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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 01 to November 15, 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
conducted under GLP conditions
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Remarks:
GC-FID
Details on sampling:
Analysis of the Test Item Concentrations:
For the determination of the actual test item concentrations, duplicate samples were taken from
each treatment at the start of the test.
After 24 and 48 hours and at the end of the test (after 72 hours), stability samples (containing
algae) were taken in duplicate from the test concentration and from the control.
The stability samples at 24 and 48 hours (duplicates 50 mL samples) 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 this sampling at
24 and 48 hours, a set of additional flasks containing the corresponding test medium (with
algae) were incubated under conditions identical to the test.
In order to investigate possible losses of test item due to volatility when the test item vessels
were opened on a daily basis, a fourth replicate vessel was prepared for the single test
concentration at 0 hours and incubated alongside the other exposure vessels. This additional
replicate remained unopened until termination of the test.
An additional test vessel with test medium and algae at the loading rate of 100 mg/L was
incubated alongside the other exposure vessels but in the dark to demonstrate possible
photolytic processes.
The samples were analyzed immediately after sampling.
The concentrations of SCENTAURUS JUICY were analytically measured in one of the
duplicate samples taken from the single test concentration and the control from all sampling
dates.

- Storage
At all the sampling timepoint during the Biological-Phase of the study, 50 mL of each test
sample was sampled and directly extracted with 1 mL of internal standard solution (see Section
1.4.6). The organic phase was analyzed directly after extraction.
Details on test solutions:
Dosage:
At the start of the main test, the undiluted equilibrated test medium with a loading rate of
100 mg/L was prepared following the slow-stirring method (see Section 5.5) with a stirring
period of 72 hours at room temperature in the dark. For this, 256.7 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 0.8958 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 single test concentration. Additionally, a control (test water only) was run
in parallel.
The test medium was prepared just before the start of the test.
The test medium was clear with no evidence of undissolved test item.
The preparation of the test medium was based on the OECD Guidance Document No. 23
(Second Edition) on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals,
2019.
The measured water saturation concentrations obtained for the 100 mg/L loading rate undiluted
filtrate in the dose range finding experiment were very similar to that obtained in the final Limit
Test (Table 5 and Table 6 of the Analytical Appendix to Report) and demonstrate the
reproducibility of the slow-stir preparation approach for this test item, despite the very low
water solubility.

Glass stoppered 50 mL Erlenmeyer flasks were used (closed system) completely filled with
about 60 mL of test medium, minimizing the air-space in the flasks and avoiding potential
losses of test item.
The test flasks were labeled with the study number and all necessary additional information to
ensure unique identification.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella 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 treatment 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 20210289 was 1.0 mg/L (May 2021)
and showed that the sensitivity of the test system was comparable to the range recommended
by the guideline (72-hour EC50 for the growth rate 0.92-1.5 mg/L).
The test method and the test species are recommended by the test guidelines.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Hardness:
The calculated water hardness of the test water was 0.15 mmol/L (= 15 mg/L as CaCO3).
Test temperature:
22°C
pH:
between 7.5 and 7.8
Nominal and measured concentrations:
At the start of the test (Day 0) a concentration of 6.41 μg/L was detected. Subsequent
measurements after 24, 48 and 72 hours were below the limit of quantification (LOQ) of
0.461 μg/L. Therefore the biological
endpoints were based on the initially measured concentration of 6.41 μg/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
72 μE s-1 m-2 (range: 70 to 73 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.

Study Design for Main Test:
Based on these results and in agreement with the Sponsor, a limit test was performed in
accordance with the test guidelines to demonstrate that the test item has no toxic effect on the
algae up to its limit of solubility in the test water. Thus, a single loading rate of the test item of
100 mg/L was tested. Additionally, a control (test water without test item) was tested in parallel.
The test design included six replicates of the single test concentration and six replicates of 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).
A static test design was applied. The duration of the test was 72 hours.

Determination of 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 single 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.

Monitoring of 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 medium was visually controlled and
recorded daily during the exposure period.
Reference substance (positive control):
yes
Remarks:
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.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 6.41 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Remarks on result:
other: No effects observed up to and including water saturation (equivalent to a loading rate of 100 mg/L test item)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 6.41 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Remarks on result:
other: No effects observed up to and including water saturation (equivalent to a loading rate of 100 mg/L test item)
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 6.41 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Remarks on result:
other: No effects observed up to and including water saturation (equivalent to a loading rate of 100 mg/L test item)
Details on results:
A limit test was performed in accordance with the test guidelines to demonstrate that the test
item has no toxic effect on the test organisms up to the loading rate of 100 mg/L, and equivalent
to the water saturation concentration.

At the start of the test (Day 0) a concentration of 6.41 μg/L was detected. Subsequent
measurements after 24, 48 and 72 hours were below the limit of quantification (LOQ) of
0.461 μg/L (see analytical results and Table 6 in Appendix 1 of the study report attached). Therefore the biological endpoints were based on the initially measured concentration of 6.41 μg/L.
The analytical results of the additional replicates incubated in parallel for determination of
possible losses through volatility and/or photolytic process could not be interpreted since all
treatments showed analytical results below the LOQ after 72 hours.
The impact of the test item on the growth of the algae is shown in Table 1 and Table 4 and in
Figure 1 of the study report attached.
The test item had no significant inhibitory effect on the growth rate μ and yield Y of the algae
after the test period of 72 hours at the initially measured test item concentration of 6.41 μg/L,
(results of two-sample t-test, one-sided smaller,  = 0.05).
Therefore, the NOEC for yield and growth rate was determined to be at the initially measured
test item concentration of 6.41 μg/L, 100 mg/L loading rate, and equivalent to the water
saturation concentration. This value might even be higher, but concentrations of the test item
exceeding the loading rate 100 mg/L, above the solubility limit in the test water were not tested,
in accordance with the test guidelines. The 72-hour LOEC was higher than 6.41 μg/L, the
maximum concentration of the test item which could be dissolved in the test water.
The NOEC, LOEC and ECx-values for growth rate and yield are summarized in 'Any other information on results inc. tables section below.
The microscopic examination of the algal cells at the end of the test showed no difference
between the algae growing in the treatment group 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 test
concentration.
The test medium was a clear solution throughout the test period.
The pH in the control was between 7.5 and 7.8 throughout the test 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 medium in the Limit Test incubates was
in the range of 7.6 to 7.8 during the test period. The water temperature during the test
was maintained at 22°C.
Results with reference substance (positive control):
The 72-hour EC50 for growth rate in the reference test IES Study Number 20210289 was 1.0 mg/L (May 2021) and showed that the sensitivity of the test system was comparable to the range recommended by the guideline (72-hour EC50 for the growth rate 0.92-1.5 mg/L.
Reported statistics and error estimates:
The values for the validity criteria of the test were calculated by the statistical software program
ToxRat Professional®.
The test was valid since the following performance criteria (according to OECD 201) were met.
- In the control, the biomass increased by a factor of 75 over 72 hours. (Criterion: increase
by at least a factor of 16 within three days).
- The mean coefficient of variation of the daily growth rates in the control (section-bysection
growth rates) during 72 hours was 32 %. (Criterion: must not be higher than
35 %).
- The coefficient of variation of the average specific growth rates in the replicates of the
control after 72 hours was 1.5 %. (Criterion: must not be higher than 7 %).

The impact of the test item SCENTAURUS JUICY on the growth of the freshwater green algal species Pseudokirchneriella subcapitata in a closed system is summarized in the table below.
The results are based on the initially measured concentration and loading rate of the test item:



















































Parameter 0-72hGrowth rateYield
Initial measured (microg/L)Loading rate (mg/L)Initial measured (microg/L)Loading rate (mg/L)
EC10> 6.41> 100> 6.41> 100
EC20> 6.41> 100> 6.41> 100
EC50> 6.41> 100> 6.41> 100
NOEC> 6.41> 100> 6.41> 100
LOEC> 6.41> 100> 6.41> 100
Validity criteria fulfilled:
yes
Conclusions:
In conclusion, the test item SCENTAURUS JUICY had no toxic effects on Pseudokirchneriella
subcapitata during the 72-hour test period up to and including the initially measured
concentration of 6.41 μg/L, 100 mg/L loading rate, which is the maximum concentration which
could be maintained under the conditions of the test.

In accordance with the test guidelines, concentrations of the test item above the solubility limit
in the test water were not tested.
Executive summary:

The impact of the test item SCENTAURUS JUICY 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.



A limit test was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the algae up to the loading rate of 100 mg/L, and equivalent to the water saturation concentration. Thus, a single loading rate of the test item of 100 mg/L was tested. A control was run in parallel (test water without addition of the test item). Loading rates of the test item exceeding 100 mg/L were not tested, in accordance with the test guidelines.



As the test item may be a volatile substance, the test was performed using glass Erlenmeyer completely filled (without headspace) with test medium that were tightly sealed with glass stoppers to avoid losses of test item by evaporation (closed system).



As the test item is a liquid with low water solubility, the slow-stirring method (to avoid formation of micro-droplets) was applied for preparation of a saturated test item solution. For preparation of the single concentration of test medium, the test item was carefully applied (pipetted) onto the surface of the test water at a loading rate of 100 mg/L. Thereafter slow-stirring was applied for 72 hours in a closed vessel.



After this treatment the lower part of the equilibrated test medium was carefully harvested from the stirring vessel through a tap at the bottom of the vessel. This equilibrated aqueous phase with a loading rate of 100 mg/L, containing dissolved test item only, was used as the test medium.



The preparation of the test medium was based on the OECD Guidance Document No. 23 (Second Edition) on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals,2019.



The concentrations of SCENTAURUS JUICYwere analytically determined in the test medium and the control every 24h period.



At the start of the test (Day 0) a concentration of 6.41 μg/L was detected. Subsequent measurements after 24, 48 and 72 hours were below the limit of quantification (LOQ) of 0.461 μg/L. Therefore the biological endpoints were based on the initially measured concentration of 6.41 μg/L.



The test item had no significant inhibitory effect on the growth rate μ and yield Y of the algae after the test period of 72 hours at the initially measured test item concentration of 6.41 μg/L.



Therefore, the NOEC for yield and growth rate was determined to be at the initially measured test item concentration of 6.41 μg/L, 100 mg/L loading rate and equivalent to the water saturation concentration. The EC values were determined to be higher than 6.41 μg/L, 100 mg/L loading rate.


The results are based on the initially measured concentration and loading rate of the test item:



















































Parameter 0-72hGrowth rateYield
Initial measured (microg/L)Loading rate (mg/L)Initial measured (microg/L)Loading rate (mg/L)
EC10> 6.41> 100> 6.41> 100
EC20> 6.41> 100> 6.41> 100
EC50> 6.41> 100> 6.41> 100
NOEC> 6.41> 100> 6.41> 100
LOEC> 6.41> 100> 6.41> 100

Validity Criteria:
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.

Description of key information

The impact of the test item SCENTAURUS JUICY 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.


The test item had no significant inhibitory effect on the growth rate μ and yield Y of the algae after the test period of 72 hours at the initially measured test item concentration of 6.41 μg/L.


Therefore, the NOEC for yield and growth rate was determined to be at the initially measured test item concentration of 6.41 μg/L, 100 mg/L loading rate and equivalent to the water saturation concentration. The EC values were determined to be higher than 6.41 μg/L, 100 mg/L loading rate.

Key value for chemical safety assessment

Additional information

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).


According to the EU CLP regulation (No 1272/2008 and its adaption 286/2011), Scentaurus Juicy doesn't need to be classified as Hazardous to the Aquatic Environment Acute 1 classification (Acute results greater than the aqueous saturation concentration of the test item).