Registration Dossier

Ecotoxicological information

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:
March 06, 2017 to July 07, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
TEST MATERIAL
Name ( stated on the report) : Hexenyl-3-cis Isobutyrate
Appearance: Colourless to pale yellow liquid
Batch: VE00450298
Expiry date: July 15, 2017

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
For the determination of the actual test item concentrations, duplicate samples were taken from each treatment at the start of the test.
After 24, 48 hours and at the end of the test (after 72 hours), stability samples (containing algae) were taken in duplicate from all test concentrations and from the control.
The stability samples (duplicate 10 mL samples) at 24 and 48 hours could not be taken from the test vessels itself, as the principle of a closed system is, that the test vessels have to remain completely filled with test medium during the entire test period. Therefore, for these samplings at 24 and 48 hours, two set of additional flasks containing the corresponding test medium (with algae) was incubated under conditions identical to the test.
For sampling at the end of the test, the test medium of the treatment replicates was pooled. Additional test vessels with test medium at a concentration of 3.2 mg/L were incubated under the following conditions:
- Test medium without algae under light conditions in order to document possible adsorption of the test item onto algae.
- Test medium without algae

Immediately after sampling, acetonitrile (3 mL acetonitrile per 10 mL sample volume) was added to each sample in order to stabilize the latter during the storage period. Thereafter, all samples were stored deep-frozen (at -20 ± 5 °C). Based on pre-experiments for investigation of the storage stability, the test item was found to be stable in the test water under these storage conditions.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test organism used for the study was Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum, occasionally also listed as Raphidocelis subcapitata), Strain No. 61.81 SAG, supplied by the Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany). The algae were cultivated at IES Ltd Laboratories under standardized conditions according to the test guidelines.
An inoculum culture was set up three days before the start of the exposure. The algae were cultivated under the test conditions and were kept in the exponential growth phase until inoculation of the test solutions.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Hardness:
15 mg/L as CaCO3
Test temperature:
22°C
pH:
7.5 - 7.8
Nominal and measured concentrations:
Nominal concentrations: 0.32, 1.0, 3.2, 10, 32 and 100 mg/L
Mean Measured concentrations: 0.13, 0.39, 0.99, 2.5, 13 and 77 mg/L
Details on test conditions:
The test flasks were incubated in a temperature controlled orbital shaker (Multitron-Pro, Infors HT, Bottmingen/Switzerland) at a temperature of 22 °C.
The test flasks were positioned randomly and repositioned daily. They were continuously illuminated by LED light installed above the test flasks.
The light intensity at the level of the test solutions was approximately 66 μE s-1 m-2 (range: 65 to 68 μE s-1 m-2, measured at nine places in the experimental area). The light intensity over the HEXENYL-3-CIS ISOBUTYRATE IES Study 20160308 Report Page 13 of 55 incubation area was within a ±15%-deviation from the average light intensity as recommended by the guideline.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
5.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
2.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
The measured concentrations of HEXENYL-3-CIS ISOBUTYRATE in the test media of all test concentrations were between 80 and 99% of the nominal values at the start of the test (see analytical results and Table 7 in Appendix 1). Thus, the correct dosage of the test item could be verified.
At the end of the test period, the test item concentrations in the test media were between The analytical results of the additional test vessels (without algae, in the dark and under light conditions) could not clarify potential adsorption and photolytic processes (as observed in the range finding test at 10 mg/L) due to the high decrease of test item concentration at the lower concentration level of 3.2 mg/L (see Table 7, Appendix 1). The biological results are based to the mean measured concentrations. These were calculated as the geometric mean of the concentrations measured at all sampling dates (0, 24, 48 and 72 hours). A clear concentration-response relationship was observed for both biological endpoints growth rate and yield during the exposure period.

The test item had a statistically significant inhibitory effect on the growth rate μ and yield Y of the algae after the test period of 72 hours at the mean measured concentration of 2.5 mg/L and at all higher test concentrations.

Therefore, the NOEC for growth rate and yield was determined to be at the mean measured concentration of 0.99 mg/L.

The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of 10 mg/L (mean measured 2.5 mg/L) and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration. No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.
Results with reference substance (positive control):
The 72-hour EC50 for growth rate in the reference test IES Study Number 20160277 was 0.9 mg/L (October 2016) and showed that the sensitivity of the test system was within the range recommended by the guideline (72-hour EC50 for the growth rate 0.9-1.5 mg/L).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
The validity criteria for increase of biomass, mean coefficient of variation of the daily growth rates and coefficient of variation of the average specific growth rates were fulfilled for the control
Conclusions:
The impact of the test item HEXENYL-3-CIS ISOBUTYRATE on the growth of the freshwater green algal species Pseudokirchneriella subcapitata is summarized in the table below. The results are based on the mean measured concentrations of the test item:
ErC50(72h) = 5.3 mg/L
ErC10 (72h) = 2.6 mg/L
Executive summary:

The impact of the test item HEXENYL-3-CIS ISOBUTYRATE on the growth of the freshwater green algal species Pseudokirchneriella subcapitata was investigated in a 72-hour static test according to the OECD Guideline 201 (2006, corrected 2011) and the Commission Regulation (EU) No 2016/266, C.3.

As the test item is a volatile substance, the test was performed using glass stoppered Erlenmeyer flasks completely filled (without headspace) with test medium that were tightly sealed with glass stoppers to avoid losses of test item by evaporation (closed system). The nominal test item concentrations tested were 0.32, 1.0, 3.2, 10, 32 and 100 mg/L.

Additionally, a control (test water without test item) was tested in parallel. The test media was prepared by completely dissolving the test item in test water using intense stirring for 24 hours in a closed vessel. The mixing vessel was nearly completely filled (including a small headspace) and tightly sealed with glass stoppers.

The measured concentrations of HEXENYL-3-CIS ISOBUTYRATE in the test media of all test concentrations were between 80 and 99% of the nominal values at the start of the test. Thus, the correct dosage of the test item could be verified.

At the end of the test period, the test item concentrations in the test media were between <LOQ (analytical limit of quantification, LOQ = 0.205 mg/L) and 66% of the nominal values.

The biological results are based on the mean measured concentrations. These were calculated as the geometric mean of the concentrations measured at all sampling dates (0, 24, 48 and 72 hours).

A clear concentration-response relationship was observed for both biological endpoints growth rate and yield after the exposure period of 72 hours. A statistically significant inhibitory effect on the growth rate and yield of the algae was observed at the end of the test at the mean measured concentration of 2.5 mg/L.

ErC50(72h) = 5.3 mg/L

ErC10 (72h) = 2.6 mg/L