Terpenes and Terpenoids, sinpine

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:

10 March 2014 - 7 May 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Lime Oxide

IUPAC Name:

Lime Oxide

Test material form:

liquid

Specific details on test material used for the study:

Identification: Lime Oxide
Chemical name: 1,6-octadien-3-ol, 3,7-dimethyl-, acid-isomerized
IUPAC name: Reaction mass of (E)-5-(but-2-en-2-yl)-2,2-dimethyltetrahydrofuran and 1-isopropyl-4-methylcyclohexa-1,3-diene and 1-isopropyl-4-methylcyclohexa-1,4-diene and 1-methyl-4-(propan-2-ylidene)cyclohex-1-ene
Empirical formula:
C10H16 and C10H18O*
Molecular weight:
136.2 g/mol and 154.2 g/mol*
EC No.:
277-225-8
CAS No.:
73018-51-6
Physical state/Appearance:
Clear colorless liquid
Batch:
SC00009577
Purity:
61.8%, sum of 4 major peaks (see certificate of analysis)**

* From information provided by the Sponsor, the test item is predominately a mixture of unsaturated monoterpene hydrocarbons (C10H16, MW 136.2 g/mol) and cyclic monoterpene ethers (C10H18O, 154.2 g/mol)
** Although the purity is stated as 61.8% (sum of 4 peaks), all known impurities are similar to the main constituents.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each loading rate WAF test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. In cases where the test item is a complex mixture and is poorly soluble in water, an approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC 1996, OECD 2000 and Singer et al 2000), is to expose organisms to a Water Accommodated Fraction (WAF) of the test item. Using this approach, aqueous media are prepared by mixing the test item with water for a prolonged period. At the completion of mixing and following a settlement period, the test item phase is separated by siphon and the test organisms exposed to the aqueous phase or WAF (which may contain dissolved test item and/or leachates from the test item). Exposures are expressed in terms of the original concentration of test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of test item in the WAF.

Test organisms

Test organisms (species):

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

Details on test organisms:

The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland. Master cultures were maintained in the laboratory by the periodic replenishment of culture medium. The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1 °C.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Test conditions

Test temperature:

Temperature was maintained at 24 ± 1 ºC throughout the test

pH:

The pH of the control and each test concentration was determined at initiation of the test and after 72 hours exposure. The pH was measured using a Hach HQ30d Flexi handheld meter. The temperature within the incubator was recorded daily.
t=0
pH = 7.0 to 8.1
t= 72 h
pH= 8.0 to 9.8

Nominal and measured concentrations:

The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 10 and 100 mg/L for a period of 72 hours.
Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 10, 18, 32, 56 and 100 mg/L.

Details on test conditions:

GROWTH MEDIUM
The culture medium used for the definitive test, range-finding test and for maintenance of the stock cultures was ASTM. However, as the definitive test was conducted in completely filled and sealed vessels the culture medium was modified by the addition of 500 mg/L of sodium bicarbonate to provide a sufficient supply of CO2 and to counteract the increase in pH due to algal growth in an enclosed system (Herman et al 1990).

Reference substance (positive control):

yes

Remarks:

Positive control tests (Study Number 41403074 and 41403365) used potassium dichromate as the reference item.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Loading rate WAF; 95% confidence limits

Remarks:

14-16mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Loading rate WAF

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Loading rate WAF

Details on results:

Since the test item consists of multiple components with different water solubility (i.e. unsaturated monoterpene hydrocarbons and oxygen-containing monoterpenes), the test was performed using Water Accomodated Fractions (WAF) and the effect parameters based on nominal loading rates. Chemical analysis at 0 and 72h demonstrated that exposure to the dissolved test item concentration (i.e. the WAF) was maintained throughout the exposure period.

Observations on the test media were carried out during the mixing and testing of the WAFs.
At both the start and end of the mixing period, and following a 1-Hour standing period, all loading rate WAFs were observed to have formed clear colorless media columns with an oily slick of test item floating at the media surface. Microscopic examination of the WAFs showed there to be no micro-dispersions of test item present.
At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control and 10 mg/L loading rate WAF test cultures were observed to be bright green dispersions whereas the 18, 32 and 56 mg/L loading rate WAF test cultures were observed to be clear colorless solutions. The 100 mg/L loading rate WAF cultures were clear colorless solutions with light blue precipitate suspended in solution.

Effects based on yield were also reported (see attached full study report). However, the preferred observational endpoint in the algal inhibition study 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. Thus only the effects based on growth rate are presented in the above "effects concentration" table. Furthermore, 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 have been selsected as the key results and used for classification purposes.

Results with reference substance (positive control):

Positive control studies using potassium dichromate as the reference item were conducted in a conventional test system (Study Number 41403074) and in a modified test system using completely filled and sealed vessels (Study Number 41403365).
Exposure conditions for the modified positive control were similar to those in the definitive test (i.e. completely filled and sealed vessels). The data evaluation for both positive control tests was similar to those in the definitive test. Both tests were conducted using ASTM culture medium, however for the modified test the culture medium contained 500 mg/L of sodium bicarbonate to provide a sufficient supply of CO2 and to counteract the increase in pH due to algal growth in an enclosed system (Herman et al 1990).
The results from the positive control test with potassium dichromate in a conventional test system were within the normal ranges for this reference item. Additionally, the results of the positive control test with potassium dichromate in a modified test system were comparable with the conventional test system.
It is evident from these results that the use of a modified test system to reduce losses of test item through volatility (completely filled and sealed vessels) is expected to give results consistent with that obtained in a conventional system. As such it can be considered that the results obtained from the definitive test give a reliable result for the test item.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and gave the EL50 (growth rate) of 15 mg/L and EL10 (growth rate) of 11mg/L as key results.

Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 761/2009.

Methods

Information provided by the Sponsor indicated that the test item is a complex mixture composed of constituents with different water solubility (i.e. unsaturated monoterpene hydrocarbons and oxygen-containing monoterpenes). 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). The test item was suspected to be volatile and hence testing was conducted in completely filled, stoppered test vessels in order to minimize possible losses due to volatilization. Following the recommendations of published data (Herman et al 1990) in order to prevent inhibition of growth due to the restriction of gaseous exchange, additional sodium bicarbonate was added to the culture medium to provide a source of carbon dioxide for algal growth. Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 10, 18, 32, 56 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.

Results

Analysis of the test preparations at 0 hours showed measured test concentrations in the range of 0.92 mg/L (10 mg/L loading rate WAF) to 23 mg/L (100 mg/L loading rate WAF). Measured test concentrations in the range of 0.77 to 22 mg/L were observed at 72 hours (84 to 96% of initial measured concentrations), indicating that the dissolved test item concentration had been maintained throughout the exposure period. Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and gave the EL50 (growth rate) of 15 mg/L and EL10 (growth rate) of 11 mg/L as key results.