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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 September 2017 to 08 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted in accordance with international guidelines and in accordance with GLP. All relevant validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
(2011)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
Council Regulation (EC) No. 266/2016 Method C.3 (2016)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All concentration levels and the control were analytically verified via LC-MS after 0 (start of exposure), 24, 48 and 72 hours (end of the exposure). Therefore, the test item 2-Methyldecan-1-al and the corresponding transformation product 2-Methyldecanoic acid were analyzed and evaluated.
- Sampling method: Separate replicates for each test item concentration and control for the test item analysis at the beginning of the exposure were prepared with algae. The samples for the test item analysis after 24, 48 and 72 hours were prepared at the beginning of the exposure phase with algae and incubated under test conditions.
Additionally, the highest concentration level was analytically verified via analysis of total organic carbon (TOC, according to DIN EN 1484) at the start and the end of the exposure. Separate replicates for the TOC measurement were prepared without algae and without MES buffer and incubated under test conditions.
- Sample storage conditions before analysis: All samples were stored at room temperature until the start of analysis, if necessary. Prepared samples were stored at room temperature until analysis
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A saturated solution with a nominal loading of 100 mg/L was saturated solution prepared with dilution water 72 ± 1 hour prior to the start of the exposure. The test item was placed on to the surface of the dilution water with a pipette. A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out with a magnetic stirrer at room temperature for 72 ± 1 hour. After stirring the saturated solution was allowed to settle for 4 hours and was then taken from the homogeneous phase and used for testing. Additionally, a saturated solution was prepared as described above with dilution water (without the compounds MES and NaHCO3). This saturated solution was used for analysis of the total organic carbon (TOC, according to DIN EN 1484) and Tyndall effect, which was negative.
The saturated solution and four dilution levels out of the saturated solution was tested in a geometrical series with a dilution factor of 2: 6.25 - 12.5 - 25.0 - 50.0 - 100% saturated solution.
- Eluate: Test medium in accordance with OECD 201
- Differential loading: Not applicable
- Controls: Six replicates (without test item) were exposed under the same conditions as the test concentrations.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: HINDÁK, SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Preculture, prepared in dilution water, was used as inoculum

ACCLIMATION
- Acclimation period: Not applicable (laboratory culture)
- Culturing media and conditions (same as test or not): Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux for 24 hours per day. Nutrient medium Z according to LÜTTGE et al. (1994)
- Any deformed or abnormal cells observed: None reported
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
The medium has a nominal hardness of 0.24 mmol Ca+Mg/L
Test temperature:
Nominal range: 21 - 24 °C, controlled at ± 2 °C
pH:
As per guideline
Dissolved oxygen:
Not applicable
Salinity:
Not applicable
Conductivity:
Not applicable
Nominal and measured concentrations:
Refer to data tables
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile headspace flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: volume: 59 mL, with aluminium tops with PTFE seals.
- Aeration: Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Not applicable
- Initial cells density: Nominal: approximately 5 x 103 - 104 cells/mL, Actual: 6839 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): Not applicable

GROWTH MEDIUM
- Standard medium used: yes (OECD 201)
- Detailed composition if non-standard medium was used: Not applicable

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Not reported

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: No
- Photoperiod: No (24 hours/day light)
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120 µE*m-2*s-1
- Salinity (for marine algae): Not applicable

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : EC10-, EC20- and EC50-value with confidence intervals of growth rate and yield inhibition after 72 hours was calculated by sigmoidal dose-response regression.

- Determination of cell concentrations: The cell density was measured daily via Chlorophyll-a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal. No self-fluorescence was found in the definitive test at the saturated solution. The NOEC / LOEC was determined by calculation of statistical significance of growth rate and yield.
- Other: Microscopic evaluation of the cells at the start and end of the incubation was carried out. The cells were checked for any unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers or aggregation of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study
- Test concentrations: 1, 10 and 100 % saturated solution
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
yes
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
30.5 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: transformation product
Remarks:
2-methyldecanoic acid
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
9.4 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: Transformation product
Remarks:
2-methyldecanoic acid
Basis for effect:
other: yield
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
6.15 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: Transformation Product
Remarks:
2-methyldecanoic acid
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
< 0.248 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
other: Transformation Product
Remarks:
2-methyldecanoic acid
Basis for effect:
other: yield
Details on results:
Microscopic evaluation of the cells at the start and end of exposure revealed no morphological abnormalities. All effect values are given based on the time weighted average test item concentrations.
The environmental conditions (pH-value, room temperature, light intensity) were determined to be within the acceptable limits. The test item solutions were concentration related turbid throughout the exposure phase.
The concentrations of the test item after derivatization with 2,4 Dinitrophenylhydrazine (DNPH) and the metabolite 2-methyldecanoic acid were determined in the fresh media (0 h), after 24 hours, after 48 hours and in 72 hours old media of all tested concentration levels and the control via LC-MS/MS.
The measured concentrations of the test item after derivatisation were < LOQ of the nominal value at the start of the exposure (0 hours), after 24 hours, after 48 hours and at the end of the exposure (72 hours). Therefore, the concentrations of the transformation product 2 Methyldecanoic acid were analyzed and calculated as 2-Methyldecan-1-al concentrations by conversion with a factor of 0.914 (based on the ration of the molar weights of the test item and its transformation product). The time weighted average concentrations of the test item were calculated based on these concentrations to be: 0.248 – 0.877 – 11.5 – 34.3 – 74.9 mg/L.
The total organic carbon content (TOC) of the saturated solution was 13.8 mg C/L at test initiation. After 72 h it was 13.7 mg C/L.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50:
72h ErC50 = 0.559 mg/L (with headspace), 0.811 mg/L (without headspace)
72h EyC50 = 0.320 mg/L (with headspace), 0.387 mg/L (without headspace)
- Other:
Reported statistics and error estimates:
EC-values and statistical analyses:
EC10-, EC20- and EC50-value with confidence intervals of growth rate and yield inhibition after 72 hours was calculated by sigmoidal dose-response regression.

NOEC, LOEC Analyses:
The NOEC / LOEC was determined by calculation of statistical significance of growth rate and yield.

The following statistical tests were conducted:
Shapiro-Wilk’s test on normal distribution was done with a significance level of 0.01.
Levene’s test on variance homogeneity was done with a significance level of 0.01.
Monotonicity of Concentration/Response was assessed by trend analysis by contrasts (significance level: 0.05).
Williams Multiple t-test was done with a significance level of 0.05.

Software
The data for the tables in this report were computer-generated and have been rounded for presentation from the fully derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.

Calculations were carried out using software
Excel, MICROSOFT CORPORATION
SigmaPlot, SPSS INC.
GraphPad Prism, GRAPHPAD SOFTWARE, INC.
ToxRat Professional, Version 3.2.1, TOXRAT SOLUTIONS GMBH

Cell Densities

Time weighted average test item concentration

Replicate

Cell density [cells/mL]

[mg/L]

No.

0 hours

24 hours

48 hours

72 hours

74.9

1

6839

4510

5806

4943

2

6839

4873

4621

4914

3

6839

4320

4952

4621

Mean

6839

4568

5126

4826

34.3

1

6839

7527

8287

54568

2

6839

6456

7061

63820

3

6839

6883

8697

46094

Mean

6839

6955

8015

54827

11.5

1

6839

13753

41038

280051

2

6839

15099

40365

327792

3

6839

11763

38030

313232

Mean

6839

13538

39811

307025

 0.877

1

6839

16451

132394

572230

2

6839

19339

83108

643215

3

6839

20863

106270

698224

Mean

6839

18884

107257

637890

 0.248

1

6839

23140

153531

686520

2

6839

23023

132452

580832

3

6839

19193

148288

669373

Mean

6839

21785

144757

645575

Control

1

6839

34519

282228

716131

2

6839

42355

263162

776348

3

6839

37173

321425

789105

4

6839

36251

259604

684237

5

6839

45240

274457

696761

6

6839

33925

193079

754169

Mean

6839

38244

265659

736125

 

Evaluation after 72 hours

Statistically significant differences of growth rates and yield compared to
control values are marked (+), not significant differences are marked (-).

Time weighted average test item concentration

Replicate

Growth rate

Inhibition of growth rate

Yield

Inhibition of yield

[mg/L]

No.

[d-1]

[%]

[cells/mL]

[%]

74.9

1

 

-0.108

100

 

-1896

100

2

 

-0.110

100

 

-1925

100

3

 

-0.131

100

 

-2218

100

Mean

(+)

-0.116

100

(+)

-2013

100

34.3

1

 

0.692

56

 

47729

93

2

 

0.744

52

 

56981

92

3

 

0.636

59

 

39255

95

Mean

(+)

0.691

56

(+)

47988

93

11.5

1

 

1.24

21

 

273212

63

2

 

1.29

17

 

320953

56

3

 

1.28

18

 

306393

58

Mean

(+)

1.27

19

(+)

300186

59

 0.877

1

 

1.48

5

 

565391

22

2

 

1.52

3

 

636376

13

3

 

1.54

1

 

691385

5

Mean

(+)*

1.51

3

(+)

631051

13

 0.248

1

 

1.54

1

 

679681

7

2

 

1.48

5

 

573993

21

3

 

1.53

2

 

662534

9

Mean

(+)*

1.52

3

(+)

638736

12

Control

1

 

1.55

 

 

709292

 

2

 

1.58

 

 

769509

 

3

 

1.58

 

 

782266

 

4

 

1.54

 

 

677398

 

5

 

1.54

 

 

689922

 

6

 

1.57

 

 

747330

 

Mean

 

1.56

 

 

729286

 

* = considered biologically not significant

Section-by-Section and Average Specific Growth Rates of the Control Group

(0 - 72 hours)

 

Replicate No.

Specific growth rate [d-1]

Mean

(0 - 72 hours)

SD

±

CV
[%]

Mean CV [%]

section-by-section

0 - 24 hours

24 - 48 hours

48 - 72 hours

Control

1

1.62

2.10

0.931

1.55

0.588

37.9

30.9

2

1.82

1.83

1.08

1.58

0.429

27.2

3

1.69

2.16

0.898

1.58

0.637

40.2

4

1.67

1.97

0.969

1.54

0.513

33.4

5

1.89

1.80

0.932

1.54

0.530

34.4

6

1.60

1.74

1.36

1.57

0.191

12.2

 

 

 

Mean

1.56

 

 

 

 

 

SD ±

0.02

 

 

 

 

CV [%]

1.26

 

 

SD = Standard deviation        CV = Coefficient of variation

Environmental Conditions

Time weighted average test item concentration

pH-value

[mg/L]

Start; 0 hours

End; 72 hours

74.9

7.40

7.47

34.3

7.79

7.74

11.5

7.91

8.00

 0.877

8.01

8.56

 0.248

8.03

8.96

Control

8.05

9.42

Room temperature [°C]:

min.: 21.0

max.: 23.5

mean value: 22.3

 

Light intensity
[lux]

n = 27

mean value: 5804

range of the measured values: 5075 to 6308

equalling -12.6 to 8.68 %

CV [%]: 6.21

CV = Coefficient of variation             n = number of measuring points

Measured Concentrations of the Nominal Concentration of2‑Methyldecan-1-al

Sampling date

0 hours

24 hours

48 hours

72 hours

 

Nominal concentration of the test item
[%

of saturated solution]

2‑Methyldecan-1-al

 

Meas.

conc.

[mg/L]

Calc.

conc. [mg/L]

Meas.

conc.

[mg/L]

Calc.

conc. [mg/L]

Meas.

conc.

[mg/L]

Calc.

conc. [mg/L]

Meas.

conc.

[mg/L]

Calc.

conc. [mg/L]

Time weighted average1)

[mg/L]

 100

84.7

78.2

83.0

76.6

79.7

73.6

77.0

71.1

74.9

50.0

42.5

39.2

36.7

33.9

37.1

34.2

33.6

31.0

34.3

25.0

23.1

21.4

15.9

14.7

12.4

11.4

4.19

3.87

11.5

12.5

11.9

11.0

4.34

4.01

< LOQ

< LOQ

0.877

6.25

5.17

4.77

< LOQ

< LOQ

< LOQ

0.248

Control

< LOQ

< LOQ

< LOQ

< LOQ

 

Meas. conc = Measured concentration of the metabolite 2-Methyldecanoic acid, dilution and weighing factor was taken into account

Calc. conc.  = equivalent concentration of2‑Methyldecan-1-alcalculated from the measured metabolite concentration (conversion factor: 0.914, based on the ratio of molar weights of2‑Methyldecan‑1‑al: 2-Methyldecanoic acid, 170.3 g/mol : 186.3 g/mol)

LOQMetabolite = limit of quantification of the analytical method for the transformation product 2-methyldecanoic acid (0.3 mg 2-methyldecanoic acid/L)

LOQTest item = limit of quantification of the analytical method for the test item (0.1 mg test item/L)

= ½ x LOQMetabolite (corresponding to 0.137 mg test item/L) was taken into account

Validity criteria fulfilled:
yes
Conclusions:
In this study, 2-Methyldecan-1-al was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitataafter 72 hours with the following effect values (based on time weighted average test item concentrations): The EC50-values with 95% confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 30.5 (28.9 – 32.1) mg/L and 9.40 (6.44 – 11.0) mg/L. The EC10-values with 95% confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 6.15 (4.78 – 7.71) mg/L and < 0.248 mg/L.
Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016 Method C.3.  The aim of the study was the determination of NOEC, LOEC, EC10- , EC20- and EC50-values of the growth rate and the yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 6839 cells/mL. A saturated solution with a nominal loading of 100 mg/L was prepared with dilution water (see Table 2) 72 ± 1 hour prior to the start of the exposure. The test item was placed on to the surface of the dilution water with a pipette. A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out with a magnetic stirrer at room temperature for 72 ± 1 hour. After stirring the saturated solution was allowed to settle for 4 hours and was then taken from the homogeneous phase and used for testing. Additionally, a saturated solution was prepared as described above with dilution water (without the compounds MES and NaHCO3). This saturated solution was used for analysis of the total organic carbon (TOC, according to DIN EN 1484) and Tyndall effect, which was negative. The saturated solution and four dilution levels out of the saturated solution were tested in a geometrical series with a dilution factor of 2: 6.25 - 12.5 - 25.0 - 50.0 - 100% of the saturated solution. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

The test media were concentration related turbid throughout the test period. The concentrations of the test item after derivatization with 2,4‑Dinitrophenylhydrazine (DNPH) and the metabolite 2-methyldecanoic acid were determined in the fresh media (0 h), after 24 hours, after 48 hours and in 72 hours old media of all tested concentration levels and the control via LC-MS/MS. The results are presented in Table 8. Details of the analytical method are presented in part 13.

The measured concentrations of the test item after derivatisation were < LOQ of the nominal value at the start of the exposure (0 hours), after 24 hours, after 48 hours and at the end of the exposure (72 hours). Therefore, the concentrations of the transformation product 2‑Methyldecanoic acid were analyzed and calculated as 2-Methyldecan-1-al concentrations by conversion with a factor of 0.914 (based on the ration of the molar weights of the test item and its transformation product). The time weighted average concentrations of the test item were calculated based on these concentrations to be: 0.248 – 0.877 – 11.5 – 34.3 – 74.9 mg/L.

In this study, 2-Methyldecan-1-al was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitataafter 72 hours with the following effect values (based on time weighted average test item concentrations): The EC50-values with 95% confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 30.5 (28.9 – 32.1) mg/L and 9.40 (6.44 – 11.0) mg/L. The EC10-values with 95% confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 6.15 (4.78 – 7.71) mg/L and < 0.248 mg/L.

Description of key information

72h ErC50 = 30.5 mg/L (meas.), 72h ErC10 = 6.15 mg/L (meas.); OIECD 202; Anon, 2017

Key value for chemical safety assessment

EC50 for freshwater algae:
30.5 mg/L
EC10 or NOEC for freshwater algae:
6.15 mg/L

Additional information

In a single key study, the effects of the test item to freshwater algae were determined at the test facility according to OECD 201.

To account for degradation of the parent substance aldehyde to acid or further degradation products, preliminary testing was conducted to assess the stability of the test item under test conditions. After 24 hours of slow stirring, followed by a 1 hour settlement phase, all measured concentrations via LC-MS/MS of test item were below LOQ. A second stability assessment was conducted with analysis of the test item (parent) and the corresponding transformation product (2 -methyldecanoic acid) using a saturated solution with a nominal concentration of 100 mg/L. Slow stirring was applied to the test solution. Analytical samples were taken at 0, 24, 48 and 72 hours after initiation of stirring.

Measured concentrations of the parent substance were <LOQ at all time points. The highest concentration of transformation product was found after 72 hours of stirring. The measured amount of transformation product was calculated as the corresponding amount of parent. This preliminary work was used to design the medium preparation method for this study. In accordance with OECD Series on Testing and Assessment No. 23 (2000), when chemical substances have a very short half-life, it is more relevant to assess the toxicity of the major transformation product(s) as the toxicity of this entity is more relevant for actual environmental exposure. Therefore, the acute toxicity testing on aquatic invertebrates is based on the measured amount of transformation product expressed as the corresponding amount of parent.

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016 Method C.3.  The aim of the study was the determination of NOEC, LOEC, EC10- , EC20- and EC50-values of the growth rate and the yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 6839 cells/mL. A saturated solution with a nominal loading of 100 mg/L was prepared with dilution water (see Table 2) 72 ± 1 hour prior to the start of the exposure. The test item was placed on to the surface of the dilution water with a pipette. A slow stirring procedure was applied. Gentle stirring (to avoid formation of an emulsion) was carried out with a magnetic stirrer at room temperature for 72 ± 1 hour. After stirring the saturated solution was allowed to settle for 4 hours and was then taken from the homogeneous phase and used for testing. Additionally, a saturated solution was prepared as described above with dilution water (without the compounds MES and NaHCO3). This saturated solution was used for analysis of the total organic carbon (TOC, according to DIN EN 1484) and Tyndall effect, which was negative. The saturated solution and four dilution levels out of the saturated solution were tested in a geometrical series with a dilution factor of 2: 6.25 - 12.5 - 25.0 - 50.0 - 100% of the saturated solution. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

The test media were concentration related turbid throughout the test period. The concentrations of the test item after derivatization with 2,4‑Dinitrophenylhydrazine (DNPH) and the metabolite 2-methyldecanoic acid were determined in the fresh media (0 h), after 24 hours, after 48 hours and in 72 hours old media of all tested concentration levels and the control via LC-MS/MS. The results are presented in Table 8. Details of the analytical method are presented in part 13.

The measured concentrations of the test item after derivatisation were < LOQ of the nominal value at the start of the exposure (0 hours), after 24 hours, after 48 hours and at the end of the exposure (72 hours). Therefore, the concentrations of the transformation product 2‑Methyldecanoic acid were analyzed and calculated as 2-Methyldecan-1-al concentrations by conversion with a factor of 0.914 (based on the ration of the molar weights of the test item and its transformation product). The time weighted average concentrations of the test item were calculated based on these concentrations to be: 0.248 – 0.877 – 11.5 – 34.3 – 74.9 mg/L.

In this study, 2-Methyldecan-1-al was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitataafter 72 hours with the following effect values (based on time weighted average test item concentrations): The EC50-values with 95% confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 30.5 (28.9 – 32.1) mg/L and 9.40 (6.44 – 11.0) mg/L. The EC10-values with 95% confidence intervals for inhibition of growth rate (ErC10) and yield (EyC10) after 72 hours were 6.15 (4.78 – 7.71) mg/L and < 0.248 mg/L.

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