Caryophyllene

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:

08 December 2016 - 23 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, 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

Details on sampling:

- Concentrations: One of the duplicate samples from the single test concentration and from the control from all sampling dates.
- Sampling method: Duplicate samples without algae were taken at the start of the test and duplicate samples with algae were taken after 24 and 48 hours and at the end of the test (72 hours). As the test was conducted in a closed system, a set of additional flasks containing the corresponding test medium (with algae) were incubated under conditions identical to the test for sampling at 24 and 48 hours. For each sampling date one set was harvested and then disposed. For sampling at the end of the test, the test media of the treatment replicates were pooled.
- Sample storage conditions before analysis: Immediately after sampling, 10 mL ethylacetate (with internal standard) were added to each sample to stabilize the latter during the storage period. Thereafter, all samples were deep-frozen (at about -20 °C).

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

- Method: The preparation of the test media was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000. Pre-experiments were conducted to determine the optimum time period to reach equilibrium within a reasonable time period. The test solution at a nominal concentration of 100 mg/L was prepared following the slow-stirring method. At the start of the test 263 μL of test item were carefully added on the surface of 2370 mL of test water and slowly stirred for 96 hours at room temperature in the dark. After stirring, the lower equilibrated aqueous phase of the solution was carefully separated from the non-dissolved test item swimming on the water surface and was used as the single test concentration.
- Controls: Test water without test item
- Evidence of undissolved material: No remarkable observations were made concerning the appearance of the test medium. The test medium was clear solution throughout the test period.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: Strain No. 61.81 SAG
- Age of inoculum (at test initiation): Exponential growth phase
- Method of cultivation: Under standardised conditions according to the test guidelines

ACCLIMATION
- Acclimation period: 4 days
- Culturing media and conditions: Same as test

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

15 mg/L as CaCO3

Test temperature:

22 °C

pH:

Test start: 7.6
Test end: 7.8

Nominal and measured concentrations:

Loading rate: 100 mg/L
Geometric mean measured concentration: 0.033 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL glass stoppered Erlenmeyer flasks
- Type: Closed
- Material, size, headspace, fill volume: Completely filled, without headspace
- Aeration: No
- Initial cells density: 5000 cells/mL nominal, corresponding to 2.1E4 relative fluorescence units
- Control end cells density: 248.7E4 relative fluorescence units
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: AAP medium, prepared according to the test guidelines, but modified according to the International Standard ISO 14442 for a closed test system

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Sterile purified water
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH was recorded in each treatment at the start and end of the test. The temperature in the incubator was monitored and recorded continuously. The appearance of the test media was also visually controlled and recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: Sterile purified water was used to prepare the test solutions
- Adjustment of pH: Yes, using 1 M NaOH
- Photoperiod: Continuously illuminated
- Light intensity and quality: LED light, 62 to 68 μ/Em2

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: Fluorescence measurements taken daily, excitation of 440 nm and emission of 680 nm
- Other: The shape and size of the algal cells were visually inspected at the end of the test

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: Limit test
- Range finding study: Yes
- Test concentrations: Equilibrated test item solution with a loading rate of 100 mg/L (measured concentration 0.092 mg/L) and 1:5 and 1:50 dilutions thereof
- Results used to determine the conditions for the definitive study: Inhibition of average growth after 72 hours was -0.5, -1.6 and -1.2% for the 1:50 dilution, 1:5 dilution and 100 mg/L loading rate concentrations, respectively, and the inhibition of yield after 72 hours was -2.4, -7.9 and -5.8%, respectively

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.033 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.033 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.033 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.033 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.033 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

>= 0.033 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

- Exponential growth in the control: Yes
- Unusual cell shape: The shape and size of the algal cells were obviously not affected by the test item up to at least this test concentration
- Any observations that might cause a difference between measured and nominal values: Test item losses were considered to be due to adsorption of the test item onto the algae

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: 0.9 mg/L based on 72-hour growth rate
- Other: Positive control tested twice a year, last tested in October 2016

Reported statistics and error estimates:

The 72-hour EC10, EC20 and EC50 values of the test item could not be determined because of the absence of a significant inhibitory effect of the test item on the algal growth at the tested concentration. For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentration were compared to the control values by the Student t-test (one-sided smaller, α = 0.05).

Table 1. Analytical measured concentrations of the test item

Loading rate* (mg/L)	Analytical measured concentrations (mg/L)				Geometric mean measured concentration** (mg/L)
	0 hours	24 hours	48 hours	72 hours
100	0.097	0.055	< LOQ	< LOQ	0.033

LOQ = 0.0294 mg test item/L

* Equilibrated test item solution with a loading rate of 100 mg/L

** If the measured concentration was below LOQ, half of the LOQ (½ LOQ = 0.0147 mg/L) was used to calculate the mean measured concentration

Table 2. Average growth rates (μ)

Geometric mean measured concentration (mg/L)	Average growth rate μ (/day) and inhibition of μ (I_r)
	0 -24 h		0 -48 h		0 -72 h
	μ	I_r (%)	μ	I_r (%)	μ	I_r (%)
Control	1.748	0.0	1.699	0.0	1.593	0.0
0.033	1.737	0.6	1.681	1.1	1.585	0.5

No statistically significant effect at the test concentration compared to control (according to Student t-test, one-sided smaller, α = 0.05)

Table 3. Yield (Y)

Geometric mean measured concentration (mg/L)	Yield Y (x10000) and inhibition of Y (I_y)
	0 -24 h		0 -48 h		0 -72 h
	Y	I_Y (%)	Y	I_Y (%)	Y	I_Y (%)
Control	9.91	0.0	60.43	0.0	246.57	0.0
0.033	9.83	0.8	58.14	3.8	240.39	2.5

No statistically significant effect at the test concentration compared to control (according to Student t-test, one-sided smaller, α = 0.05)

Table 4. Section-by-section growth rates

Geometric mean measured concentration (mg/L)	Section-by-section growth rate μ (/day) and inhibition of μ (I_r)
	0 -24 h		0 -48 h		0 -72 h
	μ	I_r (%)	μ	I_r (%)	μ	I_r (%)
Control	1.748	0.0	1.651	0.0	1.380	0.0
0.033	1.737	0.6	1.624	1.6	1.393	-0.9

Validity criteria fulfilled:

yes

Remarks:

The control biomass increased by a factor of 119 over 72 hours (must be ≥16) and the mean coefficients of variation during 72 hours were 13% for the section-by-section growth rates (must be <35%) and 1.1% the average specific growth rates (must be <7%).

Conclusions:

The 72-hour EC10, EC20, EC50 and LOEC values were determined to be > 0.033 mg/L and the NOEC was ≥ 0.033 mg/L for both growth rate and yield.

Executive summary:

The impact of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was investigated in a 72-hour static limit test according to the OECD Guideline 201 and EU C.3. As the test item is a volatile substance, the test was performed in a closed system. Due to the low water solubility of the test item, the test solution was prepared by slow-stirring method at a loading rate of 100 mg/L, based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000. During the test period the test item concentration in presence of algae decreased below the limit of quantification of the analytical method. The losses were considered to be due to adsorption of the test item onto the algae. The geometric mean measured concentration was determined to be 0.033 mg/L. Additionally, a control (test water without test item) was tested in parallel. The 72-hour EC10, EC20, EC50 and LOEC values were determined to be > 0.033 mg/L and the NOEC was ≥ 0.033 mg/L for both growth rate and yield. This study is reliable without restriction (Klimisch 1) as it was GLP-compliant and was conducted according to guideline methods.

Description of key information

For the freshwater green alga Pseudokirchneriella subcapitata, the 72-hour EC10, EC20, EC50 and LOEC values were determined to be > 0.033 mg/L and the NOEC was ≥ 0.033 mg/L for both growth rate and yield.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.033 mg/L

EC10 or NOEC for freshwater algae:

0.033 mg/L

Additional information

The impact of the test item on the growth of the freshwater green algae Pseudokirchneriella subcapitata was investigated in a 72-hour static limit test according to the OECD Guideline 201 and EU C.3 (2017). As the test item is a volatile substance, the test was performed in a closed system. Due to the low water solubility of the test item, the test solution was prepared by slow-stirring method at a loading rate of 100 mg/L, based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000. During the test period the test item concentration in presence of algae decreased below the limit of quantification of the analytical method. The losses were considered to be due to adsorption of the test item onto the algae. The geometric mean measured concentration was determined to be 0.033 mg/L. Additionally, a control (test water without test item) was tested in parallel. The 72-hour EC10, EC20, EC50 and LOEC values were determined to be > 0.033 mg/L and the NOEC was ≥ 0.033 mg/L for both growth rate and yield. This study is reliable without restriction (Klimisch 1) as it was GLP-compliant and was conducted according to guideline methods.