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Short-term toxicity to fish

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Endpoint:
short-term toxicity to fish
Type of information:
calculation (if not (Q)SAR)
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, and documentation / justification is limited
Remarks:
ECOSAR typically does not provide reliable quantitative aquatic predictions for organic peroxides (as evidenced by a large experimental REACH database), but it is used to qualitatively compare the trophic levels to estimate their relative sensitivities
Principles of method if other than guideline:
calculation
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 11.778 - < 18.143 mg/L
Remarks on result:
other: ECOSAR v1.11 calculation, Neutral Organic SAR Class (11.778) vs Peroxy Ester Class (18.143)
Sublethal observations / clinical signs:

Calculated short-term toxicity to aquatic invertebrates is 5.185 -7.942 mg/L (Peroxy Esters Class - Neutral Organic SAR Class).

Calculated toxicity to aquatic algae and cyanobacteria is 3.569 - 5.810 mg/L (Peroxy Esters Class - Neutral Organic SAR Class).

Based on these calculations, algae is expected to be the most sensitive species, which is supported by the experimental results.

Validity criteria fulfilled:
not applicable
Conclusions:
The calculated (ECOSAR v1.11) short-term toxicity to fish LC50 (96h) is 11.778 mg/L (Neutral Organic SAR Class) - 18.143 mg/L (Peroxy Ester Class).

Calculated short-term toxicity to aquatic invertebrates is 5.185 -7.942 mg/L (Peroxy Esters Class - Neutral Organic SAR Class).
Calculated toxicity to aquatic algae and cyanobacteria is 3.569 - 5.810 mg/L (Peroxy Esters Class - Neutral Organic SAR Class).

Based on these calculations, algae is the most sensitive species and more importantly, that fish is NOT the most sensitive species, which is supported by the experimental results.
Executive summary:

The calculated (ECOSAR v1.11) short-term toxicity to fish LC50 (96h) is 11.778 mg/L (Neutral Organic SAR Class) - 18.143 mg/L (Peroxy Ester Class).

Endpoint:
fish embryo acute toxicity (FET)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 236 (Fish embryo acute toxicity (FET) test)
Version / remarks:
No chemical analysis was conducted
5 embryos were tested per well in order to maximize statistical significance of this screening data
The test wells were covered during the test with a lid to further reduce loss by volatilization
GLP is not claimed for this data
In addition to the standard guideline criteria for mortality severe malformations that would have without doubt resulted in ultimate embryo mortality have been counted as mortalities for the purpose of this study
Well rinsing took place for all tests to minimize test substance loss to the wells
Limited screening concentrations aligned with GHS cutoffs were tested
In the third control for the third week of testing a control + Acetone was not tested. Instead two control replicates in DSW only were tested.
Deviations:
yes
Remarks:
See versions/remarks
GLP compliance:
no
Remarks:
GLP is not claimed for this data
Specific details on test material used for the study:
Chemical name: tert-amyl peroxypivalate
CAS no: 29240-17-3
Lot/Batch: 1511442890
Hydrolytical stability: Stable
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Replacement method: 200mg/L Stock, Semi Static

Solubility indication: 504 mg/L
The test material was weighed accurately and a stock solution was made in the normal manner and diluted with test medium to reach the desired test concentrations.
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 2-4 hours old when added to the test solutions. This was confirmed by microscopic observation. Tests were not rescored a few hours after start in order to replace / correct unfertilized eggs due to the number of tests conducted simultaneously.

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
Limit test:
no
Total exposure duration:
96 h
Hardness:
200 mg of CaCl2·2H2O, 180 mg of MgSO4·7H2O, 100 mg of NaHCO3 and 20 mg of KHCO3 per liter
Test temperature:
26 ºC +/- 1 ºC
pH:
8.2
Dissolved oxygen:
>80% of saturation
Conductivity:
550-650 µS/cm
Nominal and measured concentrations:
Test concentrations: 0.1,1,10,100 mg/L
Details on test conditions:
Tests were conducted with methodology as close as possible to the existing fish test data. A stock of 200 mg/L was made and subsequent dilution approach was used for generating the test concentrations.

Test vessels
Greiner “Bio-One” 24 well sterile suspension culture plates were used as test vessels. Each well contained a maximum volume 3 mL and each plate was closable to reduce evaporation of the test media. Each test plate contained 5 embryos per well totaling 25 embryos per concentration and 20 embryos as an internal plate control. For the controls 30 embryos per control were used.

In general semi-static replenishment was used as with a standard fish test after 48 hours for substances of sufficient stability. Testing was otherwise conducted according to the OECD 236 guideline with daily observations.

Every 24 hours, observations were recorded. All atypical effects on the embryo in comparison to the controls were noted. At the end of the exposure period, acute toxicity was determined based on a positive outcome in any of the four lethality observations as detailed in the OECD 236 guideline. The LC50 was then estimated where possible. For other observations there is as yet no finalized guidance on how to interpret any non-lethal findings from this assay. Other non-lethal findings were therefore recorded only at this stage.

Previous work using the OECD 236 guideline for predicting the effects of organic peroxides on adult fish showed a good level of concordance with existing adult fish data. It was noted however during this work that, in order to provide a sufficiently conservative estimation of an adult fish LC50
non-lethal effects should ideally also be included in the prediction. For this reason fish considered to have severe malformations that would ultimately result in their death were considered dead in order to make a worst case LC50 prediction for adult fish.

Acute toxicity is usually expressed as EC20,50,80 (Effect Concentration) values. The ECn values are the concentrations of the test substance showing n% reduction in survival relative to the controls. Depending on the test results obtained, the LOEC (Lowest Observed Effect Concentration) and NOEC (No Observed Effect Concentration) can also be determined. The LOEC is defined as the lowest tested concentration which survival is significantly reduced compared to the control. The NOEC is defined as the highest tested concentration at which survival shows no significant difference relative to the control. Endpoints are usually calculated using a validated statistical software program using the William’s and Trimmed Spearman-Karber / probit methods as appropriate. Dependent on the data generated statistical calculations cannot usually be carried out in screening studies due to the limited concentration range, in which case an estimation of the endpoint range has been made. In the event that the test substance is very poorly soluble, instable or a mixture, loading concentrations; Effect loading concentration (ELn) No observed effect loading rate (NOELR) were used to express the toxicity endpoints.

Test room
The test took place in a temperature controlled incubator set at 26 ºC. The test plates were scored outside of the incubator but were returned as soon as possible after scoring.

Test medium
The test medium Dutch Standard Water (DSW) was used for testing. DSW has a pH of 8.2, 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 an automatic dosing system and aerated before being used in the test.

Solution replenishment
The stocks made at the start of the test were re-used to make new dilutions for the solution replenishment in the same manner as at the start of the test. Stocks were sealed and refrigerated while not in use. Refreshments took place half way through the test after approximately 48 hours.
Old liquid was removed as much liquid as possible from each well (while avoiding drying of the embryos) using a pipette. After which the appropriate test solution was added to the test wells.

Addition of 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.
Reference substance (positive control):
yes
Remarks:
3,4-dichloro aniline
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
The substance was tested for acute toxicity to zebra fish embryos. From this data a prediction for adult fish toxicity was made.
The following validity criteria for all conducted tests were met:

• The overall fertilization rate of all of the eggs was >70%
• Temperature was maintained at 26 ºC using an alarmed incubator
• Survival in 3 of the 3 controls exceeded 90% (when corrected for fertilization loss)
• Hatching in 2 of the three controls exceeded 80% (remaining 3rd control was ended slightly before 96h)


The following validity criteria for all conducted tests were not met:
• Oxygen saturation was not measured in all controls. This had previously been demonstrated to remain acceptable when the test medium is thoroughly saturated before use. It was therefore not measured.
An occasional malformation (1-2 embryos in 60) was observed in the control replicates. This has also been the case in historical controls. This was considered when concluding on test substance related effects.

Results with reference substance (positive control):
Exposure to positive controls resulted in a minimum of 30% mortality in all 3 controls.
Sublethal observations / clinical signs:

Scoring

                       

Abbreviation

Meaning

OK

Okay (not hatched unless (H))

H

Hatched

HOK

Hatched & Okay

IC

Internal Plate Control

DEL

Delayed

BT

Severe tail bend

M

Severe malformation

ED

Edema (usually heart)

C

Coagulated Egg

D

Death/Mortality

Mob

Effects on mobility Reduced/increased

S

Reduced size

CV

Curved tail

DS

Development stalled (Coagulated)

ST

Short tail

T

Tail

 

Test substance (29240-17 -3) 96h

 


Test

1

2

3

4

5

6

% Survival

Predicted Adult Fish LC50

(% mortality)

0.1 mg/L

3HOK 1OK 1M*

2HOK

2OK 1M*

3HOK 2OK

3HOK
2OK

4HOK

1C

4HOK

1C

 

12

1  mg/L

3OK 2HOK

2HOK

1M* 2C

4HOK 1C

5HOK

4HOK 1M*

4HOK 1C

 

20

10 mg/L

3HOK

1M* 1C

3HOK 1M* 1C

5HOK

3HOK

2C

5HOK

3HOK

2C

 

24

100 mg/L

1H S*2

4C

3HOK

2S*2

3OK 1HBT 1S*2

3HOK 2OK

4C

1OK

4HOK 1M

 

36

Plate Control

 

 

 

 

 

   75

 

*= Severe tail malformations

*2 Short Tail  (recognizable not considered severe) 

LC50= >100 mg/L

 

 

RAW DATA(96h only)

Controls (20/03/17)


Test

1

2

3

4

5

6

Survival %

Malformations

Severe %

Hatching pooled

%

DSW

3HOK

2OK

2HOK

3OK

4HOK

1C

4OK

1HOK

4HOK

1C

2HOK

3OK

93

0

 

 

70

DSW+ ACETONE

2HOK

2OK

1C

4HOK

1C

5HOK

 

3HOK

2OK

3HOK

1OK

1M

4HOK

1OK

 

90

 

3

3,4, DCA

 

4M

1C

 

5M

3M

2C

4M

1C

3C

2M

3M

2C

 

0

 

70

 

0

Note:Hatching slightly low due to earlier test termination all unhatched embryos appear normal and are in the process of hatching.

Controls (10/04/17)(96h only)


Test

1

2

3

4

5

6

Survival %

Malformations

Severe %

Hatching Pooled %

DSW

5HOK

4HOK 1OK

3HOK

2OK

3HOK

2C

4HOK

1OK

3HOK

1C 1HS

90

0

 

 

93

DSW+ ACETONE

5HOK

3HOK 2C

5HOK

5HOK

5HOK

4HOK

1C

90

0

3,4, DCA

 

4M 1C

3M 2C

4M 1OK

3M 2C

3ED 1OK 1C

2C

3M

7

93

0

 

Controls (15/05/17)(96h only)


Test

1

2

3

4

5

6

Survival %

Malformations

Severe %

Hatching Pooled %

DSW I

4HOK 1C

5HOK

4HOK 1OK

3HOK 2C

4HOK

1OK

3OK

1M 1C

83

4

 

80

DSW II

3HOK 2OK

4OK 1HOK

3HOK

2OK

3HOK

2OK

3HOK 2C

4HOK

1C

90

0

3,4, DCA

 

2M 3C

2M 3C

2M 3C

2M 3C

3M 2C

3M C

0

46

0

Note: Had DSWI has not been corrected for non-fertilized eggs. Had this been the case survival would also have exceeded 90%.

Validity criteria fulfilled:
yes
Conclusions:
An estimation of the LC50 for adult fish species was made the material tested. Together with existing GLP fish data as well as the existing investigations of FET test applicability for organic peroxides the author considers it possible to reduce animal testing using this data. Either by a weight of evidence approach in conjunction with QSAR calculations or by demonstrating the suitability of read across to an existing GLP fish endpoint of an analogue or otherwise related substance.
The validity criteria were primarily 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 substance had a predicted adult fish LC50 of > 100 mg/L.

Executive summary:

The objective of this study was to screen the effects of the tested chemicals for their effects on newly fertilized zebra fish eggs and hatchlings over an exposure period of 96 hours according to the OECD 236 guideline. Data for each group of organic peroxides has been generated previously and the concordance with fish data was considered acceptable when comparing data to available adult fish test data. Screening tests covering a broader range of test concentrations but covering the familiar GHS cutoffs for aquatic toxicity have therefore been used to allow an indication of the expected toxicity range to fish species. Together with the existing adult fish data for similar substances and by using QSAR calculations it is the intention to fill existing data gaps and support read across with this data as an alternative to additional animal testing.  

The test substance had a predicted adult fish LC50 of > 100 mg/L.

Description of key information

An estimation of the LC50 for adult fish species was made. The effects on newly fertilized zebra fish eggs and hatchlings over an exposure period of 96 hours according to the OECD 236 were determined. The test substance has a predicted adult fish LC50 of > 100 mg/L. 100 mg/L shall be used as worst case for chemical safety assessment. ECOSAR is used as qualitative weight of evidence that fish is not the most sensitive endpoint, algae is the most sensitive endpoint (experimentally and qualitatively from ECOSAR).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
100 mg/L

Additional information