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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

GC-MS

Details on sampling:

For the determination of the actual test item concentrations, duplicate samples were taken from
each treatment at the start of the test.
After 24, 48 hours and at the end of the test (after 72 hours), stability samples (containing algae)
were taken in duplicate from all test concentrations and from the control.
The stability samples at 24 and 48 hours (duplicates 50 mL samples) could not be taken from
the test vessels itself, as the principle of a closed system is, that the test vessels have to remain
completely filled with test medium during the entire test period. Therefore, for this sampling at
24 and 48 hours, a set of additional flasks containing the corresponding test medium (with
algae) was incubated under conditions identical to the test.
For sampling at the end of the test, the test medium of the treatment replicates was pooled.
Additional test vessels with test medium at the dilution 1:2.3 were incubated under the
following conditions:
- Test medium without algae under light conditions in order to document possible
adsorption of the test item onto algae.
- Test medium without algae in the dark in order to document a possible photolytic
degradation of the test item.
All samples were stored frozen (at -20 ± 5 °C) immediately after sampling. Based on preexperiments for investigation of the storage stability, the test item was found to be stable in the test water under these storage conditions.
The concentrations of GR-86-6599 were analytically measured in one of the duplicate samples
taken from all sampling dates. From the additional flasks without algae, one of the duplicate
samples were measured after 24 and 72 hours.

Vehicle:

no

Details on test solutions:

At the start of test, the highest test medium with a loading rate of 50 mg/L was prepared for the main test following the slow-stirring method. For this, 125 µL of test item were carefully applied (pipetted) onto the surface of 2320 mL test water. This volume is equivalent to a loading rate of 50 mg/L, considering the relative density of the test item of 0.92. No auxiliary solvent or emulsifier was used.

The resulting test medium was slowly stirred for 48 hours at room temperature and in the dark. The mixing vessel was nearly completely filled (a small headspace had to be included as the test item was floating on the water surface) and tightly sealed with glass stoppers. After this treatment, stirring was stopped for 24 hours as a precaution to allow phase separation in case test item droplets had been mixed into the water column during stirring. Following cessation of mixing and the period of settling, the lower aqueous and equilibrated phase was carefully separated from the non-dissolved upper test item phase through the tap at the bottom of the vessel. This equilibrated aqueous phase with a loading rate of 50 mg/L, containing dissolved test item only, and considered to represent the aqueous saturation concentration in test media of the test item. The highest test concentration was subsequently diluted with test water to obtain the test media with the lower test concentrations described, below :

Undiluted Test Medium (loading rate of 50 mg/L)
Dilution 1 : 1.5
Dilution 1 : 2.3
Dilution 1 : 3.4
Dilution 1 : 5.1

Additionally, a control (test water only) was run in parallel.

The test media were prepared just before the start of the test.

The preparation of the test media was based on the OECD Guidance Document No. 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

The test organism used for the study was Pseudokirchneriella subcapitata (formerly
Selenastrum capricornutum, occasionally also listed as Raphidocelis subcapitata),
Strain No. 61.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant
Physiology, University of Göttingen, 37073 Göttingen / Germany). The algae were cultivated
at IES Ltd Laboratories under standardized conditions according to the test guidelines.

An inoculum culture was set up three days before the start of the exposure. The algae were cultivated under the test conditions and were kept in the exponential growth phase until inoculation of the test solutions.

The test method and the test species are recommended by the test guidelines

Test type:

static

Water media type:

freshwater

Remarks:

Reconstituted test water (AAP Medium)

Limit test:

no

Total exposure duration:

72 h

Hardness:

15 mg/L

Test temperature:

22°C

pH:

7.6 - 7.9

Nominal and measured concentrations:

The following starting convcentrations were prepared :
Undiluted Test Medium (loading rate of 50 mg/L)
Dilution 1 : 1.5
Dilution 1 : 2.3
Dilution 1 : 3.4
Dilution 1 : 5.1

Concentrations of test item detected and the mean measured test concentration are, as follows in µg/L :

0 Hours 24 Hours 48 Hours 72 Hours Geometric Mean Concn.
1:5.1 35.1 12.3 1:3.4 57.7 19.3 5.07 1:2.3 75.2 36.7 10.8 1:1.5 94 36.0 10.3 Undiluted° 123 59.1 14.5 Test Medium

°: Undiluted equilibrated medium with a loading rate of 50 mg/L.

Details on test conditions:

Since the test item was determined to be volatile, glass stoppered Erlenmeyer flasks were used completely filled (without headspace) with about 60 mL of test medium and tightly sealed with glass stoppers to avoid losses of the volatile substance by evaporation (closed system). The test flasks were labeled with the study number and all necessary additional information to ensure unique identification.

The test flasks were incubated in a temperature controlled orbital shaker (Multitron-Pro, Infors HT, Bottmingen/Switzerland) at a temperature of 22 °C. The test flasks were positioned randomly and repositioned daily. They were continuously illuminated by LED light installed above the test flasks. The light intensity at the level of the test solutions was approximately 67 µE s-1 m-2 (range: 66 to 68 µE s-1 m-2, measured at nine places in the experimental area). The light intensity over the incubation area was within a ±15%-deviation from the average light intensity as recommended by the guideline.

The main test was performed in a closed system. The test design included three replicates per
test concentration and six replicates for the control.

The test was started using a nominal algal cell density of 5000 cells/mL. The initial cell density
was selected according to the recommendations of the OECD test guideline. The algal cell
density in the pre-culture was determined using an electronic particle counter (Coulter
Counter, Model Z2).

A static test design was applied. The duration of the test was 72 hours.

Determination of Algal Biomass :
A small volume (200 µL) of the algal suspension was withdrawn daily from each test flask for the measurement of the biomass, and was not replaced.

The algal biomass in the samples was determined by fluorescence measurement (SpectraMax I3x, Molecular Devices Ltd, Wokingham Berkshire/UK). The measurements were performed at least in duplicate at an excitation of 440 nm and emission of 680 nm.

At the end of the test, a sample was taken from the control and from the dilution 1:2.3 to determine a potential influence of the test item on the algal cells. The shape and size of the algal cells were visually inspected. This test concentration was chosen since the algal cell density at the two highest dilutions was too low for a reliable examination.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 22 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

This exposure concentration represents the maximum water saturation concentration of the test item

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 22 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

This exposure concentration represents the maximum water saturation concentration of the test item

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 22 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other:

Remarks:

This exposure concentration represents the maximum water saturation concentration of the test item

Details on results:

Growth rate :
At the lowest test concentration of 6.8 µg/L there was no statistically significant difference from the control (results of Williams t-test, one-sided smaller, alpha = 0.05).

Up to and including the highest mean measured concentrations of 22.0 µg/L, the aqueous saturation concentration in test media of the test item, the mean inhibition compared to the control was in the range of 0.8 to 3.9% and was statistically significantly different from the control. This statistical significance was caused by the very low variability between replicates within each concentration (coefficient of variation in the range of 0.2 to 0.6%). However these statistically significant findings were not estimated as biologically relevant toxic effects, since the mean inhibition compared to the control was below 4%.

Therefore, the NOEC for growth rate was determined to be at the highest mean measured concentration of 22.0 µg/L. The EC10 for growth rate could not be calculated due to inhibition values lower than 4% and was therefore determined greater than 22.0 µg/L.

No effects observed on algal growth rate up to and including the highest tested exposure concentration, equivalent to the saturation concentration.

Yield :
At the lowest test concentration of 6.8 µg/L there was no statistically significant difference from the control (results of Williams t-test, one-sided smaller, alpha = 0.05).

At the mean measured concentrations of 10.6 and 16.1 µg/L, the mean inhibition compared to the control was 4.2 and 6.5%, respectively and was statistically significantly different from the control. These statistical significances were caused by the very low variability between replicates within each concentration (coefficient of variation of 2.1 and 2.9%, respectively). However these statistically significant findings were not estimated as biologically relevant toxic effects, since the maximum level of inhibition was 6.5%.

At the two highest mean measured concentrations of 16.7 and 22.0 µg/L the mean inhibition compared to the control was 12 and 18%, respectively and was statistically significantly different from the control.

Therefore, the NOEC for yield was determined to be at the mean measured concentration of 16.1 µg/L. The EC10 for this endpoint was calculated to be 16.6 µg/L.

The EC50 for yield was not attained and is greater than 22.0 µg/L, the highest test concentration corresponding to the saturation concentration in test media of the test item.

The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the dilution 1:2.3 (mean measured 16.1 µg/L) and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.

No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.

The pH in the control was 7.6 at test start and 7.9 at test end fulfilling the requirement of the OECD guideline that the pH of the control medium should not increase by more than 1.5 units during the test.

The pH of the test media was in the range of 7.6 to 7.9 during the test period.

The water temperature during the test was maintained at 22 °C.

Results with reference substance (positive control):

For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions. The 72-hour EC50 for growth rate in the reference test IES Study Number 20160277 was 0.9 mg/L (October 2016) and showed that the sensitivity of the test system was within the range recommended by the guideline (72-hour EC50 for the growth rate 0.9 1.5 mg/L).

Validity criteria fulfilled:

yes

Conclusions:

Endpoint after 72 Hours Growth Rate [μg/L] Yield [μg/L]
EC10 >22.0 16.6
(95% Confidence Interval) n.d. (15.7-17.5)
EC20 >22.0 >22.0
EC50 >22.0 >22.0
NOEC > = 22.0 16.1
LOEC n.d 16.7
n.d.: Not determined.

Executive summary:

The impact of the test item GR-86-6599 on the growth of the freshwater green algal species

Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to the

OECD Guideline 201 (2006, corrected 2011) and the Commission Regulation (EU)

No 2016/266, C.3.

As the test item is a volatile substance, the test was performed using glass stoppered Erlenmeyer

flasks (closed system) completely filled with test medium, minimizing the air space in the flasks

and avoiding potential losses of test item by evaporation.

As the test item is a liquid with low water solubility, the slow stirring method was applied for

preparation of a saturated test item solution. For preparation of the highest concentrated test

medium, the test item was carefully applied (pipetted) onto the surface of the test water at a

loading rate of 50 mg/L. Thereafter slow stirring was applied for 48 hours in a closed vessel to

reach a maximum concentration of dissolved test item in the test water. The mixing vessel was

nearly completely filled (a small headspace had to be included as the test item was floating on

the water surface) and tightly sealed with glass stoppers. After this treatment, stirring was

stopped for 24 hours as a precaution to allow phase separation in case test item droplets had

been mixed into the water column during stirring. Following cessation of mixing and the period

of settling, the lower aqueous and equilibrated phase was carefully separated from the nondissolved

upper test item phase through the tap at the bottom of the vessel. This equilibrated

aqueous phase with a loading rate of 50 mg/L, containing dissolved test item only, was used as

the highest test concentration and considered to represent the aqueous saturation concentration

in test media of the test item. The highest test concentration was subsequently diluted with test

water to obtain the following test media: the dilutions 1:5.1, 1:3.4, 1:2.3 and 1:1.5. Additionally,

a control (test water without test item) was tested in parallel.

The preparation of the test media was based on the OECD Guidance Document No. 23 on

Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

No significant algacidal effects of the test item were observed on the growth rate component at

test concentrations up to and including the highest exposure concentration which corresponded

to the saturation concentration in test media of the test item. Only minor statistically significant

effects on the yield of algae were observed in this study with only 18% inhibition occurring at

the saturation exposure concentration

Description of key information

The impact of the test item GR-86-6599 on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to the

OECD Guideline 201 (2006, corrected 2011) and the Commission Regulation (EU) No 2016/266, C.3.

 

No significant algacidal effects of the test item were observed on the growth rate component at test concentrations up to and including the highest exposure concentration which corresponded to the saturation concentration in test media of the test item.

72h ErC50 > 22 μg/L

72h ErC10 > 22 μg/L

NOErC > = 22 μg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

22 µg/L

EC10 or NOEC for freshwater algae:

22 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).