Caryophyllene, Reaction product with Acetic anhydride and acetic acid

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 04 - October 09 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

[1R-(1α,2α,5β,8β)]-4,4,8-trimethyltricyclo[6.3.1.02,5]dodecan-1-yl acetate

EC Number:

260-555-1

EC Name:

[1R-(1α,2α,5β,8β)]-4,4,8-trimethyltricyclo[6.3.1.02,5]dodecan-1-yl acetate

Cas Number:

57082-24-3

Molecular formula:

C17H28O2

IUPAC Name:

4,4,8-trimethyltricyclo[6.3.1.0~2,5~]dodec-1-yl acetate

Constituent 2

Reference substance name:

1-((1S,2S,5R)-1,4,4-trimethyltricyclo[6.3.1.02,5]dodec-8-en-9-yl)ethan-1-one

Molecular formula:

C17H26O

IUPAC Name:

1-((1S,2S,5R)-1,4,4-trimethyltricyclo[6.3.1.02,5]dodec-8-en-9-yl)ethan-1-one

Constituent 3

Reference substance name:

(3S,3aS,7R,9aS)-1,1,7-trimethyldecahydro-3a,7-methanocyclopenta[8]annulen-3-yl acetate

Cas Number:

77346-43-1

Molecular formula:

C17H28O2

IUPAC Name:

(3S,3aS,7R,9aS)-1,1,7-trimethyldecahydro-3a,7-methanocyclopenta[8]annulen-3-yl acetate

Constituent 4

Reference substance name:

1-((1S,2S,5R,9S)-1,4,4-trimethyltricyclo[6.3.1.02,5]dodec-7-en-9-yl)ethan-1-one

Cas Number:

Not applicable

Molecular formula:

C17H26O

IUPAC Name:

1-((1S,2S,5R,9S)-1,4,4-trimethyltricyclo[6.3.1.02,5]dodec-7-en-9-yl)ethan-1-one

Constituent 5

Reference substance name:

4,4,8-trimethyltricyclo[6.3.1.02,5]dodecan-1-ol

EC Number:

207-458-2

EC Name:

4,4,8-trimethyltricyclo[6.3.1.02,5]dodecan-1-ol

Cas Number:

472-97-9

Molecular formula:

C15H26O

IUPAC Name:

4,4,8-trimethyltricyclo[6.3.1.0~2,5~]dodecan-1-ol

Constituent 6

Reference substance name:

(3S,3aS,7R,9aS)-1,1,7-trimethyldecahydro-3a,7-methanocyclopenta[8]annulen-3-ol

Cas Number:

62560-57-0

Molecular formula:

C15H26O

IUPAC Name:

(3S,3aS,7R,9aS)-1,1,7-trimethyldecahydro-3a,7-methanocyclopenta[8]annulen-3-ol

Constituent 7

Reference substance name:

2,2,4,7a-tetramethyl-1,2,3,6,7,7a-hexahydro-1,3a-ethanoindene

Cas Number:

4545-68-0

Molecular formula:

C15H24

IUPAC Name:

2,2,4,7a-tetramethyl-1,2,3,6,7,7a-hexahydro-1,3a-ethanoindene

Constituent 8

Reference substance name:

not applicable

Cas Number:

not applicable

Molecular formula:

not applicable

IUPAC Name:

not applicable

Test material form:

liquid

Specific details on test material used for the study:

Name (as stated in the report): VETYNAL
Batch: VE00473596
Expiration date: March 05, 2019
Purity / Composition Type: UVCB (about 90 %, sum of main constituents)

Sampling and analysis

Analytical monitoring:

yes

Remarks:

GC-FID

Details on sampling:

The test item gave a chromatographic profile consisting of several peaks, thereof two main
peaks which represent about 75 % of the test item (area %). The analytical characterization of
the WAFs was based on these two peaks.

For this, duplicate samples were taken from each treatment at the start of the test.
At the end of the test (after 72 hours), stability samples (containing algae) were taken in
duplicate from all loading rates and from the control. For sampling at the end of the test, the
test medium of the treatment replicates was pooled.

An additional test vessel with test medium at the WAF 3.2 mg/L was incubated under the
following conditions:
- Test medium with algae under light conditions but remain closed over the entire test
duration as a volatility control.

All samples were stored frozen (at -20 ± 5 °C) immediately after sampling. Based on preexperiments
for investigation of the storage stability, the analyzed components of the test item
were found to be stable in the test water under these storage conditions.

The concentrations of two main constituents of VETYNAL were analytically measured in at
least one of the duplicate samples taken from all treatments and both sampling dates. From the
volatility control one of the duplicate samples was measured after 72 hours.

Test solutions

Vehicle:

no

Details on test solutions:

Based on the results of the range-finding test and in agreement with the Sponsor the following concentrations of VETYNAL were selected for the main test: Water Accommodated Fractions (WAFs) with the loading rates of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L.

surface of test water. The test item volumes were calculated, considering the density of the test
item of 0.9896 g/cm3 (at 20 °C).

A stirring period of 48 hours at room temperature in the dark was applied. Based on the results
of a stirring experiment study (IES Study 20170072), 48 hours was considered to be an
appropriate stirring period to reach a sufficient concentration of dissolved test item in the test
media.

The equilibrated aqueous phases with the respective loading rates, containing dissolved test
item only (clear solutions), were tested as WAFs. Due to technical reasons (operational
constraints on the precision of direct weighing of test item), the WAFs with the lowest loading
rates of 0.32 and 1.0 mg/L were prepared as dilutions of the equilibrated aqueous phase of the
WAF with the loading rate of 3.2 mg/L. Additionally, a control (test water only) was run 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 [3].

Test organisms

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, 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.

Nygaard et al. recommended describing the taxa within the Genus Raphidocelis HINDAK as:
Raphidocelis subcapitata (KORSHIKOV) nov. comb.
Basionym: Ankistrodesmus subcapitatus KORSHIKOV
Syn.: Kirchneriella subcapitata KORSHIKOV
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. In the latest reference test (IES Study Number 20170077 from May 2017), the 72-hour EC50 for growth rate was 1.0 mg/L 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).

Study design

Test type:

static

Water media type:

other: synthetic test water (AAP Medium)

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

0.15 mmol/L (= 15 mg/L as CaCO3)

Test temperature:

24 °C

pH:

7.8-8.1

Nominal and measured concentrations:

0.32, 1.0, 3.2, 10, 32 and 100 mg/L

Details on test conditions:

The algae were cultivated and tested in synthetic test water (AAP Medium), prepared according
to the test guidelines, but modified according to the International Standard ISO 14442 as a
closed test system was applied. Analytical grade salts were dissolved in sterile purified water.

The concentration of NaHCO3 was increased by 235 to 250 mg/L (as carbon source for the algal
growth) and 6 mmol/L HEPES-buffer (corresponding to 1430 mg/L) were added to keep the
pH of the test media as constant as possible. The pH of the test water was adjusted to 7.5. The
calculated water hardness of the test water was 0.15 mmol/L (= 15 mg/L as CaCO3).

The test flasks were incubated in a temperature controlled orbital shaker (Multitron-Pro, Infors HT, Bottmingen/Switzerland) at a temperature of 24 °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 65 μE s-1 m-2 (range: 63 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.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (K2Cr207)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Based on Loading Rate - Test item is a UVCB

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Based on Loading Rate - Test item is a UVCB

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Based on Loading Rate - Test item is a UVCB

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

28 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: Based on Loading Rate - Test item is a UVCB

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.39 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: Based on Loading Rate - Test item is a UVCB

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Remarks on result:

other: Based on Loading Rate - Test item is a UVCB

Details on results:

The analytical characterization of the WAFs was based on the sum of peaks of the two main constituents. At the start of the test, the measured test item concentrations in the test media with the loading rates of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L were 0.11, 0.33, 1.0, 1.2, 1.3, 1.1 mg/L, respectively. At the four highest loading rates, the analyzed test item concentrations were in the same range of 1.0 to 1.3 mg/L, showing a plateau and demonstrating that the maximum concentration (sum of peaks) of dissolved test item was reached under the conditions of the test. At the lowest WAFs, the analyzed test item concentrations reflected the gradation of the WAF with the spacing factor of 3.2. At the end of the test 63 to 88 % of the initially measured values were determined.

The analytical results of the additional test vessel at the loading rate of 3.2 mg/L (with algae,under light conditions, remain closed over the entire test duration) showed that there are no additional losses of test item due to the opening of the test vessels for sampling after 24 and 48 hours test duration.

Since VETYNAL consists of several constituents with different solubilities and is characterized as a UVCB (a chemical substance of Unknown or Variable Composition, complex reaction products and Biological materials) and the analytical method is based only on the two main constituents, the absolute amount of test item could not be analytically determined and the measured test item concentrations should be regarded as approximated values. Therefore, all reported biological results were based on the loading rates of the test item.


Growth rate
At the loading rate of 1.0 mg/L the mean inhibition compared to the control was 1.1% (results
of Williams t-test, one-sided smaller, α = 0.05). This statistical significance was
caused by the very low variability between replicates within this loading rate and the control
(coefficient of variation of 0.4 and 0.6 %, respectively). However, this statistically significant
finding was not considered as a biologically relevant toxic effect, since the mean inhibition
compared to the control was below 10 %. Moreover, the EL10 for growth rate was calculated to
be 2.0 mg/L.
At the higher loading rate of 3.2 to 100 mg/L, the mean inhibition compared to the control was
in the range of 13 to 20 % and was statistically significantly different from the control.
Therefore, the NOELR for growth rate was determined to be at the loading rate of 1.0 mg/L

Yield
At the loading rates of 1.0 to 100 mg/L, the mean inhibition compared to the control was in the
range of 5.2 to 63 % (results of Williams t-test, one-sided smaller, α = 0.05) and was
statistically significantly different from the control. The EL10 for this endpoint was calculated
to be 0.39 mg/L.
Therefore, the NOELR for yield was determined to be at the loading rate of 0.32 mg/L.

The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the loading rate of 100 mg/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 loading rate.
All test media were clear solutions throughout the test period.

Results with reference substance (positive control):

In the latest reference test (IES Study Number 20170077 from May 2017), the 72-hour EC50 for growth rate
was 1.0 mg/L 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).

Reported statistics and error estimates:

Growth rate:
At the loading rate of 1.0 mg/L the mean inhibition compared to the control was 1.1% (results of Williams t-test, one-sided smaller, α = 0.05, Table 2 of the report). This statistical significance was caused by the very low variability between replicates within this loading rate and the control (coefficient of variation of 0.4 and 0.6 %, respectively). However, this statistically significant finding was not considered as a biologically relevant toxic effect, since the mean inhibition compared to the control was below 10 %. Moreover, the EL10 for growth rate was calculated to be 2.0 mg/L.
At the higher loading rate of 3.2 to 100 mg/L, the mean inhibition compared to the control was in the range of 13 to 20 % and was statistically significantly different from the control.
Therefore, the NOELR for growth rate was determined to be at the loading rate of 1.0 mg/L.
Yield:
At the loading rates of 1.0 to 100 mg/L, the mean inhibition compared to the control was in the range of 5.2 to 63 % (results of Williams t-test, one-sided smaller, α = 0.05, Table 3) and was statistically significantly different from the control. The EL10 for this endpoint was calculated to be 0.39 mg/L.
Therefore, the NOELR for yield was determined to be at the loading rate of 0.32 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

All validity criteria for increase of biomass, mean coefficient of variation of the daily growth rates and coefficient of variation of the average specific growth rates were fulfilled for the control.

Conclusions:

A clear concentration-response relationship was observed for both biological endpoints growth
rate and yield after the exposure period of 72 hours. A statistically significant inhibitory effect
on the yield and on the growth rate of the algae was observed at the end of the test at the loading
rates of 1.0 and 3.2 mg/L, respectively.

Results based on Growth Rate Observations :
ErL50 (72 hrs) > 100 mg/L
ErL10 (72 hrs) = 2.0 mg/L (95% CI 1.7 - 2.3 mg/L)
NOErLR (72 hrs) = 1.0 mg/L

Results based on yield of algal cells :
EyL50 (72 hrs) = 28 mg/L (95% CI 24 - 32 mg/L)
EyL10 (72 hrs) = 0.39 mg/L (95% CI 0.33 - 0.45 mg/L)
NOEyLR (72 hrs) = 0.32 mg/L

Executive summary:

The impact of the test item VETYNAL 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.

VETYNAL consists of several constituents with different solubilities and is characterized as a UVCB (a chemical substance of Unknown or Variable Composition, complex reaction products and Biological materials). Therefore, Water Accommodated Fractions (WAFs) with the loading rates of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L were tested to assess the toxicity of the test item to algae.

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.

Since the test item is a liquid with low water solubility, the slow-stirring method (to avoid formation of micro emulsions) was applied for preparation of the individual saturated test item solutions. For this, the test item with the density of 0.9896 g/cm3 (at 20 °C) was carefully applied (pipetted) on to the surface of the test water at the loading rates of 3.2, 10, 32 and

100 mg/L. Thereafter, slow-stirring was applied for 48 hours in closed vessels to reach a maximum concentration of dissolved test item in the test water. The stirring vessels were 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 to avoid losses of the volatile test item during stirring.

After this treatment the lower parts of the equilibrated test media were carefully harvested from the stirring vessel through a tap at the bottom of the vessels and tested as WAFs. The WAFs with the loading rates of 0.32 and 1.0 mg/L were prepared as dilutions of the WAF with the loading rate of 3.2 mg/L since it was technically infeasible to directly weight the required amount of test item. 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.

The test item gave a chromatographic profile consisting of several peaks, thereof two main peaks which represent about 75 % of the test item (area %). The analytical characterization of the WAFs was based on the sum of these two peaks.

At the start of the test, the measured test item concentrations (based on the measurement of the two main peaks) in the test media with the loading rates of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L were 0.11, 0.33, 1.0, 1.2, 1.3, 1.1 mg/L, respectively. At the four highest loading rates, the analyzed test item concentrations were in the same range of 1.0 to 1.3 mg/L, showing a plateau and demonstrating that the maximum concentration (sum of peaks) of dissolved test item was

reached under the conditions of the test. At the lower WAFs (loading rates 0.32 and 1.0 mg/L), the analyzed test item concentrations reflected the gradation of the WAF with the spacing factor of 3.2. At the end of the test 63 to 88 % of the initially measured values were determined.

Since VETYNAL consists of several constituents with different solubilities and the analytical method is based only on the two main constituents, the absolute amount of test item could not be analytically determined and the measured test item concentrations should be regarded as approximated values. Therefore, all reported biological results were based on the loading rates of the test item. This procedure of evaluation of data of WAFs is based on recommendations of the OECD Guidance Document No. 23 on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

A clear concentration-response relationship was observed for both biological endpoints growth rate and yield after the exposure period of 72 hours. A statistically significant inhibitory effect on the yield and on the growth rate of the algae was observed at the end of the test at the loading rates of 1.0 and 3.2 mg/L, respectively.

Endpoint after 72 Hours	Growth Rate [mg/L]	Yield [mg/L]
EL10	2.0	0.39
(95 % Confidence Interval)	(1.7-2.3)	(0.33-0.45)
EL20	>100	1.7
(95 % Confidence Interval)	(n.d.)	(1.5-1.9)
EL50	>100	28
(95 % Confidence Interval)	(n.d.)	(24-31)
NOELR	1.0	0.32
LOELR	3.2	1.0