Reaction mass of 5,7-dimethoxy-3-(4-(sec-C10-C13-alkyl)-benzoyl)-2H-chromen-2-one

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:

10 May 2019 - 21 Jun 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2006; Annex 5 corrected 28 July 2011. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: Esacure 3644
Physical Description: Light yellow solid
Purity/Composition: UVCB
Storage Conditions: At room temperature protected from light

Analytical monitoring:

yes

Details on sampling:

Samples for possible analysis were taken from all test concentrations and the control according to the schedule below.
Stock solutions in MeOH used for preparation of test solutions were sampled as well.

Frequency: 0, 24, 72 h
Volume: 2.0 mL from the approximate centre of the test vessels.
Storage: Not applicable, samples were transferred to the analytical laboratory at the Test Facility and analysed on the day of sampling.

At the end of the exposure period, the replicates with algae were pooled at each test concentration and the solvent control before sampling.
For the blank control, one of the replicates was sampled separately, since the algal cell densitywas considerably lower in this replicate compared to the remaining replicates.
The remaining replicates at this test group were pooled and subsequently also sampled.

Compliance with the quality criteria regarding maintenance of actual concentrations was checked by running a test vessel at the WAF prepared at a loading rate of 0.10 mg/L but without algae and samples for analysis were taken at the start, after 24 hours of exposure and at the end of the test period.

Additionally, reserve samples of 2.0 mL were taken from all test solutions for possible analysis. If not already used, these samples were stored in a freezer (≤-15°C) for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.

Vehicle:

no

Details on test solutions:

PREPARATION OF TEST SOLUTIONS

The batch of Esacure 3644 tested was a light yellow solid UVCB and not completely soluble in test medium at the loading rates initially prepared. No correction was made for the purity/composition of the test item. Preparation of test solutions was performed with minimized exposure to light.

RANGE-FINDING TEST

Preparation of test solutions started with loading rates individually prepared at 1.0 to 100 mg/L. A three-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. Thereafter, the aqueous Water Soluble Fractions (WSFs) were collected by filtration through a 0.45 µm membrane filter (RC55, Whatman) and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure. After preparation, volumes of 50 mL were added to each replicate of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.

FINAL TEST

Based on the results obtained in the range-finding test, the preparation procedure was adjusted. Water Accommodated Fractions (WAFs) were prepared by using solutions in methanol (MeOH; Merck, Darmstadt, Germany) with nominal concentrations of 0.00050, 0.0016, 0.0050, 0.016 and 0.050 mg/mL. Empty test vessels were individually spiked with 1.0 mL of the respective nominal concentration of the methanol solution. Each replicate of the solvent control received 1.0 mL MeOH. The solvent was completely evaporated overnight before addition of the test medium to avoid modification of the WAF-composition due to presence of a water-miscible solvent. For the evaporation, the test vessels were placed in a fume hood at room temperature in the dark, complete evaporation was confirmed visually. Thereafter, 50 mL test medium was added to each vessel. Test solutions, including those of the blank control and the solvent control, were agitated for a period of two days to ensure maximum dissolution of test item in test medium. Vessels were sealed during agitation to prevent vaporization of test medium. All test solutions were clear and colourless at the end of the preparation procedure. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 104 cells/mL.
Any residual volumes were discarded.

Test organisms (species):

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

Details on test organisms:

Species: Raphidocelis subcapitata, strain: NIVA CHL 1
Source: In-house laboratory culture.
Reason for selection: This system is a unicellular algal species sensitive to toxic items in the aquatic ecosystem and has been selected as an internationally accepted species.
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 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA) with the following composition:

NaNO3 500 mg/L
K2HPO4 39.5 mg/L
MgSO4.7H2O 75 mg/L
Na2CO3 20 mg/L
C6H8O7.H2O 6 mg/L
NH4NO3 330 mg/L
CaCl2.2H2O 35 mg/L
C6H5FeO7.xH2O 6 mg/L
H3BO3 2.9 mg/L
MnCl2.4H2O 1.81 mg/L
ZnCl2 0.11 mg/L
CuSO4.5H2O 0.08 mg/L
(NH4)6Mo7O24.4H2O 0.018 mg/L

Pre-culture 3 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 10E4 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 201 Guideline, formulated using Milli-RO water and with the following composition:

NH4Cl 15 mg/L
MgCl2.6H2O 12 mg/L
CaCl2.2H2O 18 mg/L
MgSO4.7H2O 15 mg/L
KH2PO4 1.6 mg/L
FeCl3.6H2O 64 µg/L
Na2EDTA.2H2O 100 µg/L
H3BO3 185 µg/L
MnCl2.4H2O 415 µg/L
ZnCl2 3 µg/L
CoCl2.6H2O 1.5 µg/L
CuCl2.2H2O 0.01 µg/L
Na2MoO4.2H2O 7 µg/L
NaHCO3 50 mg/L
Hardness (Ca+Mg) 0.24 mmol/L (24 mg CaCO3/L)
pH 8.1 ± 0.2

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Remarks on exposure duration:

No remarks

Post exposure observation period:

No post exposure observation

Hardness:

0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

21-24°C

pH:

8.1 ± 0.2

Dissolved oxygen:

Not measured

Salinity:

Not measured

Conductivity:

Not measured

Nominal and measured concentrations:

Loading rates (mg/L) Measured (µg/L)
0.010 -
0.032 0.98
0.10 9.0
0.32 65
1.0 134

Details on test conditions:

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:

- Three replicates of exponentially growing algal cultures were exposed to a WSFs individually prepared at loading rates of 1.0 to 100 mg/L increasing by a factor of 10 and to a control.
- Cell densities were recorded only at the end of the exposure period.
- pH was only measured in the control and the highest test concentration.
- At the end of the test algae were not observed under a microscope to verify a normal and healthy appearance.

FINAL TEST

TEST CONCENTRATIONS
Esacure 3644: WAFs individually prepared at loading rates of 0.010, 0.032, 0.10, 0.32, and 1.0 mg/L.
Control(s): Test medium without test item or other additives (blank control) and test medium without test item but with the additive used in the treatment of the stock solutions (solvent control).
Replicates: 3 replicates of each test concentration,
6 replicates of each control,
1 extra replicate of each test group for sampling purposes after 24 hours of exposure,
1 or 2 replicates of each test concentration without algae.

TEST PROCEDURE AND CONDITIONS

Test duration: 72 hours
Test type: Static
Test vessels: 100 mL, all-glass with aluminium caps, perforated for ventilation, containing 50 mL of test solution
Test Medium: M2
Cell density: An initial cell density of 1 x 104 cells/mL.
Illumination: Continuously using TLD-lamps with a light intensity within the range of 85 to 88 µE.m-2.s-1.
Incubation: Vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking

Reference substance (positive control):

yes

Remarks:

K2Cr2O7 (potassium dichromate)

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 33 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 33 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

33 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

33 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 33 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 33 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

Samples taken from the stock solutions of Esacure 3644 in methanol used in the preparation of test solutions were analysed. The analysed concentrations were at 94 – 110% relative to the nominal concentrations, indicating proper preparation of the stocks.

In addition, samples taken from all test concentrations and the controls were analysed. Small responses were observed at the retention time of the test item in the control and the lowest test concentration at the start and at the end of the test. These responses were below the response of the lowest calibration standard and probably caused by carry-over in the analytical system.

At the start of the test, the measured concentrations in WAFs prepared at 0.032, 0.10, 0.32 and 1.0 mg/L were 0.98, 9.0, 65 and 134 µg/L, respectively. These concentrations decreased during the test. The two highest test concentrations decreased to 3% of the initial concentrations at the end of the test, while the lower test concentrations had decreased below the level of the lowest calibration standard used in the analysis of the samples.

In contrast, the concentrations measured in the samples taken from the solution incubated without algae increased over time. A reason for this could not be identified.

Since the test item is a UVCB, the effect parameters were based on loading rates. Indicative effect parameters were determined based on the measured concentrations. The TWA concentration at the highest concentration tested was calculated to be 33 µg/L.

Results with reference substance (positive control):

The 72h-EC50 for growth rate inhibition (ERC50) was 1.11 mg/L with a 95% confidence interval ranging from 1.10 to 1.13 mg/L.
The 72h-ERC50 was within the expected range of 0.92 and 1.46 mg/L as specified in ISO International Standard 8692, February 2012, and the historical range of 0.86 and 2.3 mg/L, which is based on reference tests performed at the Test Facility during the last ten years.

Reported statistics and error estimates:

Equations shown in additional information

EXPERIMENTAL CONDITIONS

The table below shows the pH recorded at the beginning and the end of the test. The pH was within the limits prescribed by the study plan (6-9, preferably not varying by more than 1.5 unit).

Esacure 3644	pH
Loading rate (mg/L)	t=0h	t=72h
Pooled control	8	7.9
0.01	7.9	7.8
0.032	7.9	7.8
0.1	7.9	7.8
0.32	7.9	7.8
1.0 [33]	7.9	7.8

During the exposure period the temperature measured in the incubator was maintained between 22 and 23°C. Temperature remained within the limits prescribed by the study plan (21 24°C, constant within ±1°C).

POSITIVE CONTROL

The objective of the study was to evaluate Potassium dichromate for its ability to generate toxic effects in Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) (strain: NIVA CHL-1) during an exposure period of 72 hours and, if possible, to determine the EC50 for inhibition of growth rate (Test Facility Study No. 20196511).

Start of first exposure: 29 Apr 2019

Completion last exposure: 02 May 2019

The study procedures described in this report were based on the OECD guideline No. 201, Adopted March 23, 2006; Annex 5 corrected 28 July 2011 and ISO Standard 8692, Third edition, 15 February 2012.

This reference test was carried out to check the sensitivity of the test system used by the Test Facility to potassium dichromate (Merck KGaA, Art. 1.04864, Batch K50664264).

Algae were exposed for a period of 72 hours to nominal K2Cr2O7 (potassium dichromate) concentrations of 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L and to a control. The initial cell density was 1.0 x 104 cells/ml.

Overview of % inhibition of growth rate in the reference test:

K2Cr2O7	Mean	Std. Dev.	n	%Inhibition
Nominal conc. (mg/L)
Control	1.755	0.0108	3
0.18	1.677	0.0023	3	4.4
0.32	1.516	0.0286	3	14
0.56	1.268	0.017	3	28
1	0.889	0.0201	3	49
1.8	0.571	0.023	3	67
3.2	0.4	0.0188	3	77

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

Potassium dichromate inhibited growth rate of this fresh water algae species at nominal concentrations of 0.18 mg/L and higher.

The 72h-EC50 for growth rate inhibition (ERC50) was 1.11 mg/L with a 95% confidence interval ranging from 1.10 to 1.13 mg/L.

The 72h-ERC50 was within the expected range of 0.92 and 1.46 mg/L as specified in ISO International Standard 8692, February 2012, and the historical range of 0.86 and 2.3 mg/L, which is based on reference tests performed at the Test Facility during the last ten years.

In conclusion, the sensitivity of this culture of Raphidocelis subcapitata was in agreement with ISO International Standard 8692, February 2012 and the historical data collected at the Test Facility.

The study plan, raw data and report of this study are kept in the Test Facility’s archives.

The test described above was performed under GLP conditions.

COMPARISON OF AVERAGE GROWTH RATES

The average specific growth rate for a specific period is calculated as the logarithmic increase in the biomass from the equation for each single vessel of controls and treatments:

µ_i-j = (ln X_j - ln X) / (t_j - t_i) (day^-1)

Where:       µ_i-j  = the average specific growth rate from time i to j

       X_i = the biomass at time i

       X_j = the biomass at time j

The average growth rate at each test item concentration is then compared with the control value and the percentage inhibition in growth rate is calculated:

%I_r  = ((µ_C - µ_T) / µ_C) x 100

Where:       %I_r  = percent inhibition in average specific growth rate

       µ_C = mean value for average specific growth rate in the control group

       µ_T = average specific growth rate for the treatment replicate

YIELD

The percent inhibition in yield is calculated for each treatment replicate as follows:

%I_y = ((Y_C - Y_T) / Y_C) x 100

Where:       %I_y  = percent inhibition of yield

      Y_C = mean value for yield in the control group

      Y_T = value for yield for the treatment replicate

DETERMINATION OF AVERAGE EXPOSURE CONCENTRATIONS

Average exposure concentrations were calculated as:

24 x √(C_t=0 x C_t=24) + 48 x √(C_t=24 x C_t=72) , being the Time Weighted Average (TWA) of the concentrations of Esacure 3644 measured in the samples taken at the start (C_t=0), after 24 hours (C_t=24) and the end of the test (C_t=72).

In case concentrations measured were below the concentration of the lowest calibration solution, the final exposure concentration(s) were taken as a factor of 2 below this limit. This procedure is based on the OECD “Guidance document on the use of the harmonised system for the classification of chemicals which are hazardous for the aquatic environment”.

DETERMINATION OF THE EFFECT PARAMETERS

A student t-Test (parametric, α=0.05, two-sided) was performed to test for presence of statistically significant differences in algal growth between the blank and solvent control.

For determination of the effect parameters (i.e. NOELR, NOEC, ELx, and ECx-values) the approaches recommended in the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the controls (pooled data) revealed significant inhibition of growth rate or inhibition of yield (Multiple Sequentially-rejective U-test after Bonferroni-Holm correction, α=0.05, one-sided, smaller).

The ELx and ECx-values could not be determined since the observed effects were below 10%.

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

COMPUTERIZED SYSTEMS

Critical computerized systems used in the study are listed below or presented in the appropriate Phase Report.  All computerized systems used in the conduct of this study have been validated; when a particular system has not satisfied all requirements, appropriate administrative and procedural controls were implemented to assure the quality and integrity of data.

System name	Version No.	Description of Data Collected and/or Analyzed
Deviation Information Library	2.1	Deviations
REES Centron	SQL 2.0	Temperature, relative humidity and/or
		atmospheric pressure monitoring
Shimadzu Spectrophotometer	2.52	System control, data acquisition and processing
UV-1800 including 1cm UVProbe

Validity criteria fulfilled:

yes

Conclusions:

In conclusion, under the conditions of the present study with Raphidocelis subcapitata, no inhibition of growth rate or inhibition of yield was recorded at any of the concentrations of Esacure 3644 tested.

The 72h-EL50 for growth rate inhibition (ERL50) and yield inhibition (EYL50) exceeded the loading rate of 1.0 mg/L.

The 72h-EC50 for growth rate inhibition (ERC50) and yield inhibition (EYC50) was beyond the time weighted average concentration of 33 µg/L, being considered as the maximum soluble concentration of test item in test medium at a loading rate of 1.0 mg/L.

The 72h-NOELR for both growth rate inhibition and yield inhibition was 1.0 mg/L

The 72h-NOEC for both growth rate inhibition and yield inhibition was 33 µg/L

Executive summary:

The objective of the study was to evaluate Esacure 3644 for its ability to generate toxic effects in Raphidocelis subcapitata during an exposure period of 72 hours and, if possible, to determine the NOELR, EL10, EL20 and EL50 for both inhibition of growth rate and inhibition of yield.

The study procedures described in this report were based on the OECD guideline No. 201, 2006; Annex 5 corrected 28 July 2011. In addition, procedures were based on the test methods described in the OECD series on testing and assessment number 23, 2019.

The batch of Esacure 3644 tested was a light yellow solid UVCB and not completely soluble in test medium at the loading rates initially prepared. Water Accommodated Fractions (WAFs) were individually prepared at loading rates ranging between 0.010 and 1.0 mg/L and used as test concentrations. Test solutions were prepared by using stock solutions in methanol (MeOH). Since the test item is a UVCB, the solvent was completely evaporated addition of test medium to avoid modification of the WAF-composition due to presence of a water-miscible solvent. Additionally, the preparation of test solutions was performed with minimized exposure to light.

A final test was performed based on the results of a preceding range-finding test. Six replicates of exponentially growing algal cultures per group were exposed to a blank control and a solvent control. Three replicates per test concentration were exposed to WAFs individually prepared at loading rates of 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L. The initial algal cell density was 104 cells/mL. The total exposure period was 72 hours and samples for analytical confirmation of exposure concentrations were taken at the start, after 24 and 72 hours of exposure.

Samples taken from the nominal concentration solutions of Esacure 3644 in methanol, used in the preparation of test solutions, were analysed. The analysed concentrations were at 94 – 110% relative to the nominal concentrations, indicating proper preparation of the methanol solutions.

In addition, samples taken from all test concentrations and the controls were analysed. Small responses were observed at the retention time of the test item in the control and the lowest test concentration at the start and at the end of the test. These responses were below the response of the lowest calibration standard and probably caused by carry-over in the analytical system. At the start of the test, the measured concentrations in WAFs prepared at 0.032, 0.10, 0.32 and 1.0 mg/L were 0.98, 9.0, 65 and 134 µg/L, respectively. These concentrations decreased during the test. The two highest test concentrations decreased to 3% of the initial concentrations at the end of the test, while the lower test concentrations had decreased below the level of the lowest calibration standard used in the analysis of the samples.

Since the test item is a UVCB, the effect parameters were expressed in terms of loading rates.

Stimulation instead of inhibition of growth rate and yield was recorded at all test concentrations. No statistically significant differences were found between test item treatments and the pooled controls. The study met the acceptability criteria prescribed by the study plan and was considered valid.

The effect parameters (based on loading rates) obtained in this study are summarized in the tables below.

Parameter (mg/L)	NOELR	EL10	EL50
Growth rate	1	>1.0	>1.0
Yield	1	>1.0	>1.0

Description of key information

Study performed to recognised OECD guidlines with GLP certification

Additional information

EC50 = > 1.0 mg/mL (loading rate) / > 33 µg/mL (measured)