the test media was siphoned off by means of a pump and was slowly bubbled back into the test medium at the bottom of the test flasks. Thus, the dissolved oxygen concentrations in the test media were kept suficiently high, and additionally evaporated test item was dosed back into the test medium.
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): daily test medium renewal
- 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: The loading rate was 0.40 g fish wet weight per liter test medium.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: in-house prepared by a water purification system (Millipore)
- Total organic carbon: no data
- Particulate matter: no data
- Metals: no data
- Pesticides: no data
- Chlorine: no data
- Alkalinity: 0.4 mmol/L
- Ca/mg ratio: no data
- Conductivity: no data
- Culture medium different from test medium: no
- Intervals of water quality measurement: no data
- other: Appearance of the Freshly Prepared and Old Test Media = no remarkable observations, clear test medium.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: A 16-hour light to 8-hour dark, with a 30 minute transition period was used.
- Light intensity: light intensity during the light period was approximately within the range of 100 to 480 Lux.

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: About 2
- Justification for using less concentrations than requested by guideline: no data
- Range finding study: yes
- Test concentrations of the range finding study : 0, 0.981 and 9.81 mg/L
- Results used to determine the conditions for the definitive study: not specified but the selection of the test concentrations was based on the results of a range finding test and on results of a pre-experiment to determine the stability of the test item (non-GLP).
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CI = 0.68-3.9 mg/L
Key result
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
2.7 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.72 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
1.5 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: Apathy and fish mainly at the water surface were observed during the study.
- Observations on body length and weight: no data
- Other biological observations: See table 2 in "Any other information on results incl. tables" field.
- Mortality of control: no
- Other adverse effects control: no
- Abnormal responses: no data
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
The LC50 at the observation intervals after 24 and 48 hours could not be calculated by Probit Analysis or Moving Average Interpolation due to the steep concentration-effect relationship. Instead, the LC50 values were determined as a geometric mean value of the two consecutive test concentrations with 0 and 100% mortality, and the 95% confidence limits for the LC50 as the test concentrations with 0 and 100% mortality.

Table 2 : Mortality and visible abnormalities observed in the test fish

Nominal test item concentration (mg/L

Number of abnormal and dead fish / number of dead fish

Type of visible abnormalities

Observation time

3 hours

24 hours

48 hours

72 hours

96 hours

Control

0.46 (n.a)

1.0 (0.72)*

2.2 (1.5)*

4.6 (2.7)*

 

10 (2.1)*

0/0

0/0

0/0

0/0

7/0

AP

7/0

AP, OB

0/0

0/0

0/0

0/0

7/7

 

7/7

0/0

0/0

0/0

0/0

-/-

 

-/-

0/0

0/0

0/0

3/3

-/-

 

-/-

0/0

0/0

0/0

3/3

-/-

 

-/-

LC50**

95% C.I

--

--

2.0

1.5-2.7

2.0

1.5-2.7

1.6

0.68-3.9

1.6

0.68-3.9

AP : Apathy

OB : Fish mainly at the water surface

-/- : All fish dead

* : Mean measured test concentration

n.a : not analysed since below NOEC

** : LC50 based on mean measured test item concentrations

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

Validity criteria fulfilled:
yes
Remarks:
All validity criteria were fulfilled.
Conclusions:
A study was realised in agreement with OECD Guideline 203 and EU Commission Directive 92/69/33C, Part C.1. The validity criteria were all fulfilled. The LC50 (96h) = 1.6 mg/L (95% C.I = 0.68-3.9 mg/L).
Executive summary:

The acute toxicity of the test item tert-Butyl peroxybenzoate to zebra fish Brachydanio rerio (Source : RCC's laboratories) was determined in a 96 -hour semi-static test with a daily test medium renewal according to the EU Commission Directive 92/69/ECC, Part C.1 and the OECD Guideline for Testing of Chemicals No. 203, (1992). The mean body length of the fish was 3.2 +/- 0.12 cm, and the mean body wet weight was 0.28 +/- 0.01 g. Prior to test start, the test fish were acclimated for one week to the test water and temperature. About test conditions, the validity criteria were all fulfilled according to OECD Guideline No. 203. During the last two weeks prior to the test, no fish died in the test fish batch and all fish were healthy.

The nominal test item concentrations tested were 0.46, 1.0, 2.2, 4.6 and 10 mg/L. Additionally, a control group was tested in parallel. At the start of the test, 7 fish were introduced into each test vessel in a random order. One replicate per concentration was used.

In the analyzed test medium samples from the start of the test medium renewal periods, the measured test item concentrations ranged between 88 and 98% of the nominal values. This shows the correct dosage of the test item. During the test medium renewal periods of 24 -hours a decrease of the test item concentration in the test media was determined. At the end of the test medium renewal periods, 38 -64% of the nominal values were found, due to the loss of the substance. The mean measured test item concentration were n.a, 0.72, 1.5, 2.7 and 2.1 mg/L. The reported biological results are related to the mean measured test item concentrations. In the control and at the test concentrations up to and including 0.72 mg/L, all fish survived until the end of the test and no visible abnormalities were observed in the tested fish. At the next higher test concentration of 1.5 mg/L, 3 of the 7 test fish died but no signs of intoxication were observed in the surviving fish. At the 2 highest test concentrations of 2.7 and 2.1 mg/L, all fish were dead already after 24 hours of test duration. The biological test results after 96 hours test duration were : 96 -hour LC50 = 1.6 mg/L; 96 -hour LC100 = 2.7 mg/L; 96 -hour NOEC = 0.72 mg/L and 96 -hour LOEC = 1.5 mg/L.

Description of key information

The 96h-LC50 for fish is 1.6 mg/L based on measured concentrations of the test substance

Key value for chemical safety assessment

LC50 for freshwater fish:
1.6 mg/L

Additional information

Two 96 h fish acute studies were available, however only one is considered valid as it was performed under semi-static conditions with analytical verification of the test solutions. The 96h-LC50 for this study was 1.6 mg/L based on measured concentrations with no observed effect at 0.72 mg/L