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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:
10 Oct 1995 to 25 Oct 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
1984
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
The test concentrations were verified by chemical analysis of the test substance at 0 and 72 hours. The test media of the treatment replicates were pooled.
Vehicle:
no
Details on test solutions:
A stock solution with a nominal concentration of 100 mg test substance/L was prepared by mixing 200.2 mg of the test item with the test medium and making up to 2000 mL. Appropriate volumes of the stock solution were diluted to give the test concentration series.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: Strain No. 61.81 SAG
- Source: Continuous laboratory cultures, originally obtained from Collection of Algal Cultures
- Method of cultivation: An aliquot of test solution was placed into each test vessel and the test was started by inoculation of 9,700 algal cells per mL of test medium.

ACCLIMATION
- Acclimation period: Not specified
- Culturing media: Reconstituted water
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23 - 25 °C
pH:
- Test start: 7.9 to 8.0
- Test end: 9.5 to 9.9
Nominal and measured concentrations:
- Nominal concentrations: 0 (control), 0.8, 1.6, 3.2, 6.4, 12.8, 25.6, 50 and 100 mg/L
- Mean measured concentrations at t= 0h: 95.6 to 104.5 % of the nominal values
- Mean measured concentrations at t=72h: An overview of the analytical results is presented in 'Any other information on materials and methods incl. tables'.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL glass Erlenmeyer flasks
- Type: closed with cotton stoppers.
- Fill volume: 50 mL
- Aeration: No
- Initial cells density: 9700 cells/mL
- Control end cells density: Not reported
- No. of vessels per concentration: 3
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH was measured at the start and at the end of the test. The water temperature was measured continuously.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No
- Light intensity and quality: Continuous illumination at 111 µ E/m2 sec (approximately 8000 lux)

EFFECT PARAMETERS MEASURED: algal cell density
Small volumes of all test concentrations and controls were taken from all test flasks after 24, 48, and 72 hours of exposure. The algal cell densities were measured at 24, 48 and 72 hours and the mean biomass and growth rate calculated.
- Determination of cell concentrations: The algal cell densities in these samples were determined by counting with a “TOA” cell counter.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 81.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
81.8 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.:
> 81.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
81.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
No significant inhibition in growth rate or biomass was observed throughout the test in all treatments. Additional details are provided in 'any other information on results incl. tables'.
Reported statistics and error estimates:
The 72-hour EbC50 and ErC50 values were calculated using logit and probit model. For determination of the NOEC values, a modified Dunnett’s Multiple Comparison Test was used to identify significant differences in the calculated mean biomass and growth rate of test item treatments compared to the control.

Table: Mean values at each concentration of the test substance for the growth rate integral at 72 hours for Selenastrum capricornutum and relevant endpoints

Measured concentrations at test termination (mg test substance/L)

Mean growth rate (1/day) 0 – 72 hrs

Percentage inhibition

Control

66

0.0

0.66

68

0.0

0.93

67

0.0

1.92

69

0.0

4.50

65

1.5

9.91

65

1.0

20.6

65

1.1

45.2

62

5.8

81.8

66

0.3

ErC50 (mg test substance/L)

> 81.8

NOEC (mg test substance/L)

81.8

Table: Mean values at each concentration of the test substance for the biomass integral at 72 hours for Selenastrum capricornutum and relevant endpoints

Measured concentrations (mg test substance/L)

Mean biomass integral (x 104) 0 – 72 hrs

Percentage inhibition

Blank

2389

0.0

0.66

2751

0.0

0.93

2611

0.0

1.92

2955

0.0

4.50

2364

1.0

9.91

2473

0.0

20.6

2363

1.1

45.2

2029

15.1

81.8

2347

1.7

EbC50 (mg test substance/L)

> 81.8

NOEC (mg test

substance/L)

81.8

Validity criteria fulfilled:
yes
Remarks:
See 'Any other information on materials and methods incl. tables'.
Conclusions:
Based on the measured concentrations at test termination, the 72-hour ErC50 and EbC50 were > 81.8 mg test substance/L. The NOEC at 72 hours, based on growth rate and biomass integral, was 81.8 mg test substance/L, which is the higest concentration tested.
Executive summary:

The toxicity to aquatic algae was determined in a study according to OECD TG 201 and in compliance with GLP criteria. In this study, cultures of freshwater green algae (Pseudokirchneriella subcapitata) at an initial concentration of9700 cells/mLwere exposed to the test substance at nominal concentrations 0 (control), 0.8, 1.6, 3.2, 6.4, 12.8, 25.6, 50 and 100 mg/L for 72 hours. Test concentrations were analytical verified at the start and the end of the test and found not to remain within ± 20% of nominal concentrations. Therefore, the corresponding mean measured concentrations of <LOD, 0.66, 0.93, 1.92, 4.5, 9.91, 20.56, 45.24 and 81.78 mg/L were used to express the effect values. Cell densities were measured throughout the test and growth rate and biomass (as Areas Under the Curve) determined. No significant (dose-response) inhibition in growth rate or biomass was observed in all treatments throughout the test. Based on these findings, the 72-h EC50 values for growth rate and biomass were determined at >81.8 mg/L. The 72-h NOEC values for growth rate and biomass were determined to be 81.8 mg/L, which is the highest dose tested.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 Oct 2013 to 01 November 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2006
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
At exposure initiation (0 hour) and exposure termination (96 hours), samples were removed from each test concentration and the control. Samples analyzed at 0 hour were removed from the test and control solutions prior to division into the replicate test vessels. Samples analyzed at 96 hours were removed from composited replicate solutions of each treatment level and the control. All test samples removed at 96 hours were centrifuged prior to analysis to remove algal biomass, as required by the protocol and OCSPP Guideline 850.4500. Additional archive samples were collected at 0 and 96 hours, stored frozen and discarded, if additional analyses were not required. Archive samples collected at 0 hour were non-centrifuged samples and the samples collected at 96 hours included both the centrifuged est and QC samples, as well as the non-centrifuged QC samples. At 96 hours of exposure, a sample was also removed from the replicate flask which did not contain algae (for comparison with sample containing algae). Samples removed from the exposure system were centrifuged in Nalgene centrifuge tubes at 3000 rpm for 15 minutes. Nine quality control (QC) samples were prepared (three at 0 hour and six at 96 hour). Three of the six QC samples at 96 hours were centrifuged prior to analysis. This additional set of centrifuged QC samples was used to evaluate potential losses of test substance due to centrifugation. All QC samples remained with the set of exposure solution samples throughout the analytical process. These QC samples were prepared in dilution water at concentrations of the test substance to the test concentration range. Results of the analyses of the QC samples were used to judge the precision and quality control maintained during the analysis of exposure solution samples.
Vehicle:
no
Details on test solutions:
A 100 mg/L primary stock solution was prepared prior to exposure initiation by placing 0.2003 g of test substance in a 2.0 L volumetric flask and bringing it to volume with AES medium. The resulting stock solution was observed to be clear and light yellow in color with a large amount of undissolved test substance. Following approximately 15 minutes of mixing using a Teflon®-covered stir bar and magnetic stir plate and approximately 10 minutes of sonication, the resulting stock solution was observed to be clear and light yellow in color with no visible undissolved test substance. The 100 mg/L primary stock solution was used as the high-level exposure solution and to prepare the remaining exposure solutions. Additional untreated AES medium (800 mL) was used to prepare the control. All test and control solutions were observed to be clear and light yellow in color with no visible undissolved test substance following preparation.
Test organisms (species):
Skeletonema costatum
Details on test organisms:
TEST ORGANISM
- Common name: Marine diatom
- Strain: CCMP 1332
- Class: Bacillariophyceae

CULTURING
- Culture 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 (2002). In addition, a representative sample of AES medium was analyzed monthly for total organic carbon (TOC) concentration.
- TOC concentration: The TOC concentration was 4.9 and 2.3 mg/L
- Salinity: Salinity of the medium was within the range of 30 ± 2 g/L.
- Temperature: 20 ± 2 °C; Temperature was controlled using an environmental chamber.
- Photoperiod: 14 hours of light:10 hours of darkness
- Light intensity: 3700 - 4900 lux; Lighting was supplied by fluorescent bulbs.

ACCLIMATION
The inoculum used to initiate the toxicity test 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:
21 - 22 °C
pH:
- Test start: 7.9 to 8.0
- Test end: 8.5 to 8.6
Salinity:
30 ± 2 g/L
Conductivity:
45 - 46 mS/cm at the test start
Nominal and measured concentrations:
- Nominal concentrations: 0 (control), 6.3, 13, 25, 50 and 100 mg/L (based on preliminary testing)
- Mean measured concentration: NA (control), 5.8, 12, 24, 48 and 99 mg/L (92 to 99% of nominal).
The individual measurements are presented in 'Any other information on materials and methods incl. tables'.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL flasks which
- Fill volume: 100 mL
- Type: All test vessels were fitted with stainless steel caps which permitted gas exchange.
- Fill volume: 100 mL
- Initial cells density: Approximately 1.0E+004 cells/mL.
- Control end cells density: 3.341E+05 cells/mL at t=72h.
- No. of vessels per concentration: 4
- No. of vessels per control: 8
- No. of vessels per biomass control: 1

EXPOSURE INITIATION
After the test solutions were added to the test flasks (100 mL per flask), a 0.749 mL inoculum of Skeletonema costatum cells, at a density of approximately 133.47E+04 cells/mL, was aseptically introduced into each flask. This inoculum provided the required initial (0-hour) cell density of approximately 1.0E+04 cells/mL.

BIOMASS CONTROL
In order to estimate the impact that the presence of algal biomass had on test substance concentration, an additional replicate flask of the 25 mg/L (nominal) treatment level was prepared. This flask, which was not inoculated with algae, was analyzed at 96 hours of exposure for the test substance concentration. The result of this analysis was compared with the result for the 25 mg/L solution containing algae.

GROWTH MEDIUM
- Standard medium used: Yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Artificially Enriched Seawater (AES) medium
- Culture medium different from test medium: No (as described under 'Details on test organisms')

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 8.0 ± 0.1 with dilute hydrochloric acid or sodium hydroxide prior to use.
- Photoperiod: A photoperiod of 14 hours of light:10 hours of darkness was maintained.
- Light intensity and quality: 3700 - 4900 lux; The photosynthetically active radiation (PAR) of the test area at exposure initiation ranged from 54 to 62 μE/m2/s.

WATER QUALITY MEASUREMENTS
- 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.
- Light intensity: Light intensity was measured at four locations around the perimeter of the test area with a Traceable light meter at 0 hour and at each 24-hour interval during the exposure period. The PAR of the test area was measured at exposure initiation using a photometer and a radiation sensor probe.
- pH: Measurement of pH was conducted at exposure initiation and at exposure termination. At exposure termination, after cell counts were completed, the replicate solutions were composited by treatment level or control for pH measurements. Test solution pH was measured with a pH meter and conductivity was measured with a conductivity meter.
- Conductivity: Conductivity of the test solutions was measured at exposure initiation.

EFFECT PARAMETERS MEASURED: cell count, algal health
At each subsequent 24-hour interval, a single cell count was conducted on 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, except at 96-hours of exposure an additional sample was collected and cell counts taken for each control replicate. One or more hemacytometer fields, each 0.10 × 0.10 cm in surface area, 0.010-cm deep and containing 0.00010 mL of test 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 test solutions (i.e., precipitation, clarity, material adhering to the sides of the test vessels) were made at each 24-hour interval.

PRELIMINARY TEST
A preliminary range-finding exposure was conducted at nominal concentrations of 0.10, 1.0, 10, 100 and 1000 mg/L and a control. Test solutions were prepared in a similar manner as described for the definitive test. Two exposure vessels were established for each concentration and the control. Following 96 hours of exposure, cells exposed to all treatment levels tested and the control were observed to be normal. Cell densities in the 0.10, 1.0, 10, 100 and 1000 mg/L treatment levels averaged 30.63E+04, 36.88E+04, 31.50E+04, 12.63E+04 and 0.75E+04 cells/mL, respectively. The control averaged 22.13E+04 cells/mL. Based on these results, nominal concentrations of 6.3, 13, 25, 50 and 100 mg/L and a control were selected for the definitive exposure.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 99 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 99 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
99 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.:
> 99 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
8.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% C.I.: ND - 75 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
99 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
Detailed results on cell density, yield and average specific growth rate are presented in 'Any other information on results incl. tables'.
- Density: The 72-hour cell density in the control averaged 33.41E+04 cells/mL. Cell density in the 5.8, 12, 24, 48 and 99 mg/L treatment levels averaged 35.19, 27.06, 29.81, 25.81 and 24.44 E+04 cells/mL, respectively. The 96-hour cell density in the control averaged 63.28 E+04 cells/mL. Cell density in the 5.8, 12, 24, 48 and 99 mg/L treatment levels averaged 62.50, 71.25, 61.50, 58.50 and 54.88 E+04 cells/mL, respectively.
- Area under growth curve (AUGC): Following 72-hours of exposure, AUGC in the control averaged 24.28 E+04 cells ×·days/mL. The 72-hour AUGC in the 5.8, 12, 24, 48 and 99 mg/L treatment levels averaged 25.41, 18.99, 21.59, 18.37 and 16.43 E+04 cells × days/mL, respectively. Following 96-hours of exposure, AUGC in the control averaged 71.30 E+04 cells × days/mL. The 96-hour AUGC in the 5.8, 12, 24, 48 and 99 mg/L treatment levels averaged 72.93, 66.81, 65.94, 59.24 and 54.82 E+04 cells × days/mL, respectively.
- Yield: The 72-hour yield in the control averaged 32.41 E+04 cells/mL. The 72-hour yield in the 5.8, 12, 24, 48 and 99 mg/L treatment levels averaged 34.19, 26.06, 28.81, 24.81 and 23.44 E+04 cells/mL, respectively. Following 96 hours of exposure, the yield in the control averaged 62.28 E+04 cells/mL. The 96-hour yield in the 5.8, 12, 24, 48 and 99 mg/L treatment levels averaged 61.50, 70.25, 60.50, 57.50 and 53.88 E+04 cells/mL, respectively.
- Average specific growth rate: The 72-hour growth rate in the control averaged 1.20 days-1. The 72-hour average specific growth rate in the 5.8, 12, 24, 48 and 99 mg/L treatment levels averaged 1.22, 1.13, 1.17, 1.11 and 1.10 days-1, respectively. Following 96 hours of exposure, average specific growth rate in the control averaged 1.07 days-1. The 96-hour average specific growth rate in the 5.8, 12, 24, 48 and 99 mg/L treatment levels averaged 1.06, 1.09, 1.05, 1.04 and 1.02 days-1, respectively.
- Appearance: All cells were observed to be normal throughout the exposure period.
Reported statistics and error estimates:
Based on the results of statistical analysis performed for 72- and 96-hour AUGC, yield and average specific growth rate, the NOEC and LOEC values were determined. The data were first checked for normality using Shapiro-Wilk’s Test (U.S. EPA, 2002) and for homogeneity using Bartlett's Test (U.S. EPA, 2002). If the data sets passed the tests for homogeneity and normality, then Dunnett’s Multiple Comparison Test (U.S. EPA, 2002) was used to determine the NOEC. If the data did not pass the tests for homogeneity and normality, then the NOEC was determined using an appropriate non-parametric statistical test (U.S. EPA, 2002). All statistical determinations were made at the 95% level of certainty, except in the case of Shapiro-Wilk’s and Bartlett's Tests, where the 99% level of certainty was applied. The 72-hour EC10, EC20 and EC50 values (concentrations of test substance which reduced AUGC, yield and average specific growth rate by 10, 20 and 50%, respectively), the 96-hour EC50 values and corresponding 95% confidence intervals were determined by linear interpolation of response using the ICp method (Norberg-King, 1993). If less than the required response was observed (e.g., <10, 20 or 50% response), the EC value was empirically estimated to be greater than the highest mean measured concentration tested. CETIS™ versions 1.8 (Ives, 2013) were used to perform all statistical analyses.

Table: Cell density of Skeletonema costatum after 24, 48, 72 and 96 hours of exposure to the test substance

Mean

Measured

Concentration

(mg/L)

Replicate

Cell Density (E+04 cells/mL)

Observation Interval (hours)

24

48

72

96

Control

A

1.50

6.50

46.00

59.75

B

3.50

11.50

21.75

56.00

C

1.00

5.00

32.00

56.00

D

1.00

7.25

19.25

67.13

E

2.00

7.75

39.50

71.75

F

2.75

10.00

31.25

64.25

G

1.50

9.25

44.50

67.25

H

3.50

8.25

33.00

64.13

Mean (SD)(a)

2.09 (1.03)

8.19 (2.05)

33.41 (9.72)

63.28 (5.64)(b)

5.8

A

2.00

5.50

45.50

56.25

B

3.00

14.75

23.75

39.50

C

2.25

4.75

36.00

73.25

D

4.50

5.50

35.50

81.00

Mean (SD)

2.94 (1.13)

7.63 (4.76)

35.19 (8.91)

62.50 (18.49)

12

A

2.25

7.00

36.75

55.25

B

2.50

5.50

19.50

82.25

C

2.00

5.00

33.00

58.50

D

2.25

6.00

19.00

89.00

Mean (SD)

2.25 (0.20)

5.88 (0.85)

27.06 (9.15)

71.25 (16.88)

24

A

2.00

6.25

37.25

48.75

B

3.50

8.25

28.75

73.25

C

2.25

6.50

26.25

48.25

D

1.50

7.25

27.00

75.75

Mean (SD)

2.31 (0.85)

7.06 (0.90)

29.81 (5.07)

61.50 (15.05)

48

A

3.25

3.00

26.50

57.50

B

2.50

7.25

22.00

42.50

C

1.00

8.50

38.00

82.00

D

2.00

5.00

16.75

52.00

Mean (SD)

2.19 (0.94)

5.94 (2.44)

25.81 (9.05)

58.50 (16.85)

99

A

0.75

4.00

32.50

50.50

B

0.00

9.50

19.75

46.25

C

1.00

6.75

25.75

36.00

D

2.25

3.00

19.75

86.75

Mean (SD)

1.00 (0.94)

5.81 (2.93)

24.44 (6.07)

54.88 (22.10)

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

b) Cell density for each control replicate, mean and standard deviation at 96 hours was based on the mean of two cell counts (see Deviations from the protocol).

 

Table: Yield of Skeletonema costatum after 24, 48, 72 and 96 Hours of Exposure to the test substance

Mean

Measured

Concentration

(mg/L)

Replicate

Yield (E+04 cells/mL)

Observation Interval (hours)

0-24

0-48

0-72

72-Hour

Inhibition(a,b)

(%)

0-96

96-Hour

Inhibition(a,b)

(%)

Control

 

 

A

0.50

5.50

45.00

 

58.75

 

B

2.50

10.50

20.75

55.00

C

0.00

4.00

31.00

55.00

D

0.00

6.25

18.25

66.13

E

1.00

6.75

38.50

70.75

F

1.75

9.00

30.25

63.25

G

0.50

8.25

43.50

66.25

H

2.50

7.25

32.00

63.13

Mean (SD)(b)

1.09 (1.03)

7.19 (2.05)

32.41 (9.72)

NA

62.28 (5.64)

NA

5.8

A

1.00

4.50

44.50

 

55.25

 

B

2.00

13.75

22.75

38.50

C

1.25

3.75

35.00

72.25

D

3.50

4.50

34.50

80.00

Mean (SD)

1.94 (1.13)

6.63 (4.76)

34.19 (8.91)

-5

61.50 (18.49)

1

12

A

1.25

6.00

35.75

 

54.25

 

B

1.50

4.50

18.50

81.25

C

1.00

4.00

32.00

57.50

D

1.25

5.00

18.00

88.00

Mean (SD)

1.25 (0.20)

4.88 (0.85)

26.06 (9.15)

20

70.25 (16.88)

-13

24

A

1.00

5.25

36.25

 

47.75

 

B

2.50

7.25

27.75

72.25

C

1.25

5.50

25.25

47.25

D

0.50

6.25

26.00

74.75

Mean (SD)

1.31 (0.85)

6.06 (0.90)

28.81 (5.07)

11

60.50 (15.05)

3

48

A

2.25

2.00

25.50

 

56.50

 

B

1.50

6.25

21.00

41.50

C

0.00

7.50

37.00

81.00

D

1.00

4.00

15.75

51.00

Mean (SD)

1.19 (0.94)

4.94 (2.44)

24.81 (9.05)

23

57.50 (16.85)

8

99

A

-0.25

3.00

31.50

 

49.50

 

B

-1.00

8.50

18.75

45.25

C

0.00

5.75

24.75

35.00

D

1.25

2.00

18.75

85.75

Mean (SD)

0.00 (0.94)

4.81 (2.93)

23.44 (6.07)

28

53.88 (22.10)

13

a) Percent inhibition relative to the control.

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

NA = Not Applicable.

 

Table: Average specific growth rates of Skeletonema costatum after 24, 48, 72 and 96 Hours of Exposure to the test substance

Mean

Measured

Concentration

(mg/L)

Replicate

Average Specific Growth Rate (per day)

Observation Interval (hours)

0-24

0-48

0-72

72-Hour

Inhibition(a,b)

(%)

0-96

96-Hour

Inhibition(a,b)

(%)

Control

A

0.44

0.99

1.32

 

1.05

 

B

1.37

1.29

1.07

1.04

C

0.00

0.85

1.20

1.04

D

0.00

1.05

1.02

1.08

E

0.76

1.08

1.27

1.10

F

1.11

1.21

1.19

1.07

G

0.44

1.17

1.31

1.08

H

1.37

1.11

1.21

1.07

Mean (SD)(b)

0.69 (0.56)

1.09 (0.14)

1.20 (0.11)

NA

1.07 (0.02)

NA

5.8

A

0.76

0.90

1.32

 

1.04

 

B

1.20

1.42

1.10

0.95

C

0.89

0.82

1.24

1.11

D

1.65

0.90

1.23

1.13

Mean (SD)

1.13 (0.40)

1.01 (0.28)

1.22 (0.09)

-2

1.06 (0.08)

1

12

A

0.89

1.03

1.25

 

1.03

 

B

1.00

0.90

1.03

1.14

C

0.76

0.85

1.21

1.05

D

0.89

0.95

1.02

1.16

Mean (SD)

0.89 (0.10)

0.93 (0.08)

1.13 (0.12)

6

1.09 (0.06)

-2

24

A

0.76

0.97

1.25

 

1.00

 

B

1.37

1.11

1.16

1.11

C

0.89

0.99

1.13

1.00

D

0.44

1.05

1.14

1.11

Mean (SD)

0.87 (0.39)

1.03 (0.07)

1.17 (0.06)

2

1.05 (0.06)

1

48

A

1.29

0.58

1.13

 

1.04

 

B

1.00

1.05

1.07

0.97

C

0.00

1.13

1.26

1.13

D

0.76

0.85

0.97

1.02

Mean (SD)

0.76 (0.55)

0.90 (0.24)

1.11 (0.12)

8

1.04 (0.07)

3

99

A

-0.32

0.73

1.20

 

1.01

 

B

0.00(c)

1.19

1.03

0.99

C

0.00

1.01

1.12

0.92

D

0.89

0.58

1.03

1.15

Mean (SD)

0.14 (0.52)

0.88 (0.27)

1.10 (0.08)

8

1.02 (0.10)

5

a) Percent inhibition relative to the control.

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

c) Where an average specific growth rate value could not be mathematically calculated from an observed cell density of 0.00E+04 cells/mL, an average specific growth rate value of zero was used in all subsequent calculations.

NA = Not Applicable.

ADDITIONAL EFFECT VALUES

Area under growth curve (AUGC):

72-h EC10: 8.7 mg/L (95% C.I.: 4.0 - 35 mg/L)

72-h EC20: 28 mg/L (95% C.I.: ND - 87 mg/L)

72-h EC50: > 99 mg/L

72-h NOEC: 48 mg/L

72-h LOEC: 99 mg/L

96-h EC50: > 99 mg/L

96-h NOEC: 48 mg/L

96-h LOEC: 99 mg/L

Yield:

72-h EC20: 30 mg/L

72-h LOEC: > 99 mg/L

96-h EC50: > 99 mg/L

96-h NOEC: 99 mg/L

96-h LOEC: > 99 mg/L

Average specific growth rate:

72-h EC20: > 99 mg/L

72-h LOEC: > 99 mg/L

96-h EC50: > 99 mg/L

96-h NOEC: 99 mg/L

96-h LOEC: > 99 mg/L

Validity criteria fulfilled:
yes
Remarks:
See 'Any other information on materials and methods incl. tables'.
Conclusions:
The 72-h ErC50 and 72-h ErC10 values are determined to be > 99 mg/L for the marine diatom Skeletonema costatum.
Executive summary:

This study was performed to determine the effects of the test substance on growth of Skeletonema costatum, a marine diatom, under static conditions following OCSPP Guideline 850.4500 and in compliance with GLP. In addition, the procedures were modified to include the requirements of OECD Guideline 201. Cell density was monitored at 24, 48, 72 and 96 hours of exposure. Area under the growth curve (AUGC), yield and average specific growth rate were calculated as test endpoints. Four replicate flasks for each treatment level (i.e., 0 (control), 6.3, 13, 25, 50 and 100 mg/L nominal, 5.8, 12, 24, 48 and 99 mg/L mean measured) and eight replicate flasks for the control were established and maintained in an environmental chamber designed to maintain the test conditions: a temperature of 20 ± 2 °C, a photoperiod of 14 hours of light:10 hours of darkness, a light intensity range of 3700 to 4900 lux and a photosynthetically-active radiation range of 51 to 69 μE/m2/s. Test vessels were hand shaken once daily. In order to estimate the impact the presence of algal biomass had on the test item under test conditions, an additional replicate was initiated at 25 mg/L (nominal). This vessel was not inoculated with algae and was analyzed the 96 hour timepoint. The result of this analysis was compared with the analytical result of the equivalent concentration containing algae. Samples of all test solutions and the control were removed at 0 and 96 hours of exposure for analysis of test substance concentration. Analytical verification showed that the measured test concentrations remained within ± 20% of the nominal. No effects on average specific growth rate were observed. Based on mean measured concentrations, the 72-hour ErC50 and EyC50 were determined to be > 99 mg/L. The 72-hour ErC10 and EyC10 were determined to be > 99 mg/L and 8.8 mg/L, respectively.

Description of key information

All available data were assessed and the studies representing the worst-case effects were included as key or weight-of-evidence studies. Other studies are included as supporting information. The key studies are considered to be worst-case and were selected for the CSA.

No quantitative acute effect values (ErC50 values) can be determined as no ≥50% effect was reached in any of the available studies.

The 72-h ErC50 and NOErC values are >81.8 mg/L and 81.8 mg/L, respectively in freshwater green algae (P. subcapitata), OECD 201, Grade 1996

The 72-h ErC50 and ErC10 values are both >99 mg/L in marine diatom (S. costatum), OECD 201, Staggs 2014

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
81.8 mg/L
EC10 or NOEC for marine water algae:
99 mg/L

Additional information

Table: Overview of available data on the toxicity to aquatic algae

Species

Guideline / GLP

Endpoint

Effect value

Comment

Reference

Pseudokirchneriella subcapitata (Freshwater)

OECD 201/ GLP

72-h ErC50

72-h NOErC

>81.8 mg/L

81.8 mg/L

Static regime. Measured concentrations remained within 20% of nominal.

Some test conditions were not reported. The minor deviations from the guideline (pH and microscopic observation at end of test) do not effect the reliability of the study. The validity criteria were met.

Grade, 1996

Skeletonema costatum (salt water)

OECD 201/ GLP

72-h ErC50

72-h ErC10

>99 mg/L

>99 mg/L

Static marine regime. Measured concentrations remained within 20% of nominal. One validity criterion not met, however this is not unusual for this species.

 

Staggs, 2014