ethyl 2-acetyl-4-methyltridec-2-enoate

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:

November 16, 2020 to March 29, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Version / remarks:

2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

GC/MS

Details on sampling:

For measurement of the actual concentration of the test item in this semi-static test with daily test medium renewal, duplicate 50 mL samples were taken from the test medium and the control in small glass vials at the start and the end of each test medium renewal period.
Each sample was stored frozen (at –20 ± 5 °C) and protected from light immediately after sampling until analysis. Sample storage stability had been confirmed under these storage conditions in a previous non-GLP study (IES Study No. 20160270).
The concentrations of the test item in the test medium and the control were analytically measured in one of the duplicate samples from all sampling times. The other duplicate sample remained as back-up stored frozen.

Vehicle:

no

Details on test solutions:

As the test item is a liquid with low water solubility, the slow-stirring method (to avoid formation of micro-droplets) was chosen for the preparation of the test medium. The test item was carefully applied at a loading rate of 50 mg/L to the surface of the test water in four 5 L laboratory glass bottles. The vessels were nearly completely filled (a small headspace had to be included as the test item was floating on the water surface) and tightly sealed with Teflon stoppers to avoid possible test item losses by evaporation during stirring. The dissolving of the test item in the test water was facilitated by slow stirring for 48 hours at room temperature in the dark.

After this treatment, stirring was stopped for 24 hours as a precaution to allow phase separation in case test item droplets were mixed into the water column during stirring. Following cessation of mixing and the period of settling, a Teflon hose was used to carefully siphon the equilibrated phase from the middle of the water body avoiding the non-dissolved upper test item phase in the bottle. Approximately 3.5 L of this equilibrated aqueous phase was collected from each of the four bottles, combined and mixed into a large glass vessel. A part of this solution, which was considered to represent the saturation concentration in test water under these conditions, was used as the test medium for this limit test. No auxiliary solvent or emulsifier was used. No filtration step was applied.

This procedure was repeated at each renewal interval of 24 hour for the preparation of fresh test medium.

The principle of the slow-stirring procedure is the following: The liquid test item with a specific gravity of < 1.0, is floating on the surface of the test water during the equilibration process while the water phase is gently stirred. In this way, the formation of micro-droplets of test item in the water phase is avoided, as can occur when intensive mixing is applied. Since during slow-stirring the undissolved test item is not physically mixed into the water column, it is assumed, that the water phase is containing dissolved test item only and can be used as test medium without further treatment (e.g. filtration). This remains, nonetheless, a delicate operation particularly for hydrophobic organic test substances with very low water solubility, as per SCENTAURUS CLEAN (solubility in pure water is 0.04 mg/L at 20 °C (OECD 105)).

The preparation of the test medium was based on the OECD Guidance Document No. 23 on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals, 2019.

At the start and end of each test medium renewal period in the freshly prepared and aged test medium, the test medium was visually controlled.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

The fish were obtained from a breeding culture at IES Laboratories and were from the same batch. They all were juveniles of the same age and of normal appearance and behavior throughout the acclimatization time. Prior to test start, the test fish were acclimated for at least one week to the test water and temperature.
During holding and acclimatization until one day before the start of the test, the fish were fed with a commercial fish diet (TetraMin® Hauptfutter, supplied by TETRA-Werke, 49324 Melle / Germany) at least six days of the week.
During the last three weeks prior to the test, no fish died in the test fish batch and all fish were healthy. No medication was applied during holding and acclimatization of the fish.
From the acclimated test fish batch, a representative sub-batch of 10 fish was measured at the start of the test. The mean total body length (measured from the mouth to the end of the tail fin) of the fish was 1.85 ± 0.05 cm (Mean ± SD), with the longest fish at 1.9 cm and the shortest at 1.8 cm. The mean body wet weight was 0.06 ± 0.01 g (Mean ± SD). The measured fish were not used for the test.

Test type:

semi-static

Water media type:

freshwater

Remarks:

Reconstituted test water

Limit test:

yes

Total exposure duration:

96 h

Hardness:

The calculated water hardness of this reconstituted test water was 125 mg/L (as CaCO3), i.e. in the recommended range of 40-250 (preferably <180) mg/L (as CaCO3).

Test temperature:

The water temperature was measured to be in the range of 21.6 to 22.1 °C during the test period.

pH:

The pH values measured in the test medium and the control were in the range of 7.1 to 7.3 during the test period.

Dissolved oxygen:

The dissolved oxygen concentration was in the range of 8.6 to 8.7 mg/L at the start of the test medium renewal periods and slightly decreased to 7.6 – 8.2 mg/L (i.e. 85 – 92 % saturation) in the closed system during the 24-hour renewal periods. Therefore, the oxygen concentration never dropped below 60 % of the air saturation value.

Nominal and measured concentrations:

The measured initial concentrations at the start of the renewal periods were in the range of 52.6 to 66.6 μg/L.
This shows that the slow-stirring method applied for the daily preparation of the test medium was sufficiently reproducible. The concentrations at the end of the renewal periods were in the range of 39 to 58 % of the initial measured concentrations. Thus, even by applying a closed system and a semi-static test design concentration a loss or degradation of the test item could not be completely avoided.

Geometric Mean measured concentration = Water saturation concentration = 39.3 μg/L.

Concn. at Start Concn. at End
of Renewal (microg/L) of Renewal (microg/L)
0h / 53.1 24h / 20.9
24h / 52.6 48h / 22.2
48h / 66.6 72h / 31.0
72h / 58.4 96h / 33.8

Details on test conditions:

As a closed exposure system was applied, the test was performed semi-static with a test medium renewal every 24 hours. This was necessary to keep the oxygen concentrations in the closed system sufficiently high during the renewal periods of 24 hours. Furthermore, in this way also the test item concentration in the test medium was kept as constant as possible during the test period of 96 hours.

At the start of the test, seven fish were introduced into each test vessel in a random order. The loading rate of the fish was 0.08 g fish wet weight per liter medium. Every 24 hours, the surviving fish of the test item treatment and the control were carefully transferred into a new test vessel with freshly prepared test medium and test water, respectively.

The fish were checked twice every 24-hour period (in the morning and the afternoon) for mortality and visible abnormalities. The first and second checks were approximately 2 and 5 hours after the fish were introduced into the test medium, i.e. the test start.

The NOEC and LC0 were determined directly from the Raw Data. The LOEC, LC50 and LC100 at the observation times could not be quantified due to the absence of a toxic effect, lethal or sub-lethal, of the test item at the tested concentration.

A 16-hour light to 8-hour dark photoperiod, with a 30-minute transition period was used. Since the test item is suspected to be potentially light sensitive, the test was performed under dimmed light conditions. The light intensity during the light period was measured to be < 1 μE s-1 m-2.

Key result

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 39.3 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: This exposure concentration represents the maximum water saturation concentration of the test item.

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 39.3 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Observation for sub-lethal effects : behaviour and appearance.

Remarks on result:

other: This exposure concentration represents the maximum water saturation concentration of the test item.

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

>= 39.3 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: This exposure concentration represents the maximum water saturation concentration of the test item.

Details on results:

The measured initial concentrations at the start of the renewal periods were in the range of 52.6 to 66.6 μg/L and were reproducible. This shows that the slow-stirring method applied for the daily preparation of the test medium was sufficiently reproducible. The concentrations at the end of the renewal periods were in the range of 39 to 58 % of the initial measured concentrations. Thus even by applying a closed system and a semi-static test design a loss or degradation of the test item could not be completely avoided.
Due to the decrease of the test item concentration during the renewal period, the mean concentrations of the test item during the renewal periods of 24 hours were calculated as the geometric means of the concentrations measured at the start and the end of each of the four test medium renewal periods. From the four geometric mean values obtained, the mean measured test item concentration during the test period of 96 hours was calculated as arithmetic mean. The resulting mean measured test item concentration was 39.3 μg/L.

The biological results are based on the mean measured concentration of 39.3 μg test item/L
Therefore, the 96-hour NOEC and LC0 of SCENTAURUS CLEAN to Zebra fish were both determined to be ≥ 39.3 μg/L, i.e. at the water saturation under these test conditions. The 96-hour LOEC, LC50 and LC100 could not be quantified due to the absence of toxicity of SCENTAURUS CLEAN at the tested concentration and were clearly higher than 39.3 μg/L.

Results with reference substance (positive control):

The test was considered valid as no fish died in the control and the tested threshold concentration during the 96-hour exposure period.

The oxygen concentration in the test medium and the control did not drop below 60 % of the air saturation during the test.

Validity criteria fulfilled:

yes

Conclusions:

The test item SCENTAURUS CLEAN had no acute toxic effects on juvenile Zebrafish (Danio rerio) at the sole tested concentration of 39.3 μg/L (mean measured concentration) under the tested conditions (i.e. semi-static test with 24 h renewal periods in a closed system). Therefore, the 96-hour LC50 for fish was determined to be > 39.3 μg/L (the water saturation concentration under these conditions).
According to the threshold approach, it was demonstrated that the test organism fish is not more sensitive to the SCENTAURUS CLEAN compared to daphnia and algae, where acute exposure results in no effects at saturation for the three trophic levels. According to present data in the REACH dossier EC 816-455-0, the 72 h EC50 for algae was determined to be > 22.0 μg/L, based on both growth rate and yield (OECD 201) and the 48 h EC50 for daphnia was determined to be > 123 μg/L (OECD 202). The respective EC50 values represent the saturation concentration of the test item.
This valid test was performed according to the OECD Guideline for the Testing of Chemicals, No. 203, Fish, Acute Toxicity Test, 2019.

Executive summary:

The acute toxicity of the test item SCENTAURUS CLEAN to juvenile fish of the species Zebrafish (Danio rerio) was determined in a 96-hour test according to the OECD Guideline for the Testing of Chemicals, No. 203, Fish, Acute Toxicity Test, 2019.

A limit test with one test concentration (at water saturation) was performed to demonstrate that the test organism fish is not the most sensitive test organism, under acute exposure conditions, to SCENTAURUS CLEAN compared to algae and daphnia. The test method is based on the threshold approach as developed for chemical substances at the European Commission’s Joint Research Centre.

As the test item is a liquid with low water solubility, the slow-stirring method was chosen for the preparation of the test medium. The slow stirring method is an alternative method in case filtration is not possible due to specific properties of the test item, i.e. oily liquid. The test item was carefully applied to the surface of the test water in the stirring vessel at a loading rate of 50 mg/L. The stirring vessel was nearly completely filled and tightly sealed with glass stopper to avoid possible test item losses by evaporation during stirring. Thereafter slow-stirring was applied for 48 hours in the dark to reach the maximum possible concentration of the test item in the test water. After a settling period of 24 hours, the middle part of the water column was collected and used for the test.

A semi-static test design with daily test medium renewals was applied, since the test item was expected to degrade during the exposure period (results of previous OECD 201 and OECD 202 studies), in order to maintain the test item concentration during the exposure period of 96 hours as much as possible and to provide sufficient oxygen saturation in the closed system used.

The test design was based on the OECD Guidance Document No. 23 on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals, 2019.

Analytical results

The measured concentrations of the test item SCENTAURUS CLEAN in the test medium at the start and end of the four test medium renewal periods are shown in the table on the next page. The measured initial concentrations at the start of the renewal periods were in the range of 52.6 to 66.6 μg/L. This shows that the slow-stirring method applied for the daily preparation of the test medium was sufficiently reproducible. The concentrations at the end of the renewal periods were in the range of 39 to 58 % of the initial measured concentrations. Thus, even by applying a closed system and a semi-static test design concentration a loss or degradation of the test item could not be completely avoided.

Due to the decrease of the test item concentration during the renewal period, the mean concentrations of the test item during the renewal periods of 24 hours were calculated as the geometric means of the concentrations measured at the start and the end of each of the four test medium renewal periods. From the four geometric mean values obtained, the mean measured test item concentration during the test period of 96 hours was calculated as arithmetic mean. The resulting mean measured test item concentration was 39.3 μg/L.

Renewal period (24 h each)	Analytically Measured Concentrations of the Test Item SCENTAURUS CLEAN* at the start of the renewal period (μg/L)	Analytically Measured Concentrations of the Test Item SCENTAURUS CLEAN* at the end of the renewal period (μg/L)	Analytically Measured Concentrations of the Test Item SCENTAURUS CLEAN* at the end of the renewal period (% of initial)	Mean Measured Concentration for each Renewal Period (geometric mean)   (μg/L)	Mean Measured Concentration for the Total Test Period (arithmetic mean)   (μg/L)
1	53.1	20.9	39	33.3	39.3
2	52.6	22.2	42	34.2
3	66.6	31.0	47	45.4
4	58.4	33.8	58	44.4

*: in equilibrated test medium prepared from the aqueous phase at a loading rate of 50 mg/L after 48 h of slow-stirring and a settling period of 24 h.

Biological results

At the start of the test, seven fish were introduced into each test vessel (i.e. test item treatment and control) in a random order. Every 24 hours, the fish were carefully transferred into a new test vessel with freshly prepared test medium. The fish were observed for visible abnormalities and mortality twice every 24-hour period.

All fish in the test survived until the end of the 96 hours exposure period and no toxic effect, lethal or sub-lethal, on the test organism fish were observed.

The biological results after 96 hours of exposure to SCENTAURUS CLEAN, based on the mean measured concentration of 39.3 μg test item/L are presented in the table below:

Endpoints after 96 Hours Test Period	Based on Mean Measured Concentration of the Test Item SCENTAURUS CLEAN [μg/L]
96-hour LC50	> 39.3
96-hour LC0	>= 39.3
96-hour LC100	> 39.3
96-hour NOEC	>= 39.3
96-hour LOEC	> 39.3

In conclusion, the test item SCENTAURUS CLEAN did not cause mortality or sub-lethal effects in zebrafish at the sole tested concentration of 39.3 μg/L, i.e. at the water saturation under these test conditions. Thus, the 96-hour LC50 for fish was determined to be > 39.3 μg/L. The test demonstrated that the test organism fish is not more sensitive to the test item compared to daphnia and algae, where acute exposure results in no effects at saturation for the three trophic levels. According to present data in the REACH dossier EC 816-455-0, the 72 h EC50 for algae was determined to be > 22.0 μg/L, based on both growth rate and yield (OECD 201) and the 48 h EC50 for daphnia was determined to be > 123 μg/L (OECD 202). The respective EC50 values represent the saturation concentration of the test item.

Validity Criteria:

The test was considered valid as no fish died in the control and the tested threshold concentration during the 96-hour test period. The oxygen concentration in the test medium and the control did not drop below 60 % of the air saturation during the test.

Note:

A first limit test was conducted, which resulted in toxic effects and mortality for the fish. This result triggered, according to the guideline, the performance of a full test. However, the analytical and biological results of the full test, with a more than 10 times lower test item concentration in the saturated test medium and no toxicity to fish, were inconsistent with the results of the first limit test.

The discrepancy between the results of these two tests questioned the informative value of the study. Therefore, a confirmatory third test, designed again as a limit test, was performed.

The inspection of the test procedures applied for the first two tests and the observations made during the third test, revealed that the higher test item concentration obtained in the first limit test was caused by minimal technical deficiencies during the test media preparation procedure. A possible contamination of the saturated test medium with fractions of undissolved test item, which may have induced the toxic effect to the fish, could not be excluded.

As a consequence of these observations the results of the first limit test were not taken into consideration for the evaluation of the present study. Instead, the results of the third test, i.e. the limit test with valid test media preparation, were applied for the study evaluation and are in-line with the findings of the dose-response test also.

Description of key information

The acute toxicity of the test item SCENTAURUS CLEAN to juvenile fish of the species Zebrafish (Danio rerio) was determined in a 96-hour test according to the OECD Guideline for the Testing of Chemicals, No. 203, Fish, Acute Toxicity Test, 2019 under GLP conditions.

 

The test item SCENTAURUS CLEAN did not cause mortality or any sub-lethal effects in zebrafish at the sole tested concentration of 39.3 μg/L, i.e. at the water saturation under these test conditions. Thus, the 96-hour LC50 for fish was determined to be > 39.3 μg/L, LC0 and NOEC > or = 39.3 μg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Dose descriptor:

LC50

Effect concentration:

> 39.3 µg/L

Additional information

According to the EU CLP regulation (No 1272/2008 and its adaption 286/2011), Scentaurus Clean doesn't need to be classified as Hazardous to the Aquatic Environment Acute 1 nor Chronic classifications (Acute results represent saturation concentration of the test item).