Reaction mass of rel-(2R,3aR,6R,7aR)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran and rel-(2R,3aR,6S,7aR)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran and rel-(2R,3aS,6S,7aS)-2,6-dimethyl-3a-(propan-2-yl)octahydro-1-benzofuran

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:

The study was conducted between 23 January 2015 and 22 February 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 201 using a growth inhibition method and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For measurement of the actual concentrations of the test item, duplicate samples were taken from the test media of all test concentrations at the start of the test (without algae) and at the end of the test (containing algae). At the same sampling times, duplicate samples were also taken from the control.
For the sampling at the end of the test, additional flasks containing the test medium with algae were incubated for each treatment under the test conditions. This was necessary as the volume of test solution of the treatment replicates (3 x 50 mL) was too small to perform the analyses. A volume of 100 mL per sample was necessary for analytical purposes.
All samples were stored deep-frozen (at about -20 °C) immediately after sampling until analysis. In GLP – pre-experiments for investigation of the storage stability of the samples, the test item proved to be stable under these storage conditions.
The concentrations of the test item IFF 215 (Floriane) were determined in one of the duplicate test medium samples from all treatments. From the control samples, one of the duplicate samples was analyzed per sampling time.

Vehicle:

no

Details on test solutions:

Due to the low solubility of the test item in test water, a dispersion with the loading rate of 100 mg/L was prepared at the start of the test by dispersing 232.82 mg of the test item in
2320 mL of test water. This preparation was supported by intense stirring by magnetic stirrers over 24 hours at room temperature in the dark, to dissolve a maximum amount of the test item in the dispersion. No auxiliary solvent or emulsifier was used.
The stirring period of 24 hours was selected according to the results of a GLP study with the same test item conducted at Harlan Laboratories Ltd., Shardlow, UK (Harlan Study Number 41401231).

After the 24-hour stirring period, the dispersion of the test item was filtered through a membrane filter (Whatman, Type NC20, pore size 0.2 μm) after preconditioning of the filter with about 200 mL of the dispersion. The negative pressure of the filtration unit was reduced as far as possible as a precaution to avoid possible losses of the test item by evaporation during filtration.
The undiluted filtrate was used as the highest concentrated test medium and as a stock solution for preparation of the test media with lower test concentrations. For this preparation, the filtrate was diluted with test water. The test media were prepared just before the start of the test (= start of exposure).
The test method is based on the OECD series on testing and assessment No. 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000 [OECD, 2000].

Test organisms (species):

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

Details on test organisms:

The test organism used for the study was Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum), 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 Harlan Laboratories under standardized conditions according to the test guidelines.
Nygaard et al. [Nygaard, 1986] recommended describing the taxa within the Genus Raphidocelis HINDAK as:
Raphidocelis subcapitata (KORSIKOV) nov. comb.
Basionym: Ankistrodesmus subcapitatus KORSIKOV
Syn.: Kirchneriella subcapitata KORSIKOV
Syn.: Selenastrum capricornutum PRINTZ
Syn.: NIVA-CHL 1
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.
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 result of the latest positive control test performed in August 2014 (72-hour EC50 for the growth rate:
1.1 mg/L, Harlan Study Number D94966) showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate from 2000 to 2014: 0.71-1.7 mg/L). The test method and the test species are recommended by the test guidelines.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

15 mg/L as CaCO3

Test temperature:

The water temperature was measured and recorded daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. The water temperature during the test was between 23 and 24 °C

pH:

The pH was measured and recorded in each treatment at the start and end of the test.

Nominal and measured concentrations:

Dilution 1:100, 1:32, 1:10, 1:3.2 and the undiluted filtrate, equivalent to 0.34, 1.1, 3.5, 11 and 36 mg/L, respectively (measured concentration)

Details on test conditions:

Material
As a precaution to avoid potential losses of test item, glass stoppered 50 mL Erlenmeyer flasks were used (closed system) completely filled with about 60 mL of test medium, to minimize the air-space in the flasks [ISO, 1999]. The test flasks were labeled with the study number and all necessary additional information to ensure unique identification. During exposure, the test solutions were continuously stirred by magnetic stirrers.

Experimental Conditions
The test flasks were incubated in a temperature-controlled water bath at a temperature between 23 and 24 °C and illuminated by fluorescent tubes (Philips TLD 36W-1/840), installed above the test flasks. The test flasks were positioned randomly and repositioned daily. The mean measured light intensity at the level of the test solutions was approximately 7200 Lux (range: 6690 to 7980 Lux, 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.


Study Design
The selection of the test concentrations was based on the results of a range-finding test. The following treatments were tested: Dilution 1:100, 1:32, 1:10, 1:3.2 and the undiluted filtrate. Additionally, a control was tested in parallel (test water without test item).
The enlarged spacing factor of approximately 3.2 between the test concentrations was chosen based on the results of the range-finding test, which documented a rather flat concentration effect relationship. Therefore, a large concentration range had to be tested.
The test design included three replicates per test concentration and six replicates of 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.


Dosage
Due to the low solubility of the test item in test water, a dispersion with the loading rate of 100 mg/L was prepared at the start of the test by dispersing 232.82 mg of the test item in 2320 mL of test water. This preparation was supported by intense stirring by magnetic stirrers over 24 hours at room temperature in the dark, to dissolve a maximum amount of the test item in the dispersion. No auxiliary solvent or emulsifier was used.
The stirring period of 24 hours was selected according to the results of a GLP study with the same test item conducted at Harlan Laboratories Ltd., Shardlow, UK (Harlan Study Number 41401231).

After the 24-hour stirring period, the dispersion of the test item was filtered through a membrane filter (Whatman, Type NC20, pore size 0.2 μm) after preconditioning of the filter with about 200 mL of the dispersion. The negative pressure of the filtration unit was reduced as far as possible as a precaution to avoid possible losses of the test item by evaporation during filtration. The undiluted filtrate was used as the highest concentrated test medium and as a stock solution for preparation of the test media with lower test concentrations. For this preparation, the filtrate was diluted with test water. The test media were prepared just before the start of the test (= start of exposure). The test method is based on the OECD series on testing and assessment No. 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000 [OECD, 2000].


Evaluations
Determination of Algal Biomass
A small volume 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 (BIOTEK ® Multi-Detection Microplate Reader, Model FLx800, wavelength: excitation 440 nm, emission 680 nm). The measurements were performed at least in duplicate.
At the end of the test, a sample was taken from the control and from the dilution 1:3.2 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 was too low for a reliable examination at the highest test concentrations (undiluted filtrate).


Determination of Algal Growth Inhibition and EC Values
Inhibition of algal growth was determined from the following growth parameters:
a) the specific growth rate (μ)
b) the yield (Y)
using the following equations:

a) Specific growth rate (µ):

µ= (1nNn – 1nN1) / (tn – t1)

Where:
µ: average specific growth rate from time t1 to tn
N1 = cell concentration at t1
Nn = cell concentration at tn
t1 = time of first measurement
tn = time of nth measurement

The average growth rate over the test duration and the section-by-section growth rates (daily growth rates between the sampling times) were calculated.

Inhibition of growth rate (Ir):
Ir = ((µc - µt) / µc) • 100 %

Where:
Ir = percent inhibition in average specific growth rate
µc = mean value for average specific growth rate in the control group
µt = average specific growth rate for the treatment replicate

b) Yield (Y)

Y = Nt – Nn

Where:
Y = yield
Nn = cell concentration (nominal value at the start of the test)
Nt = cell concentration at time t (at the end of the test)

Inhibition of yield (Iy)

Iy = ((Yc – Yt) / Yc) • 100 %

Where:
Iy = percent inhibition of yield
Yc = mean value for yield in the control group
Yt = value for yield for the treatment replicate

Growth rate and yield were calculated for each test flask. The mean values for growth rate and yield were calculated for each treatment. The tabulated values represent rounded results obtained by calculation using the exact raw data.
The biological results were related to the mean measured test item concentrations calculated as the geometric means of the concentrations measured at the start and end of the test [Sachs, 1984].

The 72-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated Weibull Analysis [Christensen, 1984a, b].
For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by Dunnett t-test [Dunnett, 1955; Dunnett, 1964].
All statistical calculations were performed using ToxRat Professional, ToxRat Solutions GmbH, Version 2.10.05 (released 20.02.2010).

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

16 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence intervals of 15 - 17 mg/L.

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

10 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence intervals of 9.7 - 10 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

7.5 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence intervals of 6.9 - 7.9 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

11 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

At the start of the test, the analytically determined concentrations of IFF 215 (Floriane) in the test media (dilutions 1:100, 1:32, 1:10, 1:3.2 and the undiluted filtrate) were 0.34, 1.1, 3.5, 11 and 36 mg/L, respectively. During the test period of 72 hours, a slight decrease of test item concentration in the test media occurred. At the end of the test, 70 to 93% of the initial measured concentrations were found.

The test item had a significant inhibitory effect on the growth rate µ of the algae after the test periods of 72 hours at the concentration of 11 mg/L and at the highest test concentration (results of Dunnett t-test, one-sided smaller, α = 0.05).
The yield Y of the algae was statistically significantly reduced first at the test concentration of 0.93 mg/L.

The 72-hour NOEC based on the growth rate µ was determined to be 3.0 mg/L, since up to and including this test concentration the growth rate of the algae after 72 hours was not significantly lower than in the control.

The NOEC and ECX-values for growth rate are summarized in the conclusion. Additionally, the corresponding values for yield are given.


The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the dilution 1:3.2 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.
In the control, the biomass increased by a factor of 362 over 72 hour. The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates) during 72 hours was 22%. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35%. Thus, the validity criterion was fulfilled. The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 1.1%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled.

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

The pH rose in the control from 7.7 at test start to 8.4 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 8.5 during the test period.

The water temperature during the test was between 23 and 24 °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 result of the latest positive control test performed in August 2014 (72-hour EC50 for the growth rate: 1.1 mg/L, Harlan Study Number D94966) showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate from 2000 to 2014: 0.71 1.7 mg/L).

Reported statistics and error estimates:

Growth rate and yield were calculated for each test flask. The mean values for growth rate and yield were calculated for each treatment. The tabulated values represent rounded results obtained by calculation using the exact raw data.
The biological results were related to the mean measured test item concentrations calculated as the geometric means of the concentrations measured at the start and end of the test [Sachs, 1984].

The 72-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated Weibull Analysis [Christensen, 1984a, b].
For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by Dunnett t-test [Dunnett, 1955; Dunnett, 1964].
All statistical calculations were performed using ToxRat Professional, ToxRat Solutions GmbH, Version 2.10.05 (released 20.02.2010).

The biological results were related to the mean measured test item concentrations calculated as the geometric means of the concentrations measured at the start and end of the test:

 

Treatment	Mean measured concentration of the test item (geometric mean)
	[mg/L]
Dilution 1:100	0.31
Dilution 1:32	0.93
Dilution 1:10	3.0
Dilution 1:3.2	11
Undiluted filtrate	34

Average Growth Rates(µ)

Treatment / dilution	Mean measured concentration	Average growth rate µ (day^-1) and inhibition of µ (Ir)
		0-24 h		0-48 h		0-72 h
	[mg/L]	µ	Ir[%]	µ	Ir[%]	µ	Ir[%]
Control	---	2.370	0.0	2.192	0.0	1.964	0.0
1:100	0.31	2.281	3.8	2.183	0.4	1.944	1.0
1:32	0.93	2.251	5.0	2.151	1.9	1.934	1.5
1:10	3.0	2.067*	12.8	2.054	6.3	1.919	2.3
1:3.2	11	1.463*	38.3	1.434*	34.6	1.484*	24.4
Undiluted filtrate with loading rate 100 mg/L	34	-0.781*	133.0	-0.801*	136.6	-0.728*	137.1

*: mean value statistically significantly lower than in the control
(according to Dunnett t-test one-sided smaller,a= 0.05)

Note: Percentage inhibition values in excess of 100% are obtained when the biomass
at the end of the interval is lower than at the start of the interval.

 

 

Yield(Y)

Treatment / dilution	Mean measured concentration	Yield Y (x 10^3) and inhibition of Y (Iy)
		0-24 h		0-48 h		0-72 h
	[mg/L]	Y	Iy[%]	Y	Iy[%]	Y	Iy[%]
Control	---	5.0	0.0	40.7	0.0	185.9	0.0
1:100	0.31	4.5*	9.5	40.0	1.8	174.7	6.0
1:32	0.93	4.4*	12.5	37.5*	7.9	169.6*	8.8
1:10	3.0	3.5*	28.9	30.8*	24.4	162.5*	12.6
1:3.2	11	1.7*	65.8	8.5*	79.0	43.6*	76.5
Undiluted filtrate with loading rate 100 mg/L	34	-0.3*	105.5	-0.4*	101.0	-0.4*	100.2

*: mean value statistically significantly lower than in the control
(according to Dunnett t-test one-sided smaller, a= 0.05)

Note: Percentage inhibition values in excess of 100% are obtained when the biomass
at the end of the interval is lower than at the start of the interval.

Section-by-Section Growth Rates

Treatment / dilution	Mean measured concentration	Section-by-section growth rates (day^-1) and inhibition of the growth rates (Ir)
		0-24 h		24-48 h		48-72 h
	[mg/L]	µ	Ir[%]	µ	Ir[%]	µ	Ir[%]
Control	---	2.370	0.0	2.013	0.0	1.508	0.0
1:100	0.31	2.281	3.8	2.085	-3.5	1.465	2.8
1:32	0.93	2.251	5.0	2.051	-1.9	1.499	0.6
1:10	3.0	2.067	12.8	2.041	-1.4	1.650	-9.4
1:3.2	11	1.463	38.3	1.405	30.2	1.585	-5.1
Undiluted filtrate with loading rate 100 mg/L	34	-0.781	133.0	-0.821	140.8	-0.582	138.6

Note:    Percentage inhibition values in excess of 100% are obtained when the biomass
at the end of the interval is lower than at the start of the interval.

 

pH Values in theTreatments

Treatment / dilution	pH values
	Start	End
Control	7.7	8.4
1:100	7.6	8.4
1:32	7.6	8.5
1:10	7.6	8.4
1:3.2	7.6	7.9
Undiluted filtrate	7.6	7.8

 

Water Temperature During the Test Period

	Temperature [°C]
Day 0 (Start)	23
Day 1	23
Day 2	23
Day 3 (End)	24

Validity criteria fulfilled:

yes

Conclusions:

The impact of the test item IFF 215 (Floriane) on the growth of the freshwater green algal species Pseudokirchneriella subcapitata can be summarized as follows (based on mean measured concentrations of the test item):
The 72-hour growth rate EC50 was 16 mg/L with 95 % confidence intervals of 15 - 17 mg/L.
The 72-hour growth rate EC20 was 10 mg/L with 95 % confidence intervals of 9.7 - 10 mg/L
The 72-hour growth rate EC10 was 7.5 mg/L with 95 % confidence intervals of 6.9 - 7.9 mg/L
The growth rate NOEC was 3.0 mg/L
The growth rate LOEC was 11 mg/L

The 72-hour yield EC50 was 7.1 with 95 % confidence intervals of 6.3 - 7.9 mg/L.
The 72-hour yield EC20 was 3.6 with 95 % confidence intervals of 2.8 - 4.3 mg/L.
The 72-hour yield EC10 was 2.3 with 95 % confidence intervals of 1.6 - 3.0 mg/L.
The yield NOEC was 0.31 mg/L
The yield LOEC was 0.93 mg/L

Executive summary:

The toxicity to aquatic algae of the test substance, 215 (Floriane), was assessed according to OECD Test Guideline 201 using a growth inhibition method. The 72-hour EC50 (growth rate) was 16 mg/L with 95 % confidence intervals of 15 - 17 mg/L. The NOEC was 3.0 mg/L and the LOEC was 11 mg/L.

Description of key information

The toxicity to aquatic algae of the test substance, IFF 215 (Floriane), was assessed according to OECD Test Guideline 201 using a growth inhibition method. The 72-hour EC50 (growth rate) was 16 mg/L with 95 % confidence intervals of 15 - 17 mg/L. The NOEC was 3.0 mg/L and the LOEC was 11 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

16 mg/L

EC10 or NOEC for freshwater algae:

3 mg/L