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Short-term toxicity to fish

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Endpoint:
fish embryo acute toxicity (FET)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1.12.2015 - 4.12.2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study was conducted to OECD 236 guideline. Study is non GLP intended as support for the existing GLP adult fish study. Data is considered reliable for use as supporting evidence but not as a key study.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 236 (Fish embryo acute toxicity (FET) test)
Deviations:
yes
Remarks:
Daily refreshment was used, No chemical analysis, More embryos, Less test concentrations
GLP compliance:
no
Remarks:
Conducted in a GLP facility under non GLP
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
N/A
Analytical monitoring:
no
Details on sampling:
N/A
Vehicle:
no
Details on test solutions:
A 1000 mg/L stock solution was made by direct addition of the test substance to the test medium followed by rapid stirring. While still under agitation the stock was further diluted in test medium to generate the desired test concentrations. The stock was intentionally generated above the water solubility of 171 mg/L so as to allow comparison with the existing fish data. After use the stock was sealed and refrigerated until use the following day. The thermal and hydrolytical stability of the test substance is sufficient to allow this without significant deterioration of the stock solution.
The stock was sealed to prevent volatilization.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Fertilized zebra fish wild type embryos were sourced at Wageningen UR Animal sciences group 6708 WG Wageningen the Netherlands. Fertilized embryos were between 2-3 hours old when added to the test solutions. This was confirmed by microscopic observation.
Test type:
other: Daily refreshment
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
None
Hardness:
The test medium Dutch Standard Water (DSW) was used for testing. DSW has a pH of 8.2, conductivity of 550-650µs/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 an automatic dosing system and was continually aerated before being used in the test.
Test temperature:
26ºC +/- 2ºC
pH:
8.2
Dissolved oxygen:
All media saturated before use
Salinity:
N/A
Nominal and measured concentrations:
10, 100 and 1000 mg/L including a control in test medium were prepared. In addition a positive control of 3,4-dichloro aniline at a concentration of 4 mg/L was prepared as indicated by the test guideline. Due to the positive control being prepared with acetone, a solvent control was also required according to the test guideline. A solvent control of 100µl/L was therefore also prepared. Test concentrations were not measured.
Details on test conditions:
The stock solution was checked for pH and adjusted with HCL or NaOH as required if pH deviated from that of the test medium. The received embryos were placed directly in beakers containing the appropriate test solution to maximize exposure prior to being added to the well plates. The well plates were then prepared. Each well was rinsed with the appropriate test solution and then discarded to reduce any adsorption effect that may take place. Each well was then filled for the second time with the appropriate test solution by transfer of 2-3 ml of the test solution using a pipette. 5 embryos per well were initially added using a glass pipette to the test plates. A total of 25 embryos were tested per test concentration. The test plate contained 15 embryos in control medium as an internal plate control. In addition a negative control plate containing 25 embryos and a positive control plate
containing 25 embryos was also tested. A solvent control replicate (solvent was used to prepare positive control) of 25 embryos was also included.
The control plate also contained and internal plate control of 15 embryos.

Observations were made at 0, 24 48, 72 and 96 hours. Coagulation of fertilized eggs, lack of somite formation, lack of detachment of the tail-bud from the yolk sac, and a lack of a heartbeat were scored were scored as primary indications of lethality. Any other clearly visible effects were also recorded. Solutions were refreshed every 24 hours by removing the majority of the liquid in the well and replacing with fresh test solutions that had been
re-made from a 1000 mg/L stock solution that was stored and sealed in the refrigerator between refreshments.
Reference substance (positive control):
yes
Remarks:
3,4 Dichloroanaline
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 - < 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Lethality as indicated by guideline criteria
Remarks on result:
other: Solutions were replaced daily and test plate was sealed
Details on results:
The LC50 to fish embryos is between 100 and 1000 mg/L.
Results with reference substance (positive control):
Exposure to the positive control (3,4 Dichloroanaline) resulted in a minimum of 30% mortality at the end of the test as required by the test guideline.
Reported statistics and error estimates:
Due to the study being a supporting study the test concentration range was broad. Interpolation between the test concentrations is therefore
inaccurate and not reccommended. It is however still clear in which range the LC50 to fish embryos would fall. Hence the endpoint has been
expressed accordingly.

Validity Criteria

The following validity criteria for the test were met:

 

·        The fertilization rate of the embryos in the batch tested was > 70%

·        The temperature should was maintained at 26ºC +/- 2 ºC during the test

·        Exposure to the positive control met the guideline requirements

·        Hatching rate in the negative control should be > 80% at the end of the test

·        Internal control success was >80%

 

 

The following validity criterion was not met:

 

·        The oxygen concentration in the control at the end of the test was not measured. The control survival exceeded the validity

requirements and hence detrimental shortage of oxygen could not have occurred. In addition the test medium was continually aerated before use and the test solutions were replaced daily. This is therefore not expected to have negatively influenced the study. 


 

Validity criteria fulfilled:
yes
Remarks:
in support of existing GLP fish endpoint
Conclusions:
The study can be considered a reliable indication of the toxicity of the test substance to fish embryos. The guideline was followed and critical quality
criteria were met and study design adapted for the test substance properties. Due to the study not being GLP it is reliable as a supporting evidence
only. The increased sensitivity of fish embryos observed in this study may be due to the higher sensitivity of the fish embryos to the test material
(Reliability of the OECD236 for use with organic peroxides is still ongoing) or due to the extra exposure created by the test design.
Executive summary:

A non GLP fish embryo study to the OECD236 guideline was conducted in support of the existing adult fish data. The existing adult fish study was not sufficiently adapted for the volatile properties of the test substance. This study was therefore conducted with daily refreshment and sealed conditions to support the existing data and prevent the underestimation of fish toxicity.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP accredited and study guideline followed but no measured concentrations. Solution refreshment did however take place and measured stability data is present in other aquatic studies that could be used to better estimate the exposure. It is also highly likely that the substance has been tested over maximum solubility (170 mg/l). Due to no physical effects on the test animals being observed the data is still of value and is valid with the mentioned restrictions. Additional fish embryo testing has been conducted in support of this endpoint to ensure that the effects to fish have not been underestimated.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
yes
Remarks:
no measured concentrations
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
Principles of method if other than guideline:
- No measurement of concentrations
-No adaption of test methodology for the volatility of the test substance.
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Water solubility: 0.018-0.025 %
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
no
Details on test solutions:
In the first range finding test the test substance was added directly to the diluting water followed by an ultrasonic treatment to improve the solubility. However, after this treatment the test substance was still visible in the test medium.
In the second range finding test the concentrations of up to 21.0 mg/l were prepared by using an acetonic stock solution, whereas the other concentrations were prepared by direct addition of the test substance. The use of acetone as an organic solvent did not improve the solubility either.
Therefore, in the definitive test the test concentrations were prepared by direct addition of the test substance to the diluting water in amounts of 210, 460 and 1000 mg/l. Due to the low solubility of the test substance in water a two phase mixture of water and the test material was obtained.
This mixture was stirred for 24 hours at room temperature to reach an equilibrium concentration of the test substance in the medium.
However, after this time and during the test the test substance remained visible as a film on the surface of the test medium at all concentrations.
Test organisms (species):
Poecilia reticulata
Details on test organisms:
The test organism was Poecilia reticulata (guppy). In contravention to the study plan they were obtained from a well-known local aquarium retailer (Rasbora). The fish were held in stock for 10 days in the Akzo Research Laboratories Arnhem, dept. CRL, in conformity with Standard Operating Procedures SOP CRL T9 and T42 to determine the acceptability of the batch. The size of the fish was approximately 2 cm.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
None
Hardness:
13 dH
Test temperature:
Measured at 0,24, 72 and 96h (22 - 23° C).
See details in Table 2 in the "Any other information on results incl. tables" rich field below.
pH:
Measured at 0, 48 and 96h (8.2 - 8.3 when freshly prepared and 7.5 - 7.9 48h old solutions).
See details in Table 1 in the "Any other information on materials and methods incl. tables" rich field below.
Dissolved oxygen:
Measured at 48 and 96h (5.8 - 7.9 mg/L).
See details in Table 1 in the "Any other information on materials and methods incl. tables" rich field below.
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: Control, 210, 460 and 1000 mg/L.
Details on test conditions:
The temperature was kept between 21.5° and 22.5°C and the light regime was 12 hours of ambient light per day. The duration of the test was 96 hours. As test vessels were used 2-L covered glass aquaria which contained 1 L of test medium. The loading of the test vessels was approximately 0.6 g biomass/L.
The surviving fish were counted daily and dead fish removed immediately upon observation. Measurements of the oxygen concentration were carried out on days 2 and 4, pH-measurements on days 0, 2 and 4. If a high mortality was observed on the other days, the oxygen concentration and pH were measured then also.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 171 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Remarks:
(test concentration up to water solubility)
Basis for effect:
mortality
Remarks on result:
other: Measured stability data from daphnia study used
Details on results:
The fish survived in the definitive test at all concentrations up to and including 1 g(1 during 96 hours.
Binomial theory dictates that when 10 fish are used showing 0% mortality, there is a 99.9% confidence that the LC50 (96 h) is greater than 1 g/L.
However, given that the test substance has a low solubility in water (171 mg/L) and that it remained visible during the test as a film layer
on the surface at all concentrations the water solubility was clearly exceeded. Considering the measured stability data from other studies it is still
likely that the LC50 is > than the water solubility limit despite the lack of analytical measurments during this study.
No other effects such as deviations in the behaviour or appearance of the fish were observed.

Details in range finding test:

Testsubstance mg/L

Number of surviving animals

0h

24h

48h

72h

96h

12.4

5

Nd

5

Nd

Nd

108

5

Nd

5

Nd

Nd

1002

5

Nd

5

Nd

Nd

Control

9

9

9

9

9

Control with acetone

10

10

10

10

0

2.1

10

10

10

10

10

4.6

10

10

10

10

9

10.0

10

10

10

10

10

21.0

10

10

10

10

10

46.0

10

10

10

10

10

100

10

10

10

10

10

details on definitive test results:

Testsubstance mg/L

Number of surviving animals

0h

24h

48h

72h

96h

Control

10

10

10

10

10

210

10

10

10

10

10

460

10

10

10

10

10

1000

10

10

10

10

10

Temperature

Nd

22 C

Nd

22 C

23 C

Nd= not determined

Validity criteria fulfilled:
no
Remarks:
No evidence of concentrations stability during test
Conclusions:
There is no evidence that tested concentrations have been maintained over 80% of nominal throughout the test. It is likely that the substance has been tested over its maximum solubility (surface layer described at all concentrations). The LC50 for the di-tert-butyl peroxide for guppy is determined after 96h to be > 1000 mg/L, the NOEC is determined to be >= 1000 mg/L based on nominal concentrations. Based on existing measured data from
the daphnia test the 96h NOEC can be corrected to >/= 489 mg/L which is more than twice the water solubility limit. The LC50 may therefore be
concluded to exceed the water solubility of the test substance.
Executive summary:

In a 96-h acute toxicity study, according with EEC method C.1 (1984) and OECD Test Guideline 203 (1984), Poecilia reticulata were exposed to di-tert-butyl peroxide at nominal concentrations of 0 (control), 210, 460 and 1000 mg/L under semi-static conditions. 

The fish survived for 96 hours at all concentrations tested up to and including 1000 mg/l. Due to the volatility of the test material and lack of analytical measurments full exposure to the test material cannot be demonstrated. Data should therefore be interpreted with care. Due to the fact that the test material was prepared many times above its solubility limit, was refreshed after 48 hours and was not aerated. It is unlikely that all of the test material left the test system. This does however remain a significant flaw to the study. A better indication of the actual exposure can be provided by the measured concentrations from the existing daphnia study. For animal welfare reasons this study was not repeated with adult fish but a supporting fish embryo study was conducted instead to strengthen this endpoint, in which daily refreshment and sealed plates were used to reduce loss of the test material from the system.

Endpoint:
short-term toxicity to fish
Type of information:
calculation (if not (Q)SAR)
Adequacy of study:
weight of evidence
Study period:
March 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: QSAR estimation with publicly available software
Justification for type of information:
QSAR prediction: migrated from IUCLID 5.6
Qualifier:
according to
Guideline:
other: ECHA TGD, Ch. R.6 - QSARs and grouping of chemicals (Chapter R.6)
GLP compliance:
not specified
Test organisms (species):
Pimephales promelas
Total exposure duration:
96 h
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
10.044 mg/L
Nominal / measured:
not specified
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: QSAR calculation used for qualitative analysis

ECOSAR Version 1.11 Results Page

 

SMILES : C(C)(C)(C)OOC(C)(C)C

CHEM  :

CAS Num: 110-05-4

ChemID1: di tert butyl peroxide

MOL FOR: C8 H18 O2

MOL WT : 146.23

Log Kow: 3.448     (EPISuite Kowwin v1.68 Estimate)

Log Kow: 3.200     (User Entered)

Log Kow:           (PhysProp DB exp value - for comparison only)

Melt Pt:           (User Entered for Wat Sol estimate)

Melt Pt: -40.00    (deg C, PhysProp DB exp value for Wat Sol est)

Wat Sol: 212.3     (mg/L, EPISuite WSKowwin v1.43 Estimate)

Wat Sol: 171       (mg/L, User Entered)

Wat Sol: 100       (mg/L, PhysProp DB exp value)

 

 

--------------------------------------

Values used to Generate ECOSAR Profile

--------------------------------------

Log Kow: 3.200     (User Entered)

Wat Sol: 171       (mg/L, User Entered)

 

 

------------------------------------------------

Available Measured Data from ECOSAR Training Set

------------------------------------------------

 

  No Data Available

 

 

--------------------------------------

ECOSAR v1.1 Class-specific Estimations

--------------------------------------

Peroxy Acids

                                                                   Predicted

ECOSAR Class                Organism           Duration End Pt  mg/L (ppm)

=========================== ================== ======== ======  ==========

Peroxy Acids              : Daphnid            48-hr    LC50       1.416

Peroxy Acids              : Daphnid                      ChV        0.095 !

 

=========================== ================== ======== ======  ==========

Neutral Organic SAR       : Fish               96-hr    LC50      10.044

(Baseline Toxicity)       : Daphnid            48-hr    LC50       6.371

                          : Green Algae        96-hr    EC50       7.504

                          : Fish                         ChV        1.119

                          : Daphnid                      ChV        0.846

                          : Green Algae                  ChV        2.515

 

 Note: * = asterisk designates: Chemical may not be soluble enough to

       measure this predicted effect. If the effect level exceeds the

       water solubility by 10X, typically no effects at saturation (NES)

       are reported.

 

 NOTE: ! = exclamation designates: The toxicity value was estimated through

           application of acute-to-chronic ratios per methods outlined in

           the ECOSAR Methodology Document provided in the ECOSAR Help Menu.

 

 

------------------------------

Class Specific LogKow Cut-Offs

------------------------------

If the log Kow of the chemical is greater than the endpoint specific cut-offs

presented below, then no effects at saturation are expected for those endpoints.

 

Peroxy Acids:

------------

Maximum LogKow: 5.0 (LC50)

Maximum LogKow: 5.0 (EC50)

Maximum LogKow: 8.0 (ChV)

 

Baseline Toxicity SAR Limitations:

---------------------------------

Maximum LogKow: 5.0 (Fish 96-hr LC50; Daphnid LC50)

Maximum LogKow: 6.4 (Green Algae EC50)

Maximum LogKow: 8.0 (ChV)

 

Validity criteria fulfilled:
not applicable
Conclusions:
The estimated 96hr LC50 to fish is 10.044 mg/L, which is higher than the estimated Daphnid 48hr LC50 of 6.371 mg/L and the Green Algae 96hr EC50 of 7.504 mg/L. The QSAR estimation of this substance supports qualitative (trend) evidence that fish is the least sensitive species for this class tert-butyl substituted volatile liquid organic peroxide. QSAR and measured results for acute toxicity for di-tert butyl peroxide and its analogue (tert-butylhydroperoxide, CAS 75-91-2) show that algae are the most sensitive organism compared to fish and Daphnia. Toxicity data for fish and Daphnia
do not show a significant difference (EC50 values do not differ by a factor >10). For animal welfare reasons, there is no justification for conducting
further acute (vertebrate) fish toxicity testing. Due to the concerns expressed with the existing adult fish endpoint further fish embryo testing was
conducted in support of this endpoint.
Executive summary:

QSAR and measured results for acute toxicity for di-tert butyl peroxide and its analogue (tert-butylhydroperoxide, CAS 75-91-2) show that algae are the most sensitive organism compared to fish and Daphnia. Toxicity data for fish and Daphnia do not show a significant difference (EC50 values do not differ by a factor >10).

Description of key information

QSAR and measured results for acute toxicity for di-tert butyl peroxide and its analogue (tert butyl hydroperoxide, CAS 75-91-2) show  that algae 
are the most sensitive organism compared to fish and daphnia. Toxicity data for fish, fish embryos and daphnia do not show a significant difference (EC50 values do not differ by a factor >10). Based on these observations, LC50 value of > 171 mg/L (or the water solubility limit) is the most correct way of expressing this endpoint. For animal welfare reasons, there is was no justification for conducting further acute (vertebrate) fish toxicity testing due to the existing study flaws. Therefore, in order to strengthen the fish data further and address the concerns raised regarding loss of the
test substance from the system, an additional fish embryo supporting study was conducted. This data also supported the existing trend that fish
species are the least sensitive but not significantly different from daphnia. Further use of existing measured stability data from the daphnia study was used to correct the adult fish endpoint for expected loss of the test substance from the test system. Data indicated that after correction that the LC50 would exceed the water solubility limit.

Key value for chemical safety assessment

LC50 for freshwater fish:
171 mg/L

Additional information

One GLP study on Poecilia reticulata following international guidelines is available. The substance was thought to have low solubility at the time and efforts were made in order to optimize exposure such as ultra-sonication and addition of a vehicle in range-finding tests. In these tests no toxicity was observed at the highest concentration tested (as a loading 1000 mg/L nominal) and a final test was conducted without using vehicle. After 96 hours, no mortality was observed in any of the test groups. However, a layer of film was observed throughout the test as an evidence of saturation limit of the medium was maintained. No sufficient efforts were made to maintain exposure concentrations, however medium was saturated and the substance does not hydrolyze (t1/2 > 1 year @ 25 oC).

Due to the test system not being adapted for the volatility of the test substance a supporting fish embryo toxicity was conducted with adaptions to maximise exposure and strengthen the existing fish data set. The supporting study indicated the LC50 for fish embryos to be between 100 and 1000 mg/L. In addition to this measured recoveries from the existing daphnia study were used to better estimate the amount of test material that would have been present in the original adult fish study. Based on both of these data points the LC50 for adult fish is concluded to exceed the water solubility limit. For the purposes of the chemical safety assessment the LC50 has been set at 171 mg/L.