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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:

August 4 to December 2, 2022

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 (Alga, Growth Inhibition Test)

Version / remarks:

23 March, 2006, with updated Annex V of 28 July, 2011.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test Item Name: GR-87-6331
Batch Number: 10
Appearance: Liquid
Chemical Name: 1-methoxy-4-(3-phenylprop-1-en-1-yl)benzene (E/Z isomers)
Purity: 96.6 % (sum of E/Z-isomers), GC-MS
Density: 1.059 g/cm3
Expiration Date: March 18, 2023
Storage Conditions: 4-25 °C

Analytical monitoring:

yes

Remarks:

GC-FID

Details on sampling:

Samples were taken for quantification of the test item from the Control and all test levels and analysed for Time 0, at 24 hours and also at 72 hours.

Vehicle:

no

Details on test solutions:

Based on range finding results, the following concentrations of GR-87-6331 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.

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 with a stirring period of 72 hours at room temperature in the dark. For this, 222 μL of test item were carefully applied (pipetted) onto the surface of 2350 mL test water. This volume is equivalent to a loading rate of 100 mg/L, considering the density of the test item of 1.059 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 Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals, 2019.

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 the test facility 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 20220010 was 0.65 mg/L (February 2022) and showed that the sensitivity of the test system was slightly higher than 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.

Test type:

static

Water media type:

freshwater

Remarks:

The algae were cultivated and tested in synthetic test water (AAP Medium), prepared according to the test guidelines, but modified according to the International Standard ISO 14442 [2] as a closed test system was applied.

Limit test:

no

Total exposure duration:

72 h

Hardness:

15 mg/L as CaCO3

Test temperature:

22 °C

pH:

7.5 to 7.6

Nominal and measured concentrations:

Based on range finding results (see 'Any other information on materials and methods incl. tables' section), the following concentrations of GR-87-6331 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.

Treatment/ Measured Concentrations (mg/L) Geometric Mean (mg/L)
Dilution 0 hr 24 hrs 72 hrs
1 : 22 0.0223 0.0172 0.0111 0.016
1 : 10 0.0390 0.0354 0.0242 0.032
1 : 4.6 0.0882 0.0835 0.0608 0.076
1 : 2.2 0.173 0.160 0.148 0.16
Undiluted 0.376 0.352 0.330 0.35

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 64 μE s-1 m-2 (range: 61 to 65 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, the following concentrations of
GR-87-6331 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).

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

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 with a stirring
period of 72 hours at room temperature in the dark. For this, 222 µL of test item were carefully
applied (pipetted) onto the surface of 2350 mL test water. This volume is equivalent to a loading
rate of 100 mg/L, considering the density of the test item of 1.059 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
Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals, 2019.


EVALUATIONS

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 9 nm and emission of 15 nm.

At the end of the test, a sample was taken from the control and from the dilution 1:2.2 to
determine a potential influence of the test item on the algal cells. The shape and size of the algal
cells were visually inspected. This test concentration was chosen since the algal cell density at
the highest concentration (undiluted test medium with a loading rate of 100 mg/L) was too low
for a reliable examination.

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 media was visually controlled and
recorded daily during the exposure period.

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 all test concentrations 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 dilution 1:10 at
0 hours and incubated alongside the other exposure vessels. This additional replicate remained
unopened until termination of the test.

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.

The concentrations of the test item were analytically measured in one of the duplicate samples
taken from all treatments from the start of the test, after 24 hours and at the end of the test
(72 hours).

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.061 mg/L

95% CI:

>= 0.053 - <= 0.07

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.12 mg/L

95% CI:

>= 0.11 - <= 0.14

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.:

0.079 mg/L

95% CI:

>= 0.071 - <= 0.089

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.032 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.089 mg/L

95% CI:

>= 0.072 - <= 0.11

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Details on results:

The test item had a significant inhibitory effect on the growth rate μ and on the yield Y of the algae over the test period of 72 hours at the mean measured concentration of 0.076 mg/L and at all higher test concentrations.

The NOEC for yield and growth rate was determined to be at the mean measured concentration of 0.032 mg/L.

The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the mean measured concentration of 0.16 mg/L and the algal cells in the control. The shape and size of the algal cells were clearly 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 7.5 at test start and 7.7 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 7.5 to 7.6 during the test period.
The water temperature during the test was maintained at 22°C.

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 77 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-by-section growth rates) during 72 hours was 9.8 %. (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 1.3 %. (Criterion: must not be higher than 7 %).

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 was 0.65 mg/L (February 2022) and showed that the sensitivity of the test system was slightly higher than recommended by the guideline (72-hour EC50 for the growth rate 0.9-1.5 mg/L).

The measured concentrations of the test item in the test media are shown in the table below. The analytical measurements from test media at the start of the test show the correct preparation of the test media, i.e. the theoretical spacing factor of 2.2 between the different test concentrations was met. Since the test item concentrations decreased over the test period, the mean measured concentrations were calculated as the time-weighted geometric mean of the concentrations measured at all sampling dates (0, 24 and 72 hours).

Treatment/dilution#	Analytically Measured Concentration of the Test Item [mg/L]			Mean Measured Concentration (Geometric Mean) [mg/L]
	0 Hours	24 Hours	72 hours
1:22	0.0223	0.0172	0.0111	0.016
1:10	0.0390	0.354	0.0242	0.032
1:4:6	0.0882	0.0835	0.0608	0.076
Undiluted test medium*	0.376	0.352	0.330	0.35

*: Undiluted equilibrated test medium with a loading rate of 100 mg/L.
#: Dilutions of the equilibrated test medium with a loading rate of 100 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

The impact of the test item on the growth of the freshwater green algal species Pseudokirchneriella subcapitata in a closed system is summarized below.

The results are based on the mean measured concentrations of the test item:

72 hr ErC50 = 0.12 mg/L
72 hr ErC10 = 0.079 mg/L
72 hr NOErC = 0.032 mg/L
72 hr EyC50 = 0.089mg/L
72 hr EyC10 = 0.061 mg/L
72 hr NOEyC = 0.032 mg/L

Executive summary:

The impact of the test item 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 thought to be a volatile substance, the test was performed using glass 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).

As the test item is a liquid with relatively 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 highest 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 highest 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 without test item) was tested in parallel. The preparation of the test media was based on the OECD Guidance Document No. 23 on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals, 2019.

The measured concentrations of the test item in the test media are shown in the table below. The analytical measurements from test media at the start of the test show the correct preparation of the test media, i.e. the theoretical spacing factor of 2.2 between the different test concentrations was met. Since the test item concentrations decreased over the test period, the mean measured concentrations were calculated as the time-weighted geometric mean of the concentrations measured at all sampling dates (0, 24 and 72 hours).

Treatment/dilution#	Analytically Measured Concentration of the Test Item [mg/L]			Mean Measured Concentration (Geometric Mean) [mg/L]
	0 Hours	24 Hours	72 hours
1:22	0.0223	0.0172	0.0111	0.016
1:10	0.0390	0.354	0.0242	0.032
1:4:6	0.0882	0.0835	0.0608	0.076
Undiluted test medium*	0.376	0.352	0.330	0.35

*: Undiluted equilibrated test medium with a loading rate of 100 mg/L.

#: Dilutions of the equilibrated test medium with a loading rate of 100 mg/L.

The biological end-points results are based on the mean measured test item concentrations:

72 hr ErC50 = 0.12 mg/L
72 hr ErC10 = 0.079 mg/L
72 hr NOErC = 0.032 mg/L
72 hr EyC50 = 0.089mg/L
72 hr EyC10 = 0.061 mg/L
72 hr NOEyC = 0.032 mg/L

A clear concentration-response relationship was observed for both biological endpoints growth rate and yield over 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 0.076 mg/L and at all higher test concentrations.

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. The study is thus considered valid.

Description of key information

The impact of the test item 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 thought to be a volatile substance, the test was performed using glass 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).

As the test item is a liquid with relatively 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 highest 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 highest 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 without test item) was tested in parallel. The preparation of the test media was based on the OECD Guidance Document No. 23 on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals, 2019.

The measured concentrations of the test item in the test media are shown in the table below. The analytical measurements from test media at the start of the test show the correct preparation of the test media, i.e. the theoretical spacing factor of 2.2 between the different test concentrations was met. Since the test item concentrations decreased over the test period, the mean measured concentrations were calculated as the time-weighted geometric mean of the concentrations measured at all sampling dates (0, 24 and 72 hours).

Treatment/dilution#	Analytically Measured Concentration of the Test Item [mg/L]			Mean Measured Concentration (Geometric Mean) [mg/L]
	0 Hours	24 Hours	72 hours
1:22	0.0223	0.0172	0.0111	0.016
1:10	0.0390	0.354	0.0242	0.032
1:4:6	0.0882	0.0835	0.0608	0.076
Undiluted test medium*	0.376	0.352	0.330	0.35

*: Undiluted equilibrated test medium with a loading rate of 100 mg/L.

#: Dilutions of the equilibrated test medium with a loading rate of 100 mg/L.

The biological end-points results are based on the mean measured test item concentrations:

72 hr ErC50 = 0.12 mg/L
72 hr ErC10 = 0.079 mg/L
72 hr NOErC = 0.032 mg/L
72 hr EyC50 = 0.089mg/L
72 hr EyC10 = 0.061 mg/L
72 hr NOEyC = 0.032 mg/L

A clear concentration-response relationship was observed for both biological endpoints growth rate and yield over 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 0.076 mg/L and at all higher test concentrations.

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. The study is thus considered valid.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.12 mg/L

EC10 or NOEC for freshwater algae:

0.079 mg/L

Additional information

According to the EU CLP regulation (No 1272/2008 and its adaption 286/2011), the substance needs to be classified as Hazardous to the Aquatic Environment Acute 1 and Chronic 1 classifications (Aquatox test results below 1 mg/L). No M factor needs to be applied.