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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
Adequacy of study:
key study
Study period:
Experimental start date 02 April 2019 Experimental completion date 19 July 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Identification: 1,3-bis(tert-butylperoxyisopropyl)benzene CAS 2212-81-9
Batch: 1403454131
Purity: 91.1%
Physical state/Appearance: White flakes
Expiry Date: 01 January 2024
Storage Conditions: Room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
Water samples were taken from the control and all surviving test groups on Days 0, 6, 13, 20, 27 and 32 and from the old or expired media on Days 1, 7, 14, 21, 28 and 33. All samples were taken for immediate quantitative analysis with the exception of the Day 32 samples which were stored frozen prior to analysis.

Duplicate sets of samples were taken from each occasion and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
Information provided by the Sponsor indicated that the test item had a low water solubility and high log Kow. The quoted water solubility for the test item was 0.04 mg/L.

Based on this information the test item was categorized as being a ‘difficult substance’ as defined by the OECD Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals (OECD 2019) and therefore the test solutions were prepared using a slow-stir saturated solution method of preparation.

A nominal amount of test item (300 mg) was added to the surface of 30 liters of test water and stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours (with the exception of Day 30 where the stirring was stopped after 23.5 hours) and the mixture allowed to stand for 1 hour before the aqueous was removed by siphoning to produce the 100% v/v saturated solution. A dilution was made from the 100% v/v saturated solution to give a further test concentration of 32% v/v saturated solution.

The 32% v/v saturated solution was inverted or stirred with a magnetic stirrer to ensure adequate mixing and homogeneity.

Test organisms (species):
Pimephales promelas
Details on test organisms:
The test was carried out using freshly laid eggs of fathead minnows (Pimephales promelas). The adult fathead minnows that produced the eggs for the test were bred at Covance CRS Research Limited on 14 February 2019 and maintained in dechlorinated tap water with an activated carbon and biological filtration system.

The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. In the 7 days preceding the start of the test, the water temperature was controlled at approximately 25 °C with a dissolved oxygen content of greater than or equal to 8.2 mg O2/L. The breeding stock fish were fed frozen brine shrimp.

Each breeding tank was supplied with inverted plastic guttering for the fish to lay eggs on and be fertilized. Fertilized eggs were collected from the breeding tanks on 29 May 2019 and used for the definitive test. The eggs were visually inspected before introduction into the test system and were identified as being at early blastodisc stage.

The diet and diluent water are considered not to contain any contaminant that would affect the integrity and outcome of the study
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
33 d
Hardness:
The water hardness values were observed to range from 138 to 144 mg/L as CaCO3 at the start of the test and from 128 to 158 mg/L as CaCO3 at termination of the test.

The water hardness was measured in the bulk test preparation at the start and in each vessel on termination of the test.
Test temperature:
The temperature recorded in each test vessel before and after each media renewal ranged from 23.2 to 27.3 °C
The temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
pH:
The pH and dissolved oxygen concentration was measured using a Hach Flexi handheld meter.
Dissolved oxygen:
The dissolved oxygen concentration was maintained at least at 4.8 mg/L (equivalent to 58% ASV) and the pH ranged from 7.5 to 8.7.

The pH and dissolved oxygen concentration was measured using a Hach Flexi handheld meter
Salinity:
Freshwater study
Nominal and measured concentrations:
Nominal: 32% v/v saturated solution, made from a 100% v/v saturated solution.
Geometric mean measured: 0.0015 and 0.0062 mg/L
Details on test conditions:
Exposure Conditions
In the definitive test 1 liter glass vessels were used from Day 0 to Day 14 and from Day 15 to the end of the test, 5 liter glass vessels were used. The approximate volume of test preparation in each vessel was 400 mL from Day 0 to Day 5, 800 mL from Day 6 to Day 14 and 4000 mL from Day 15 to the end of the test. Four replicate flasks were used for each control and test concentration.

A semi static test regime was employed in the test involving renewal of the test preparations daily throughout the test, with the exception of Day 3 where there was no water change to avoid causing premature hatching of the eggs.

Twenty eggs were placed into each replicate test vessel and the vessels covered to reduce evaporation. The test vessels were maintained at 25 °C with a maximum deviation of ±1.5 °C between test chambers or between successive days with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 33 days.

The test vessels were aerated via narrow bore glass tubes from Day 13 onwards. The eggs and fish were not individually identified.

The fish were fed with freshly hatched brine shrimp from Day 7 to Day 32.
Reference substance (positive control):
no
Duration:
33 d
Dose descriptor:
LC50
Effect conc.:
> 0.006 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
number hatched
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.006 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
number hatched
Duration:
33 d
Dose descriptor:
LC50
Effect conc.:
> 0.006 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Post-hatch survival
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.006 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Post-hatch survival
Duration:
33 d
Dose descriptor:
LC50
Effect conc.:
> 0.006 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
length
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.006 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
length
Duration:
33 d
Dose descriptor:
LC50
Effect conc.:
> 0.006 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Key result
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.006 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Details on results:
Number of Eggs Hatching
The number of dead eggs observed was low throughout the test with no concentration dependent effects being observed.

The start of egg hatching was observed on Day 4 of the test and completion of hatching was observed on Day 6 of the test. There were no significant mortalities or sub lethal effects of exposure observed in
any of the test concentrations.

Inspection of the hatching data at Day 6 based on the geometric mean measured test concentrations gave the following results:
LC10 (Hatching): >0.0062 mg/L
LC20 (Hatching): >0.0062 mg/L
LC50 (Hatching): >0.0062 mg/L

Statistical analysis of the hatching data was carried out for the control and all test concentrations. There were no statistically significant differences (P≥0.05), between the control and any of the test concentrations; therefore the NOEC based on the number of eggs hatching was 0.0062 mg/L. Correspondingly the LOEC based on the number of eggs hatching was not determined.

Post-hatch Survival
The number of dead fish were observed to be low throughout the duration of the test with no concentration dependent effects being observed.

There were no significant mortalities observed in any of the test concentrations.

Inspection of the post-survival hatching data based on the geometric mean measured test concentrations gave the following results:
LC10 (Post-hatch survival): >0.0062 mg/L
LC20 (Post-hatch survival): >0.0062 mg/L
LC50 (Post-hatch survival): >0.0062 mg/L

Statistical analysis of the post-hatch survival data was carried out for the control and all test concentrations. There were no statistically significant differences (P≥0.05), between the control and any of the test concentrations; therefore the NOEC based on post hatch survival was 0.0062 mg/L. Correspondingly the LOEC based on post hatch survival was not determined.

Length and Weight Data
Inspection of the body length data based on the geometric mean measured test concentrations gave the following results:

EC10 (Length): >0.0062 mg/L
EC20 (Length): >0.0062 mg/L
EC50 (Length): >0.0062 mg/L

Statistical analysis of the fish body length data was carried out for the control and all test concentrations. There were no statistically significant differences (P≥0.05), between the control and any of the test concentrations; therefore the NOEC based on body length was 0.0062 mg/L. Correspondingly the LOEC based on body length was not determined.

Inspection of the fresh weight data based on the geometric mean measured test concentrations gave the following results:
EC10 (Weight): >0.0062 mg/L
EC20 (Weight): >0.0062 mg/L
EC50 (Weight): >0.0062 mg/L
Statistical analysis of the fish fresh weight data was carried out for the control and all test concentrations. There were no statistically significant differences (P≥0.05), between the control and any of the test concentrations; therefore the NOEC based on fresh weight was 0.0062 mg/L. Correspondingly the LOEC based on wet weight was not determined.

  Range-finding Test

The results showed there was no difference between the control and all test concentrations in terms of hatching, survival and growth.

One fish in the 10% v/v saturated solution replicate 2 was observed with a bent spine on Day 6; however, given the low number of effects observed this was considered not to be significant. No other sub‑lethal effects were observed in the control and all test concentrations throughout the test.

Based on this information test concentrations of 32 and 100% v/v saturated solution were selected for the definitive test. This experimental design conforms to an "extended limit test" to confirm that at the concentrations tested, no adverse reactions to exposure were observed.

Chemical analysis of the fresh test preparations on Days 0 and 1 showed measured test concentrations of between 0.00025 and 0.014 mg/L. Measured concentrations of the 1.0, 10 and 100% v/v saturated solution old test preparations on Days 1 and 4 were observed to be 0.00031 to 0.0030 mg/L. Measured concentrations in the 0.10% v/v saturated solution old test preparations on Days 1 and 4 had increased from the corresponding Day 0 and 1 measured concentrations and were therefore considered to be anomalous.

Measured concentrations of 1.0, 10 and 100% v/v saturated solution old preparations on Days 1 and 4 taken from vessels incubated alongside the test but without fish or feed were observed to be 0.00026 to 0.0039 mg/L. No consistent pattern was apparent to suggest whether adsorption of the test item onto biomass was a significant factor from these results.

Post Range-finding Media Preparation Trial

Chemical analysis of the fresh test preparation from the post range-finding media preparation trial showed measured test concentrations in the two 100% v/v saturated solution preparations of 0.0097 and 0.011 mg/L. These results were comparable to the analytical results from the range-finding test Day 0 and 1 fresh test preparations therefore confirming the water solubility under the test conditions was slightly lower than the quoted water solubility of 0.04 mg/L.

DefinitiveTest

 Verification of Test Concentrations

Chemical analysis of the fresh test preparations on Days 0, 6, 13, 20, 27 and 32 showed measured test concentrations to range from 0.0043 to 0.034 mg/L. A decline in measured test concentration of the old test preparations on Days 1, 7, 14, 21, 28 and 33 was observed to be between less than the Limit of Quantification (LOQ) of the analytical method, determined to be 0.000066 mg/L and0.011 mg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations in order to give a “worst case” analysis of the data.

Inspection of the results from Day 0 (fresh) and Day 1 (old) suggest a sampling error had occurred as the measured concentration in the Day 0 (fresh) 100% v/v saturated solution was lower than in the 32% saturated solution; however, this pattern was not repeated in the Day 1 (old) samples. Analysis of the frozen duplicate samples confirmed the original results and therefore geometric mean measured concentrations have been calculated excluding the results from the Day 0 and Day 1 samples. The geometric mean measured test concentrations were determined to be:

Nominal Test Concentration
(% v/v Saturated Solution)

Geometric Mean Measured Test Concentration (mg/L)

32

0.0015

100

0.0062

Measured concentrations of old preparations on Days 21 and 28 taken from vessels incubated alongside the test but without fish or feed were observed to be between 0.0031 and 0.014 mg/L. Measured concentrations of old preparations taken from the test vessels including fish and feed on the corresponding days were betweenless than the Limit of Quantification (LOQ) of the analytical method, determined to be 0.000066 mg/L and0.0029 mg/L. These results suggest that a loss of the test item onto the biomass within the test system was occurring.

 Observations

One fish in Control replicate 1 was observed with a bent spine on Day 10 and one fish in 0.0015 mg/L replicate 2 was observed to be under-developed on Day 14. Given the low number of sub-lethal effects recorded, these are considered to be due to developmental defects occurring in a natural population, and not attributable to the test item.

Validation Criteria

The following validity criteria were achieved during the test:

 

Required

Achieved

1)     Dissolved oxygen

60% ASV

>60% ASV on all days with the exception of Day 13 when the lowest dissolved oxygen concentration recorded was 58% ASV

2)     Water temperature

 

 

Between test chambers

±1.5oC

± 2.1oC

Between successive days

±1.5oC

± 2.7oC

3)     Hatching success rate*

>70%

75%

4)     Post-hatch survival*

>75%

98%

Water Quality Criteria

The temperature measurements recorded in control replicate 1 by a Testo temperature logger are presented inFigure 1and were shown to have been maintained at between 24.6 and 26.6 °C. The temperature recorded in each test vessel before and after each media renewal ranged from 23.2 to 27.3 °C. The dissolved oxygen concentration was maintained at least at 4.8 mg/L (equivalent to 58% ASV) and the pH ranged from 7.5 to 8.7. Throughout the test (excluding Day 9 when no lux measurement was taken) the light intensity was observed to be in the range 352 to 826 Lux.

The water hardness values were observed to range from 138 to 144 mg/L as CaCO3at the start of the test and from 128 to 158 mg/L as CaCO3at termination of the test.

1.1.4            Observations on Test Item Solubility

After dosing all control and test concentrations were observed to be clear, colorless solutions by visual inspection.


*In control vessels

Validity criteria fulfilled:
yes
Conclusions:
This study showed that there were no toxic effects at saturation.
Executive summary:

Introduction

A study was performed to assess the effects of the test item on theearly-life stages of the fathead minnow (Pimephales promelas). The method followed that described in the OECD Guidelines for Testing of Chemicals (2013) No 210, "Fish, Early-Life Stage Toxicity Test”.

Methods

Information provided by the Sponsor indicated that the test item had a low water solubility and high log Kow. The quoted water solubility for the test item was 0.04 mg/L, therefore the test solutions were prepared using a slow-stir saturated solution method of preparation.

Based on the results of a preliminary range‑finding test,newly fertilized fathead minnow eggs(4 replicates of 20 eggs per group) were exposed to solutions of the test item at nominal concentrations of 32 and 100% v/v saturated solution for a period of 33 days at a temperature of 23 to 27 °C under semi‑static test conditions. The test solutions were renewed daily throughout the test with the exception of Day 3 where there was no water change to avoid causing premature hatching of the eggs. The test item solutions were prepared by stirring an excess (10 mg/L) of test item in test water using a propeller stirrer at approximately 1500 rpm for 23 hours. After the stirring period the preparation wasallowed to stand for 1 hour before the aqueous was removed avoiding any undissolved test itemto produce the 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the 32% v/v saturated solution test group.

The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were recorded daily until termination of the test (28 days post-hatch). At test termination the total length and fresh weight of the surviving fish were measured.

Results

Analysis of the fresh test preparations on Days 0, 6, 13, 20, 27 and 32 showed measured test concentrations to range from 0.0043 to 0.034 mg/L. A decline in measured test concentration of the old test preparations on Days 1, 7, 14, 21, 28 and 33 was observed to be between less than the Limit of Quantification (LOQ) of the analytical method, determined to be 0.000066 mg/L and0.011 mg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration in order to give a “worst case” analysis of the data. The geometric mean measured concentrations for the 32 and 100% v/v saturated solutions were determined to be 0.0015 and 0.0062 mg/L, respectively.


 Exposure of the fathead minnow early-life stages to the test item gave the following results based on the geometric mean measured test concentrations:

Endpoint

Concentration (mg/L)

Number Hatched

LC10

>0.0062

LC20

>0.0062

LC50

>0.0062

No Observed Effect Concentration

0.0062

Lowest Observed Effect Concentration

Not determined

Post-hatch Survival

LC10

>0.0062

LC20

>0.0062

LC50

>0.0062

No Observed Effect Concentration

³0.0062

Lowest Observed Effect Concentration

Not determined

Body Length

EC10

>0.0062

EC20

>0.0062

EC50

>0.0062

No Observed Effect Concentration

0.0062

Lowest Observed Effect Concentration

Not determined

Fresh Weight

EC10

>0.0062

EC20

>0.0062

EC50

>0.0062

No Observed Effect Concentration

0.0062

Lowest Observed Effect Concentration

Not determined

This study showed that there were no toxic effects at saturation.

Description of key information

A long term fish study according to OECD guideline 210 was conducted. Newly fertilized fathead minnow eggs (4 replicates of 20 eggs per group) were exposed to solutions of the test item at nominal concentrations of 32 and 100% v/v saturated solution for a period of 33 days at a temperature of 23 to 27 °C under semi‑static test conditions. The test solutions were renewed daily throughout the test. The test item solutions were prepared by stirring an excess (10 mg/L) of test item in test water, after the stirring period the preparation was allowed to stand for 1 hour before the aqueous was removed avoiding any undissolved test itemto produce the 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the 32% v/v saturated solution test group.

Exposure of the fathead minnow early-life stages to the test item gave no effects on no. hatched, post-hatch survival, body length and -weight based on the geometric mean measured test concentrations of 0.0015 and 0.0062 mg/L, respectively. Therefore, this study showed there were no toxic effects at saturation or at 10 mg/L loading.

Key value for chemical safety assessment

Additional information