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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 June 2017 to 18 Jun 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
Remarks:
see Deviations from the Protocol in 'Any other information on materials and methods incl. tables'
Qualifier:
according to guideline
Guideline:
other: EPA OCSPP 850.4500
Version / remarks:
2012
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
At exposure initiation (0 hour), 24 hours, and exposure termination (96 hours), samples were removed from each test concentration and the control. Samples analyzed at 0 hour were removed from the intermediate mixing vessels prior to division into the replicate test vessels. Samples analyzed at 24 and 96 hours were removed from composited replicate solutions of each treatment level and the controls. All test samples removed at 24 and 96 hours were centrifuged prior to analysis to remove algal biomass, as required by the protocol and OCSPP Guideline 850.4500. One sample was analyzed for the test substance concentration while the duplicate was stored frozen as an archive backup sample. Each sample was collected from the approximate midpoint of the test vessel using a pipet. At 24 and 96 hours of exposure, a sample was also removed from the replicate flask E of the nominal 0.25 mg/L test concentration, which did not contain algae.
Vehicle:
yes
Remarks:
DMF
Details on test solutions:
A 10 mg/mL primary stock solution was prepared prior to exposure initiation by placing 0.2504 g (tested as 100%) of the test substance in a volumetric flask and bringing it to volume with 25 mL of DMF. Following mixing by shaking and inversion of the volumetric flask, the resulting stock solution was observed to be clear and colorless with no visible undissolved test substance. Secondary stock solutions were prepared from dilutions of the 10 mg/mL primary stock solution. The preparation scheme is provided in Table 1 in "Any other information on materials and methods incl. tables".
Following mixing by shaking and inversion of the volumetric flasks, the secondary stock solutions were observed to be clear and colorless with no visible undissolved test substance. Prior to exposure initiation, all exposure solutions were prepared from primary and secondary stock solutions. The preparation scheme of exposure solutions is provided in Table 2 in "Any other information on materials and methods incl. tables"
All exposure solutions were observed to be clear and colorless with no visible undissolved test substance. A set of exposure solutions was prepared in this manner the day prior to exposure initiation. These solutions were used to pre-condition the intermediate mixing vessels for the overnight period prior to initiation in order to minimize potential adsorptive losses. On the day of exposure initiation, exposure solutions were freshly prepared in the above manner using the pre-conditioned volumetric flasks and these solutions were used for the toxicity test.
Replicate 250 mL sterile flasks were conditioned for the overnight period prior to exposure initiation by filling with the appropriate volume of exposure solution. On the day prior to exposure initiation, one hundred millilitres of the appropriate exposure or control solution was placed in each replicate flask. On the day of exposure initiation, the pre-conditioning solution was discarded. The pre-conditioned vessels were then refilled with the appropriate exposure solution. Four replicate flasks were maintained for each treatment level, the solvent control, and the negative control. The solvent control solution was prepared from AAP medium with 0.1 mL/L DMF. Additional untreated AAP medium was used to prepare the negative control. The control and solvent control vessels were maintained under the same conditions as the treatment vessels, but contained no the test substance. Each test flask was labelled with the test concentration, replicate, test species, and study number. All test vessels were fitted with stainless steel caps that permitted gas exchange.
Test organisms (species):
Navicula pelliculosa
Details on test organisms:
TEST ORGANISM
- Common name: Diatom
- Strain: 661
- Class: Bacillariophyceae
- Source: Maintained in stock culture at the test facility

CULTIVATION
- Culturing medium: The culture medium used was Algal Assay Procedure (AAP) medium prepared with sterile, deionized water.
- Representative samples of the dilution water source used to prepare the medium were analyzed periodically for the presence of toxic metals, pesticides, and PCBs. None of these compounds have been detected at concentrations that are considered toxic in any of the water samples analyzed, in agreement with ASTM standard practices (2007).
- Total organic carbon: A representative sample of AAP medium was analyzed monthly for total organic carbon (TOC) concentration. The TOC concentration was 0.97 mg/L for June 2017.
- Culturing condition: Stock cultures were grown in 250 mL glass flasks, each containing 100 mL of medium. The flasks were covered with stainless steel caps that permitted gas exchange.
- Temperature: 24 ± 2 °C
- Photoperiod: Continuously
- Light intensity and location: 60 to 80 µE/m2/S at the surface of the meidum
- pH: If necessary, the pH of the culture medium was adjusted to pH 7.5 ± 0.1 with dilute hydrochloric acid or sodium hydroxide.
- Culture flasks were agitated once daily by hand shaking.
- The inoculum used to initiate the toxicity test with the test substance was taken from a stock culture that had been transferred to fresh medium three days before testing.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
21 - 23 °C
pH:
7.0 - 8.5
Conductivity:
260 µS/cm
Nominal and measured concentrations:
- Nominal concentration: 0 (negative control), 0 (solvent control), 0.063, 0.13, 0.25, 0.50 and 1.0 mg/L
- Measured concentration: < MDL (negative control), < MDL (solvent control), 0.023, 0.042, 0.090, 0.21 and 0.61 mg/L respectively. See Table 3 in 'Any other information on materials and methods incl. tables'.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass flasks with stainless steel caps permitting gas exchange
- Initial cells density: 1.25 E+04 cells/mL
- No. of vessels per concentration: 4
- No. of vessels per control: 4
- No. of vessels per vehicle control: 4

GROWTH MEDIUM
- Standard medium used: No
- Composition of the medium: See Table 4 in 'Any other information on materials and methods incl. tables'
- The AAP medium used to prepare the exposure solutions was formulated in the same manner as the culture medium. Several liters of AAP medium were prepared and equilibrated to test temperature. The pH of each batch of medium was adjusted, if necessary, to a pH of 7.5 ± 0.1 with dilute hydrochloric acid or sodium hydroxide prior to use.

EXPOSURE INITIATION
- After the test solutions were added to the test flasks (100 mL per flask), a 1.473 mL inoculum of Navicula pelliculosa cells, at a density of approximately 8.487E+05 cells/mL, was aseptically introduced into each flask. This inoculum provided the required initial (0-hour) cell density of approximately 1.25E+04 cells/mL.

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: No
- Intervals of water quality measurement: Temperature was measured continuously with a minimum/maximum thermometer located in a flask of water adjacent to the test flasks in the environmental chamber. Minimum and maximum temperatures and the daily hand shaking of the test flasks were recorded daily. Measurement of pH was conducted at exposure initiation and at exposure termination. In addition, conductivity of the test solutions was measured at exposure initiation. Measurements at exposure initiation were conducted on the exposure solution remaining after the individual test flasks had been filled. At exposure termination, after cell counts were completed, the replicate solutions were composited by treatment level or control for pH measurements.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: The pH of each batch of medium was adjusted, if necessary, to a pH of 7.5 ± 0.1 with dilute hydrochloric acid or sodium hydroxide prior to use.
- Photoperiod: Continuously
- Light intensity: 60 to 80 µE/m2/S (approximately 4440 to 8880 lux)
- Test vessels were agitated by once daily hand shaking and by vigorous pipetting prior to each cell density determination.
- Test flasks were randomly placed on a shelf at exposure initiation based on computer-generated random numbers. Following each observation interval, the test flasks were assigned new random positions based on the random numbers.

EFFECT PARAMETERS MEASURED
- Algal growth: At each subsequent 24-hour interval, cell counts were determined using a Coulter Multisizer Particle Analyzer. Two samples generally were removed from each test vessel, and two counts were determined for each sample. The two counts determined for each sample were averaged, and the two average cell counts determined for each replicate were averaged. The reported cell density was the average of the four cell counts determined for each replicate. Observations of the health of the algal cells and the exposure solutions (i.e., precipitation, clarity, material adhering to the sides of the test vessels) were made at each 24-hour interval. These observations were made in an alternating replicate of each control or treatment group using a hemactyometer and compound microscope.
- Solution for algistatic/algicidal properties: At exposure termination, a composite solution was prepared from the four 1.0 mg/L (nominal) replicate exposure vessels. From this composite, a sample was removed and then diluted with freshly prepared AAP medium to prepare a subculture with a nominal concentration of 0.063 mg/L, equivalent to the lowest nominal test concentration. The subculture was incubated for up to nine days under conditions consistent with those maintained during the definitive exposure. During this period, the subculture was examined with the Beckman Coulter Multisizer 4e Particle Analyzer every other day to determine whether or not cell growth had resumed. The subculture was discontinued after a substantial increase in cell density (i.e., > 10×) was observed. If an increase in cell density was not observed after nine days, the vessel was terminated.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.26 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% C.L.: 0.22 - 0.29 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.57 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
95% C.L.: 0.55 - 0.59 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.023 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.37 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other:
Remarks:
95% C.L.: 0.076 - 0.55 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.023 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.41 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other:
Remarks:
95% C.L.: 0.37 - 0.45 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.042 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
An overciew of the result is provided in Table 5 - Table 8 in 'Any other information on results incl. tables'.

CELL DENSITY
Cells in the 1.0 mg/L nominal treatment level were observed to be bloated at 72 and 96 hours. Cells at all other treatment levels were observed to be normal throughout the exposure period.
The 72-hour cell density in the negative control and solvent control averaged 42.01 and 39.52 E+04 cells/mL, respectively. The 72-hour cell density for the pooled control was 40.76 E+04 cells/mL. Cell density in the 0.023, 0.042, 0.090, 0.21, and 0.61 mg/L geometric mean measured treatment levels averaged 39.43, 37.39, 35.41, 32.65, and 5.94 E+04 cells/mL, respectively.
The 96-hour cell density in the negative control and solvent control averaged 82.74 and 74.57 E+04 cells/mL, respectively. The 96-hour cell density for the pooled control was 78.66 E+04 cells/mL. Cell density in the 0.023, 0.042, 0.090, 0.21, and 0.61 mg/L geometric mean measured treatment levels averaged 72.08, 70.85, 66.58, 62.75, and 10.91 E+04 cells/mL, respectively.

YIELD
The 72-hour yield in the negative control and solvent control averaged 40.76 and 38.27 E+04 cells/mL, respectively (pooled control = 39.51 E+04 cells/mL). The 72-hour yield in the 0.023, 0.042, 0.090, 0.21, and 0.61 mg/L geometric mean measured treatment levels averaged 38.09, 36.14, 34.16, 31.40, and 4.69 E+04 cells/mL, respectively.
Following 96 hours of exposure, the yield in the negative control and solvent control averaged 81.49 and 73.32 E+04 cells/mL, respectively (pooled control = 77.41 E+04 cells/mL). The 96-hour yield in the 0.023, 0.042, 0.090, 0.21, and 0.61 mg/L geometric mean measured treatment levels averaged 70.83, 69.60, 65.33, 61.50, and 9.66 E+04 cells/mL, respectively.
No statistically significant difference was detected between the 72- or 96-hour negative control and solvent control data for yield. Therefore, treatment data for 72- and 96-hour yield was compared to the pooled control. Based on the results of Shapiro-Wilks’ and Bartlett’s Tests, the 72- and 96-hour data met the assumptions for normality and homogeneity of variance. Williams’ Multiple Comparison Test determined a significant reduction in 72-hour yield in the 0.042, 0.090, 0.21, and 0.61 mg/L geometric mean measured treatment levels compared to the pooled control. Therefore, the 72-hour NOEC and LOEC were determined to be 0.023 and 0.042 mg/L respectively. The 72-hour EyC50 was determined to be 0.37 mg/L with a corresponding 95% confidence interval of 0.076 to 0.55 mg/L. Williams’ Multiple Comparison Test determined a significant reduction in 96-hour yield in all the geometric mean measured treatment levels (0.023, 0.042, 0.090, 0.21, and 0.61 mg/L) compared to the pooled control. Therefore, the 96-hour NOEC and LOEC were determined to be < 0.023 and 0.023 mg/L respectively. The 96-hour EyC50 was determined to be 0.35 mg/L with a corresponding 95% confidence interval of 0.30 to 0.39 mg/L.

AVERAGE SPECIFIC GROWTH RATE
The 72-hour growth rate in the negative control and solvent control averaged 1.18 and 1.16 day-1 (pooled control = 1.17 day-1). The 72-hour average specific growth rate in the 0.023, 0.042, 0.090, 0.21, and 0.61 mg/L geometric mean measured treatment levels averaged 1.16, 1.14, 1.12, 1.10, and 0.52 day-1, respectively.
Following 96 hours of exposure, average specific growth rate in the negative control and solvent control averaged 1.06 and 1.03 day-1, respectively (pooled control = 1.04 day-1). The 96-hour average specific growth rate in the 0.023, 0.042, 0.090, 0.21, and 0.61 mg/L geometric mean measured treatment levels averaged 1.02, 1.02, 1.00, 0.99, and 0.54 day-1, respectively.
No statistically significant difference was detected between the 72- or 96-hour negative control and solvent control data for growth rate. Therefore, treatment data for 72- and 96-hour growth rate was compared to the pooled control. Based on the results of Shapiro-Wilk’s and Bartlett's Tests, the 72-hour data met the assumptions for normality and homogeneity of variance, however, the 96-hour data failed to meet the assumptions for normality and homogeneity of variance. Williams’ Multiple Comparison Test determined a significant reduction in 72-hour average specific growth rates in the 0.042, 0.090, 0.21, and 0.61 mg/L treatment levels compared to the pooled control. Therefore, the 72-hour NOEC and LOEC were determined to be 0.023 and 0.042 mg/L, respectively. The 72-hour ErC50 value was determined to be 0.57 mg/L with a corresponding 95% confidence interval of 0.55 to 0.59 mg/L. Jonckheere-Terpestra’s Step-Down Test determined a significant reduction in 96-hour average specific growth rate in all the geometric mean measured treatment levels (0.023, 0.042, 0.090, 0.21, and 0.61 mg/L) compared to the pooled control. Therefore, the 96-hour NOEC and LOEC were determined to be < 0.023 and 0.023 mg/L, respectively. The 96-hour ErC50 value was empirically estimated to be > 0.61 mg/L.

AUGC
Following 72 hours of exposure, AUGC in the negative control and solvent control averaged 33.64 and 33.89 E+04 cells·days/mL, respectively (pooled control = 33.76 E+04 cells·days/mL). The 72-hour AUGC in the 0.023, 0.042, 0.090, 0.21, and 0.61 mg/L geometric mean measured treatment levels averaged 33.97, 33.46, 31.08, 29.51, and 6.17 E+04 cells·days/mL, respectively.
Following 96 hours of exposure, AUGC in the negative control and solvent control averaged 94.13 and 89.10 E+04 cells·days/mL, respectively (pooled control = 91.62E+04 cells·days/mL). The 96-hour AUGC in the 0.023, 0.042, 0.090, 0.21, and 0.61 mg/L geometric mean measured treatment levels averaged 87.86, 85.78, 80.30, 75.48, and 13.27 E+04 cells·days/mL, respectively.
No statistically significant difference was detected between the 72- or 96-hour negative control and solvent control data for AUGC. Therefore, treatment data for 72- and 96-hour AUGC was compared to the pooled control. Based on the results of Shapiro-Wilk’s and Bartlett's Tests, the 72- and 96-hour data met the assumptions for normality and for homogeneity of variance. Williams’ Multiple Comparison Test determined a significant reduction in 72-hour AUGC in the 0.090, 0.21, and 0.61 mg/L geometric mean measured treatment levels compared to the pooled control. Therefore, the 72-hour NOEC and LOEC were determined to be 0.042 and 0.090 mg/L, respectively. The 72-hour EC50 value was determined to be 0.41 mg/L with a corresponding 95% confidence interval of 0.37 to 0.45 mg/L. Williams’ Multiple Comparison Test determined a significant reduction in 96-hour AUGC in the 0.090, 0.21, and 0.61 mg/L geometric mean measured treatment levels compared to the pooled control. Therefore, the 96-hour NOEC and LOEC were determined to be 0.042 and 0.090 mg/L, respectively. The 96-hour EC50 value was determined to be 0.37 mg/L with a corresponding 95% confidence interval of 0.33 to 0.41 mg/L. Table 11 summarizes the 72- and 96-hour EC10, EC20, and EC50 values and the 72- and 96-hour NOEC and LOEC values based on AUGC results.

ALGISTATIC/ALGICIDAL RECOVERY PERIOD
The subculture prepared for the algistatic/algicidal determination from the 1.0 mg/L (nominal) solution had the test substance concentration of 0.063 mg/L, a concentration equivalent to that of the lowest nominal test concentration, and an estimated cell density of 0.6871 E+04 cells/mL. The subculture was agitated once daily by hand shaking and was maintained at a temperature range of 22 to 23 °C, a continuous photoperiod at a light intensity of 67 to 69 µE/m2/S. After two days, a cell density of 8.573 E+04 cells/mL was measured in the subculture, corresponding to an approximately 12.5-fold increase in cell density over four days. This observation indicates that the test substance has an algistatic, rather than algicidal effect on the growth of N. pelliculosa at a nominal concentration of 1.0 mg/L.

Table 5. Cell Density of Navicula pelliculosa after 24, 48, 72, and 96 Hours of Exposure to the test substance

Geometric Mean Measured Concentration

(mg/L)

 

Cell Density (× 104 cells/mL)a

Replicate

Observation Interval (hours)

 

24

48

72

96

Negative Control

A

3.70

11.76

42.10

86.44

B

3.71

12.00

41.75

80.96

C

3.58

12.10

44.72

86.89

D

3.60

11.81

39.48

76.69

Mean (SD)b

3.65 (0.069)

11.92 (0.16)

42.01 (2.15)

82.74 (4.86)

Solvent Control

A

4.33

11.04

37.74

76.40

B

4.23

14.59

41.12

74.85

C

4.46

13.47

41.02

78.75

D

4.46

11.65

38.20

68.30

Mean (SD)

4.37 (0.11)

12.69 (1.64)

39.52 (1.80)

74.57 (4.48)

Pooled Control

Mean (SD)

4.01 (0.40)

12.30 (1.15)

40.76 (2.27)

78.66 (6.15)

0.023

A

5.37

12.95

38.50

73.55

B

3.61

13.30

43.60

72.37

C

4.31

13.07

37.32

71.89

D

4.07

12.24

37.96

70.51

Mean (SD)

4.34 (0.75)

12.89 (0.45)

39.34 (2.88)

72.08 (1.26)

0.042

A

5.42

13.76

39.25

72.55

B

4.28

12.95

37.10

70.94

C

3.61

13.26

34.41

66.55

D

4.47

13.01

38.82

73.37

Mean (SD)

4.44 ( 0.75)

13.25 (0.37)

37.39 (2.20)

70.85 (3.04)

0.090

A

4.79

12.03

34.43

67.49

B

4.53

13.73

38.35

72.92

C

3.81

12.26

35.30

64.68

D

3.88

10.27

33.55

61.22

Mean (SD)

4.25 (0.48)

12.07 (1.42)

35.41 (2.09)

66.58 (4.95)

0.21

A

6.15

11.40

32.37

62.74

B

4.08

11.22

31.74

59.53

C

3.73

11.89

33.83

65.98

D

3.95

NAc

NA

NA

Mean (SD)

4.48 (1.12)

11.50 (0.35)

32.65 (1.07)

62.75 (3.23)

0.61

A

3.00

3.53

5.62

8.74

B

2.45

3.38

5.04

7.64

C

2.50

3.49

6.13

1.29

D

2.77

4.09

6.98

1.43

Mean (SD)

2.68 (0.25)

3.62 (0.32)

5.94d(0.82)

10.91d(3.22)

Table 6. Mean values for the control and test item treatment of the test substance for the percent inhibition of growth rate, yield and AUC at 72 hours for Navicula pelliculosa

Mean measured concentrations
(mg/L)

0 to 72 h

AUC (104*day)

Percentage inhibition of AUC

Growth rate (1/day)

Percentage inhibition of growth rate

Yield (x 104)

Percentage inhibition of yield

Negative control

33.64

NA

1.18

NA

40.76

NA

Solvent control

33.89

NA

1.16

NA

38.27

NA

Pooled control

33.76

NA

1.17

NA

39.51

NA

0.023

33.97

-1

1.16

1

38.09

4

0.042

33.46

1

1.14*

2

36.14*

9

0.090

31.08*

8

1.12*

4

34.16*

14

0.21

29.51*

13

1.10*

6

31.40*

21

0.61

6.17*

82

0.52*

55

4.69*

88

NA = Not applicable

* Significantly reduced compared to the pooled control, based on Williams’ Multiple Comparison Test.

 

Table 7. Mean values for the control and test item treatment of the test substance for the percent inhibition of growth rate, yield and AUC at 96 hours for Navicula pelliculosa

Mean measured concentrations
(mg/L)

0 to 96 h

AUC
(104*day)

Percentage inhibition of AUC

Growth rate
(1/day)

Percentage inhibition of growth rate

Yield
(x 104)

Percentage inhibition of yield

Negative control

94.13

NA

1.06

NA

81.49

NA

Solvent control

89.10

NA

1.03

NA

73.32

NA

Pooled control

91.62

NA

1.04

NA

77.41

NA

0.023

87.86

4

1.02**

2

70.83*

9

0.042

85.78

6

1.02**

2

69.60*

10

0.090

80.30

12

1.00**

4

65.33*

16

0.21

75.48*

18

0.99**

5

61.50*

21

0.61

13.27*

86

0.54**

48

9.66*

88

NA = Not applicable

*Significantly reduced compared to the pooled control, based on Williams’ Multiple Comparison Test

**Significantly reduced compared to the pooled control, based on Jonckheere-Terpstra’s Step-Down Test

Table 8.Summary of biological results for toxicity of the test substance to Navicula pelliculosa after 72 and 96 hours

 

Biological Parameter

Based on Geometric Mean Measured Concentrations (mg/L)

EC10

(95% Confidence Intervals)

EC20

(95% Confidence Intervals)

EC50

(95% Confidence Intervals)

 

NOEC

 

LOEC

0 - 72-Hour

Yield

NRa

(NAb)

0.20

(0.16 – 0.24)

0.37

(0.076 – 0.55)

0.023

0.042

0 - 72-Hour

Average Specific

Growth Rate

0.26

(0.22 – 0.29)

0.34

(0.031 – 0.37)

0.57

(0.55 – 0.59)

 

0.023

 

0.042

72-Hour AUGC

0.13

(0.076 – 0.16)

0.23

(0.18- 0.28)

0.41

(0.37 – 0.45)

0.042

0.090

0 - 96-Hour

Yield

NRa

(NAb)

0.14

(0.095 – 0.18)

0.35

(0.030 – 0.39)

< 0.023

0.023

0 - 96-Hour

Average Specific Growth Rate

0.28

(0.18 – 0.35)

0.38

(0.30 – 0.44)

> 0.61 (NDc)

 

< 0.023

 

0.023

96-Hour AUGC

0.069

(0.033 – 0.099)

0.22

(0.17 – 0.25)

0.37

(0.33 – 0.41)

0.042

0.090

NR = Not reported

NA = Not applicable

ND* = Not determined; EC value is empirically estimated, therefore corresponding 95 % confidence interval could not be determined

Validity criteria fulfilled:
yes
Conclusions:
Based on mean measured concentrations, the 72-hour ErC50 was determined to be 0.57 mg/L (with 95% Confidence Intervals of 0.55 - 0.59 mg/L) and the ErC10 was determined to be 0.26 mg/L (with 95% Confidence Intervals of 0.22 - 0.29 mg/L).
Executive summary:

The toxicity of the substance to the freshwater diatom Navicula pelliculosa was investigated in a 96 hour static test. Diatoms were exposed to nominal concentrations of 0.063, 0.13, 0.25, 0.50 and 1.0 mg/L alongside culture medium and solvent controls. Based on mean measured concentrations, the 72-hour ErC50 was 0.57 mg/L and the EyC50 was 0.37 mg/L. The 96-hour ErC50 was >0.61 mg/L and the EyC50 was 0.35 mg/L.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 Apr 2017 to 11 Apr 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
Remarks:
see Deviations from the Protocol in 'Any other information on materials and methods incl. tables'
Qualifier:
according to guideline
Guideline:
other: EPA OCSPP 850.4500
Version / remarks:
2012
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
At exposure initiation (0 hour), 24 hours, and exposure termination (96 hours), samples were removed from each test concentration and the control. Samples analyzed at 0 hour were removed from the intermediate mixing vessels prior to division into the replicate test vessels. Samples analyzed at 24 and 96 hours were removed from composited replicate solutions of each treatment level and the controls. All test samples removed at 24 and 96 hours were centrifuged prior to analysis to remove algal biomass, as required by the protocol and OCSPP Guideline 850.4500. One sample was analyzed for the test substance concentration while the duplicate was stored frozen as an archive backup sample. Each sample was collected from the approximate midpoint of the test vessel using a pipet. At 24 and 96 hours of exposure, a sample was also removed from the replicate flask E of the nominal 0.25 mg/L test concentration, which did not contain algae.
Vehicle:
yes
Remarks:
DMF
Details on test solutions:
A 50 mg/mL primary stock solution was prepared prior to exposure initiation by placing 1.2501 g (tested as 100%) of the test substance in a volumetric flask and bringing it to volume with 25 mL of DMF. Following mixing by shaking and inversion of the volumetric flask and then sonication for approximately five to ten seconds, the resulting stock solution was observed to be clear and light yellow in color with no visible undissolved test substance. Secondary stock solutions were prepared from dilutions of the 50 mg/mL primary stock solution. The preparation scheme is provided in Table 1 in "Any other information on materials and methods incl. tables".
Following mixing by shaking and inversion of the volumetric flasks, the secondary stock solutions were observed to be clear and colorless with no visible undissolved test substance. Prior to exposure initiation, all exposure solutions were prepared from secondary stock solutions. The preparation scheme of the exposure solution was provided in Table 2 in 'Any other information on materials and methods incl. tables'.
All exposure solutions were observed to be clear and light yellow in color with no visible undissolved test substance. The 1.0 mg/L exposure solution was mixed for approximately five minutes with a stir bar and Corning stirrer, resulting in the same observation. Exposure and control solutions prepared in AES medium are typically observed to be light yellow in color due to the composition of the AES medium. The yellow color of the test medium is due to the presence of FeCl3, a required nutrient for the growth of S. costatum. A set of exposure solutions was prepared in this manner the day prior to exposure initiation. These solutions were used to pre-condition the volumetric flasks for the overnight period prior to initiation in order to minimize potential adsorptive losses. On the day of exposure initiation, exposure solutions were freshly prepared in the above manner using the pre-conditioned volumetric flasks and these solutions were used for the toxicity test.
The solvent control solution was prepared from AES medium with 0.1 mL/L DMF. Additional untreated AES medium was used to prepare the negative control. The negative control and the solvent control vessels were maintained under the same conditions as the treatment vessels, but contained no test substance.
Test organisms (species):
Skeletonema costatum
Details on test organisms:
TEST ORGANISM
- Common name: Marine diatom
- Strain: CCMP 1332
- Class: Bacillariophyceae
- Source: Maintained in stock culture at the test facility

CULTIVATION
- Culturing medium: The culture medium used was Artificially Enriched Seawater (AES) medium prepared with sterile, filtered, natural seawater
- Representative samples of the dilution water source used to prepare the medium were analyzed periodically for the presence of toxic metals, pesticides, and PCBs. None of these compounds have been detected at concentrations that are considered toxic in any of the water samples analyzed, in agreement with ASTM standard practices (2007).
- Total organic carbon: A representative sample of AAP medium was analyzed monthly for total organic carbon (TOC) concentration. The TOC concentration was 1.8 mg/L for April 2017.
- Salinity: 30 ± 2 g/L
- Culturing condition: Stock cultures were grown in 250 mL glass flasks, each containing 100 mL of medium. The flasks were covered with stainless steel caps that permitted gas exchange.
- Temperature: 20 ± 2 °C
- Photoperiod: 14 hours of light: 10 hours of darkness
- Light intensity: 51 to 69 µE/m2/S
- The inoculum used to initiate the toxicity test with the test substance was taken from a stock culture that had been transferred to fresh medium three days before testing.
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
19 to 23 ºC
pH:
- Test initiation: 7.8 to 8.1
- Test termination: 7.7 to 8.5
Salinity:
30 ± 2 g/L
Conductivity:
45 - 48 mS/cm
Nominal and measured concentrations:
- Nominal concentration: 0 (negative control), 0 (solvent control), 0.063, 0.13, 0.25, 0.50, and 1.0 mg/L
- Measured concentration: < MDL (negative control), < MDL (solvent control), 0.023, 0.044, 0.093, 0.21, and 0.54 mg/L, respectively. See Table 3 in 'Any other information on materials and methods incl. tables'.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL sterile flasks
- Initial cells density: 1.0E+04 cells/mL
- No. of vessels per concentration: 4
- No. of vessels per control: 8
- No. of vessels per vehicle control: 4

Exposure initiation:
- After the exposure solutions were added to the test flasks (100 mL per flask), a 1.38 mL inoculum of Skeletonema costatum cells, at a density of approximately 72.42 × 104 cells/mL, was aseptically introduced into each flask.

GROWTH MEDIUM
- The AES medium used to prepare the exposure solutions was formulated in the same manner as the culture medium. Several liters of AES medium were prepared and equilibrated to test temperature.
- Adjustment of pH: The pH of each batch of medium was adjusted, if necessary, to a pH of 8.0 ± 0.1 with dilute hydrochloric acid or sodium hydroxide prior to use.
- Salinity: Adjusted to 30 ± 2 g/L
- Standard medium used: No
- Detailed composition if non-standard medium was used: See Table 4 in "Any other information on materials and mthod incl. tables"

TEST MEDIUM / WATER PARAMETERS
- Temperature: Temperature was measured continuously with a thermometer located in a flask of water adjacent to the test flasks in the environmental chamber. Minimum and maximum temperatures and the daily hand shaking of the test flasks were recorded daily.
- pH: Measurement of pH was conducted at exposure initiation and at exposure termination.
- Conductivity: Conductivity of the exposure solutions was measured at exposure initiation.

OTHER TEST CONDITIONS
- Photoperiod: 14 hours of 14 hours of
- Light intensity: 51 to 69 µE/m2/S, equivalent to a light intensity of approximately 4300 lux, i.e., 3700 to 4900 lux
- Test vessels were agitated by once daily hand shaking and by vigorous pipetting prior to each cell density determination.
- Test flasks were randomly placed on a shelf at exposure initiation based on computer-generated random numbers. Following each observation interval, the test flasks were assigned new random positions based on the random numbers.

EFFECT PARAMETERS MEASURED:
- Algae growth: At each subsequent 24-hour interval, cell counts were conducted on one sample from each replicate treatment and control solution using a hemacytometer and a compound microscope. One sample was removed from each flask and one count was made on each sample. One or more hemacytometer fields, each 0.10 × 0.10 cm in surface area, 0.010 cm deep and containing 0.00010 mL of exposure solution, were examined for each sample until at least 400 algal cells or four fields were counted. Observations of the health of the algal cells and exposure solutions (i.e., precipitation, clarity, material adhering to the sides of the test vessels) were made at each 24-hour interval.
- Solution for algistatic/algicidal properties: At exposure termination, a composite solution was prepared from the four 1.0 mg/L (nominal) replicate exposure vessels. From this composite, a sample was removed and then diluted with freshly prepared AES medium to prepare a subculture with a nominal concentration of 0.010 mg/L, a concentration lower than the lowest nominal test concentration. The subculture was incubated for up to nine days under conditions consistent with those maintained during the definitive exposure. During this period, the subculture was examined microscopically every other day to determine whether or not cell growth had resumed. The subculture was discontinued after a substantial increase in cell density (i.e., > 10×) was observed. If an increase in cell density was not observed after nine days, the vessel was terminated.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.27 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other:
Remarks:
95% C.L.: 0.25 - 0.30 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.14 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% C.L.: 0.11 - 0.16 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.044 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.17 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other:
Remarks:
yield
Remarks on result:
other:
Remarks:
95% C.L.: 0.15 - 0.19 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.093 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other:
Remarks:
yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.17 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other:
Remarks:
95% C.L.: 0.14 - 0.19 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.044 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Details on results:
An overview of the results is provided in Table 5 - Table 8 in 'Any other information on results incl. tables'

CELL DESNITY
All cells in the controls and treatment levels were observed to be normal throughout the exposure period. The 72-hour cell density in the negative control and solvent control averaged 35.59 and 37.06 × 104 cells/mL, respectively. The 72-hour cell density for the pooled control was 36.08 × 104 cells/mL. Cell density in the 0.023, 0.044, 0.093, 0.21, and 0.54 mg/L geometric mean measured treatment levels averaged 38.81, 33.50, 29.25, 13.00, and 0.75 × 104 cells/mL, respectively.

The 96-hour cell density in the negative control and solvent control averaged 71.09 and 69.63 × 104 cells/mL. The 96-hour cell density for the pooled control was 70.60 × 104 cells/mL. Cell density in the 0.023, 0.044, 0.093, 0.21, and 0.54 mg/L geometric mean measured treatment levels averaged 60.31, 50.31, 48.06, 27.69, and 1.19 × 104 cells/mL, respectively.

YIELD
The 72-hour yield in the negative control and solvent control averaged 34.59 and 36.06 × 104 cells/mL, respectively (pooled control = 35.08 × 104 cells/mL). The 72-hour yield in the 0.023, 0.044, 0.093, 0.21, and 0.54 mg/L geometric mean measured treatment levels averaged 37.81, 32.50, 28.25, 12.00, and -0.25 × 104 cells/mL, respectively.
Following 96 hours of exposure, the yield in the negative control and solvent control averaged 70.09 and 68.63 × 104 cells/mL, respectively (pooled control = 69.60 × 104 cells/mL). The 96-hour yield in the 0.023, 0.044, 0.093, 0.21, and 0.54 mg/L geometric mean measured treatment levels averaged 59.31, 49.31, 47.06, 26.69, and 0.19 × 104 cells/mL, respectively.
No statistically significant difference was detected between the 72- or 96-hour negative control and solvent control data for yield. Therefore, treatment data for 72- and 96-hour yield was compared to the pooled control. Based on the results of Shapiro-Wilk’s and Bartlett's Tests, the 72- and 96-hour data met the assumptions for normality, and the 96-hour data met the assumption for homogeneity of variance; the 72-hour data did not meet the assumption for homogeneity of variance. Jonckheere-Terpstra’s Step-Down Test determined a significant reduction in 72-hour yield in the 0.21 and 0.54 mg/L geometric mean measured treatment levels compared to the pooled control. Therefore, the 72-hour NOEC and LOEC were determined to be 0.093 and 0.21 mg/L respectively. The 72-hour EyC50 was determined to be 0.17 mg/L with a corresponding 95% confidence interval of 0.15 to 0.19 mg/L.
Williams’ Multiple Comparison Test determined a significant reduction in 96-hour yield in all the geometric mean measured treatment levels (0.023, 0.044, 0.093, 0.21, and 0.54 mg/L) compared to the pooled control. Therefore, the 96-hour NOEC and LOEC were determined to be < 0.023 and 0.023 mg/L respectively. The 96-hour EyC50 was determined to be 0.15 mg/L with a corresponding 95% confidence interval of 0.10 to 0.23 mg/L.

AVERAGE SPECIFIC GROWTH RATE
The 72-hour growth rate in the negative control and solvent control averaged 1.20 and 1.22 day-1 (pooled control = 1.20 day-1). The 72-hour average specific growth rate in the 0.023, 0.044, 0.093, 0.21, and 0.54 mg/L geometric mean measured treatment levels averaged 1.23, 1.18, 1.14, 0.86, and 0.00 day-1, respectively.
Following 96 hours of exposure, average specific growth rate in both the negative control and solvent control averaged 1.08 day-1 (pooled control = 1.08 day-1). The 96-hour average specific growth rate in the 0.023, 0.044, 0.093, 0.21, and 0.54 mg/L geometric mean measured treatment levels averaged 1.04, 0.99, 0.98, 0.84, and -0.040 day-1, respectively.
No statistically significant difference was detected between the 72- or 96-hour negative control and solvent control data for growth rate. Therefore, treatment data for 72- and 96-hour growth rate was compared to the pooled control. Based on the results of Shapiro-Wilk’s and Bartlett's Tests, the 72-hour data met the assumptions for normality and homogeneity of variance, however, the 96-hour data failed to meet the assumptions for normality and homogeneity of variance. Williams’ Multiple Comparison Test determined a significant reduction in 72-hour average specific growth rates in the 0.093, 0.21, and 0.54 mg/L compared to the pooled control. Therefore, the 72-hour NOEC and LOEC were determined to be 0.044 and 0.093 mg/L, respectively. The 72-hour ErC50 value was determined to be 0.27 mg/L with a corresponding 95% confidence interval of 0.25 to 0.30 mg/L.
Jonckheere-Terpestra’s Step-Down Test determined a significant reduction in 96-hour average specific growth rate in all the geometric mean measured treatment levels (0.023, 0.044, 0.093, 0.21, and 0.54 mg/L) compared to the pooled control. Therefore, the 96-hour NOEC and LOEC were determined to be < 0.023 and 0.023 mg/L, respectively. The 96-hour ErC50 value was determined to be 0.29 mg/L with a corresponding 95% confidence interval of 0.12 to 0.46 mg/L.

AUGC
Following 72 hours of exposure, AUGC in the negative control and solvent control averaged 33.28 and 33.66 × 104 cells·days/mL, respectively (pooled control = 33.40 × 104 cells·days/mL). The 72-hour AUGC in the 0.023, 0.044, 0.093, 0.21, and 0.54 mg/L geometric mean measured treatment levels averaged 32.77, 29.12, 23.97, 13.43, and -0.080 × 104 cells·days/mL, respectively.
Following 96 hours of exposure, AUGC in the negative control and solvent control averaged 84.35 and 84.73 × 104 cells·days/mL, respectively (pooled control = 84.48× 104 cells·days/mL). The 96-hour AUGC in the 0.023, 0.044, 0.093, 0.21, and 0.54 mg/L geometric mean measured treatment levels averaged 80.15, 69.04, 60.71, 32.30, and -0.11 × 104 cells·days/mL, respectively.
No statistically significant difference was detected between the 72- or 96-hour negative control and solvent control data for AUGC. Therefore, treatment data for 72- and 96-hour AUGC was compared to the pooled control. Based on the results of Shapiro-Wilk’s and Bartlett's Tests, the 72- and 96-hour data met the assumptions for normality; the 72- and 96-hour data did not meet the assumption for homogeneity of variance.
Jonckheere-Terpestra’s Step-Down Test determined a significant reduction in 72-hour AUGC in the 0.093, 0.21, and 0.54 mg/L geometric mean measured treatment levels compared to the pooled control. Therefore, the 72-hour NOEC and LOEC were determined to be 0.044 and 0.093 mg/L, respectively. The 72-hour EC50 value was determined to be 0.17 mg/L with a corresponding 95% confidence interval of 0.14 to 0.19 mg/L.
Jonckheere-Terpestra’s Step-Down Test determined a significant reduction in 96-hour AUGC in the 0.044, 0.093, 0.21, and 0.54 mg/L geometric mean measured treatment levels compared to the pooled control. Therefore, the 96-hour NOEC and LOEC were determined to be 0.023 and 0.044 mg/L, respectively. The 96-hour EC50 value was determined to be 0.15 mg/L with a corresponding 95% confidence interval of 0.13 to 0.18 mg/L.

ALGISTATIC/ALGICIDAL RECOVERY PERIOD
The subculture prepared for the algistatic/algicidal determination from the 1.0 mg/L (nominal) solution had the test substance concentration of 0.010 mg/L, a concentration lower than the lowest nominal test concentration, and an estimated cell density of 0.0119 × 104 cells/mL. The subculture was agitated once daily by hand shaking and was maintained at a temperature range of 19 to 22 °C, a photoperiod of 14 hours at a light intensity of 58 to 64 µE/m2/S and 10 hours of darkness. After four days, a cell density of 1.5 × 104 cells/mL was measured in the subculture, corresponding to an approximately 126-fold increase in cell density over four days. This observation indicates that the test substance has an algistatic, rather than algicidal effect on the growth of S. costatum at a nominal concentration of 1.0 mg/L.

Table 5. Cell Density of Skeletonema costatum after 24, 48, 72, and 96 Hours of Exposure

Geometric Mean Measured Concentration

(mg/L)

Replicate

Cell density (x10^4)a

Observation interval (hours)

24

48

72

96

Negative Control

A

6.00

13.75

34.75

81.00

B

4.75

16.25

33.75

55.75

C

5.25

11.50

29.00

76.50

D

4.50

10.13

28.75

67.50

E

2.75

15.50

36.25

67.75

F

4.25

14.50

38.25

66.50

G

2.75

14.75

49.50

74.75

H

5.50

10.00

34.50

79.00

Mean (SD)b

4.47 (1.20)

13.30 (2.43)

35.59 (6.51)

71.09 (8.31)

Solvent Control

A

6.25

11.38

29.50

71.50

B

4.25

14.00

45.75

73.00

C

2.75

17.25

38.25

69.00

D

3.50

10.25

34.75

65.00

Mean (SD)

4.19 (1.51)

13.22 (3.11)

37.06 (6.82)

69.63 (3.50)

Pooled Control

Mean (SD)

4.38 (1.25)

13.27 (2.53)

36.08 (6.34)

70.60 (6.92)

0.023

A

3.75

12.75

40.50

62.88

B

3.75

11.25

36.00

61.38

C

3.50

17.25

51.25

66.88

D

2.50

7.75

27.50

50.13

Mean (SD)

3.38 (0.60)

12.25 (3.94)

38.81 (9.89)

60.31 (7.18)

0.044

A

4.75

11.75

33.75

50.50

B

3.75

8.50

33.25

46.00

C

3.25

13.00

41.75

57.00

D

3.50

10.25

25.25

47.75

Mean (SD)

3.81 (0.66)

10.88 (1.94)

33.50 (6.74)

50.31 (4.83)

0.093

A

4.75

7.00

29.50

43.25

B

2.75

9.50

22.75

48.00

C

2.00

9.75

35.00

49.75

D

2.75

8.25

29.75

51.25

Mean (SD)

3.06 (1.18)

8.63 (1.27)

29.25 (5.02)

48.06 (3.47)

0.21

A

3.50

7.75

14.50

25.50

B

3.00

6.00

15.50

24.25

C

4.00

5.50

11.75

23.75

D

3.25

4.50

10.25

37.25

Mean (SD)

3.44 (0.43)

5.94 (1.36)

13.00 (2.42)

27.69 (6.42)

0.54

A

0.50

1.00

1.00

3.00

B

1.75

0.25

1.00

0.50

C

2.00

0.75

1.00

0.50

D

1.75

0.25

0.00

0.75

Mean (SD)

1.50 (0.68)

0.56 (0.38)

0.75 (0.50)

1.19 (1.21)

a Cells were vigorously pipetted prior to each observation interval to ensure that cells were evenly distributed before counting.

b Mean and standard deviation (SD) are calculated from original raw data and not from the rounded values presented in this table.

Table 6. Mean values for the control and test item treatment of the test substance for the percent inhibition of growth rate, yield and AUC at 72 hours for Skeletonema costatum

Mean measured concentrations
(mg/L)

0 to 72 h

AUC
(104*day)

Percentage inhibition of AUC

Growth rate
(1/day)

Percentage inhibition of growth rate

Yield
(x 104)

Percentage inhibition of yield

Negative control

33.28

NA

1.20

NA

34.59

NA

Solvent control

33.66

NA

1.22

NA

36.06

NA

Pooled control

33.40

NA

1.21

NA

35.08

NA

0.023

32.77

2

1.23

-2

37.81

-8

0.044

29.12

13

1.18

2

32.52

7

0.093

23.97*

28

1.14**

6

28.25

19

0.21

13.43*

60

0.86**

29

12.00*

66

0.54

-0.080*

100

0.00**

100

-0.25*

101

NA = Not applicable

* Significantly reduced compared to the pooled control, based on Jonckheere-Terpstra’s Step-Down Test.

** Significantly reduced compared to the pooled control, based on Williams’ Multiple Comparison Test

 

Table 7. Mean values for the control and test item treatment of the test substance for the percent inhibition of growth rate, yield and AUC at 96 hours for Skeletonema costatum

Mean measured concentrations
(mg/L)

0 to 96 h

AUC
(104*day)

Percentage inhibition of AUC

Growth rate
(1/day)

Percentage inhibition of growth rate

Yield
(x 104)

Percentage inhibition of yield

Negative control

84.35

NA

1.08

NA

70.09

NA

Solvent control

84.73

NA

1.08

NA

68.63

NA

Pooled control

84.48

NA

1.08

NA

69.60

NA

0.023

80.15

5

1.04*

4

59.31**

15

0.044

69.04*

18

0.99*

8

49.31**

29

0.093

60.71*

28

0.98*

9

47.06**

32

0.21

32.30*

62

0.84*

22

26.69**

62

0.54

-0.11*

100

-0.040*

103

0.19**

100

NA = Not applicable

* Significantly reduced compared to the pooled control, based on Jonckheere-Terpstra’s Step-Down Test

** Significantly reduced compared to the pooled control, based on Williams’ Multiple Comparison Test

Table 8. Summary of biological results for toxicity of the test substance to Skeletonema costatum after 72 and 96 hours

 

Biological Parameter

Based on Geometric Mean Measured Concentrations (mg/L)

EC10

(95% Confidence Intervals)

EC20

(95% Confidence Intervals)

EC50

(95% Confidence Intervals)

 

NOEC

 

LOEC

0- to 72-Hour

Yield

0.079

(0.040 - 0.10)

0.11

(0.080 - 0.13)

0.17

(0.15 - 0.19)

0.093

0.21

0- to 72-Hour

Average Specific Growth Rate

0.14

(0.11 - 0.16)

0.18

(0.16 - 0.19)

0.27

(0.25 - 0.30)

 

0.044

 

0.093

72-Hour AUGC

NRa

(NAb)

0.087

(0.062 - 0.11)

0.17

(0.14 - 0.19)

0.044

0.093

a NR = Not Reported. EC value failed criteria for ECx determination and therefore is not reported.

b NA = Not Applicable

Validity criteria fulfilled:
yes
Remarks:
See validity criteria in 'Any other information on materials and methods incl. tables'.
Conclusions:
Based on mean measured concentrations, the 72-hour ErC50 was determined to be 0.27 mg/L (corresponding with 95% Confidence Intervals of 0.25 - 0.30 mg/L) and the ErC10 was determined to be 0.14 mg/L (corresponding with 95% Confidence Intervals of 0.11 - 0.16 mg/L).
Executive summary:

To determine the toxicity of the test substance on the growth of Skeletonema costatum, a marine diatom, the exposure was conducted under static conditions following OECD TG 201. In addition, the procedures were modified to include the requirements of OCSPP Guideline 850.4500. The test was in compliance with GLP criteria.

Cell density was monitored at 24, 48, 72, and 96 hours of exposure. Yield, average specific growth rate, and area under the growth curve (AUGC) were calculated as test endpoints. Four replicate flasks for each treatment level (i.e., 0.063, 0.13, 0.25, 0.50, and 1.0 mg/L nominal and 0.023, 0.044, 0.093, 0.21, and 0.54 mg/L geometric mean measured concentrations), the solvent control (dimethylformamide, DMF), and eight replicate flasks were established for the negative control and maintained in an environmental chamber designed to maintain the test conditions specified in the protocol: a temperature of 20 ± 2 °C, a photoperiod of 14 hours of light : 10 hours of darkness a photosynthetically-active radiation (PAR) range of 51 to 69 µE/m2/S. Test vessels were agitated once daily by hand shaking during the exposure.

At the start of the test, the initial cell density was approximately 1.0E+04 cells/mL. After 72-hours of exposure, cell density in the negative control averaged approximately 3.559E+05 cells/mL and in the solvent control averaged approximately 3.706E+05 cells/mL. At 72-hour, the averaged algal density in the 0.023, 0.044, 0.093, 0.21, and 0.54 mg/L treatment levels were 3.881, 3.350, 2.925, 1.300 and 0.075E+05 cells/mL, respectively. Following 96-hour of exposure, cell density in the negative control averaged 7.109E+05 cells/mL and in the solvent control average approximately 6.963E+05 cells/mL. The 0.023, 0.044, 0.093, 0.21, and 0.54 mg/L treatment levels showed a 6.031, 5.031, 4.806, 2.769 and 0.119E+05 cells/mL algal density, respectively, at 96-hour. The growth rate in the negative control after 72-hours of exposure, averaged approximately 1.20 per day and in the solvent control averaged approximately 1.22 per day. The growth rate at 72-hour in the 0.023, 0.044, 0.093, 0.21, and 0.54 mg/L treatment levels were 1.23, 1.18, 1.14, 0.86 and 0.00 per day, respectively. The effects of the test substance on the growth rate at 72-hour and 96-hour relative to the pooled control, ranged from -2% to 100% and from 4% to 103%, respectively. In addition, the effects of the test substance on the yield at 72-hour and 96-hour relative to the pooled control, ranged from -8% to 101% and from 15% to 100%, respectively. The effects of the test substance on the biomass at 72-hour and 96-hour relative to the pooled control, ranged from 2% to 100% and from 5% to 100%, respectively.

Based on mean measured concentrations, the 72-hour ErC50 was determined to be 0.27 mg/L (corresponding with 95% confidence intervals of 0.25 – 0.30 mg/L), the EyC50 was 0.17 mg /L (corresponding with 95% confidence intervals of 0.15 – 0.19 mg/L) and the EbC50 was 0.17 mg/L (corresponding with 95% confidence intervals of 0.14 – 0.19 mg/L). The 72-hour ErC10 was determined to be 0.14 mg/L (corresponding with 95% confidence intervals of 0.11 – 0.16 mg/L) and EyC10 was 0.079 mg/L (corresponding with 95% confidence intervals of 0.040 – 0.10 mg/L).

Description of key information

Freshwater, 72-h ErC10 = 0.26 mg/L (corresponding with 95% confidence intervals of 0.22 to 0.29 mg/L), Navicula pelliculosa, OECD TG 201, Softcheck 2018

Freshwater, 72-h ErC50 = 0.57 mg/L (corresponding with 95% confidence intervals of 0.55 to 0.59 mg/L), Navicula pelliculosa, OECD TG 201, Softcheck 2018

Saltwater, 72-h ErC10 = 0.14 mg/L (corresponding with 95% confidence intervals of 0.11 to 0.16 mg/L), Skeletonema costatum, OECD 201, Softcheck 2018

Saltwater, 72-h ErC50 = 0.27 mg/L (corresponding with 95% confidence intervals of 0.25 to 0.30 mg/L), Skeletonema costatum, OECD 201, Softcheck 2018

Key value for chemical safety assessment

EC50 for freshwater algae:
0.57 mg/L
EC50 for marine water algae:
0.27 mg/L
EC10 or NOEC for freshwater algae:
0.26 mg/L
EC10 or NOEC for marine water algae:
0.14 mg/L

Additional information

Freshwater


The toxicity of the substance to freshwater algae and cyanobacteria was tested under GLP to the relevant OECD TG 201, using three different species of green algae, diatoms and cyanobacteria. Algae were less sensitive to exposure to the substance compared to tested species from other trophic levels. The freshwater diatom Navicula pelliculosa was the most sensitive species, and the reliable and valid study with the freshwater diatom resulted in 72-hour ErC10 and ErC50 values of 0.26 mg/L and 0.57 mg/L and 96-hour ErC10 and ErC50 values of 0.28 and >0.61 mg/L, respectively.


Marine water


The toxicity of the substance to the marine diatom Skeletonema costatum was tested under GLP to the relevant OECD TG 201. The reliable and valid study with the marine diatom resulted in 72-hour ErC10 and ErC50 values of 0.14 mg/L and 0.27 mg/L, respectively. The tested marine species was slightly more sensitive to exposure to the substance compared to the tested freshwater diatom.