(1,2-dioxoethylene)bis(iminoethylene) bis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate]

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 November 2017 to 01 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

OECD Guideline for Testing of Chemicals, No. 203, "Fish, Acute Toxicity Test", adopted July 17, 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.1 (Acute Toxicity for Fish)

Version / remarks:

Commission Regulation (EU) 2017/735 of 14 February 2017 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), Annex Part C, C.1.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)

Version / remarks:

EPA Health Effects Test Guidelines, OCSPP 850.1075

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

No further details specified in the study report.

Analytical monitoring:

yes

Details on sampling:

The concentration of the test item was measured at the test concentration at the beginning and at the end of the experiment.

Vehicle:

no

Details on test solutions:

Because the test item is poorly soluble in water, a test solution was prepared using a saturated solution method according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23.
Saturated test item solution (100 mg/L nominal loading rate) was prepared by dispersing/dissolving the amount of test item into the test medium (aquarium water) two days before the start of the study. This solution was shaken for about 24 hours at approximately 30°C and then equilibrated for about 24 hours at approximately 20°C. The non-dissolved test material was removed by filtration through a fine (0.22 µm) filter to give appropriate 100 mg/L nominal loading rate. The solution was considered to be saturated.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

Species: Zebrafish (Brachydanio rerio)
Source: István Szent University, 2100 Gödöllő, Páter Károly u. 1, Hungary
Justification of species: Zebrafish (Brachydanio rerio) is one of the convenient species for acute fish toxicity test.
Number of animals: seven fish per test group
Body length of animals: 2.2 – 2.6 cm
Food and Feeding: The fish were not fed during the test
Acclimatisation: more than 12 days
Animal health: Fish were bred in a well-known fish farm, under disease- and parasite-controlled conditions. Fish were observed during the acclimatisation and test periods, the fish were healthy. There was no mortality of the population for seven days before the test.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Post exposure observation period:

No post observation period specified.

Hardness:

The hardness of the dilution water was determined as 191 mg/L (as CaCO3).

Test temperature:

The test temperature was between 20.5 and 21.5 °C.

pH:

The pH was in the range of 7.38 – 8.13.

Dissolved oxygen:

The dissolved oxygen concentration was in the range of 67 – 99 % of the air saturation value at the temperature used.

Salinity:

Not specified.

Conductivity:

Not specified.

Nominal and measured concentrations:

Because no toxic response was observed during the preliminary range-finding test, only one test concentration at 100 mg/L nominal loading rate and one control group was tested in the definitive study in a static system (based on analytical results indicated).
Test item concentration could not be detected and therefore measured concentration was below the Limit of Detection (LOD = 0.01 mg/L) at each measurement during the experiment, consequently biological results were based on the nominal loading rate.

Details on test conditions:

Environmental test conditions
The water temperature, oxygen concentration and pH were measured daily during the experiment in each test aquarium.
The test temperature was between 20.5 and 21.5 °C.
The dissolved oxygen concentration was in the range of 67 – 99 % of the air saturation value at the temperature used.
Test solutions were aerated during the test to avoid decreasing of dissolved oxygen concentration below the accepted level.
The pH was in the range of 7.38 – 8.13.
The hardness of the dilution water was determined as 191 mg/L (as CaCO3).
The light-dark cycle during the test was 16 hours light and 8 hours darkness.

Description of the Test Procedure
The test duration was 96 hours. One aquarium was used for test group and for the control group, respectively. Each aquarium comprised 7 fish and 5 L test solution. The animals were not fed during the test. The loading of the test aquaria was less than 1.0 g fish/L test solution at the start of the experiment.
The choice of the test concentrations was made on the basis of the results of the preliminary range-finding test.

Preliminary Range-Finding Test
A preliminary concentration range-finding test was conducted to determine the approximate toxicity of the test item so that appropriate test concentrations could be selected for use in the definitive test. Four fish were exposed to each concentration of the test item plus a control, for 96 hours under semi-static conditions (with 24-hour renewal in absence of stability data).
During the formulation procedure the test item stock solution was prepared by similar method described above. The test solutions were prepared by appropriate diluting of this stock solution.

Observations
The observations of fish were carried out in the following intervals: at 3, 6, 24, 48, 72 and 96 hours. Mortality or any sub-lethal effects were not observed during the experiment.
The test conditions (pH, temperature, oxygen saturation) were measured daily during the test.
The body weights of the fish were recorded before the introduction of fish. The body weight of 7 fish per aquarium was registered. The loading of the aquaria was calculated on the basis of these body weights (1.0 g fish/litre testing liquid).

Reference substance (positive control):

no

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Key result

Duration:

96 h

Dose descriptor:

LC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Details on results:

MORTALITY DATA: No sublethal effect was observed.
BODY WEIGHT: There was no considerable difference observed concerning body weights between the groups.

Results with reference substance (positive control):

Reference substance not reported.

Reported statistics and error estimates:

No statistical analysis was performed because of the lack of toxic effects.
The LC50, NOEC, LOEC and LC100 were determined directly from the raw data.

Sublethal observations / clinical signs:

Results of the Preliminary Range-Finding Test

Nominal concentration [% saturated solution]	Untreated control	0.1	1	10	100
Number of treated animals	4	4	4	4	4
Number of dead animals	0	0	0	0	0

 

Cumulative mortality data in the Limit Test

Test Group	Cumulative Mortality (number of dead fish) [initial population = 7 fish]
	3h	6h	24h	48h	72h	96h
Control	0	0	0	0	0	0
100 mg/L nominal loading rate	0	0	0	0	0	0

 

Measured and calculated data of bodyweight

Test Group	Measured weight of 7 fish (g)	Calculated mean weight of 1 fish (g)	Loading of testing aquarium (g fish/L testing liquid)
Control	1.23	0.18	0.25
100 mg/L nominal loading rate	1.80	0.26	0.36

 

Temperature

Test Group	Temperature (°C)
	Measuring
	0h	24h	48	72	96
Control	21.5	20.8	20.6	20.5	20.6
100 mg/L nominal loading rate	21.5	20.8	20.6	20.6	20.5

 

Dissolved oxygen concentration

Test Group	Dissolved oxygen concentration (%)
	Measuring
	0h	24h	48	72	96
Control	87	86	98	97	99
100 mg/L nominal loading rate	67	87	97	96	98

 

Temperature

Test Group	pH
	Measuring
	0h	24h	48	72	96
Control	7.76	7.48	7.38	7.71	7.64
100 mg/L nominal loading rate	8.13	7.82	7.73	7.95	7.78

 

Body length of test fish at the end of the test

Test Group	Body length [cm]
	1	2	3	4	5	6	7
Control	2.5	2.3	2.3	2.4	2.3	2.2	2.3
100 mg/L nominal loading rate	2.2	2.6	2.4	2.4	2.3	2.5	2.4

Remark: At the end of the test, body length of test fish were measured in order to check their compliance with the size range recommended for the species by the test guideline [OECD Guideline for Testing of Chemicals, No. 203, “Fish, Acute Toxicity Test”, adopted July 17, 1992].

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of NAUGARD® XL-1 was assessed with acute fish toxicity test on Zebrafish (Brachydanio rerio), over an exposure period of 96 hours in a static system.

Under the conditions of the acute fish toxicity study on Zebrafish (Brachydanio rerio) the observed and calculated endpoints for the effect of NAUGARD® XL-1 were the following:

The 24h, 48h, 72h and 96h LC50 value: > 100 mg/L nominal loading rate
The 96h LC100 value: > 100 mg/L nominal loading rate
The 96h No-Observed Effect Concentration (NOEC): 100 mg/L nominal loading rate
The 96h Lowest Observed Effect Concentration (LOEC): > 100 mg/L nominal loading rate

Executive summary:

The acute toxicity of the test item was assessed with acute fish toxicity test on Zebrafish (Brachydanio rerio), over an exposure period of 96 hours in a static system.

 

Because no toxic response was observed during the preliminary range-finding test, only a Limit test was carried out using only one test concentration at 100 mg/L nominal loading rate (100 % saturated solution) and one control group in the definitive study under static conditions.

 

The test concentration was analytically determined at the start and at the end of the experiment. Test item concentration could not be detected and therefore measured concentration was below the Limit of Detection
(LOD = 0.01 mg/L) for all samples during the experiment. The biological results are based on the nominal loading rate.

 

One aquarium was used in the test group and one in the control group. Each aquarium comprised seven fish and five litre test solution.

 

All achievable validity criteria were met during this study.

 

Under the conditions of the acute fish toxicity study on Zebrafish (Brachydanio rerio) the observed and calculated endpoints for the effect of NAUGARD® XL-1 were the following:

 

The 24h, 48h, 72h and 96h LC50 value: > 100 mg/L nominal loading rate

The 96h LC100 value: > 100 mg/L nominal loading rate

The 96h No-Observed Effect Concentration (NOEC): 100 mg/L nominal loading rate

The 96h Lowest Observed Effect Concentration (LOEC): > 100 mg/L nominal loading rate

Description of key information

The 96h No-Observed Effect Concentration (NOEC): 100 mg/L nominal loading rate

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

100 mg/L

Additional information

The acute toxicity of the test item was assessed over an exposure period of 96 hours in a static system.

Under the conditions of the acute fish toxicity study on Zebrafish (Brachydanio rerio) the observed and calculated endpoints for the effect of NAUGARD® XL-1 were the following:

 

The 24h, 48h, 72h and 96h LC50 value: > 100 mg/L nominal loading rate

The 96h LC100 value: > 100 mg/L nominal loading rate

The 96h No-Observed Effect Concentration (NOEC): 100 mg/L nominal loading rate

The 96h Lowest Observed Effect Concentration (LOEC): > 100 mg/L nominal loading rate