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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:
April 2014 to March 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
impurity
Test material form:
other: liquid, may crystalize to white solid
Specific details on test material used for the study:
Test substance name: Pivacyclene
Chemical name: 3a,4,5,6,7,7a-Hexahydro-4,7-methano-1H-inden-5(6)-yl-pivalate
Molecular Formula: C15H22O2
Molecular Weight: 234.3
Purity: 93.0% (sum of the isomers)
Vapour Pressure: 0.361 Pa at 20 deg C and 0.554 Pa at 25 deg C
Water Solubility: 3.59 mg/L
Octanol water partition co-efficient: 5.38
Batch number: SC0004118
Receipt date: 10 March 2014 and 21 March 2014
Re-test expiry date: 14 March 2015
Storage conditions: Refrigerated (2 - 8 °C)

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Test vessel sampling: At approximately 24-hour intervals after the start of the incubation period, pre-determinded volumes of test media were removed from each incubated test vessel, and transferred to individually identified cell counting vials. The contents of each vial were diluted to a 10 mL final volume with an electrolyte solution. The cell density of the vial contents was them determined using a particle counter.
At each sampling occasion, duplicate samples were taken. One for chemical analysis and one as a 'back up' sample should further analysis be required. in addition, 4 mL of hexane was added immediately to each sample to stabilise the sample.

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

Test solutions

Vehicle:
no
Details on test solutions:
The most suitable method of preparation for the test substance was to prepare a saturated solution using a loading rate of 100 mg/L, stirred for ca 24 hours and filtered to give a nominal test concentration of 100% saturated solution. Analysis of the filtrates showed measured concentrations of 1.39 - 2.07 mg/L, indicating this to be the solubility limit in the test media.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The test organism was obtained from the Culture Collection of Algae and Protozoa (CCAP) and is a representative species of the freshwater aquatic phytoplankton.
Prior to testing, duplicate starter clutures were prepared and incubated under test conditions to obtain sufficient algal cells in exponential growth and to achieve a starting alga cell density of 1 x 10^4 cells/mL.

Study design

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

Test conditions

Test temperature:
The laboratory temperature was set within the range 21 - 24 °C and maintained within ± 2°C for the duration of the test. The temperature was recorded continuously using a digital logger.
Test vessels were incubated at 21 - 24 °C (under 4440 - 8880 Lux light intensity)
pH:
At the start of the test, the pH of freshly prepared test media was determined. The pH in each test vessel was also determined at the end of the test. The
OECD guideline states that the pH in the control should not vary by more than 1.5 units during the test. At the end of the test elevated pH values were observed in some of the control vessels. This is an indicator of good algal cell growth, whereby the release of oxygen from the algal cells during normal photosynthetic activity promotes an alkali pH in the surrounding test media. It was considered that the elevated pH levels was due to good algal cell growth and therefore considered not to affect the validity of the test.
Nominal and measured concentrations:
The definitive test was conducted at nominal test concentrations of 10, 18, 32, 56 and 100% saturated solution (0.25, 0.46, 0.79, 1.4 and 2.5 mg/L based on initial measured concentrations).
The geometric mean measured concentrations were 0.043, 0.081, 0.17, 0.29, and 0.53 mg/L.
Details on test conditions:
Light intensity at 0 hours (lux): 4740
Light intensity at 72 hours (lux:) 4560
Reference substance (positive control):
not specified

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
2.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Estimated by extrapolation using non-linear regression analysis
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 0.53 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.:
0.32 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.17 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield, biomass and 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). 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.

Analysis of the test sample at 0 hours showed measured concentrations to range from 0.252 to 2.45 mg/L. A gradual decline in measured concentrations was observed in the 24, 48 and 72 hour samples with measured concentrations at 72 hours ranging from 0.0125 to 0.160 mg/L. Given this decline in measured concentration over the 72-hour test period, it was considered justifiable to base the results on geometric mean measured concentrations.

Based on geometric mean measured test concentrations, the 72-hour EyC50 and the 0-72 hour EbC50 and ErC50 values were calculated to be 0.41, 0.41 and >0.53 mg/L, respectively. The 72-hour EyC10 and the 0-72 hour EbC10 and ErC10 values were calculated to be 0.05, 0.05 and 0.32 mg/L, respectively. The ErC50 was estimated to be 2.1 mg/L by extrapolation using non-linear regression analysis. The corresponding NOEC values for yield, biomass and specific growth rate after 72 hours were 0.17 mg/L.

The results differed slightly from those observed in the range-finding test which suggested that all EC50 values would be >100% saturated solution. This was considered to be possibly due to natural variation in the response of the algae and/or the increased sensitivity of the definitive test due to increased replication. Although a flat, and slightly variable, dose response was observed at the 0.081 to 0.29 mg/L test concentrations, a clear response was observed at the highest test concentration therefore the results are considered to be valid.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The effects of pivacyclene on the growth of the unicellular green alga, Pseudokirchneriella subcapitata, were determined during a 72-hour growth inhibition toxicity test conducted in accordance with OECD Chemicals Testing Guideline No. 201 Alga, Growth Inhibition Test (adopted 23 March 2006) (Annex 5 corrected 28 July 2011).

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

Based on geometric mean measured test concentrations, the 72-hour EyC50 and the 0-72 hour EbC50 and ErC50 values were calculated to be 0.41, 0.41 and >0.53 mg/L, respectively. The 72-hour EyC10 and the 0-72 hour EbC10 and ErC10 values were calculated to be 0.05, 0.05 and 0.32 mg/L, respectively. The ErC50 was estimated to be 2.1 mg/L by extrapolation using non-linear regression analysis. The corresponding NOEC values for yield, biomass and specific growth rate after 72 hours were 0.17 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 > 0.53 (estimated to be 2.1 mg/L by extrapolation using non-linear regression) and 0.32 mg/L respectively.