Reaction mass of rel-{(1R,2S)-1-methyl-2-[(2R)-5-methylhex-4-en-2-yl]cyclopropyl}methanol and rel-{(1S,2R)-1-methyl-2-[(2R)-5-methylhex-4-en-2-yl]cyclopropyl}methanol

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:

November, 2014 - May, 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2011

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

GR-50-1408 is the Givaudan identification code which was employed for ROSYFOLIA during the early, developmental and testing period.

Analytical monitoring:

yes

Remarks:

GC-FID

Details on sampling:

Samples were taken for substance specific analytics from the Control and each of the test concentrations at Time 0 and 72 hours.

Vehicle:

no

Details on test solutions:

Range-finding Test :
The range-finding test was conducted at nominal test substance concentrations of
0.10, 1.0, 10 and 100% saturated solution. The 100% saturated solution was prepared
by stirring an excess (200 mg/L) of test substance for ca 24 hours. After stirring, the
preparation was filtered through a 0.45 μm filter to give the 100% saturated solution
from which serial dilutions were prepared, in EC medium, to give the remaining test
concentrations. A control group was also included. Duplicate test vessels were
prepared for the control and each test concentration. Based on nominal
concentrations, the results of the range-finding test suggested that the
72-hour ExC50 value would be between 1.0 and 100% saturated solution.

Definitive Test :
Based on the results of the range-finding test, for which the key results only have been
reported, the definitive test was conducted at nominal test concentrations of 0.32, 1.0,
3.2, 10 and 32% saturated solution (0.19, 0.66, 2.0, 7.2 and 23 mg/L based on
geometric mean measured concentrations). A control group was also included.

At the start of the test, the 100% saturated solution test concentration was prepared by
weighing ca 200 mg of test substance and adding to 1000 mL of EC medium. This
was stirred slowly for ca 24 hours and then filtered through a 0.45 μm filter to give
the 100% saturated solution test concentration. Dilutions were then prepared from the
100% saturated solution, in EC medium, to give the nominal test concentrations of
0.32, 1.0, 3.2, 10 and 32% saturated solution. A control treatment was prepared by
adding EC medium only to the control vessels.

Appearance of Test Media :
The appearance, colour and behaviour of the test substance in the test media were
recorded at the start and end of the test.

Test organisms (species):

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

Details on test organisms:

The test organism, Pseudokirchneriella subcapitata (Strain 278/4), was originally obtained from the Culture Collection of Algae and Protozoa (CCAP) and is a representative species of the freshwater aquatic phytoplankton. This species is recommended for testing in accordance with the OECD regulatory guidelines.

Culturing of Pseudokirchneriella subcapitata and Water Quality

Introduction :

Semi-axenic cultures of Pseudokirchneriella subcapitata were maintained in liquid
culture. These were used to inoculate starter liquid cultures, which in turn were used
to inoculate test vessels containing control and test media at the start of each test.
Source

The axenic strain of Pseudokirchneriella subcapitata (CCAP 278/4) was obtained
from a concentrated liquid slope culture from the Culture Collection of Algae and
Protozoa (CCAP), SAMS Research Services Ltd., Oban, UK.

On receipt from the CCAP, the slope culture was stored in the fridge at 2 -8°C.
A typical shelf life for each slope was 8 months, after which the slopes were
discarded.

Preparation of Algal Medium :
To prepare the alga culture medium, stock solutions containing the various nutrients
were prepared with reverse osmosis (RO) water. Aliquots of each of the stock
solutions were then added to RO water. The alga culture medium was autoclaved.

Once cooled, an aliquot of a stock solution of NaHCO3 was added to the alga culture
medium through a sterile filter (0.2 m filter pore size).

Inoculation of Test Cultures :
Prior to each test, a sub-sample (ca 200 L) of a current liquid slope culture was added
to two starter cultures (conical flasks) containing 100 mL EC medium.
Each culture was incubated (under the same conditions as used in the test) for at least
72 hours prior to the start of each test. Following incubation, the cell density of a single
starter culture was established using a particle counter (Z2 Coulter Counter®) and
confirmed manually using a haemocytometer.

An inoculum volume was then calculated and added to each test vessel, to achieve a
starting alga cell density of 1 x 104 cells/mL. All flasks for a particular test were
inoculated from a single starter culture. The second (unused) starter culture (incubated
as a contingency) was discarded after the test was started.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

EC Medium used. No value provided in the report for Hardness..

Test temperature:

Temperature range (°C) 22.4 – 23.1

pH:

At the start of the test, the pH of freshly prepared test media was determined. The pH in each test vessel was also determined at the end of the test.

0 hours 72 hours
Control 7.61 10.73
0.32% Saturated Soln. 7.64 10.82
1.0% Saturated Soln. 7.66 10.71
3.2% Saturated Soln. 7.65 10.64
10% Saturated Soln. 7.64 9.09
32% Saturated Soln. 7.62 8.29

Nominal and measured concentrations:

In general, the concentrations remained stable over the 72-hour test period however some variation was observed therefore results have been based on geometric mean measured concentrations.
These were 0.19, 0.66, 2.0, 7.2 and 23 mg/L for the 0.32, 1.0, 3.2, 10 and 32% saturated solution, respectively.

Details on test conditions:

Test Vessel Preparation :
The test vessels were sterile autoclaved glass 250 mL Erlenmeyer (conical) flasks. To
minimise potential losses through volatility, the test vessels contained sufficient
media to leave as minimum a headspace as possible. The vessels were then loosely
capped and a marble added to aid stirring. This system constituted a ‘closed’ system.
Three test vessels were prepared for each test concentration as well as six control
vessels (algal nutrient medium only). Each test and control vessel was inoculated with
sufficient Pseudokirchneriella subcapitata cells to achieve a starting algae cell concentration of 1 x 10^4 cells/mL. An additional inoculated vessel was prepared for
the control and each test concentration for initial water quality analysis.
A media blank (EC media only) was prepared to establish background counts on each
sampling occasion. Background counts were subtracted from the cell counting results
for each of the inoculated test vessels. The resulting cell counts were then used to
calculate the area under the growth curves, yield and the corresponding specific
growth rates.

Test Vessel Sampling :
At approximately 24-hour intervals after the start of the incubation period,
pre-determined volumes of test media were removed from each incubated test vessel,
and transferred to individually identified cell counting vials. The contents of each vial
were diluted to a 10 mL final volume with an electrolyte solution. The cell density of
the vial contents was then determined using a particle counter (Z2 Coulter Counter®).

All flasks were incubated under a light bank in a temperature controlled laboratory.
The vessels were placed on an orbital shaker (ca 100 rpm) under conditions of constant light (4440 to 8880 Lux), using cool white fluorescent tube lights, emitting white light across the visible portion of the spectrum (400 - 700 nm).
At the start of the test, the pH of freshly prepared test media was determined. The pH in each test vessel was also determined at the end of the test.
The laboratory temperature was set within the range 21 to 24°C and maintained within ± 2°C for the duration of the test. The temperature was recorded continuously using a digital temperature logger.
The light intensity within the test area was monitored at the start and end of the test.

All environmental conditions were within those specified in the study protocol. The OECD guideline states that the pH in the control should not vary by more than 1.5 units during the test. At the end of the test elevated pH values were observed in some of the vessels, at and below the 3.2% dilution (2.0 mg/L geometric mean measured concentration), corresponding to treatment levels at and below the NOErC and NOEbC. This is an indicator of good algal cell growth (cell density increase of 42 to 58 over the 72-hour test period), whereby the release of oxygen from the algal cells during normal photosynthetic activity promotes an alkali pH in the surrounding test media. It was considered that the elevated pH levels was due to good algal cell growth and therefore considered not to affect the validity of the test.

Reference substance (positive control):

yes

Remarks:

Zinc chloride

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: 95% CI : 3.0 - 4.5 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: 95% CI : 0.48 - 1.6 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI : 5.8 - 10 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI : 2.0 - 2.8 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The test system used was a ‘closed’ system with minimal headspace in the test vessels.
This is a modified test system used to minimise losses of test substance through
volatility. This system reduces the gaseous exchange and as such reduces the amount of
growth observed during a test. A non-GLP positive control test was conducted using
completely filled and sealed vessels to determine the sensitivity of the test species under
the conditions of the test.
This test showed that the use of a modified test system to reduce losses of test substance
through volatility (completely filled and sealed test vessels) had a slightly lowering
effect on ECx values obtained compared with that in a conventional test system. As
such it can be considered that the results obtained from the definitive test give a worst
case result for the test substance.

Chemical Analysis :
The limit of quantification (LOQ) for GR-50-1408 in EC medium using this method
was 0.05 mg/L.
Analysis of the 100% saturated solution, used to prepare the test concentration at
0 hours, showed a measured concentration of 58.4 mg/L. Analysis of the test samples
at 0 hours showed measured concentrations to range from 0.195 (0.32% dilution) to
19.0 mg/L (32% dilution). At 72 hours, analysis of the test samples showed measured
concentrations to range from 0.186 (0.32% dilution) to 28.1 mg/L (32% dilution).
In general, the concentrations remained stable over the 72-hour test period however
some variation was observed therefore results have been based on geometric mean
measured concentrations. These were 0.19, 0.66, 2.0, 7.2 and 23 mg/L for the 0.32,
1.0, 3.2, 10 and 32% saturated solution, respectively.

Environmental Conditions :
All environmental conditions were within those specified in the study protocol. The
OECD guideline states that the pH in the control should not vary by more than 1.5
units during the test. At the end of the test elevated pH values were observed in some
of the vessels, at and below the 3.2% dilution (2.0 mg/L geometric mean measured
concentration), corresponding to treatment levels at and below the NOErC and
NOEbC. This is an indicator of good algal cell growth (cell density increase of 42 to
58 over the 72-hour test period), whereby the release of oxygen from the algal cells
during normal photosynthetic activity promotes an alkali pH in the surrounding test
media. It was considered that the elevated pH levels was due to good algal cell growth
and therefore considered not to affect the validity of the test.

Validity Criteria :
The control cell density increased by a factor of 49. Therefore the validity criterion of
achieving an increase in control cell density by at least a factor of 16 was achieved,
and the environmental conditions of the test were considered acceptable.

The mean coefficient of variation for section-by-section specific growth rates (0-24,
24-48 and 48-72 hours) in the control culture was 24.45% and therefore did not
exceed 35%.

The coefficient of variation for average specific growth rate during the whole test
period in replicate control cultures was 4.13% which did not exceed 7% and therefore
the validity criteria were met.

Results with reference substance (positive control):

Results with Zinc Chloride :
Yield – EyC50 – 0.045 mg Zn/L
Biomass – EbC50 – 0.055 mg Zn/L (EbC50 range using conventional test system – 0.035 – 0.18 mg Zn/L (historical reference test data)
Growth rate – ErC50 – 0.11 mg Zn/L (ErC50 range using conventional system – 0.10 – 0.31 mg Zn/L)

Validity criteria fulfilled:

yes

Conclusions:

Based on geometric mean measured test concentrations, the 72-hour end-points were established, as follows :

ErC50 = 8.6 mg/L
ErC10 = 2.4 mg/L
NOErC = 2.0 mg/L

EyC50 = 3.8 mg/L
EyC10 = 1.2 mg/L
NOEyC = 2.0 mg/L

Description of key information

 

The effects of GR-50-1408 on the growth of the unicellular green alga, Pseudokirchneriella subcapitata, were determined during a 72-hour growth inhibition toxicity test conducted in accordance with OECD Chemicals Testing Guideline No. 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test (adopted 23 March 2006) (Annex 5 corrected 28 July 2011).

Based on geometric mean measured test concentrations, the 72-hour EyC50, EbC50 and ErC50 values were calculated to be 3.8, 3.7 and 8.6 mg/L, respectively.

Based on geometric mean measured test concentrations, the 72-hour EyC10, EbC10 and ErC10 values were calculated to be 1.2, 1.0 and 2.4 mg/L, respectively

The corresponding NOEC values for yield, biomass and specific growth rate after 72 hours were 2.0, 0.66 and 2.0 mg/L, respectively

Key value for chemical safety assessment

EC50 for freshwater algae:

8.6 mg/L

EC10 or NOEC for freshwater algae:

2.4 mg/L