Reaction mass of Terpenes and Terpenoids, turpentine-oil, limonene fraction, 1-methyl-4-(1-methylethenyl)cyclohexene and turpentine-oil beta-pinene fraction terpenes, dimers and Terpenes and Terpenoids, turpentine-oil, limonene fraction, 1-methyl-4-(1-methylethenyl)cyclohexene and turpentine-oil beta-pinene fraction terpenes, trimers

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:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Principles of method if other than guideline:

Due to the low aqueous solubility and complex nature of the test item, the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item as recommended by regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al. 2000).

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: Terpenes and terpenoids, turpentine oil, alpha-pinene fraction oligomers (70750-57-1)
Description: Extremely viscous pale amber colored liquid
Batch:0910002624
Purity:Not applicable – complex mixture
Expiry date:Indefinite Storage conditions:Room temperature in the dark

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples were taken from the control and the 100 mg/L loading rate WAF test vessel from the bulk preparation at 0 hours nd from the pooled replicates at 72 hours.
- Sample storage conditions before analysis: Samples were stored frozen prior to analysis.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item. An amount of test item (200 mg) was added to the surface of 2L of culture medium to give the 100 mg/L loading rate. After the addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 h and the mixture allowed to stand for 1 h. A wide bore glass tube, covered at one end with Nescofilm was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was inserted into the glass tube and pushed through the Nescofilm seal. The aqueous phase or WAF was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 100 mg/L loading rate WAF.

- Controls: negative control (medium without test item), positive control (potassium dichromate)
- Evidence of undissolved material: Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. After siphoning and for the duration of the test, the 100 mg/L loading rate was observed to be a clear, colorless solution.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd., Scottish Marine Institute, Oban, Argyll, Scotland
- Age of inoculum (at test initiation): Pre-culture conditions gave an algal suspension in log phase characterised by a cell density of 1.03 x 10 6 cells/mL. Initial nominal cell density after inoculation the test medium was 5 x 10 3 cells/mL.
- Method of cultivation: Cultures were maintained in the laboratory by the periodic replenishement of culture medium.

ACCLIMATION
- Acclimation period: yes
- Culturing media and conditions (same as test or not): Culturing media were the same as during the test.
- Any deformed or abnormal cells observed: not reported

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Hardness:

Not reported.

Test temperature:

24 ± 1°C

pH:

7.5 - 7.8

Dissolved oxygen:

Not reported.

Nominal and measured concentrations:

A single nominal loading rate of 100 mg/L Water Accommodated Fraction was used. Measured concentrations were less than the limit of quantification (LOQ) at 0 and 72 hours.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks plugged with polyurethane foam bungs
- Material, size, headspace, fill volume: Flasks were filled with 100 mL of test preparation.
- Aeration: no
- Initial cells density: 5 x 10 3 cells/mL
- Control end cells density: 6.47 x 10 5 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, AAP medium was used.

OTHER TEST CONDITIONS
- Adjustment of pH: pH of the AAP medium was adjusted to 7.5 with 0.1N NaOH or HCl.
- Photoperiod: continuous illumination
- Light intensity and quality: approximately 7000 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Cells densities were determined at 0, 22, 46 and 72 hours using a Coulter Multisizer Particle Counter. the shape and size of the algal cells was inspected microscopically and any abnormalities recorded in the control and test culture at the end of the test.

TEST CONCENTRATIONS
- Range finding study: yes
- Test concentrations: 10 and 100 mg/L loading rate WAF (nominal)
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 100 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 100 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 other: mg/L loading rate WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

There were no abnormalities in the control or test cultures at 72 hours. The growth rate and yield were not affected by the presence of the test item over the 72-hour exposure period.

Results with reference substance (positive control):

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
ErC50 (0-72 h): 1.1 mg/L, 95% CL: 0.91 - 1.2 mg/L
EyC50 (0-72 h): 0.51 mg/L, 95% CL: 0.45 - 0.59 mg/L
NOEC: 0.25 mg/L (based on growth rate and yield)
LOEC: 0.5 mg/L (based on growth rate and yield)

Reported statistics and error estimates:

There were no statistically significant differences (p≥0.05) between the control and 100 mg/L loading rate WAF test group based on growth rate or yield data in a Student's t-test incorporating Bartlett's test for homogenicity of variance.

Validity criteria fulfilled:

yes

Remarks:

The cells concentration of the control increased by a factor of 139 after 72 hours. the mean CV for section by section specific growth rate for the control was 5%. The CV for average specific growth rate for the control over the 72 hours was 3%.

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and gave EL50 values greater than 100 mg/L loading rate WAF. The NOELR was 100 mg/L loading rate WAF.

Executive summary:

A GLP-compliant study following OECD guideline 201 and EU Method C.3 was conducted using the test material Terpenes and terpenoids, turpentine oil, alpha-pinene fraction oligomers

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). Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to a Water Accommodated Fraction (WAF) of the test item at single nominal loading rate of 100 mg/L (six replicates) for 72 hours, under constant illumination and shaking at a temperature of approximately 24 °C. Samples of the algal populations were removed daily and cell concentrations were determined for each control and treatment group, using a Coulter Multisizer Particle Counter.

In conclusion, the exposure of Pseudokirchneriella subcapitata to the test item gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

Description of key information

A GLP-compliant study following OECD guideline 201 and EU Method C.3 was conducted using the test materialTerpenes and terpenoids, turpentine oil, alpha-pinene fraction oligomers

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). Following a preliminary range-finding test,Pseudokirchneriella subcapitatawas exposed to a Water Accommodated Fraction (WAF) of the test item at single nominal loading rate of 100 mg/L (six replicates) for 72 hours, under constant illumination and shaking at a temperature of approximately 24 °C. Samples of the algal populations were removed daily and cell concentrations were determined for each control and treatment group, using a Coulter Multisizer Particle Counter.

In conclusion, the exposure ofPseudokirchneriella subcapitatato the test item gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L