Ethyl 4-methyl-2-oxocyclohexanecarboxylate

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:

June 6, 2017 to January 11, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Reliability 1 is assigned because the study conducted according to OECD TG 201 in compliance with GLP, without deviations that influence the quality of the results.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

FRET 13-0545
4-methyl-2-oxocyclohexanecarboxylic acid, ethyl ester
lot number PDJ378-68
Purity 93.2%
Expiration date June 2018

Analytical monitoring:

yes

Details on sampling:

Samples of the test solutions were collected at approximately 0 and 72 hours of exposure to
measure concentrations of the test substance. At the start of the test, samples were collected from the
individual batches of test solution prepared for each treatment and control group prior to distribution into
the test chambers. At 72 hours of exposure, the remaining biological replicates from each respective
treatment and control group sacrificed at that time interval were pooled, and then sampled. Samples were
also collected from the abiotic replicate included in the nominal 25 mg a.i./L treatment group. All
samples were collected in Teflon extraction tubes pre-rinsed with hexane for the immediate analysis
samples and plastic scintillation vials for the refrigerated samples. All samples were acidified with 10%
phosphoric acid immediately after collection, to stabilize the test substance. An additional set of samples
were collected at each sampling interval and stored refrigerated for potential future analysis.

Vehicle:

no

Details on test solutions:

Test solutions were prepared at target nominal concentrations of 6.3, 13, 25, 50, and
100 mg a.i./L by weighing calculated masses of FRET 13-0545 on an analytical balance and qualitatively
transferring the test substance with freshwater AAP medium with additional sodium bicarbonate added
into 4000 mL glass aspirator bottles. The bottles were filled to the maximum volume of each bottle with
freshwater AAP medium with additional sodium bicarbonate added, leaving no headspace. The test
solutions were stirred for approximately 3.5 hours to mix. All test solutions appeared clear and colorless
and were otherwise unremarkable after mixing. The negative control solution consisted of freshwater
AAP medium with additional sodium bicarbonate added. Two aspirator bottles were used for the
negative control to provide adequate test solution for the required number of negative control replicates.

Test organisms (species):

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

Details on test organisms:

The freshwater green alga, Raphidocelis subcapitata, was selected as the test species for this
study. Algal cells used in this test were obtained from EAG Laboratories cultures that had been actively growing in culture medium for at least two weeks prior to test initiation. Algal cells for this study were taken from a culture that had been transferred to fresh media four days prior to test initiation.
Prior to test initiation, the cell density of R. subcapitata culture 17-01 was determined to be 1.20 x 106 cells/mL using a hemacytometer and microscope. In order to achieve a nominal concentration of approximately 5,000 cells/mL at test initiation 1.25 mL of culture was added to each biotic replicate test vessel.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23.8-24.05 C

pH:

7.8-10.5
Although the pH increased by greater than 1.5 units over the course of the test, this is not considered to be detrimental to the study as all control validity criteria were achieved.

Nominal and measured concentrations:

Nominal concentrations selected for use in this study were 6.3, 13, 25, 50, and 100 mg a.i./L
Measured concentrations of FRET 13-0545 in samples collected from the nominal 6.3, 13, 25, 50, and
100 mg a.i./L treatment groups at exposure initiation were 91.4, 84.6, 84.8, 91.5, and 80.6% of nominal,
respectively. Measured concentrations of FRET 13-0545 in samples collected from the 6.3, 13, 25, 50,
and 100 mg a.i./L treatment groups at the end of exposure (72 hours) were 27.0, 32.6, 40.0, 55.6, and
72.8% of nominal, respectively. The measured concentration of FRET 13-0545 in the abiotic replicate
included in the 25 mg a.i./L treatment group at test termination was 69.6% of nominal. The measured
concentration of FRET 13-0545 in the abiotic sample included in the 25 mg a.i./L treatment group at test
termination was higher than the measured concentration in the biotic replicates included in the same
treatment group. The more substantial decline observed in the biotic solutions indicates that the test
substance may have been adsorbed by the test organism. Measured concentrations of FRET 13-0545
declined over the 72 hour exposure period indicating that the test substance was not stable under the
conditions of the test.Since the measured concentrations of FRET 13-0545declined substantially over the exposure period the results of the study were based on geometric mean,measured concentrations of 3.1, 6.8, 15, 36, and 77 mg a.i./L, equivalent to 49.2, 52.3, 60.0, 72.0, and 77.0% of nominal, respectively.

Details on test conditions:

Due to the closed-bottle test design, additional sodium bicarbonate was added
to achieve a total concentration of 50 mg NaHCO3/L in the medium, to provide a sufficient supply of CO2
for algal growth per the OECD guidance on the testing of difficult substances and mixture. The pH
was adjusted to 7.5 using 10% hydrochloric acid and 0.1 N sodium hydroxide, as necessary, and the
medium was then sterilized by filtration (0.22 m) prior to use.Test chambers were held in an environmental chamber at a temperature of 24 ± 2ºC and with continuous cool-white fluorescent lighting at an intensity of 6,000 lux ± 10%.

Test chambers were gas-tight, sterile, 300-mL glass bottles sealed with glass stoppers. The
bottles were completely filled, leaving no headspace. Two glass marbles were present within each bottle
to promote mixing. The test chambers were labeled with the project number, concentration and replicate,
and were indiscriminately positioned daily on mechanical shakers in an environmental chamber designed
to maintain the desired test temperature throughout the test. The test chambers were shaken continuously
at 100 rpm.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

22 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other:

Remarks:

cell density /yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

55 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

6.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other:

Remarks:

cell density/growth rate/yield

Details on results:

After 72 hours of exposure, inhibition of cell density in the 3.1, 6.8, 15, 36, and 77 mg a.i./L
treatment groups (based on geometric mean, measured concentrations) was 3, 9, 34, 73, and 97%,
respectively, relative to the negative control response. Inhibition of growth rate at the 0-72 hour interval
in 3.1, 6.8, 15, 36, and 77 mg a.i./L treatment groups was 0, 2, 8, 26, and 70%, respectively, relative to the
negative control. Inhibition of yield after 72 hours of exposure in the 3.1, 6.8, 15, 36, and 77 mg a.i./L
treatment groups was 3, 9, 34, 74, and 98%, respectively, relative to the negative control. Reductions in
cell density, growth rate and yield were statistically significant (Dunnett’s test; p < 0.05) in the 15, 36,
and 77 mg a.i./L treatment groups when compared to the negative control response. The 72-hour NOEC
was determined to be 6.8 mg a.i./L, based on statistically significant reductions in cell density, growth
rate and yield after 72 hours of exposure.
After 72 hours of exposure, there were no signs of noticeable aggregation or flocculation of cells
in the negative control or in any treatment groups. Cells were not observed to be adhering to the test
vessels in any of the treatment groups or in the negative control. R. subcapitata cells in the 77 mg a.i./L
treatment group appeared enlarged when compared the negative control. Cells in all other FRET 13-0545
treatment groups appeared normal when compared to the negative control. No particulates or
surface-slicks were observed in the test solutions at test termination.
Conditions for the Validity of the Test
The mean cell density in the negative control replicates increased by a factor of 148 after three
days, exceeding the 16X growth criteria listed in the study protocol. The mean coefficient of variation for
section-by-section specific growth rates was 29.8%, which did not exceed the 35%
criterion. The coefficient of variation for mean average specific growth rates in the negative control
replicates at the 0-72 hour interval was 1.6%, which did not exceed 7%. Based on the
response of the control replicates this study was considered valid.

A non-GLP range-finding test was conducted prior to the definitive test at nominal concentrations

of 10 and 100 mg/L and yielded -2 and 87% inhibition of cell density after 72 hours of exposure.

Temperatures remained within the 24 ± 2°C range established for the test. The pH in all of

the test solutions was 7.8 at test initiation, and ranged from 7.8 to 10.5 at test termination. The pH of the

abiotic replicate was 7.8 at test termination. The observed increase in pH is typical for tests conducted

with R. subcapitata and is attributed to the photosynthetic activity of the algae. Although the pH

increased by greater than 1.5 units over the course of the test, this is not considered to be detrimental to

the study as all control validity criteria were achieved. The light intensity ranged from 5,410 to 6,570 lux,

which was within the desired range of 6,000 lux ± 10%.

Validity criteria fulfilled:

yes

Remarks:

OECD TG guideline, mean cell densitiy in the negative control replicates, mean coefficient of variation for section-by-section specific growth grates, coefficient of variaton for mean average specific growth rates all met the validity criteria.

Conclusions:

The freshwater green alga, Raphidocelis subcapitata, was exposed to five concentrations of
FRET 13-0545, with geometric mean, concentrations ranging from 3.1 to 77 mg a.i./L. The alga were
exposed in a closed-system to prevent loss of the test substance due to volatilization. Effects were
evaluated based on cell density, growth rate and yield. All control validity criteria were achieved. The
72-hour EC50, ErC50 and EyC50 values, based on cell density, growth rate and yield, were 22, 55, and
22 mg a.i./L, respectively. The 72-hour NOEC value, based on effects on cell density, growth
rate and yield, was determined to be 6.8 mg a.i./L.

Executive summary:

Following OECD TG 201, the freshwater green alga, Raphidocelis subcapitata, was exposed to five concentrations of

FRET 13-0545 for 72 hr, with geometric mean, concentrations ranging from 3.1 to 77 mg a.i./L.

The alga were exposed in a closed-system to prevent loss of the test substance due to volatilization. Nine replicate test chambers in each treatment group, and eighteen replicate test chambers in the control group, were initiated on Day 0. Three replicates from each treatment group, and six negative control replicates, were sacrificed at each 24-hour interval (24, 48 and 72 hours) and were sampled to provide cell density data. An additional abiotic replicate was included in the 25 mg a.i./L nominal test concentration for analytical sampling at 72 hours of exposure. Nominal test concentrations selected were 6.3, 13, 25, 50 and 100 mg active ingredient (a.i.)/L.

Effects were evaluated based on cell density, growth rate and yield. All control validity criteria were achieved. The

72-hour EC50, ErC50 and EyC50 values, based on cell density, growth rate and yield, were 22, 55, and 22 mg a.i./L, respectively (Table 7). The 72-hour NOEC value, based on effects on cell density, growth rate and yield, was determined to be 6.8 mg a.i./L.

Description of key information

Following OECD TG 201, the freshwater green alga, Raphidocelis subcapitata, was exposed to five concentrations of

FRET 13-0545 for 72 hr, with geometric mean, concentrations ranging from 3.1 to 77 mg a.i./L.

The alga were exposed in a closed-system to prevent loss of the test substance due to volatilization. Nine replicate test chambers in each treatment group, and eighteen replicate test chambers in the control group, were initiated on Day 0. Three replicates from each treatment group, and six negative control replicates, were sacrificed at each 24-hour interval (24, 48 and 72 hours) and were sampled to provide cell density data. An additional abiotic replicate was included in the 25 mg a.i./L nominal test concentration for analytical sampling at 72 hours of exposure. Nominal test concentrations selected were 6.3, 13, 25, 50 and 100 mg active ingredient (a.i.)/L.

Effects were evaluated based on cell density, growth rate and yield. All control validity criteria were achieved. The

72-hour EC50, ErC50 and EyC50 values, based on cell density, growth rate and yield, were 22, 55, and 22 mg a.i./L, respectively. The 72-hour NOEC value, based on effects on cell density, growth rate and yield, was determined to be 6.8 mg a.i./L.

Key value for chemical safety assessment

EC50 for freshwater algae:

55 mg/L

EC10 or NOEC for freshwater algae:

6.8 mg/L