4-[(2E)-3-(3,5-dichloro-4-fluorophenyl)-4,4,4-trifluorobut-2-enoyl]-N-[(4R)-2-ethyl-3-oxo-1,2-oxazolidin-4-yl]-2-methylbenzamide

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date: November 05, 2019; Experimental start date: November 11, 2019; Experimental completion date: January 27, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 2006, corrected 2011

Deviations:

no

Remarks:

96 h exposure duration

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of decision 2018-01-10; Swiss Ordinance relating to GLP, adopted May 18, 2005 [SR 813.112.1]. This Ordinance is based on the OECD Principles of GLP, as revised in 1997 and adopted November 26, 1997 by decision of the OECD Council [C(97)186/Final].

Test material

Test material information

Constituent 1

Reference substance name:

4-[3-(3,5-dichloro-4-fluorophenyl)-4,4,4-trifluorobut-2-enoyl]-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide

Molecular formula:

C23H18Cl2F4N2O4

IUPAC Name:

4-[3-(3,5-dichloro-4-fluorophenyl)-4,4,4-trifluorobut-2-enoyl]-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide

Test material form:

solid

Details on test material:

Off-white

Specific details on test material used for the study:

- Expiration date of the lot/batch: End of November 2022
- Storage condition of test material: < 30 °C

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control and all treatments
- Sampling method: For the determination of the test item concentrations, duplicate samples (10 mL) were taken from the test media of all test concentrations and the control at the start and at the end of the test (96 hours). For sampling at the end of the test, the contents of the replicate test vessels were combined prior to sampling. As the test item was instable in the pre-experiments, additional stability samples from all treatments in duplicate were taken after 24, 48 and 72 hours. These analytical stability samples (10 mL sample volume) could not be taken from the test vessels itself due to the limited test medium volume (30 mL). Therefore, a set of additional flasks containing the corresponding test medium (with algae) was incubated under conditions identical to the test.
- Sample storage conditions before analysis: All samples were stored frozen (-20 ± 5 °C) immediately after sampling until analysis.
- Analysed samples: The concentrations of the test item were measured in one of the duplicates for all taken samples at the start and the end of the test. The stability samples from 24, 48 and 72 hours were not analyzed, since the test item was observed to only slightly decrease over the test period of 96 hours.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: At the start of the test, the highest test concentration (undiluted filtrate) was prepared by suspending 100.12 mg of the test item in 1000 mL of test water. This preparation was supported by ultrasonic treatment for 15 minutes and intense stirring on a magnetic stirrer for three hours at room temperature in the dark. The stirring time was based on the stirring preexperiment which showed, that the maximum amount of dissolved test item was reached after this stirring time. After stirring, the suspension of the test item was filtered through a membrane filter (Whatman, Type NC45, pore size 0.45 μm). As a precaution, the filter was pre-conditioned with 200 mL filtrate to avoid losses of dissolved test item due to adsorption on the filter material. This undiluted filtrate was used as the highest tested concentration. The test medium of the highest test concentration was diluted with test water to prepare the test media of the lower test concentrations
- Controls: A control was tested in parallel (test water without test item). The test design included three replicates per test concentration and six replicates of the control. Additionally one replicate (100 ml test medium in 250 mL Erlenmeyer flask) per treatment were prepared for potential analytical samples at 24, 48 and 72 hours, which were incubated in parallel to the test.
- Evidence of undissolved material: No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the entire test duration. The test media were prepared just before the start of the exposure.

Test organisms

Test organisms (species):

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

Details on test organisms:

•TEST ORGANISM
- Common name: Green algae
- Strain: No. 61.81 SAG
- Source: Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany
- Age of inoculum (at test initiation): An inoculum culture of algae was set up three days before the start of the test.
- Method of cultivation: The algae were cultivated at IES Ltd Laboratories under standardized conditions according to the test guidelines.

•ACCLIMATION
- Acclimation period: The algae were cultivated under the test conditions and kept in the exponential growth phase until inoculation of the test solutions. The inoculum culture was incubated under the same conditions and dilution media as the test cultures. After the end of the evaluations, the algae cultures in the treatments including the control were disposed and not be used for any further testing.
- Culturing media and conditions: Same as test

Study design

Test type:

static

Water media type:

freshwater

Remarks:

Reconstituted test water (AAP Medium) prepared according to the test guideline was used for algal cultivation and testing. Analytical grade salts were dissolved in sterile purified water.

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

In agreement with the Test Guideline (TG) the normal exposure duration of 72 h was extended to 96 h because all validity criteria of in paragraph 11 of the TG could be met.

Test conditions

Hardness:

The calculated water hardness of the test water was 0.15 mmol/L (= 15 mg/L as CaCO3).

Test temperature:

The temperature during the test was maintained at 21 °C.

pH:

- The pH in the test water (AAP medium) was 7.5.
- The pH of the test media was in the range of 7.2 to 8.5 during the test period

Nominal and measured concentrations:

- Nominal concentrations: Undiluted filtrate of a suspension of the test item with the loading rate of 100 mg/L and the dilutions 1:2, 1:4, 1:8 and 1:16
- Mean measured concentrations: 3.8, 7.4, 16, 26 and 54 μg/L
- Determined test item concentrations
• in the fresh solutions (day 0, 0 h) 4.51, 8.64, 16.7, 34.1 and 60.9 μg/L
• in the aged solutions (day 4, 96 h) 3.19, 6.34, 15.3, 20.3, and 48.6 μg/L corresponding to 71, 73, 91, 60 and 80 % of the initial concentration (respectively).
• At the end of the test, 60 to 91 % of the initially measured values were found. Therefore, the biological results were calculated based on the mean measured concentrations of the test item (calculated as the geometric mean of the concentrations measured at the start and the end of the test).

Details on test conditions:

•DETAILS ON TEST CONDITIONS
•TEST SYSTEM
- Test vessel: Erlenmeyer flasks. Each test flask was loosely covered with a glass lid, thereby adequate CO2 be transferred from surrounding air to the test solution.
- Material, size, headspace, fill volume: Glass, 75 mL, not firmly closed, 30 mL
- Aeration: No
- Initial cells density: 5000 algal cells/mL, corresponding to 0.94 ∙ 10^4 relative fluorescence units. The algal cell density in the pre-culture was determined using an electronic particle counter (Cell Counter CASY TT, OLS, Bremen/Germany).
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

•GROWTH MEDIUM
- Standard medium used: Yes, AAP-Medium

•TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Sterile purified (reconstituted) tap water
- Macro-nutrients:
•Ingredient / Concentration
• NaHCO3 / 15.0 mg/L
• K2HPO4 / 1.044 mg/L
• MgSO4 × 7 H2O / 14.6 mg/L
• MgCl2 × 6 H2O / 12.16 mg/L
• CaCl2 × 2 H2O / 4.41 mg/L
• NaNO3 / 25.5 mg/L
- Trace elements:
•Ingredient / Concentration
• H3BO3 / 186.0 µg/L
• MnCl2 × 4 H2O / 415.0 µg/L
• ZnCl2 / 3.27 µg/L
• CoCl2 × 6 H2O / 1.43 µg/L
• CuCl2 × 2 H2O / 0.012 µg/L
• Na2MoO4 × 2 H2O / 7.26 µg/L
• FeCl3 × 6 H2O / 160.0 µg/L
• Na2EDTA × 2 H2O: 300.0 µg/L

- Culture medium different from test medium: No

•OTHER TEST CONDITIONS
- Sterile test conditions: No
- Adjustment of pH: No
- Photoperiod: Continuously illuminated
- Light intensity and quality: ca. 68 µE/sm² (range: 67 to 70), LED, 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.

•EFFECT PARAMETERS MEASURED
- Fluorescence (or algal cell density) as surrogate measure for algal biomass (Fluorimeter) at 0, 24, 48, 72 and 96 hours.
- Microscopic examination of the algal cells at the end of the test

•TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 2. The analytical results show the correct preparation of the test media, i.e. the theoretical spacing factor of two between the different test concentrations was met.

•RANGE-FINDING STUDY
- Test concentrations: 0.1 / 1.0 / 10 / 100 μg/L
- Results used to determine the conditions for the definitive study:
•Dilution / Inhibition of Average Growth after 96 hours [%] / Inhibition of Yield after 96 hours [%]
• 1:1000 / 0.1 / 0.8
• 1:100 / -0.2 / -1.0
• 1:10 / -0.8 / -4.2
• Undiluted Filtrate / -0.5 / -2.9

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (CAS 7778-50-9), Batch No. MKBR3876V), obtained from Sigma-Aldrich

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 54 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

100 mg/L LR

Basis for effect:

growth rate

Remarks on result:

not determinable

Remarks:

95% CI n.d.

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

16 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

100 mg/L LR corresponding to 54 µg/L

Basis for effect:

growth rate

Remarks:

and also for Area Under Curve (AUC) and biomass / yield

Remarks on result:

other:

Remarks:

Williams t-test, one-sided smaller, α = 0.05

Effect concentrations 3

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 54 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

100 mg/L LR

Basis for effect:

growth rate

Remarks on result:

not determinable

Remarks:

95% CI n.d.; inhibition rate at 54 µg/L was 5.5 %

Effect concentrations 4

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

26 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

100 mg/L LR corresponding to 54 µg/L

Basis for effect:

growth rate

Remarks:

and also for Area Under Curve (AUC) and biomass / yield

Remarks on result:

other:

Remarks:

Williams t-test, one-sided smaller, α = 0.05

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 54 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

100 mg/L LR

Basis for effect:

growth rate

Remarks on result:

not determinable

Remarks:

95% CI n.d.

Effect concentrations 6

Duration:

96 h

Dose descriptor:

EC10

Effect conc.:

> 54 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

100 mg/L LR

Basis for effect:

growth rate

Remarks on result:

not determinable

Remarks:

95% CI n.d.; inhibition rate at 54 µg/L was 1.1 %; NOEC ≥54 µg/L (Williams t-test, one-sided smaller, α = 0.05); LOEC >54 µg/L.

Effect concentrations 7

Duration:

96 h

Dose descriptor:

EC20

Effect conc.:

> 54 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

100 mg/L LR

Basis for effect:

growth rate

Remarks on result:

not determinable

Remarks:

95% CI n.d.

Effect concentrations 8

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 54 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

100 mg/L LR

Basis for effect:

growth rate

Remarks on result:

not determinable

Remarks:

95% CI n.d.

Effect concentrations 9

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

19 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

100 mg/L LR corresponding to 54 µg/L

Basis for effect:

biomass

Remarks:

yield

Remarks on result:

other:

Remarks:

95% CI 6.8 – 28 µg/L (Probit analysis); NOEC 16 µg/L (Williams t-test, one-sided smaller, α = 0.05); LOEC 26 µg/L.

Effect concentrations 10

Duration:

96 h

Dose descriptor:

EC10

Effect conc.:

> 54 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

100 mg/L LR

Basis for effect:

biomass

Remarks:

yield

Remarks on result:

not determinable

Remarks:

95% CI n.d.; NOEC ≥54 µg/L (Williams t-test, one-sided smaller, α = 0.05); LOEC >54 µg/L.

Effect concentrations 11

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

47 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

100 mg/L LR corresponding to 54 µg/L

Basis for effect:

biomass

Remarks:

yield

Remarks on result:

other:

Remarks:

95% CI 32 – >54 µg/L (Probit analysis)

Effect concentrations 12

Duration:

96 h

Dose descriptor:

EC20

Effect conc.:

> 54 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

100 mg/L LR

Basis for effect:

biomass

Remarks:

yield

Remarks on result:

not determinable

Remarks:

95% CI n.d.

Effect concentrations 13

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 54 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

100 mg/L LR

Basis for effect:

biomass

Remarks:

yield

Remarks on result:

not determinable

Remarks:

95% CI n.d.

Effect concentrations 14

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 54 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

100 mg/L LR

Basis for effect:

biomass

Remarks:

yield

Remarks on result:

not determinable

Remarks:

95% CI n.d.

Effect concentrations 15

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

37 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

100 mg/L LR corresponding to 54 µg/L

Basis for effect:

biomass

Remarks:

calculated as Area Under Curve (AUC)

Remarks on result:

other:

Remarks:

95% CI 18 – >54 µg/L (Probit Analysis); NOEC 16 µg/L (Williams t-test, one-sided smaller, α = 0.05); LOEC 26 µg/L.

Effect concentrations 16

Duration:

96 h

Dose descriptor:

EC10

Effect conc.:

69 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

extrapolation, 100 mg/L LR corresponding to 54 µg/L

Basis for effect:

biomass

Remarks:

calculated as Area Under Curve (AUC)

Remarks on result:

other:

Remarks:

95% CI 31 – >54 µg/L (Probit Analysis); NOEC 26 µg/L (Williams t-test, one-sided smaller, α = 0.05); LOEC 54 µg/L.

Effect concentrations 17

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

> 54 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

100 mg/L LR

Basis for effect:

biomass

Remarks:

calculated as Area Under Curve (AUC)

Remarks on result:

not determinable

Remarks:

95% CI n.d.

Effect concentrations 18

Duration:

96 h

Dose descriptor:

EC20

Effect conc.:

> 54 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

100 mg/L LR

Basis for effect:

biomass

Remarks:

calculated as Area Under Curve (AUC)

Remarks on result:

not determinable

Remarks:

95% CI n.d.

Effect concentrations 19

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 54 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

100 mg/L LR

Basis for effect:

biomass

Remarks:

calculated as Area Under Curve (AUC)

Remarks on result:

not determinable

Remarks:

95% CI n.d.

Effect concentrations 20

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 54 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (dissolved fraction)

Remarks:

100 mg/L LR

Basis for effect:

biomass

Remarks:

calculated as Area Under Curve (AUC)

Remarks on result:

not determinable

Remarks:

95% CI n.d.

Details on results:

- Exponential growth in the control: Yes
- Observation of abnormalities:The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the undiluted filtrate (54 μg/L mean measured) and the algal cells in the control. The shape and size of the algal cells were not affected by the test item at any of the test concentrations tested.
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50: 1.3 mg potassium dichromate/L with a 95 % confidence intervall of 1.2 to 1.4 mg/L for growth rate and 0.47 mg potassium dichromate/L with a 95 % confidence intervall of 0.44 to 0.49 for yield
- Other: IES Study 20190393 (Experimental Starting Date: November 01, 2019; Experimental Completion Date: November 08, 2019)

Reported statistics and error estimates:

AUC, growth rate and yield were calculated for each test flask. The mean values for AUC, growth rate and yield were calculated for each treatment. The 72 and 96-hour EC10, EC20 and EC50 values for the inhibition of AUC, average growth rate and yield and their 95 % confidence intervals were calculated, where possible, by Probit Analysis using linear maximum likelihood regression. For the determination of the LOEC and NOEC, the AUC, average growth rate and yield at the test concentrations were compared to the control values by Williams t-test. Statistical analysis was performed using ToxRat Professional®.

Any other information on results incl. tables

The biological results of the study (based on nominal concentrations) are summarized in the table below:

Parameter	after 72 hours			after 96 hours
	AUC	Growth rate	Yield	AUC	Growth rate	Yield
	[µg/L]
EC10	37^a	>54^b	19^a	69^a	>54^b	>54^b
95% CI	18 – >54	n.d.	6.8 – 28	31 – >54	n.d.	n.d.
EC20	>54^b	>54^b	47^a	>54^b	>54^b	>54^b
95% CI	n.d.	n.d.	32 – >54	n.d.	n.d.	n.d.
EC50	>54^b	>54^b	>54^b	>54^b	>54^b	>54^b
95% CI	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.
NOEC*	16	16	16	26	54	54
LOEC	26	26	26	54	>54	>54

a: Probit Analysis

b: EC_X values of the test item could not be calculated because of the low inhibitory effect of the test item on the algal growth at the tested concentrations.

^*: Williams t‑test, one-sided smaller, α = 0.05

95% CI: 95% confidence interval

n.d.: Could not be determined

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test substance had a weak inhibitory effect on the growth rate of freshwater green algae over the study period of 72 hours.

Executive summary:

The acute toxicity of the test item on the growth of a freshwater green algae (Raphidocelis subcapitata) was investigated in a 96-hour static test according to the OECD TG 201, adopted 2006 (corrected 2011). The validity criteria were met.

Due to the low water solubility of the test item, a suspension of the substance with the loading rate of 100 mg/L was prepared by using ultrasonic treatment for 15 minutes and intensive stirring for 3 hours to reach a maximum concentration of dissolved test item in test water. The stirring time was based on a pre-experiment. After stirring, the suspension was filtered through a 0.45 μm membrane filter. Based on the range-finding test results, this undiluted filtrate and the dilutions 1:2, 1:4, 1:8 and 1:16 were used as test media. A control (test medium without test item) was tested in parallel. The preparation of the test medium was based on the OECD Guidance Document on Aqueous-Phase Aquatic Toxicity Testing of Difficult Test Chemicals, 2019. AAP-medium was used as test medium.

Erlenmeyer flasks (glass, size 75 mL, fill volume 30 mL), loosely covered with a glass lid allowing adequate carbon dioxide transfer from surrounding air to the test solution, served as test vessels. In agreement with the Test Guideline the normal exposure duration of 72 h was extended to 96 h. Duplicate samples (10 mL) were taken from the test media of all test concentrations and the control at the start and end of the test (96 hours). For sampling at the end of the test, the contents of the replicate test vessels were combined prior to sampling. All samples were stored frozen (-20 ± 5 °C) immediately after sampling until analysis. As the test item was instable in the pre-experiments, additional duplicate stability samples from all treatments were taken after 24, 48 and 72 hours. These analytical stability samples (10 mL sample volume) could not be taken from the test vessels itself due to the limited test medium volume (30 mL). Therefore, a set of additional flasks containing the corresponding test medium (with algae) was incubated under conditions identical to the test. The concentrations of the test item were measured in one of the duplicates for all samples at the start and the end of the test. The stability samples from 24, 48 and 72 hours were not analyzed, since the test item was observed to only slightly decrease over the test period of 96 hours. At the end of the test, 60 to 91% of the initially measured values were found. Therefore, the biological results were calculated based on the mean measured concentrations of the test item (calculated as the geometric mean of the concentrations measured at the start and the end of the test), which were 3.8, 7.4, 16, 26 and 54 μg/L.

The effect parameter measured was fluorescence (or algal cell density) as surrogate measure for algal biomass at 0, 24, 48, 72 and 96 hours. Microscopic examination of the algal cells was performed at the end of the test. 

All test media were clear solutions throughout the entire test duration. The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the undiluted filtrate (54 μg/L mean measured) and the algal cells in the control. The shape and size of the algal cells were not affected by the test item at any of the test concentrations tested.

After 72 hours, the effects on algal growth rate were determined and the ErC50 value was >54 μg/L and the NOEC and LOEC values were 16 and 26 μg/L. For the biomass-based endpoints, the 72-hour EC10 values were 19 μg/L for yield and 37 μg/L for AUC. The 72-hour NOEC and LOEC values were 16 and 26 μg/L for yield and AUC.