2-(3,5-dimethylhex-3-en-2-yloxy)-2-methylpropyl cyclopropanecarboxylate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 21 September 2020 to 18 December 2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Deviations of the study Guideline: The test medium was supplemented with sodium bicarbonate to increase the CO2 concentration in the test medium to achieve maximum algal growth rates without pH drift. A closed-bottle test system was used to reduce losses of volatile components of the test item. A lower initial cell density than recommended by the guideline (0.25×10^4 algal cells per mL) was used to allow exponential growth throughout the incubation period without CO2 depletion caused by a sealed system (closed-bottle system), furthermore the lower cell density may also prevent a significant pH-drift throughout the exposure period. Since the algae showed normal growing behaviour and all validity criteria were met in this study, this deviation has no impact on the integrity of the study results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

yes

Remarks:

See details on deviations in "Rationale for reliability incl. deficiencies". Deviations has no impact on the integrity of the study results.

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of inspection: 12 June 2019 / Date of signature: 26 August 2019

Specific details on test material used for the study:

- Solubility in water: 14 mg/L at 20°C
- Vapour pressure: 0,0046 hPa at 20°C (OECD 104)
- Conditions of storage: In a closed container in a dry, well ventilated dark location at ambient temperature/10–30°C

Batch No. : SC00029369
Purity : 97.6%
Date of Expiry : 19 May, 2022

Analytical monitoring:

yes

Remarks:

GC-MS

Details on sampling:

- Concentrations: Samples of stock solution and test solutions were taken to determine the actual test item concentrations in comparison to the nominally applied concentrations.
- Sampling method: Control and test solutions were sampled in duplicate, except the reserve samples taken after 24 and 48 hours of exposure and the samples without algae at the end of the exposure (72 hours). The duplicate samples were kept separately as a reserve.
- Sample storage conditions before analysis: After sampling and before shipment, all samples were stored in glass bottles in the dark at a temperature of ≤ -18 °C. A non-GLP storage stability test was performed at the analytical test site prior to the conduct of the definitive biological phase, which confirmed sample integrity through frozen storage.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: See details in "Any other information on materials and methods, incl. tables".

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: SAG 61.81
- Source (laboratory, culture collection): The organisms were originally supplied by Sammlung von Algenkulturen, Albrecht-vonHaller-Institut, Universität Göttingen, Germany.
- Age of the pre-culture: 4 days

ACCLIMATION
To adapt the algae to the test conditions a pre-culture was inoculated by a liquid algal culture and incubated under test conditions.
- pH-value of the algal medium: 7.9
- Culture medium: OECD medium (OECD 201, EN ISO 8692)
- Volume of liquid stock culture per pre-culture vessel: 100±5 mL
- Pre-culture vessels: 300 mL Erlenmeyer flasks
- Number of replicates: 2
- Light: Permanent illumination (24/0 h light/dark); Econlux LED Sunstrip “daylight”
- Light intensity: 60–120 µE m-2s-1; (measured: 74.2–75.3 µE m-2s-1)
- Shaker: 100±5 oscillations/min
- Temperature in the test room: 21–24 C; (measured: 22.6–23.1 °C)

Test type:

static

Water media type:

freshwater

Total exposure duration:

72 h

Test temperature:

22.5–23.2°C, mean 22.8 °C

pH:

8.1–9.4

Nominal and measured concentrations:

- Nominal test concentrations: 3.13, 6.25, 12.5, 25.0, 50.0 and 100% of a saturated solution at a loading rate of 100 mg/L.
- Geometric mean measured concentrations: 0.119, 0.240, 0.435, 0.964, 1.90 and 4.15 mg test substance/L. See details in table 6.1.5/3 in "Any other information on results incl. tables".

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL glass vials, air-tightly closed by a screwcap with PTFE lining
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 60 mL (nominal), filled to the rim
- Shaker: 100±5 oscillations/min; the test vessels were placed randomly on a horizontal shaker, so that each test vessel was rotated around its own axis
- Renewal rate of test solution (frequency/flow rate): none (static system)
- Initial cells density: initial cell concentration of approximately 0.25×10^4 cells/mL.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels for potential chemical analyses, after 24 and 48 hours of exposure (replicates): 1 per treatment level and sampling day
- No. of vessels for stability check, without algae (replicates): 2 (treatment level: C4, 25% dilution)

GROWTH MEDIUM
- Standard medium used: yes
The growth medium was OECD medium as described in the test guideline, modified to optimise growth of Pseudokirchneriella subcapitata. The OECD growth medium was supplemented with sodium bicarbonate (300 mg/L NaHCO3 instead of 50 mg/L NaHCO3).
- Justification for modification of growth medium: The use of a sealed system result in culture growth being limited by CO2 depletion, thus additional NaHCO3 is used to serve as a source of CO2.

OTHER TEST CONDITIONS
- Sterile test conditions: yes. The algal medium was sterilised (filtered, pore size 0.2 µm) before use. The vessels used were sterilised by autoclaving for 20 minutes at 121°C.
- Adjustment of pH: Before use, the pH of the medium was adjusted to 8.1±0.2.
- Photoperiod: Permanent illumination (24 h light/0 h dark)
- Light intensity and quality: 75.5–88.0 µE m–2s–1; mean 80.0 µE m–2s–1 (Econlux LED Sunstrip “daylight”)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Inhibition of growth in relation to control (growth rate & yield)

TEST CONCENTRATIONS
- Range finding study: Yes. A preliminary non-GLP range finding test was conducted at 1.56, 6.25, 25.0 and 100% dilutions of a saturated solution at a loading rate of 100 mg/L, in the same test conditions of the definitive test.
- Results used to determine the conditions for the definitive study:
72h-ErC50 > 4.97 mg/L based on mean measured concentrations.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.964 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 4.15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: The highest attainable test concentration (100% saturated solution).

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: 95% CI : 0.665 - 1.95 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.964 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 4.15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: The highest attainable test concentration (100% saturated solution)

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 4.15 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: The highest attainable test concentration

Details on results:

See tables in "Any other information on results incl. tables".
- Analytical findings: Samples from the test solutions were analysed using GC-MS to determine actual levels of the test item at start of exposure period (time 0h) and following 72 hours of exposure. At time 0 hours the measured concentrations ranged between 92.2–102.1% of the measured reference concentration and slightly decreased after 72 hours exposure to values between 70.3% and 91.7% of the initial measured concentrations. Therefore, the test item concentration based on initial measured concentrations were not stable within ±20% at the end of the exposure period. As a consequence, and since all concentration levels were measured, the biological results are calculated and reported based on geometric mean measured concentrations at each concentration level.
- Biological findings: The ErC50 of Pseudokirchneriella subcapitata was determined to be >100 % of a saturated solution at a loading rate of 100 mg test item/L based on nominal concentrations, equivalent to >4.15 mg test substance/L based on measured concentrations. The NOEC based on nominal concentration was 25.0 % of a saturated solution at a loading rate of 100 mg test item/L, equivalent to 0.964 mg test substance/L based on measured concentrations.
In none of the concentration levels (C0 and C1–C4, up to 25.0 % of a saturated solution at a loading rate of 100 mg test item/L), deformed and/or damaged algal cells were observed during microscopic inspection. At the two highest concentration levels (C5 and C6, 50.0 and 100 % of a saturated solution at a loading rate of 100 mg test item/L) an increasing number of algae cells showing predominantly enlargement were observed, i.e. 30% and 70% of algae cells, respectively.

Results with reference substance (positive control):

Two reference tests using potassium dichromate (K2Cr2O7) as reference item were performed in separate studies. One was performed using a standard test (study number: IAO2008, August 2020) design and the other using a closed-bottle system (study number: IAO2005-CB, May 2020). The latter was performed to confirm that that the use of a modified test system to reduce losses of test item through volatility (completely filled and sealed vessels) is expected to give results consistent with that obtained in a conventional system.

Result:
- Standard: Growth rate 72h-ErC50 = 0.855 mg/L (95% CL: 0.821 – 0.889 mg/L).
- Closed-Bottle: Growth rate 72h-ErC50 = 0.792 mg/L (95% CL: 0.773 – 0.811 mg/L).

Based on an international ring test mentioned in OECD guideline 201, the ErC50 (72h)-values for potassium dichromate obtained from different laboratories was 1.19 mg/L with a standard deviation of 0.27 mg/L.
The ErC50 value for the toxic reference item, potassium dichromate, was determined as 0.855 mg/L (standard system) and 0.792 mg/L (closed system). These values are within the historical range of the general reference test results (standard open test system) of our laboratory (mean ErC50 value = 1.051 ± 0.338 mg/L) and within the narrow upper (1.384 mg/L) and lower (0.721 mg/L) warning limits. The narrow warning limits were calculated as one standard deviation from the historical mean of the EµC50 values. The calculation of the warning limits was not conducted in compliance with Good Laboratory Practice Principles (all calculated EµC50 using the log concentration; according to Environment Canada (2005), Guidance Document on Statistical Methods for Environmental Toxicity Tests, Method Development and Applications Section, Environment Canada, Ottawa, ON, Report EPS 1/RM/46). Furthermore, the EµC50 value (standard system) is within the refined range of 1.10 ± 0.48 mg/L, recommended based on an international ring test (Pattard, M.; Römbke, J.; Moser, T. (2009). Range of Reference Tests in Aquatic Tests. [in] Moser, H.; Römbke, J. “Ecotoxicological Characterization of Waste. Results and Experiences of an International Ring Test”, chapter 5, pp. 61-70.). Therefore, the results of this reference test are acceptable and the test conditions are reliable.

Table 6.1.5/3: Summary of measured concentrations at the beginning and at the end of the test

Conc. level	Test period (d)	Nominal concentration (%*)	Measured concentration (mg test substance/L)	% of reference/initial concentration** (%)	Geometric mean measured concentration over 72h (mg test substance/L)
Control C1 C2 C3 C4 C5 C6	0 0 0 0 0 0 0	0 3.13 6.25 12.5 25.0 50.0 100	<LOD 0.125 0.276 0.519 1.02 2.21 4.33	n.a. 92.2 102.0 95.9 94.2 102.1 100.0	- - - - - - -
Control C1 C2 C3 C4 C5 C6	3 3 3 3 3 3 3	0 3.13 6.25 12.5 25.0 50.0 100	<LOD 0.113 0.209 0.365 0.911 1.63 3.97	n.a. 90.4 75.7 70.3 89.3 73.8 91.7	n.d. 0.119 0.240 0.435 0.964 1.90 4.15
C4 (without algae)	3	25.0	1.44	-	-

* % of a saturated solution at a loading rate of 100 mg test item/L
LOQ = Limit of Quantification (0.05 mg/L) n.d.: not determined
LOD = Limit of Detection (0.015 mg/L) n.a.: not applicable
** Note: Recoveries were calculated based on the measured concentration of the highest test concentration taken at start of the exposure period (C6, 100% of a saturated solution at a loading rate of 100 mg test item/L, 4.33 mg test substance/L), and based on measured initial concentration for the aged
test solution measured at day 3 of exposure separate for each concentration level.

 

Table 6.1.5/4: Cell number per mL (divided by 10^4) in Pseudokirchneriella subcapitata as dependent on concentration of the test item and time

	Concentrations (%*)	Control	3.13	6.25	12.5	25.0	50.0	100
0h	Replicates Mean [cells]**	6 0.25	3 0.25	3 0.25	3 0.25	3 0.25	3 0.25	3 0.25
24h	Replicates Mean [cells] Std. Dev. [cells] CV %	6 1.41 0.217 15.4	6 1.48 0.138 9.3	3 1.32 0.031 2.3	3 1.29 0.075 5.8	3 1.05 0.086 8.2	3 1.14 0.061 5.3	3 0.93 0.095 10.3
48h	Replicates Mean [cells] Std. Dev. [cells] CV %	6 11.67 1.332 11.4	3 12.95 0.988 7.6	3 12.02 1.201 10.0	3 12.49 0.311 2.5	3 11.33 0.787 6.9	3 9.56 0.202 2.1	3 8.45 1.16 13.7
72h	Replicates Mean [cells] Std. Dev. [cells] CV %	6 73.23 4.742 6.5	3 80.23 7.915 9.9	3 76.86 5.633 7.3	3 74.41 2.452 3.3	3 71.56 1.901 2.7	3 59.68 2.695 4.5	3 47.79 3.465 7.3

* % of a saturated solution at a loading rate of 100 mg test item/L
** The statistical analyses were performed using the nominal initial cell number of 0.25×10^4 cells/mL
Mean: arithmetic mean; Std. Dev.: standard deviation; CV: coefficient of variation

 

Table 6.1.5/5: Inhibition of growth rate: Growth rate of P. subcapitata in relation to concentration of the test item and %inhibition caused by the test item, after 72 hours

Nominal concentration (%)*	Measured concentration (mg/L)	Mean growth rate (day-1)	Std. Dev. (day-1)	n	% inhibition	Significant difference from control (Williams-t test; p<=0.05)
Control 3.13 6.25 12.5 25.0 50.0 100	n.d. 0.119 0.240 0.435 0.964 1.90 4.15	1.8927 1.9227 1.9088 1.8985 1.8855 1.8249 1.7504	0.02192 0.03268 0.02407 0.01089 0.00879 0.01526 0.02438	6 3 3 3 3 3 3	0.0 -1.6 -0.9 -0.3 0.4 3.6 7.5	- No No No No Yes Yes

* % of a saturated solution at a loading rate of 100 mg test item/L
Mean: arithmetic mean; Std.Dev.: standard deviation; n: number of replicates

Validity criteria fulfilled:

yes

Remarks:

Mean biomass increase in the control: factor >16 within 72h (=293); Mean CoV for section-by-section specific growth rates in the control: up to 35% (=11.9%); CoV of average specific growth rates during test period in replicate controls: up to 7% (=1.2%).

Conclusions:

A slight concentration-response relationship was observed for growth rate during the exposure period but not sufficient to determine ErC10 and ErC50 values, after 72 hours of exposure. The ErC10 and ErC50 of Pseudokirchneriella subcapitata were determined to be >4.15 mg test substance/L based on geometric measured concentrations, which was considered to be the maximum solubility of the test substance in OECD-medium.

72 hour ErC50 > 4.15 mg/L
72 hour ErC10 > 4.15 mg/L
72 hour NOErC = 0.964 mg/L

72 hour EyC50 > 4.15 mg/L
72 hour EyC10 = 1.14 mg/L
72 hour NOEyC = 0.964 mg/L

Executive summary:

This study was performed to assess the growth inhibition of the test substance to the green algae Pseudokircheriella subcapitata according to OECD Guideline 201 and GLP compliance.

Based on the results of a preliminary non-GLP range finding test, the following concentrations were tested in the definitive test: 100, 50.0, 25.0, 12.5, 6.25 and 3.13% of a saturated solution at a loading rate of 100 mg/L. The test period (exposure of test organisms to the test solutions in a static system) was 72 hours. Additionally, the test organisms were exposed under control conditions (untreated test medium, mod. OECD medium), for the same period of time. The test item was applied once at the beginning of the exposure period. Three replicate vessels were used per test item concentration, and six replicate vessels for the control. Additional test vessels with algae were prepared for potential chemical analysis of all test item concentrations at 24 and 48 hours of the test. Additional test vessels without algae were prepared for potential chemical analysis of one intermediate test item concentration (25% dilution) at the end of the test, in order to assess test item stability during exposure without the presence of algae.

The study met acceptability criteria prescribed by the protocol and was considered valid.

Samples of the test solutions were analysed, using GC-MS method, to determine the actual levels of the test item at start of exposure period and following 72 hours of exposure. The limit of quantification (LOQ) was 0.05 mg/L. The limit of detection (LOD) was defined as 30% of the LOQ (i.e. 0.015 mg/L). At time 0 hours the measured concentrations ranged between 92.2–102.1% of the measured reference concentration and slightly decreased after 72 hours exposure to values between 70.3% and 91.7% of the initial measured concentrations. Therefore, the test item concentration based on initial measured concentrations were not stable within ±20% at the end of the exposure period. As a  consequence, and since all concentration levels were measured, the biological results are calculated and reported based on geometric mean measured concentrations at each concentration level: 0.119, 0.240, 0.435, 0.964, 1.90 and 4.15 mg test item/L.

Significant growth rate inhibitions were observed after 72 hours at the two highest tested exposure concentrations: 3.6% and 7.5% inhibition at the measured concentrations of 1.90 and 4.15 mg/L, respectively. This slight concentration-response relationship observed is not sufficient to determine ErC10 and ErC50 values, after 72 hours of exposure.

In conclusion, the 72h-ErC10 and 72h-ErC50 of Pseudokirchneriella subcapitata were determined to be >4.15 mg test substance/L based on geometric measured concentrations, which was considered to be the maximum solubility of the test substance in OECD-medium.  The 72 hour NOErC was determined to be 0.964 mg/L.

Reference 2

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 02 March to 15 May 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Limit test, spiked with solvent at a nominal test concentration of 14 mg/L (maximum water solubility), but only 5.7 mg/L was detected as initial exposure concentration. The test substance reduced growth rate of this fresh water algal species significantly at 5.7 mg/L. However, the 48h-ErC50 for growth rate reduction was beyond the range tested, i.e. exceeded 5.7 mg/L. No chronic end-points (EC10, NOEC) determined or reported. 48 hour duration of study.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

yes

Remarks:

test period: 48h instead of 96h; during the range-finding test, the difference between the temperature of the medium at the start of the test and the highest temperature recorded in the incubator was larger than the allowed 2°C.

Qualifier:

equivalent or similar to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

not specified

GLP compliance:

yes

Specific details on test material used for the study:

- Storage conditions: At room temperature in the dark
- Stability under storage conditions: Stable
- Solubility in water: 14 mg/L

Batch No. : LDP0800001
Purity : 97.0%
Date of Expiry : 01 May, 2010

Analytical monitoring:

yes

Remarks:

GC-FID

Details on sampling:

- Sampling method: Samples for possible analysis were taken from both the limit concentration and the controls at t=0h, t=24h and t=48h. Volume = 15 mL. Compliance with the quality criteria regarding maintenance of actual concentrations was demonstrated by running a test vessel at the limit concentration but without algae and samples for analysis were taken at the start, after 24h of exposure and at the end of the test period. Additionally, singular reserve samples of 15 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer for a maximum of three months.
- Sample storage conditions before analysis: Samples were stored in a freezer until analysis.

Vehicle:

yes

Remarks:

DMSO at a concentration of 0.1 mL/L.

Details on test solutions:

- Preparation of the test solution: Preparation of test solutions started with stock solutions in DMSO of 1.4, 14 and 140 mg/mL (range finding test) or 140 mg/mL only (limit test). In the range-finding test, 0.05 mL of the stock solution was added per 0.5 litre and in the final test, 0.1 mL of the stock solution was added per litre of test medium in order to reach the test concentrations. After a 15-minute stirring period, the clear and colourless solutions were siphoned off and used for the test. Directly after preparation, volumes of 75 mL were added to each replicate of the respective test concentration. Subsequently, 1.5 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL and the test vessels were airtight closed with a septum-containing cap. Note that during the entire formulation all vessels incubated with test solution were closed directly after opening and/or filling to minimise any possible loss by volatilisation. Controls: Test medium without test substance or other additives (blank control) and one control containing the DMSO used in the treatment of the stock solutions (solvent control).

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: strain NIVA CHL 1
- Source (laboratory, culture collection): in house laboratory culture

FRESH WATER ALGAE CULTURE
- Stock culture: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
- Light intensity: 60 to 120 µE/m²/s when measured in the photosynthetically effective wavelength range of 400 to 700 nm.
- Stock culture medium: M1, according to the NPR 6505, formulated using Milli-Ro water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA).
- Pre-culture: 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Pre-culture medium: M2, according to the OECD Guideline 201, formulated using Milli-Ro water (tap-water purified by reverse osmosis (milli-RO) and subsequently passed over activated carbon and ion-exchange cartridges: Milli-Q water, Millipore Corp., Bedford, Mass., USA) preventing precipitation.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Hardness:

no data

Test temperature:

The temperature of the test medium was 22.2°C at the start of the test. During the exposure period, the temperature measured in the incubator was maintained between 22.2 and 22.6°C. Temperature remained within the limits prescribed by the protocol (21-24°C, constant within 2°C).

pH:

From 7.3 to 9.2.
The pH of the controls slightly exceeded the upper limit (6.0-9.0) at the end of the test due to a relatively high algal growth rate.

Dissolved oxygen:

no data

Salinity:

not applicable

Conductivity:

no data

Nominal and measured concentrations:

Nominal test concentration: 14 mg/L.
Analysis of the sample taken from the limit concentration at the start of the test showed a measured concentration of 5.7 mg/L. The measured concentration had not decreased by more than 20% at the end of the 48h period. Therefore, effect parameters were based on the initial exposure concentration. See table 6.1.5/1 in "Any other information on results incl. tables"

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL all glass containing 75 mL of test solution and airtight closed with a screw cap containing a septum
- Type (delete if not applicable): closed
- Initial cells density: 1 x 10^4 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Illumination: continuously using TLD-lamps of the type "cool-white" of 30 Watt, with a light intensity with the range of 92 to 97 µE/m²/s.
- Other: during incubation the algal cells were kept in suspension by continuously shaking

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 720 nm using a Varian Cary 50 single beam spectrophotometer with cuvettes. Algal medium was used as blank.
At the end of the limit test microscopic observations were performed to verify a normal and healthy appearance of the inoculum culture and to observe for any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Preliminary data: A non-GLP pretest was performed by Givaudan. This experiment shows that a solution of the test substance in ISO medium does not remain stable (65.8-70.3% of initial) without agitation at least for 96 hours in closed glass containers (full or not). When stored in an open vessel, the substance had evaporated out of the solution almost totally after 96 hours (1.4% initial).
- Range-finding test: A range-finding test was performed to provide information about the range of concentrations to be used in the final test. Test procedure and conditions were similar to those applied in the final test with the following exceptions: Exponentially growing algal cultures were exposed to concentrations of 0.14, 1.4 and 14 mg/L and to a blank and solvent control; three replicates were tested per concentration and three replicates in each of the control groups; one extra test vessel per concentration without algae was used as background for the determination of the algal cell density at each time interval; pH was only measured in the control groups and the highest test concentration; at the end of the test, algae were not observed to verify a normal and healthy appearance; no sampling for determination of actual test concentrations was performed.
No significant effects were observed on algal cell growth and it was thus decided to continue the project with a limit test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

ca. 5.7 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

yield

Remarks on result:

other: Reported as "The EC50 for yield inhibition (EyC50: 0 - 48 h) approcimated 5-7 mg/L. Average yield inhibition at this limit-test concentration was 54.5%.

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 5.7 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Mean cell densities: See table 6.1.5/2 in "Any other information on results incl. tables".
Growth rates of algae exposed to the limit concentration was signficantly reduced. See table 6.1.5/3 in "Any other information on results incl. tables". Microscopic observations at the end of the test revealed a normal and healthy appearance of the exposed cells when compared to the control.
No EC50 values could be calculated (EC50 > maximum concentration tested).

Results with reference substance (positive control):

- Results with reference substance valid? yes
The 72h-ErC50 for growth rate reduction was 1.6 mg/L (95% CI: 1.2 - 2.1 mg/L). The historical ranges for growth rate reduction lie between 0.82 and 2.3 mg/L. Hence, the 72h-ErC50 for the present batch corresponds with this range.

Table 6.1.5/1: Concentrations of the test substance in test medium (limit test)

Time of sampling (hours)	Date of sampling (dd-mm-yy)	Date of sample pretreatment* (dd-mm-yy)	Concentration
			Nominal (mg/L)	Analysed (mg/L)	Relative to nominal (%)	Relative to initial (%)
0	31-03-09	06-04-09	0 14 14**	n.d. 5.68 5.78	n.a. 41 41
24	01-04-09	06-04-09	0 14 14**	n.d. 6.15 6.27	n.a. 44 45	108 109
48	02-04-09	06-04-09	0 14 14**	n.d. 4.64 5.60	n.a. 33 40	82 97

* Samples were stored in the freezer until the day of analysis

** without algae

n.d. Not detected

n.a. Not applicable

Table 6.1.5/2: Individual cell densities

Number of inoculated cells at t= 0: 1 x 10^4 cells/mL
Initial test concentration (mg/L)	Vessel number	Exposure time (hours)
		0	24	48
Solvent control	1 2 3 4 5 6	1.00 1.00 1.00 1.00 1.00 1.00	6.58 6.55 6.57 6.64 6.01 6.17	26.85 25.60 24.64 23.37 23.76 24.50
Control	1 2 3 4 5 6	1.00 1.00 1.00 1.00 1.00 1.00	7.55 6.24 5.96 5.72 6.47 6.14	25.56 25.86 24.29 25.18 25.59 25.55
5.7	1 2 3 4 5 6	1.00 1.00 1.00 1.00 1.00 1.00	9.01 5.66 5.45 5.31 5.19 5.26	12.66 13.01 10.79 12.54 12.35 9.58

Table 6.1.5/3: Calculation of growth rate

Initial test concentration (mg/L)	Vessel number	Growth rate (µ)	Growth rate reduction (%)
		0-48h	0-48h
Solvent control	1 2 3 4 5 6	0.06855 0.06755 0.06676 0.06566 0.06600 0.06664
	Mean CV	0.06686 2%
Control	1 2 3 4 5 6	0.06752 0.06776 0.06646 0.06721 0.06755 0.06751	-1.0 -1.4 0.6 -0.5 -1.0 -1.0
	Mean	0.06734	-0.7
5.7	1 2 3 4 5 6	0.05288 0.05345 0.04955 0.05269 0.05237 0.04708	21 20 26 21 22 30
	Mean	0.05134	23.2

Validity criteria fulfilled:

yes

Remarks:

See Overall remarks

Conclusions:

The test substance reduced growth rate of this fresh water algal species significantly at 5.7 mg/L. However, the 48h-ErC50 for growth rate reduction was beyond the range tested, i.e. exceeded 5.7 mg/L. Further additional end-points (EC10, NOEC) could not be established from this study.

Executive summary:

This study was performed to assess the toxicity of the test substance on the fresh water algal growth inhibition according to the EPA Guideline OPPTS 850.5400 with GLP compliance. In addition, the procedures were designed to meet the test methods of the ISO 8692 Guideline.

Based on the results of a range-finding test, a limit test was performed. Preparation of test solutions started with a stock solution in DMSO of 140 mg/mL. Subsequently, 0.1 mL of the stock solution was added per litre of test medium in order to reach the nominal test concentration of 14 mg/L. After a 15-minute stirring period, the clear and colourless solution was siphoned off and used for the test. Exponentially growing algal cultures were exposed to a blank control, a solvent control and the nominal test concentration of 14 mg/L. The total test period was 48 hours and the initial algal cell density was 10^4 cells/mL. Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 hours and at the end of the test period.

The study met the acceptability criteria prescribed by the protocol and was considered valid.

At the start of the test, the actual test concentration was 5.7 mg/L at the expected nominal concentration of 14 mg/L. The measured concentration had not decreased by more than 20% at the end of the 48h period. Based on these results, the effect parameters were expressed in terms of initial concentrations.

The test substance reduced growth rate of this fresh water algal species significantly at 5.7 mg/L. However, the 48h-ErC50 for growth rate reduction was beyond the range tested, i.e. exceeded 5.7 mg/L.

Description of key information

OECD Guideline 201, GLP, key study, validity 2:

72h-ErC10 (Pseudokirchneriella subcapitata) > 4.15 mg/L (the highest attainable test concentration), based on geometric mean measured concentrations.

72h-ErC50 (Pseudokirchneriella subcapitata) > 4.15 mg/L (the highest attainable test concentration), based on geometric mean measured concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:

4.15 mg/L

EC10 or NOEC for freshwater algae:

4.15 mg/L

Additional information

To assess the toxicity of the registered substance to aquatic algae, two experimental studies are available.

 

The more recent study (ECT, 2020), determined as the key study, was performed according to OECD Guideline No. 201 with GLP compliance to assess the growth inhibition of the registered substance to the green algae Pseudokirchneriella subcapitata. Following a preliminary range-finding test, exponentially growing algal cultures were exposed to a control group and to 100, 50.0, 25.0, 12.5, 6.25 and 3.13% of a saturated solution at a loading rate of 100 mg test substance/L, for a period of 72 hours under static and closed test conditions. Samples of the test solutions were analysed, by GC-MS method, to determine the actual levels of the test item at start and the end of the test. The test item concentrations were not stable within +/-20% at the end of the exposure period. As a consequence, and since all concentration levels were measured, the biological results were calculated and reported based on the geometric mean measured concentrations, determined at: 0.119, 0.240, 0.435, 0.964, 1.90 and 4.15 mg test item/L. A slight concentration-response relationship was observed on the growth rare inhibition but not sufficient to determine ErC10 and ErC50 values, after 72 hours of exposure. The 72h-ErC10 and 72h-ErC50 values were determined to be above 4.15 mg/L, respectively, based on geometric measured concentrations, which was considered to be the maximum solubility of the test substance in adapted OECD-medium, under the test conditions.

 

The other study (NOTOX, 2009), determined as a supporting study, was performed according to the EPA Guideline OPPTS 850.5400 with GLP compliance to assess the growth inhibition of the registered substance to the green algae Pseudokirchneriella subcapitata. Based on the results of a range-finding test, a limit test was performed with a nominal test concentration of 14 mg/L (prepared with DMSO as solvent). Exponentially growing algal cultures were exposed to a blank control, a solvent control and the nominal test concentration of 14 mg/L for a period of 48 hours under static and closed test conditions. Samples for analytical confirmation of actual exposure concentrations were taken at the start, after 24 hours and at the end of the test period. Analysis of the sample taken from the limit concentration at the start of the test showed a measured concentration of 5.7 mg/L.  The measured concentration had not decreased by more than 20% at the end of the 48h period. Therefore, the effect parameters were expressed in terms of initial concentrations. The test substance reduced growth rate of this fresh water algal species significantly at 5.7 mg/L. However, the 48h-ErC50 for growth rate reduction was beyond the range tested, i.e. exceeded 5.7 mg/L, considered to be the maximum solubility of the test substance in adjusted M2 medium, under the test conditions.