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:
01 December 2017 to 22 February 2018
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

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
Test material form:
liquid
Specific details on test material used for the study:
TRASEOLIDE :
Givaudan Product Code : 9366993
Batch No. : VE00416911
Purity : 95.15% (sum of isomers)
Date of Expiry : 03 June 2019

Sampling and analysis

Analytical monitoring:
yes
Remarks:
HPLC - UV/VIS
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.

Duplicate samples were taken from the test media of all test concentrations and from the control
at the start of the test (without algae). 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. For
sampling at the end of the test, the test medium of the treatment replicates was pooled.

All samples were stored frozen (at -20 ± 5 °C) immediately after sampling. Based on preexperiments
for investigation of the storage stability, the test item was found to be stable in the
test water under these storage conditions.

The concentrations of TRASEOLIDE were analytically measured in one of the duplicate
samples taken from all treatments at the sampling times of 0 and 72 hours.

Test solutions

Vehicle:
no
Details on test solutions:
At the start of the main test, the highest concentrated test medium (i.e. the undiluted equilibrated
test medium with a loading rate of 100 mg/L) was prepared following the slow-stirring method
with a stirring period of 96 hours at room temperature in the dark. For this,
234.8 μL of test item were carefully applied (pipetted) onto the surface of 2300 mL test water.
This volume is equivalent to a loading rate of 100 mg/L, considering the density of the test item
of 0.9797 g/cm3. No auxiliary solvent or emulsifier was used.

This equilibrated aqueous phase with a loading rate of 100 mg/L, containing dissolved test item
only, was used as the highest test concentration and was diluted with test water to obtain the
dilutions 1:2.2, 1:4.6, 1:10 and 1:22. Additionally, a control (test water only) was run in parallel.

The test media were prepared just before the start of the test.

All solutions were clear with no evidence of undissolved test item.

The preparation of the test media was based on the OECD Guidance Document No. 23 on
Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

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.

Nygaard et al.recommended describing the taxa within the Genus Raphidocelis HINDAK
as:
Raphidocelis subcapitata (KORSHIKOV) nov. comb.
Basionym: Ankistrodesmus subcapitatus KORSHIKOV
Syn.: Kirchneriella subcapitata KORSHIKOV
Syn.: Selenastrum capricornutum PRINTZ
Syn.: NIVA-CHL 1

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.

After the end of the evaluations the algae cultures in the treatments including the control were
disposed.

For evaluation of the algal quality and experimental conditions, potassium dichromate is tested
as a positive control twice a year to demonstrate satisfactory test conditions. The 72-hour EC50
for growth rate in the reference test IES Study Number 20170367 was 1.1 mg/L (October 2017)
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).

The test method and the test species are recommended by the test guidelines.

Study design

Test type:
static
Water media type:
freshwater
Remarks:
AAP Medium
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
The microscopic examination of the algal cells at the end of the test showed no difference
between the algae growing at the highest mean measured concentration of 0.46 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.

Test conditions

Hardness:
15 mg/L as CaCO3
Test temperature:
23 °C
pH:
The pH of the test media was in the range of 7.6 to 7.9 during the test period.
Nominal and measured concentrations:
The concentrations determined in the freshly prepared test sample (day 0/0 hours) were 0.0173 mg/L
(dilution 1:22), 0.0424 mg/L (dilution 1:10), 0.110 mg/L (dilution 1:4.6), 0.217 mg/L (dilution
1:2.2) and 0.489 mg/L (undiluted). The measured concentrations reflect the dilution factors.
The concentrations determined in the test samples taken from the aged test media
(day 3/72 hours) were 0.0143 mg/L (dilution 1:22), 0.0352 mg/L (dilution 1:10), 0.0854 mg/L
(dilution 1:4.6), 0.181 mg/L (dilution 1:2.2) and 0.441 mg/L (undiluted).
The test item concentration was almost stable over the test period.

The concentration determined in the volatility test sample (dilution 1:4.6) taken from the aged
test media which was incubated under identical test conditions as the actual test, but without
daily sampling was found to be 0.0892 mg/L. There was no clear influence of the daily sampling
on the test item concentration.

Treatment / Dilution Mean Msd (Geometric Mean Concn. (mg/L)
Control < LOQ (0.00993 mg/L)
1 : 22 0.016 mg/L
1 : 10 0.039 mg/L
1 : 4.6 0.10 mg/L
1 : 2.2 0.20 mg/L
Undiluted Test Medium (WSF) 0.46 mg/L

The biological results are reported based on both the Time 0 starting concentrations as well as the Geometric Mean measured concentration.
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 23 °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
67 μE s-1 m-2 (range: 65 to 69 μE s-1 m-2, measured at nine places in the experimental area).

The light intensity over the incubation area was within a ±15 %-deviation from the average
light intensity as recommended by the guideline.

Based on the results of the stirring experiment and the range-finding test and in agreement with
the Sponsor, the following concentrations of TRASEOLIDE were selected for the main test:
The undiluted equilibrated test medium with a loading rate of 100 mg/L and the dilutions 1:2.2,
1:4.6, 1:10 and 1:22. Additionally, a control (test water without test item) was tested in parallel.

The main test was performed in a closed system to avoid losses of the test item by evaporation.
The test design included three replicates per test concentration and six replicates for the control.
The test was started using a nominal algal cell density of 5000 cells/mL. The initial cell density
was selected according to the recommendations of the OECD test guideline. The algal cell
density in the pre-culture was determined using an electronic particle counter (Cell Counter
CASY TT, OLS, Bremen/Germany).

Determination of the Algal Biomass :
A small volume (100 μL per sample) of the algal suspension was withdrawn daily from each
test flask for the measurement of the biomass, and was not replaced.

The algal biomass in the samples was determined by fluorescence measurement (SpectraMax
I3x, Molecular Devices Ltd, Wokingham Berkshire/UK). The measurements were performed
at least in duplicate at an excitation of 440 nm and emission of 680 nm.

At the end of the test, a sample was taken from the control and from the highest concentration
(undiluted equilibrated test medium with a loading rate of 100 mg/L) to determine a potential
influence of the test item on the algal cells. The shape and size of the algal cells were visually
inspected.

A static test design was applied. The duration of the test was 72 hours.

Monitoring of the Experimental Conditions :
The light intensity was measured at the start of the test. The pH was measured and recorded in
each treatment at the start and end of the test. The temperature in the incubator was monitored
and recorded continuously. The appearance of the test media was also visually controlled and
recorded daily during the exposure period.

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.:
> 0.46 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: ErC50 greater than water saturation concentration of test item (14.1% growth rate inhibition observed at saturation)
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.28 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% Confidence Interval : 0.26 - 0.31 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.039 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.:
0.43 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: 95% Confidence Interval : 0.40 - 0.47 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.078 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Remarks on result:
other: 95% Confidence Interval : 0.066 - 0.089 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.039 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Yield
Details on results:
At the mean measured concentrations of 0.016 and 0.039 mg/L, the mean inhibition values for
growth rate and yield were not statistically significantly different from the control. The test item
had a significant inhibitory effect on the growth rate and yield of the algae after the test period
of 72 hours at the test concentrations of 0.10 to 0.46 mg/L (results of Williams t-test, one-sided
smaller, α = 0.05).

Therefore, the NOEC for yield and growth rate was determined to be at the mean measured
concentration of 0.039 mg/L.

The microscopic examination of the algal cells at the end of the test showed no difference
between the algae growing at the highest mean measured concentration of 0.46 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.

All test media were clear solutions throughout the test period.

The pH in the control was 7.7 at test start and 7.9 at test end fulfilling the requirement
of the OECD guideline that the pH of the control medium should not increase by more than 1.5
units during the test. The pH of the test media was in the range of 7.6 to 7.9 during the test
period. The water temperature during the test was maintained at 23 °C.

The values for the validity criteria of the test were calculated by the statistical software program
ToxRat Professional®.

In the control, the biomass increased by a factor of 211 over 72 hours. The validity criterion of
increase of biomass by at least a factor of 16 within three days was fulfilled.

The mean coefficient of variation of the daily growth rates in the control (section-by-section
growth rates) during 72 hours was 10 %. According to the OECD test guideline, the mean
coefficient of variation must not be higher than 35 %. Thus, the validity criterion was fulfilled.

The coefficient of variation of the average specific growth rates in the replicates of the control
after 72 hours was 0.6 %. According to the OECD test guideline, the coefficient of variation
must not be higher than 7 %. Thus, the validity criterion was fulfilled.

All validity criteria were thus fulfilled for the control.
Results with reference substance (positive control):
For evaluation of the algal quality and experimental conditions, potassium dichromate is tested
as a positive control twice a year to demonstrate satisfactory test conditions. The 72-hour EC50
for growth rate in the reference test IES Study Number 20170367 was 1.1 mg/L (October 2017)
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
Conclusions:
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 0.10 mg/L.

End-Points Based on Growth Rate :
ErC50 (72 hours) > 0.46 mg/L (14.1% growth rate inhibition at the water saturation concentration for the test item)
ErC10 (72 hours) = 0.28 mg/L
NOErC (72 hours) = 0.039 mg/L

End-Points Based on Yield :
EyC50 (72 hours) = 0.43 mg/L
EyC10 (72 hours) =0.078 mg/L
NOEyC (72 hours) = 0.039 mg/L
Executive summary:

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 0.10 mg/L.

End-Points Based on Growth Rate :

ErC50 (72 hours) > 0.46 mg/L (14.1% growth rate inhibition at the water saturation concentration for the test item)

ErC10 (72 hours) = 0.28 mg/L

NOErC (72 hours) = 0.039 mg/L

End-Points Based on Yield :

EyC50 (72 hours) = 0.43 mg/L

EyC10 (72 hours) =0.078 mg/L

NOEyC (72 hours) = 0.039 mg/L