2,6,10-trimethylundec-9-enal

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:

18 July 2014 - 24 June 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test substance name: Adoxal
Batch number: SC00010875
Expiry date: 09 April 2015

Analytical monitoring:

yes

Details on sampling:

Analysis of the test media samples was conducted at 0, 24, 48 and 72 hours.

Vehicle:

yes

Remarks:

Acetone

Details on test solutions:

The test substance is poorly water soluble. The determined water solubility of the substance is 0.67 mg/L (Smithers Viscient study number 3200705). Therefore a media preparation trial was conducted comparing solvent spike and saturated solution methods of preparation. A 0.67 mg/L concentration was prepared from an initial solvent spike (Acetone) and analysis gave a measured concentration of 0.3795 mg/L. Analysis of a saturated solution prepared from an initial 100 mg/L loading rate followed by filtration gave a measured concentration of 2.6684 mg/L. Given that the latter was greater than the determined water solubility, it was considered that undissolved liquid test item had passed through the filter. Therefore the solvent spike method of preparation was considered the most appropriate method of preparation for the test item.

Test organisms (species):

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

Details on test organisms:

The test organism, Pseudokirchneriella subcapitata (Strain 278/4), was originally obtained from the Culture Collection of Algae and Protozoa (CCAP) and is a representative species of the freshwater aquatic phytoplankton. This species is recommended for testing in accordance with the OECD regulatory guidelines.

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Nominal and measured concentrations:

Definitive test:
nominal test concentrations of 0.042, 0.084, 0.168, 0.335 and 0.67 mg/L, the latter being equivalent to the determined water solubility of the test substance.
0-hour measured concetrations of 0.0422, 0.0632, 0.1490, 0.3063 and 0.5877 mg/L, the latter representing the solublity limit in the test media
Control and solvent control groups were also included.

Details on test conditions:

Test vessels (350 mL conical flasks) were prepared which were completely filled and sealed to minimise any potential losses through volatility. Each test vessel was inoculated with 1 x 104 alga cells/mL and incubated at 21 - 24°C (under 4440 - 8880 Lux light intensity) for 72 hours with cell counts at 24-hour intervals.

Reference substance (positive control):

yes

Remarks:

Control and solvent control groups were also included.

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.588 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: solubility limit

Remarks:

31% inhibition observed

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.427 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.288 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.149 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Effects based on yield and biomass were also reported (see attached full study report and summary below). 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, OECD 201 Guidelines). The guideline includes the additional response variable of yield, to satisfy current regulatory requirements in some countries. 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.

Analytical results showed that a reduction in the measured concentrations occurred over the course of the 72 hour test period.

The OECD 201 guideline explains that "The alga growth inhibition test is a more dynamic test system than most other short-term aquatic toxicity tests. As a consequence, the actual exposure concentrations may be difficult to define, especially for adsorbing substances tested at low concentrations. In such cases, disappearance of the test substance from solution by adsorption to the increasing algal biomass does not mean that it is lost from the test system. When the result of the test is analysed, it should be checked whether a decrease in concentration of the test substance in the course of the test is accompanied by a decrease in growth inhibition. If this is the case, application of a suitable model describing the decline of the concentration of the test substance may be considered. If not, it may be appropriate to base the analysis of the results on the initial (nominal or measured) concentrations."

Adoxal has a high log Kow of > 6 and as such is expected to adsorb to the increasing algal biomass and although, a decline in measured concentration was observed over the 72-hour test, it was not accompanied by a significant decrease in growth inhibition. Furthermore, the test was conducted in sealed vessels, with no head space in an effort to prevent loss due to volatility and maintain the maximum exposure concentrations. Therefore, the results have been based on the initial 0-hour measured concentrations. These were 0.0422, 0.0632, 0.1490, 0.3063 and 0.5877 mg/L (100, 75, 89, 91 and 88% of nominal respectively).

Based on initial measured concentrations, the 72-hour EyC50 and the 0-72 hour EbC50 and ErC50 values were calculated to be 0.433, 0.429 and greater than 0.5877 mg/L, respectively. The latter is considered to be the solubility limit of the test item under test conditions. The corresponding NOEC values for yield, biomass and specific growth rate after 72 hours was 0.149 mg/L, respectively. The 72-hour ErC10 value was 0.288 mg/L.

Validity criteria fulfilled:

yes

Remarks:

All validity criteria were met therefore the test was considered valid.

Conclusions:

Based on initial measured concentrations, the 72-hour EyC50 and the 0-72 hour EbC50 and ErC50 values were calculated to be 0.433, 0.429 and greater than 0.5877 mg/L, respectively. The latter is considered to be the solubility limit of the test item under test conditions. The corresponding NOEC values for yield, biomass and specific growth rate after 72 hours was 0.149 mg/L, respectively. The 72-hour ErC10 value was 0.288 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 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 > 0.5877 (solubility limit, 31% inhibition) and 0.288 mg/L respectively. The EbC50 of 0.429 mg/L was also consider as supporting data for the acute classification of Adoxal given that the EC50 based on growth rate was greater than the solubility limit.

Executive summary:

The objective of the study was to determine the effects of Adoxal on the growth of the green alga, Pseudokirchneriella subcapitata, during a 72-hour growth inhibition test. The test was conducted in accordance with OECD Chemicals Testing Guideline No. 201. Alga, Growth Inhibition Test (adopted 23 March 2006) (Annex 5 corrected 28 July 2011). The test substance was shown to have limited solubility in the test medium. Initial media preparation trials indicated that the most appropriate method of preparation for the test substance was via an auxiliary solvent. The definitive test was conducted at nominal test concentrations of 0.042, 0.084, 0.168, 0.335 and 0.67 mg/L, the latter being equivalent to the determined water solubility of the test substance (Smithers Viscient study number 3200705). The concentrations used were based on the results of a range-finding test, for which only the key findings have been reported. Control and solvent control groups were also included. Test vessels (350 mL conical flasks) were prepared which were completely filled and sealed to minimise any potential losses through volatility. Each test vessel was inoculated with 1 x 104 alga cells/mL and incubated at 21 - 24°C (under 4440 - 8880 Lux light intensity) for 72 hours with cell counts at 24-hour intervals. Analysis of the test media samples was conducted at 0, 24, 48 and 72 hours. Analytical results showed that a reduction in the measured concentrations occurred over the course of the 72 hour test period. Analysis of the test samples at 0 hours showed measured concentrations to range from 0.0422 – 0.5877 mg/L. At 24 hours, analysis showed measured concentrations to range from below the limit of quantification (LOQ) of the analytical method (assessed as 0.02 mg/L) to 0.2312 mg/L. Analysis at 48 hours showed no measured concentrations above the LOQ with the exception of the top concentration which showed a concentration of 0.1519 mg/L. Analysis at 72 hours showed no measured concentrations above the LOQ. Given this decline in measured test concentrations, results were based on the geometric mean measured test concentrations in order to give a "worst case" analysis of the data. In order to calculate the geometric mean measured concentrations a value of ½ the LOQ (0.01 mg/L) was substituted in place of the below the LOQ recoveries. The geometric mean measured concentrations were 0.0143, 0.0220, 0.0324, 0.0464 and 0.1199 mg/L (34, 26, 19, 14 and 18% of nominal respectively). However, the test was conducted in sealed vessels, with no head space in an effort to prevent loss due to volatility and maintain the maximum exposure concentrations. Although a decline in measured concentration was observed over the 72-hour test period it was not accompanied by a significant decrease in growth inhibition; therefore the results have also been based on the initial 0-hour measured concentrations as recommended in the OECD 201 Test Guideline. These were 0.0422, 0.0632, 0.1490, 0.3063 and 0.5877 mg/L (100, 75, 89, 91 and 88% of nominal respectively). Based on geometric mean measured concentrations, the 72-hour EyC50 and the 0-72 hour EbC50 and ErC50 values were calculated to be 0.0807, 0.0793 and greater than 0.1199 mg/L, respectively. The corresponding NOEC values for yield, biomass and specific growth rate after 72 hours was 0.0324 mg/L, respectively. and ErC50 values were calculated to be 0.433, 0.429 and greater than 0.5877 mg/L, respectively. The latter is considered to be the solubility limit of the test item under test conditions. The corresponding NOEC values for yield, biomass and specific growth rate after 72 hours was 0.149 mg/L, respectively. All validity criteria were met therefore the test was considered valid.

Description of key information

ErC50 (72h): > 0.5877 mg/L (solubility limit, 31% inhibition)

Key value for chemical safety assessment

EC50 for freshwater algae:

0.588 mg/L

EC10 or NOEC for freshwater algae:

0.288 mg/L

Additional information

The effects of Adoxal on the growth of the green alga, Pseudokirchneriella subcapitata, were determined during a 72-hour growth inhibition test. Initial media preparation trials indicated that the most appropriate method of preparation for the test substance was via an auxiliary solvent. The definitive test was conducted at nominal test concentrations of 0.042, 0.084, 0.168, 0.335 and 0.67 mg/L, the latter being equivalent to the determined water solubility of the test substance (Smithers Viscient study number 3200705). Control and solvent control groups were also included. Test vessels were prepared which were completely filled and sealed to minimise any potential losses through volatility. Analysis of the test media samples was conducted at 0, 24, 48 and 72 hours.

The OECD 201 guideline explains that "The alga growth inhibition test is a more dynamic test system than most other short-term aquatic toxicity tests. As a consequence, the actual exposure concentrations may be difficult to define, especially for adsorbing substances tested at low concentrations. In such cases, disappearance of the test substance from solution by adsorption to the increasing algal biomass does not mean that it is lost from the test system. When the result of the test is analysed, it should be checked whether a decrease in concentration of the test substance in the course of the test is accompanied by a decrease in growth inhibition. If this is the case, application of a suitable model describing the decline of the concentration of the test substance may be considered. If not, it may be appropriate to base the analysis of the results on the initial (nominal or measured) concentrations."

Adoxal has a high log Kow of > 6 and as such is expected to adsorb to the increasing algal biomass and although, a decline in measured concentration was observed over the 72-hour test, it was not accompanied by a significant decrease in growth inhibition. Furthermore, the test was conducted in sealed vessels, with no head space in an effort to prevent loss due to volatility and maintain the maximum exposure concentrations. Therefore, the results have been based on the initial 0-hour measured concentrations. These were 0.0422, 0.0632, 0.1490, 0.3063 and 0.5877 mg/L (100, 75, 89, 91 and 88% of nominal respectively). Based on initial measured concentrations, the 72-hour EyC50 and the 0-72 hour EbC50 and ErC50 values were calculated to be 0.433, 0.429 and greater than 0.5877 mg/L, respectively. The latter is considered to be the solubility limit of the test item under test conditions. The corresponding NOEC values for yield, biomass and specific growth rate after 72 hours was 0.149 mg/L, respectively. The 72-hour ErC10 value was 0.288 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 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 > 0.5877 (solubility limit, 31% inhibition) and 0.288 mg/L respectively. The EbC50 of 0.429 mg/L was also consider as supporting data for the acute classification of Adoxal given that the EC50 based on growth rate was greater than the solubility limit. Since the average specific growth rate for estimating toxicity is scientifically preferred, the ErC50 of > 0.5877 mg/L is used for PNEC derivation.