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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 08 January 2019. Experimental completion date 25 June 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Deviations:
yes
Remarks:
Please see any other information on materials and methods section.
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Identification: Tert-butyl perbenzoate (CAS# 614-45-9)
CAS number: 614-45-9
Batch: 1802465400
Purity: 98.8%
Physical State/Appearance: Clear colorless liquid
Expiry Date: 01 April 2028
Storage Conditions: Room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
Range-finding Test
Samples of the test preparations were taken on Days 1, 2 and 3 of the pre dosing and Days 0, 2, 8 and 15 of the test. All samples were provided for analysis either on the day of sampling or the following day.

Initial Experiments
Samples were taken for analysis from the freshly prepared test solutions and after incubation under test conditions for 1 Day.

Definitive Test
Water samples were taken from the control and all surviving test groups from the freshly prepared bulk test preparation on Days 0, 6, 13, 20, 27 and 32 and from the old media on Days 1, 7, 14, 21, 28 and 33 (Replicates R1 to R4 pooled) for quantitative analysis. All samples were sent for analysis on the day of sampling with the exception of the freshly prepared samples on Day 33 which were stored frozen prior to analysis.
Duplicate sets of samples were taken on each occasion and stored frozen for further analysis if necessary.
Additional samples were prepared without biomass on Days 6, 13, 20, 27 and 32 and incubated alongside the test before being provided for analysis on Days 7, 14, 21, 28 and 33.
Vehicle:
yes
Remarks:
Dimethylformamide
Details on test solutions:
Range-finding Test
A nominal amount of test item (2500 mg) was dissolved in DMF and the volume adjusted to 25 mL to give a 100 mg/mL solvent stock solution from which a series of dilutions was made in DMF to give the further stock solutions of 0.10, 1.0 and 10 mg/mL. The stock solutions were freshly prepared every second day.
On Day 2, the amount of test item weighed out was recorded as 2250 mg. Although this was less than nominal, measured concentrations showed dose levels to be correct, therefore this was considered not to have had an impact on the test.
The stock solutions were inverted several times to ensure adequate mixing and homogeneity, with the exception of the 100 mg/mL on Day 4 of the pre dosing period, when no inversions were performed in error. Given that no affects were observed on the measured concentrations obtained during this period, this was considered not to have had an impact on the test.

Definitive Test
On Days 0, 1, 2 and 4 to 32, a nominal amount of test item (500 mg) was dissolved in DMF and the volume adjusted to 5 mL to give a 100 mg/mL solvent stock solution. Further dilutions were performed in DMF to give further stock solutions of 32, 10, 3.2 and 1.0 mg/mL. The stock solutions were inverted several times 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 daily.
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 22 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 156 to 160 mg/L as CaCO3 at the start of the test and from 134 to 138 mg/L as CaCO3 at termination of the test.
Test temperature:
The temperature recorded in each test vessel before and after each media renewal ranged from 25 to 26 °C
pH:
The pH ranged from 7.5 to 8.7.
Dissolved oxygen:
The dissolved oxygen concentration was maintained at least at 4.8 mg/L (equivalent to 58% ASV)
Nominal and measured concentrations:
Definitive test
Nominal test concentration: 0.050, 0.16, 0.5, 1.6, 5.0 mg/L
Geometric mean measured test concentration: 0.010, 0.031, 0.072, 1.3, 3.7 mg/L
Details on test conditions:
Range-finding Test
The test concentrations to be used in the initial experiments were determined by a preliminary range-finding test. The range-finding test was conducted by exposing newly fertilized eggs to a series of nominal test concentrations of 0.0050, 0.050, 0.50 and 5.0 mg/L for a period of 15 days.
Dynamic continuous flow test conditions were employed in the test. Dilution water was dosed at a flow rate of 120 mL per minute using Tacmina solenoid-driven metering pumps and the solvent stock solutions at a flow rate of 0.36 mL per hour using a Kd Scientific Syringe Pump. The dilution water and solvent stock solutions were combined in separate mixing vessels and delivered to the test vessels using a two way splitter at 30 mL per minute to each replicate.
Two replicate vessels were used for each control and test concentration.
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 and a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods for a period of 15 days under dynamic test conditions.
The control and the solvent control groups were maintained under identical conditions but not exposed to the test item. The solvent control group was exposed to 50 µL/L of DMF.
The number of dead eggs (up to completion of hatching), dead and live larvae and sub-lethal effects of exposure were recorded daily.
The larvae were fed freshly hatched brine shrimp nauplii from Day 6 to the end of the test.

Initial Experiments
Two initial tests were conducted using a continuous flow design at nominal test concentrations of 0.030, 0.065, 0.125, 0.25 and 0.50 mg/L; however, in the first test a microbial bloom in the 0.125 mg/L test group resulted in 100% mortality of the eggs on Day 4 of the test whereas in the second test the results of the chemical analysis showed considerable inconsistencies in the measured concentration.
Review of the data suggested that bacterial growth in the test system was affecting test item stability, and providing erroneous results. The method of preparation was amended to a semi-static design with daily renewal, which would help to alleviate the build up of bacterial blooms in the test vessels.
Prior to conducting further tests, a stability trial was performed to assess the new test method. Nominal concentrations of 0.0256, 0.16 and 1.0 mg/L were prepared by spiking solvent stock solutions into test media and stirring for approximately 5 minutes. Samples were taken for analysis from the freshly prepared test solutions and after incubation under test conditions for 1 Day. Results of the stability trial indicated that near nominal concentrations could be prepared and maintained under semi-static test conditions.
A further initial test was conducted employing a concentration range of 0.0256, 0.064, 0.16, 0.40 and 1.0 mg/L using the daily renewal regime, however, this test failed to achieve the validity criteria for post-hatch survival in the controls. Assessment of the data from the test groups indicated that the concentration range employed was not high enough to effectively capture the toxicity, therefore, the test concentrations for the definitive test were increased to 0.050, 0.16, 0.50, 1.6 and 5.0 mg/L.
The results from the initial experiments were not used for reporting purposes.

Definitive Test
Based on the results of the preliminary range-finding test and the initial experiments, newly fertilized fathead minnow eggs (4 replicates of 20 eggs per group) were exposed to aqueous solutions of the test item at nominal concentrations of 0.050, 0.16, 0.50, 1.6 and 5.0 mg/L for a period of 33 days under semi-static 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 Day 33. Four replicate flasks were used for each control and test concentration.
A semi-static test regime was employed in the test involving a renewal of the test preparations daily from the start of the test, with the exception of Day 3 in order to prevent 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 and 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 contents of the test vessels were aerated via narrow bore glass tubes from Day 12 onwards. The eggs and larvae were not individually identified.
The larvae were fed daily with brine shrimp.
Reference substance (positive control):
no
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
0.072 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
number hatched
Duration:
33 d
Dose descriptor:
LOEC
Effect conc.:
1.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
number hatched
Duration:
33 d
Dose descriptor:
LC10
Effect conc.:
0.63 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
number hatched
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
0.072 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: post hatch survival
Duration:
33 d
Dose descriptor:
LOEC
Effect conc.:
1.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: post hatch survival
Duration:
33 d
Dose descriptor:
LC10
Effect conc.:
0.101 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: post hatch survival
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.072 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
length
Remarks on result:
other: highest test concentration at the end of the test
Duration:
33 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.072 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Remarks on result:
other: highest test concentration at the end of the test
Details on results:
Range-finding Tests
The results showed there was no difference between the controls and test concentrations of 0.0050 and 0.050 mg/L in terms of hatching, survival and growth, however, differences were observed at test concentrations of 0.50 and 5.0 mg/L.
No sub-lethal effects were observed at the test concentrations of 0.0050, 0.050 and 5.0 mg/L, however, significant sub-lethal effects were observed at 0.50 mg/L. These sub-lethal effects observed were lethargy and reduced growth.
Chemical analysis of the 0.0050, 0.050, 0.50 and 5.0 mg/L test preparations on Days 0 and 2, and of the 0.0050, 0.050 and 0.50 mg/L test preparations on Days 8 and 15 showed that measured concentrations of between 0.0022 and 4.1 mg/L were obtained.

Definitive Test
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 0.016 to 4.0 mg/L. A decline in measured test concentration of the aged test preparations on Days 1, 7, 14, 21, 28 and 33 was observed to between less than the Limit of Quantification (LOQ) of the analytical method, determined to be 0.00064 mg/L and 3.5 mg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations only in order to give a “worst case” analysis of the data.
Analysis of the old samples incubated without biomass showed that results of between less than the LOQ and 0.40 mg/L were obtained.

Number of Eggs Hatching
The start of egg hatching was observed on Day 4 of the test and completion of hatching was observed on Day 5 of the test.
LC10 (Hatching): 0.63 mg/L; 95% confidence limits 0.28 to 1.4 mg/L
LC20 (Hatching): 0.96 mg/L; 95% confidence limits 0.51 to 1.8 mg/L
LC50 (Hatching): 1.8 mg/L; 95% confidence limits 1.1 to 2.9 mg/L
Statistical analysis of the hatching data was carried out for the pooled controls and all test concentrations. There were no statistically significant differences between the pooled controls and the 0.010, 0.031 and 0.072 mg/L test concentrations (P≥0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the NOEC based on the number of eggs hatching was 0.072 mg/L. Correspondingly the LOEC based on the number of eggs hatching was 1.3 mg/L.

Post-hatch Survival
The number of dead larvae were observed to be low throughout the duration of the test with no concentration dependent effects being observed.
LC10 (Post-hatch survival): 0.101 mg/L; 95% confidence limits 0.007 to 1.5 mg/L
LC20 (Post-hatch survival): 0.16 mg/L; 95% confidence limits 0.015 to 1.8 mg/L
LC50(Post-hatch survival): 0.34 mg/L; 95% confidence limits 0.035 to 3.2 mg/L
Statistical analysis of the post-hatch survival data was carried out for the pooled controls and the 0.010, 0.031, 0.072 and 1.3 mg/L test groups. There were no statistically significant differences between the pooled controls and the 0.010, 0.031 and 0.072 mg/L test concentration (P≥0.05), however the 1.3 mg/L test concentration was significantly different (P<0.05) and, therefore the NOEC based on post-hatch survival was 0.072 mg/L. Correspondingly the LOEC based on post-hatch survival was 1.3 mg/L.

Total Length and Weight Data
EC10 (Total Length): >0.072 mg/L*
EC20 (Total Length): >0.072 mg/L*
EC50 (Total Length): >0.072 mg/L*
Statistical analysis of the fish total length data was carried out for the pooled controls and the 0.010, 0.031 and 0.072 mg/L test group. There were no statistically significant differences (P≥0.05), between the control and the test groups and therefore the NOEC based on total length was 0.072 mg/L.
EC10 (Weight): >0.072 mg/L*
EC20 (Weight): >0.072 mg/L*
EC50 (Weight): >0.072 mg/L*
Statistical analysis of the fish wet weight data was carried out for the pooled controls and the 0.010, 0.031 and 0.072 mg/L test groups. There were no statistically significant differences (P≥0.05), between the pooled controls and the test groups and therefore the NOEC based on wet weight was 0.072 mg/L.
* It was not possible to calculate 95% confidence limits for the EC50 value as the data generated did not fit the models available for the calculation of confidence limits

Observations
Some larvae were observed to have bent spines. This observation was recorded from Day 18 onwards in 0.16 mg/L replicate R1 (1 larva) and Day 9 onwards in 0.50 mg/L replicate R3 (2 larva, 1 larva from Day 31).

Validation Criteria
The following validity criteria were achieved during the test:
Required Achieved
1) Dissolved oxygen 60% ASV >60% ASV on all but one occasion when a dissolved oxygen concentration of 58% ASV was recorded
2) Water temperature
Between test chambers ±1.5 °C ± 1.1 °C
Between successive days ±1.5 °C ± 1.0 °C
3) Hatching success rate* >70% 93%
4) Post-hatch survival* >75% 97%
* In control vessels

Observations on Test Item Solubility
After dosing all control, solvent control and test concentrations were observed to be clear, colorless solutions by visual inspection.
Validity criteria fulfilled:
yes
Conclusions:
Exposure of fathead minnow (Pimephales promelas) to the test item gave the following results based on the geometric mean measured test concentrations (mg/L) [95% confidence limits]:

Number Hatched
LC10 0.63 [0.28 – 1.4]
LC20 0.96 [0.51 – 1.8]
LC50 1.8 [1.1 – 2.9]
No Observed Effect Concentration 0.072
Lowest Observed Effect Concentration 1.3

Post-hatch Survival
LC10 0.101 [0.007 – 1.5]
LC20 0.16 [0.015 – 1.8]
LC50 0.34 [0.035 – 3.2]
No Observed Effect Concentration 0.072
Lowest Observed Effect Concentration 1.3

Total Length
EC10 >0.072
EC20 >0.072
EC50 >0.072
No Observed Effect Concentration ≥0.072
Lowest Observed Effect Concentration Not determined

Wet Weight
EC10 >0.072
EC20 >0.072
EC50 >0.072
No Observed Effect Concentration ≥0.072
Lowest Observed Effect Concentration Not determined
Executive summary:

Introduction

A study was performed to assess the effects of the test item on the early-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

Based on the results of a preliminary range-finding test and initial experiments, newly fertilized fathead minnow eggs (4 replicates of 20 eggs per group) were exposed to aqueous solutions of the test item at nominal concentrations of 0.050, 0.16, 0.50, 1.6 and 5.0 mg/L for a period of 33 days (28 days post-hatch) at a temperature of 25 to 26 ºC under semi-static test conditions.  The test solutions were renewed daily throughout the test with the exception of Day 3 when there was no water change to avoid causing premature hatching of the eggs.  An initial solvent stock solution was prepared by dissolving 500 mg of test item in a final volume of 5.0 mL of dimethylformamide (DMF) to give a 100 mg/mL solvent stock solution, from which a series of dilutions was prepared to give solvent stock solutions of 32, 10, 3.2 and 1.0 mg/mL.  An aliquot of each solvent stock solution was dispersed in test water with the aid of magnetic stirring for approximately 5 minutes to give the test solutions of 0.050, 0.16, 0.50, 1.6 and 5.0 mg/L.

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 wet 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 0.016 to 4.0 mg/L.  A decline in measured test concentration of the aged 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  0.00064 mg/L and 3.5 mg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration only in order to give a “worst case” analysis of the data.

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

Geometric Mean Measured Concentration (mg/L)

[95% Confidence Limits]

Number Hatched

LC10

0.63 [0.28 – 1.4]

LC20

0.96 [0.51 – 1.8]

LC50

1.8 [1.1 – 2.9]

No Observed Effect Concentration

0.072

Lowest Observed Effect Concentration

1.3

Post-hatch Survival

LC10

0.101 [0.007 – 1.5]

LC20

0.16 [0.015 – 1.8]

LC50

0.34 [0.035 – 3.2]

No Observed Effect Concentration

0.072

Lowest Observed Effect Concentration

1.3

Total Length

EC10

>0.072

EC20

>0.072

EC50

>0.072

No Observed Effect Concentration

0.072

Lowest Observed Effect Concentration

Not determined

Wet Weight

EC10

>0.072

EC20

>0.072

EC50

>0.072

No Observed Effect Concentration

0.072

Lowest Observed Effect Concentration

Not determined

Description of key information

A study was performed to assess the effects of the test item on the early-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”.

Post hatch survival turned out to be the critical endpoint in this study with an LC10 of 0.101 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
0.101 mg/L

Additional information

A study was performed to assess the effects of the test item on the early-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”.

Newly fertilized fathead minnow eggs (4 replicates of 20 eggs per group) were exposed to aqueous solutions of the test item at nominal concentrations of 0.050, 0.16, 0.50, 1.6 and 5.0 mg/L for a period of 33 days (28 days post‑hatch) at a temperature of 25 to 26 ºC under semi-static test conditions. The test solutions were renewed daily throughout the test.  

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 wet weight of the surviving fish were measured.

Analysis of the fresh test preparations showed measured test concentrations to range from 0.016 to 4.0 mg/L. A decline in measured test concentration of the aged test preparations was observed to be between less than the Limit of Quantification (LOQ) of the analytical method, determined to be 0.00064 mg/L and 3.5 mg/L and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration only in order to give a “worst case” analysis of the data. The geometric mean measured concentrations were 0.010, 0.031, 0.072, 1.3 and 3.7 mg/L.

Post hatch survival turned out to be the critical endpoint in this study. All hatched larvae died at 1.3 mg/L, which is the LOEC and all hatched larvae survived at 0.072 mg/L which is the NOEC. LC10 for this endpoint is 0.101 mg/L.