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Short-term toxicity to fish

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Endpoint:
fish embryo acute toxicity (FET)
Remarks:
Combined report in which existing fish data gaps were filled with FET screening data for multiple substances
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Collection of OECD236 data for various substances
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 236 (Fish embryo acute toxicity (FET) test)
Version / remarks:
Test conducted in a screening form with limited concentrations and no chemical analysis.
Deviations:
yes
Remarks:
Not GLP, No Chemical analysis,
Principles of method if other than guideline:
Screening method used a higher number of Embryos are used per well. Daily refreshment from a water accommodated fraction containing and excess of the parent material. When the parent material is visibly in excess and stirred for an extended period the water fraction can be considered saturated with the parent material and any accumulating degradation products or impurities at their corresponding solubility limits. Test material is of extremely low water solubility lower than the detection limit of available analytical methods. Hence daily refreshment was utilized to maximise exposure.
GLP compliance:
no
Remarks:
Conducted to simplified version of the guideline OECD236 study
Specific details on test material used for the study:
batch no.: 1702422114
Analytical monitoring:
no
Remarks:
Hydrolysis expected to be very rapid to more stable degradation products
Details on sampling:
N/A
Vehicle:
no
Details on test solutions:
Test solutions
When the solubility and stability of the material was known to be very low WAF (Water accommodated fraction) solutions were made to prepare the test material.

Slow stir WAF preparations have been shown to be capable of producing a stable concentration of dissolved parent material (as well as stabilizing agent or accumulating degradation products) when loaded in excess of the water solubility and slowly stirred in the same manner as a slow stir water solubility test.

Each WAF vessel was equipped with a Teflon tap that allows only the water accommodated fraction to be transferred without the transfer of undissolved material.

The test material for the WAF prepared substances was weighed accurately and added to each WAF vessel separately. The vessels were then carefully filled with 1 liter of test medium and stirred slowly under sealed conditions at room temperature for approximately 72 hours. Prior to the test the stirring was stopped and all WAF vessels were left to rest for 1 hour after which all of the test wells were rinsed with the appropriate test solution to minimize the absorbance of the test material. The test solutions were then discarded and refilled prior to testing. WAF vessels were then restarted and used for the daily refreshment of the solutions in the same manner.

Solution replenishment
Solution refreshment was carried out daily for WAF preparations by removing as much liquid as possible from each well (while avoiding drying of the embryos) using a pipette. After which the appropriate WAF solution was tapped off directly into the test wells. The WAF solutions (in which excess of test material remained visible) were used at the start of the test as well as for all solution replacements.

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Embryos arrived 3-4 hpf (hours post fertilization). As soon as the test solutions were made embryos were divided into bulk batches using glass pipettes at the appropriate concentration for each test chemical to prevent delay in exposure caused by preparation time. The wells were then filled with each of the stocks made for each chemical at each concentration. After a maximum of one hour the wells were emptied and refilled, after which the embryos (5 per well) were added. Plates were then incubated for the test duration and observed daily.

Test type:
semi-static
Water media type:
freshwater
Remarks:
Daily replenishment of test solutions from a WAF containing an excess of test material.
Limit test:
no
Total exposure duration:
96 h
Hardness:
The test medium Dutch Standard Water (DSW) was used for testing. DSW has a , conductivity of 550-650 µS/cm, and contains: 200 mg of CaCl2·2H2O, 180 mg of MgSO4·7H2O, 100 mg of NaHCO3 and 20 mg of KHCO3 per liter. The water was made by addition of the stocks and continual aeration before being used in the test.
Test temperature:
26ºC +/- 2ºC
pH:
pH of 8.2
Dissolved oxygen:
Continually aerated before use
Salinity:
N/A
Conductivity:
550-650 µS/cm
Nominal and measured concentrations:
0.1,1,10 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Wellplate
- Type: closed
- Material, size, headspace, fill volume: 3mL
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): 48h
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

OTHER TEST CONDITIONS
- Adjustment of pH: Not required
- Photoperiod:16 / 8 Light / Day
- Light intensity: Incubated in incubater withought light.

TEST CONCENTRATIONS

- Justification for using less concentrations than requested by guideline: screening (fish data not compulsory at registered tonnage)
Reference substance (positive control):
yes
Remarks:
Internal positive and negative controls acceptable
Duration:
96 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Extended stir parent + Degraded material
Basis for effect:
coagulation of the embryo
Details on results:
Test material can be concluded to have an LC50 / EL50 in excess of its water solubility limit for fish embryos / hatchlings.
Results with reference substance (positive control):
Acceptable according to guideline
Reported statistics and error estimates:
Range estimated in comparison to control only
Validity criteria fulfilled:
yes
Remarks:
With restrictions Not GLP and No Chemical analysis. Valid therefore as supporting evidence.
Conclusions:
An estimation of the EL50 for adult fish species was made for the material tested. The validity criteria for positive and negative controls were met and where this was not the case this was justified accordingly. The embryo batches were considered of good quality and sufficient for use in the OECD 236 test. The test material was not toxic to the test embryos or hatchlings at the water solubility limit of the test substance.
Executive summary:

Guideline study in simplified screening form without GLP and without chemical analysis. Required validity and reference criteria were met. Data considered an acceptable nominal estimation for the marketed product. Daily refreshment and well rinsing used to maximise exposure.

Endpoint:
short-term toxicity to fish
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From 13/06/2008 to 01/08/2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This semi-static study was performed according to OECD Guideline 203 with GLP statement. In presence of fish, there is a marked decrease in the test item concentrations (from 1.85-2.48 mg/L for new solutions to lower than the detection limit of the analytical method: 0.33 mg/L for old solutions) during each 24 hours period of exposure (due to bioaccumulation, adsorption and/or volatilization and hydrolysis). Nevertheless, this study allows to conclude that there is no acute toxicity up to the highest available concentration in solution (no effect up to 2.11 mg/L).
Justification for type of information:
See justification as explained in "Read Across Justification Acute Fish Target 70833-40-8 Source 34443-12-4" in section 13.2.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
yes
Remarks:
, minor deviation: five fish were used at each test concentrations and in the control, instead of at least 7.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
2008-05-06
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
- water solubility = 2.597 to 4.0874 mg/L at 25°C
- relative density at 20°C = 920 Kg/m3
Analytical monitoring:
yes
Details on sampling:
No data
Vehicle:
no
Details on test solutions:
The preliminary test was carried out in semi-static mode, wih renewal of solutions. Three were prepared by mixing 200, 100 and 50 mg of the test item per litre of dilution water in order to obtain 5 litres of each solution that was kept under stirring during 67h for the first session, then 22h for the 3 following ones. The day of the test, solutions were filtered on Millipore HVLP 14250 membrane, that was first rinsed with dilution water and 300 mL of the corresponding solution.
Based on the results of preliminary test, the definitive test was carried out in semi-static mode at the concentration 100 mg/L (limit test). Solutions were prepared the same manner.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Danio rerio (Teleostei, Cyprinidae)
- Strain: Batch n° 08/Br/01/1
- Source: Aquatrade France (44150 ANCENIS)
- Age at study initiation (mean and range, SD): the fish selected for the test were all from the same batch and were of the same age, date of birth stated by the supplier is 06/02/2008.
- Length at study initiation (length definition, mean, range and SD): 3.3 +/- 0.22 cm
- Weight at study initiation (mean and range, SD): average = 0.53 g/fish
- Feeding during test: TetraPro (Tetra GmbH, D-49304 Melle, Germany)
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Remarks on exposure duration:
none
Post exposure observation period:
None
Hardness:
No data
Test temperature:
between 22.4 +/- 0.5°C
pH:
between 7.75 and 8.13
Dissolved oxygen:
between 89 and 102% air saturation
Salinity:
Not applicable
Nominal and measured concentrations:
See table 2 in "Any other information on results incl. tables".
Details on test conditions:
The study was carried out in a thermostatically constrolled room. The temperature, which was measured on a continuous basis in a test flask, was between 22.4 +/- 0.5°C. The room was equipped with a programmed lighting system which produced a controlled lighting sequence: intensity between 400 and 800 Lux at the surface of the aquariums; photoperiod of 12 hours of darkness per 24h.
The study was performed using aquariums with a nominal capacity of 5L, covered with Altuglas cover plates drilled with holes to allow the test solutions to be aerated. Volume of test medium per aquarium was 2.5L. As 5 fish were added, this corresponding to a fish loading of 1.06 g/L.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 2.11 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: geometric mean of initial new solutions
Details on results:
The appearance of the test solutions was visually checked at the beginning and at the end of the test. Solutions were found to be clear, colourless over the period of the test. No precipitation was observed at the end of the test.
See tables in "Any other information on results incl. tables".
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
None

Table 6.1.1/1: Nominal and measured concentrations of the test item at the beginning and at the end of the stability test.

Conditions

Time of dissolution (hours)

Nominal concentration (mg/L)

Theor. Analyte (mg/L)

T0 (mg/L)

T24h (mg/L)

Without fish

68

100

100

1.93

1.29

With fish

100

 

<DL

Without fish

24

100

100

2.22

1.22

With fish

100

 

<QL

<DL: concentration lower than the detection limit of the analytical method (0.33 mg/L)

<QL: concentration lower than the quantification limit of the analytical method (1.09 mg/L)

These results in table 6.1.1/1 show that 24 or 68 hours of dissolution of the test item did not change the T0 concentration. However, the final concentration appears dependent of the presence of fish.

Table 6.1.1/2: Nominal and measured concentrations of the test item at the beginning and at the end of each 24h period (definitive test).

Nominal concentration (mg/L)

Theor. Analyte (mg/L)

D0 T0h (mg/L)

D1 T24h (mg/L)

D1 T0h (mg/L)

D1 T24h (mg/L)

D2 T0h (mg/L)

D2 T24h (mg/L)

D3 T0h (mg/L)

D3 T24h (mg/L)

0

0

<DL

<DL

<DL

<DL

<DL

<DL

<DL

<DL

0

0

<DL

<DL

<DL

<DL

<DL

<DL

<DL

<DL

100

100

2.04

<DL

2.25

<DL

1.96

<QL

ND

<QL

100

100

1.88

<DL

2.48

<DL

1.85

<QL

2.41

<DL

<DL: concentration lower than the detection limit of the analytical method (0.33 mg/L)

<QL: concentration lower than the quantification limit of the analytical method (1.09 mg/L)

As already observed in the stability test, in presence of fish, there is a marked decrease in the test item concentrations during each 24 hours period of exposure (table 6.1.1/2).

Table 6.1.1/3: Definitive test - percentage of dead fish:

Loading rate (mg/L)

Mortality (%)

24h

48h

72h

96h

100

0

0

0

0

0

0

0

0

0

No mortality of fish was observed up to a loading rate of 100 mg/L (table 6.1.1/3).

Validity criteria fulfilled:
no
Remarks:
The concentration of the substance being tested hasn't been satisfactorily maintained (at least 80 per cent of the nominal concentration) throughout the test and the results were not based on measured concentrations.
Conclusions:
No toxicity effect was detected, due to the test item or its degradation products, up to a loading rate of 100 mg/L. The 96h-LC50 was greater than 100 mg/L, corresponding to > 2.11 mg/L based on geometric mean initial measured concentration in new solutions.
Executive summary:

The acute toxicity of the test item to Danio rerio for a duration of 96 hours was assessed according to the OECD guideline 203 and GLP statement.

Fish were exposed under semi-static conditions to 100 mg/L of the test item dissolved in dilution water (limit test).

No mortality of fish was observed up to a loading rate of 100 mg/L. The 96h-LC50 was greater than the loading rate of 100 mg/L, corresponding to greater than 2.11 mg/L based on geometric mean initial measured concentration in new solutions.

Description of key information

There is one fish embryo test according to OECD 236 available for Tert-Amylperoxy 2 -ethylhexyl carbonate and one acute fish study according to OECD 203 for the closely related material Tert-Butylperoxy 2 -ethylhexyl carbonate. For additional information see the accompanying read across document. Due to the physical chemical properties of these material being similar and the toxicity to fish embryos and the toxicity to adult fish both being above the solubility limits. It is considered justified to read across to the existing adult fish endpoint for Tert-Butylperoxy 2 -ethylhexyl carbonate. This study has therefore been highlighted as the key information for this endpoint.

Both studies show no toxicity up to 100 mg/L loading, therefore no toxicity up to the water solubility limit.

Key value for chemical safety assessment

LC50 for freshwater fish:
100 mg/L

Additional information

According to REACH specific rule for adaptation from column 1, 9.1.3, the study does not need to conducted if there are mitigating factors indicating that aquatic toxicity is unlikely to occur, for instance if the substance is highly insoluble in water or the substance is unlikely to cross biological membranes. Since the substance is highly insoluble in water, and no toxicity has been observed at the limit of water solubility for the other performed aquatic toxicity test, the short term toxicity to fish test is waived.

As support to this argument, a fish test from the similar substance tert-butylperoxy 2-ethylhexyl carbonate (CAS 34443-12-4) is submitted as supporting evidence. In the conducted fish test on this substance, no toxicity was observed below the water solubility of the substance.

Conducting a new fish test would not add any new information for either classification or risk assessment, and hence, the study is waived.