Acetic acid, esters with turpentine-oil myrcene fraction terpene alcs.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult items and mixtures, OECD series on testing and assessment number 23, December 14, 2000.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

p-menth-1-en-8-yl acetate

EC Number:

201-265-7

EC Name:

p-menth-1-en-8-yl acetate

Cas Number:

80-26-2

Molecular formula:

C12H20O2

IUPAC Name:

1-methyl-1-(4-methylcyclohex-3-en-1-yl)ethyl acetate

Constituent 2

Reference substance name:

Myrcenyl acetate

EC Number:

214-262-0

EC Name:

Myrcenyl acetate

Cas Number:

1118-39-4

Molecular formula:

C12H20O2

IUPAC Name:

1,1-dimethyl-5-methylenehept-6-en-1-yl acetate

Constituent 3

Reference substance name:

(E)-2-(3,3-dimethylcyclohexylidene)ethyl acetate

EC Number:

308-183-1

EC Name:

(E)-2-(3,3-dimethylcyclohexylidene)ethyl acetate

Cas Number:

97890-04-5

Molecular formula:

C12H20O2

IUPAC Name:

(E)-2-(3,3-dimethylcyclohexylidene)ethyl acetate

Constituent 4

Reference substance name:

(Z)-2-(3,3-dimethylcyclohexylidene)ethyl acetate

EC Number:

308-184-7

EC Name:

(Z)-2-(3,3-dimethylcyclohexylidene)ethyl acetate

Cas Number:

97890-05-6

Molecular formula:

C12H20O2

IUPAC Name:

(Z)-2-(3,3-dimethylcyclohexylidene)ethyl acetate

Constituent 5

Reference substance name:

Geranyl acetate

EC Number:

203-341-5

EC Name:

Geranyl acetate

Cas Number:

105-87-3

Molecular formula:

C12H20O2

IUPAC Name:

(E)-3,7-dimethylocta-2,6-dien-1-yl acetate

Constituent 6

Reference substance name:

1-methyl-4-(1-methylethylidene)cyclohexyl acetate

EC Number:

233-564-3

EC Name:

1-methyl-4-(1-methylethylidene)cyclohexyl acetate

Cas Number:

10235-63-9

Molecular formula:

C12H20O2

IUPAC Name:

4-isopropylidene-1-methylcyclohexyl acetate

Constituent 7

Reference substance name:

4-(5-methyl-1-methylene-4-hexenyl)-1-(4-methylpent-3-enyl)cyclohexene

EC Number:

208-546-3

EC Name:

4-(5-methyl-1-methylene-4-hexenyl)-1-(4-methylpent-3-enyl)cyclohexene

Cas Number:

532-87-6

Molecular formula:

C20H32

IUPAC Name:

4-(5-methyl-1-methylenehex-4-en-1-yl)-1-(4-methylpent-3-en-1-yl)cyclohexene

Constituent 8

Reference substance name:

cis-1-methyl-4-(1-methylvinyl)cyclohexyl acetate

EC Number:

244-028-3

EC Name:

cis-1-methyl-4-(1-methylvinyl)cyclohexyl acetate

Cas Number:

20777-47-3

Molecular formula:

C12H20O2

IUPAC Name:

4-isopropenyl-1-methylcyclohexyl acetate

Constituent 9

Reference substance name:

p-menth-1-en-8-ol

EC Number:

202-680-6

EC Name:

p-menth-1-en-8-ol

Cas Number:

98-55-5

Molecular formula:

C10H16O

IUPAC Name:

2-(4-methylcyclohex-3-en-1-yl)propan-2-ol

Constituent 10

Reference substance name:

Neryl acetate

EC Number:

205-459-2

EC Name:

Neryl acetate

Cas Number:

141-12-8

Molecular formula:

C12H20O2

IUPAC Name:

(Z)-3,7-dimethylocta-2,6-dien-1-yl acetate

Constituent 11

Reference substance name:

1-(3,3-dimethylcyclohex-1-en-1-yl)ethyl acetate

Cas Number:

150461-96-4

Molecular formula:

C12H20O2

IUPAC Name:

1-(3,3-dimethylcyclohex-1-en-1-yl)ethyl acetate

Constituent 12

Reference substance name:

Tentative monoterpene diacetate

IUPAC Name:

Tentative monoterpene diacetate

Constituent 13

Reference substance name:

Tentative oxygenated monoterpene acetate

IUPAC Name:

Tentative oxygenated monoterpene acetate

Constituent 14

Reference substance name:

Tentative Diterpene hydrocarbon

IUPAC Name:

Tentative Diterpene hydrocarbon

Constituent 15

Reference substance name:

Unresolved/non-elucidated mixed terpene/diterpene/oxygenated terpene/terpene acetate derivatives

IUPAC Name:

Unresolved/non-elucidated mixed terpene/diterpene/oxygenated terpene/terpene acetate derivatives

Test material form:

liquid

Specific details on test material used for the study:

Identification: Neobergamate Forte
Appearance: Pale yellow liquid
Batch: SC00015234
Purity/Composition: See Certificate of Analysis
Test item storage: At room temperature protected from light
Stable under storage conditions until: 07 November 2016 (expiry date)
Test item handling: Use amber glassware or wrap container in aluminum-foil
CAS Number: 69103-01-1
EC Number: 273-868-3

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

not specified

Details on test solutions:

The batch of Neobergamate Forte tested was a pale yellow liquid and not completely soluble in test medium at the loading rates originally prepared. No correction was made for the purity/composition of the test item. Information provided by the Sponsor indicated that the test item is a complex mixture composed of constituents with different water solubility. Due to the low aqueous solubility and complex nature of the test item for the purposes of the test, the test item was prepared as a Water Accommodated Fraction (WAF).
Test solutions were prepared separately at loading rates ranging from 1.0 to 100 mg/L, applying 24 hours of magnetic stirring. Thereafter, the resulting mixtures were allowed to stabilize for 1 hour. Subsequently, the Water Accommodated Fractions (WAFs) were siphoned off. Due to the light sensitivity of the test item, preparation of test solutions was performed under dimmed light and used glassware was wrapped in aluminum foil. All final test solutions were clear and colourless.
After preparation, volumes of 110 mL were added to each replicate of the respective test concentration containing 2.2 mL of an algal suspension providing a cell density of 104 cells/mL. Subsequently, 0.66 mL HEPES were added to each replicate.

Test organisms

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata, strain: NIVA CHL 1
Source: In-house laboratory culture.
Reason for selection: This system is a unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.

Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
Light intensity: 60 to 120 μE/m2/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
Stock culture medium: M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tapwater purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:
NaNO3 500 mg/L
K2HPO4.3H2O 52 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3.10H2O 54 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L
Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 104 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was
measured immediately before use.
Pre-culture medium: Adjusted M2; Standard M2 according to the OECD 201 Guideline but with a larger amount of NaHCO3, the addition of HEPES buffer and a lower pH. This medium will be formulated using Milli-RO water and will have the following composition:
NH4Cl 15 mg/l
MgCl2.6H2O 12 mg/l
CaCl2.2H2O 18 mg/l
MgSO4.7H2O 15 mg/l
KH2PO4 1.6 mg/l
FeCl3.6H2O 64 μg/l
Na2EDTA.2H2O 100 μg/l
H3BO3 185 μg/l
MnCl2.4H2O 415 μg/l
ZnCl2 3 μg/l
CoCl2.6H2O 1.5 μg/l
CuCl2.2H2O 0.01 μg/l
Na2MoO4.2H2O 7 μg/l
NaHCO3 300 mg/l
HEPES buffer 6 mmol/l
Hardness (Ca+Mg) 0.24 mmol/l (24 mg CaCO3/l)
pH 7.1 ± 0.3

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test conditions

Test temperature:

Continuously in a temperature control vessel. During the exposure period the temperature measured in the incubator was maintained between 21 and 22°C. Temperature remained within the limits prescribed by the study plan (21-24°C, constant within 2°C).

pH:

Measured at the beginning and at the end of the test in all test concentrations and the control. The pH was within the limits prescribed by the study plan (6.0-9.0, preferably not varying by more than 1.5 units)

Nominal and measured concentrations:

Neobergamate Forte: WAFs prepared at loading rates of 1.0, 3.2, 10, 32 and 100 mg/L.
Controls Test: medium without test item or other additives.
Replicates:
6 replicates of the control.
3 replicates of each test concentration.
1 extra replicate of each test concentration for sampling
purposes after 24 hours.
2 replicates of each test concentration without algae.
1 replicate of each test concentration without algae and without
HEPES buffer for TOC analysis.

At the start of the test, the actual test concentrations in the solutions incubated with algae were 0.53, 1.8, 5.4, 20 and 37 mg/L in WAFs prepared at 1.0, 3.2, 10, 32 and 100 mg/L, respectively. The measured concentrations decreased throughout the test duration and were at 27-29% of initial at the end of the test. A decrease in measured concentrations was also observed for samples without algae (29-49% of initial at the end of the test), indicating that the loss seen in the samples with algae was not due to adsorption to the increasing algal biomass but either chemical instability or evaporation into the minimum headspace.

Details on test conditions:

Test duration: 72 hours
Test type: Static
Test vessels: 120 mL, all-glass, containing 110 mL of test solution, with minimum headspace and closed airtight.
Medium: Adjusted M2 medium for volatile test items
Cell density: An initial cell density of 1 x 104 cells/mL.
Illumination: Continuously using TLD-lamps with a light intensity within the range of 80 to 84 μE/m2/s.
Incubation: Capped vessels were distributed at random in the incubator and as such were daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

14 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval ranging from 12 to 17 mg/L

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

45 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval ranging from 4.2 to 10 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Given that only a fraction of the total mass of a multi-component substance may be present in the WAF, that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole and that the test was performed in airtight vessels to prevent loss due to volatilization, the effect parameters were based on loading rates initially prepared.

Under the conditions of the present study with Pseudokirchneriella subcapitata, Neobergamate Forte reduced growth rate of this fresh water algae species significantly in WAFs prepared at 32 mg/L and higher.
The EL10 for growth rate inhibition (72h-ERL10) was 14 mg/L with a 95% confidence interval ranging from 12 to 17 mg/L.
The EL10 for yield inhibition (72h-EYL10) was 7.4 mg/L with a 95% confidence interval ranging from 4.2 to 10 mg/L.
The EL50 for growth rate inhibition (72h-ERL50) was 45 mg/L with a 95% confidence interval ranging from 41 to 49 mg/L.
The EL50 for yield inhibition (72h-EYL50) was 18 mg/L with a 95% confidence interval ranging from 15 to 23 mg/L.
The 72h-NOEL for growth rate inhibition was 10 mg/L and the NOEL for yield inhibition was 3.2 mg/L.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, Neobergamate Forte reduced growth rate of this fresh water algae species significantly in WAFs prepared at 32 mg/L and higher.
The EL10 for growth rate inhibition (72h-ERL10) was 14 mg/L with a 95% confidence interval ranging from 12 to 17 mg/L.
The EL50 for growth rate inhibition (72h-ERL50) was 45 mg/L with a 95% confidence interval ranging from 41 to 49 mg/L.
The 72h-NOEL for growth rate inhibition was 10 mg/L.

The preferred observational end point in the algal growth inhibition test is growth rate because it is not dependent on the test design (ECHA guidance Chapter R.7b v1.1). The EU CLP regulation (No 1272/2008 and its adaption 286/2011) also states that classification should be based on the ErC50. The preferred observational endpoint in long-term studies is the EC10 value because it is derived from the dose response curve. In contrast the NOEC strongly depends on the experiment design (e.g. the concentrations used in the test). Thus the 72-h EL50 and EL10 based on growth rate are used for classification purposes, which were determined in this study to be 45 mg/L and 14 mg/L respectively.