6,6-dimethoxy-2,5,5-trimethylhex-2-ene

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

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)

Specific details on test material used for the study:

Test item: 205601/B
Identification: Methyl Pamplemousse
Appearance: Colourless to pale yellow liquid
Batch: AS00239830
Test item storage: At room temperature protected from light
Stable under storage conditions until: 11 March 2017 (expiry date)

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.

Frequency at t=0 h, t=24 h, t=48h and t=72 h
Volume 4.0 mL
Storage Samples were stored in a freezer (≤ -15°C) until analysis

At the end of the exposure period, samples were taken from one of the replicates of each concentration.
Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at an intermediate item concentration but without algae and samples for analysis were taken at the start, after 24, 48 and 72 hours of exposure.
Additionally, reserve samples of 4.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤ -15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Details on test solutions:

Range Finding
A range-finding test was performed to provide information about the range of concentrations to be used in the final test. Test procedure and conditions were similar to those applied in the final test with the following exceptions:
- Exponentially growing algal cultures were exposed to nominal concentrations of 1.0, 10 and 100 mg/L;
- Three replicates were tested per concentration and three replicates in the control group; One extra test vessel per concentration without algae was used as background for the determination of the algal cell density at each time interval;
- Cell densities were recorded at 24-hour intervals in the control and the 100 mg/L concentration. Lower concentrations were measured only at the end of the exposure period;
- pH was only measured in the control and the highest test concentration;
- At the end of the test algae were not observed to verify a normal and healthy appearance.
- Samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure;
- Replicates without algae were not sampled

Final Test
Methyl Pamplemousse: 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L
Control: Test medium without test item or other additives
Replicates: 3 replicates of each test concentration; 6 replicates of the control; 2 extra replicates of each test concentration and the control for sampling after 24 and 48 hours of exposure; 3 replicates of each test concentration without algae for sampling after 24, 48 and 72 hours of exposure.

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 an unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.

Fresh water algae culture
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.
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

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Test temperature:

Measured continuously in a temperature control vessel.

pH:

Measured at the beginning and at the end of the test.

Nominal and measured concentrations:

Final Test Nominal concentration: 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L

Measured test item concentrations
Replicates with algae
No detectable test item concentrations were found in the samples taken from nominal concentrations up to and including 1.0 mg/L. Analysis of the samples taken at the start of the test showed measured concentrations of 1.9, 6.2, 21 and 74 mg/L at nominal concentrations of 3.2, 10, 32 and 100 mg/L, respectively. These concentrations decreased during the test period.

Replicates without algae
No detectable test item concentrations were found in the samples taken from nominal concentrations up to and including 1.0 mg/L. Analysis of the samples taken at the start of the test showed measured concentrations of 2.2, 6.4, 23 and 73 mg/L at nominal concentrations of 3.2, 10, 32 and 100 mg/L, respectively. These concentrations decreased during the test period.

The decrease of test item concentrations over time in the replicates without algae was only slightly lower than in the replicates with algae. The measured concentration in the replicate of the highest test concentration with algae after 48 hours of exposure was unexpectedly low and might have been caused by the respective test vessel not being completely air-tight. However, taking a worst-case scenario into account effect parameters were expressed in terms of the average exposure concentrations derived from the replicates with algae. All measurements of nominal concentrations of 3.2 mg/L and higher (with algae) were used in the calculation of these average exposure concentrations , i.e. 0.84, 2.4, 6.6 and 26 mg/L.

Details on test conditions:

Test duration: 72 hours
Test type: Static
Test vessels: 120 mL, airtight closed with no headspace to prevent any loss of the test item due to volatilization.
Medium: Adjusted M2
Cell density: An initial cell density of 1 x 10^4 cells/mL.
Illumination: Continuously using TLD-lamps with a light intensity within the range of 84 to 88 μE.m-2.s-1.
Incubation: Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

Appearance of the cells: Measured at the end of the final test microscopic observations were performed on the control and the 32 mg/L concentration to observe for any abnormal appearance of the algae.
Recording of cell densities: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with cuvettes (path length =10 mm. Algal medium was used as blank.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval ranging from 12 to 13 mg/L.

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

5.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence interval ranging from 5.1 to 6.0 mg/L.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.84 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.:

5.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% confidence interval ranging from 4.5 to 6.0 mg/L.

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% confidence interval ranging from 0.85 to 1.5 mg/L.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.84 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study with Pseudokirchneriella subcapitata, Methyl Pamplemousse inhibited both growth rate and yield of this fresh water algae species significantly at average exposure concentrations of 2.4 mg/L and higher.
The EC10 for growth rate inhibition (72h-ERC10) was 5.5 mg/L with a 95% confidence interval ranging from 5.1 to 6.0 mg/L.
The EC50 for growth rate inhibition (72h-ERC50) was 13 mg/L with a 95% confidence interval ranging from 12 to 13 mg/L.
The 72h-NOEC for growth rate inhibition was 0.84 mg/L.
The EC10 for yield inhibition (72h-EYC10) was 1.2 mg/L with a 95% confidence interval ranging from 0.85 to 1.5 mg/L.
The EC50 for yield inhibition (72h-EYC50) was 5.2 mg/L with a 95% confidence interval ranging from 4.5 to 6.0 mg/L.
The 72h-NOEC for yield inhibition was 0.84 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 v2.0, OECD 201 Guideline). 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 EC50 and EC10 based on growth rate are used for classification purposes, which were determined in this study to be 13 mg/L and 5.5 mg/L respectively.

Description of key information

The effects of the test item on the growth of the green alga, Pseudokirchneriella subcapitata, were determined during a 72-hour growth inhibition test. The study was performed according to GLP and OECD Guideline 201. The study met the acceptability criteria prescribed by the study plan and was considered valid.

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 EC50 and EC10 based on growth rate, which were determined in this study to be 13 mg/L and 5.5 mg/L respectively, are used for classification purposes and have been selected as the key values for chemical safety assessment.

Key value for chemical safety assessment

EC50 for freshwater algae:

13 mg/L

EC10 or NOEC for freshwater algae:

5.5 mg/L

Additional information

Based on the results of a range-finding test, the definitive test was conducted at nominal concentrations of 0.10, 0.32, 1.0, 3.2, 10, 32 and 100mg/L. Preparation of test solutions started with a loading rate of 100 mg/L applying one day of magnetic stirring in closed vessels to reach the maximum dissolution of the test item in the test medium. All lower test concentrations were prepared by subsequent dilutions of the saturated solution. The test item was suspected to be volatile and hence testing was performed in closed airtight vessels with a reduced headspace.

Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24, 48 and 72 hours of exposure. For each test concentration extra replicates without algae were incubated and sampled. No detectable test item concentrations were found in the samples taken from nominal concentrations up to and including 1.0 mg/L. Analysis of the samples taken at the start of the test showed measured concentrations of 1.9, 6.2, 21 and 74 mg/L at nominal concentrations of 3.2, 10, 32 and 100 mg/L, respectively. All concentrations decreased during the test period. The decrease of test item concentrations over time in the replicates without algae was only slightly less than in the replicates with algae. Taking a worst-case scenario into account effect parameters were expressed in terms of the average exposure concentrations derived from the replicates with algae. All measurements of nominal concentration of 3.2 mg/L and higher (with algae) were used in the calculation of these average exposure concentrations, i.e. 0.84, 2.4, 6.6 and 26 mg/L.

Under the conditions of the present study with Pseudokirchneriella subcapitata, Methyl Pamplemousse inhibited both growth rate and yield of this fresh water algae species significantly at average exposure concentrations of 2.4 mg/L and higher. The effect parameters obtained are summarised in the endpoint record table.