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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:
supporting study
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)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Sampling method: At the start of the study, samples were taken of each test solution, using the excess remaining after filling the test vessels. At the end of the study each blank solution was sampled and analyzed in the same manner.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Each test solution was prepared by the addition of aliquots of the stock solution to sterile culture medium. All additions were made below surface, gradually, by microlitre syringe, into solutions vigorously stirred by magnetic follower. The dilution water control consisted of culture medium only. Using aseptic techniques, 100 ml volumes of the appropriate test solution were dispensed to each test and blank vessel.
- Controls: culture medium control and a solvent control
- Chemical name of vehicle: dimethylformamide
- Concentration of vehicle in test medium: 0.10 mL/L
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Unicellular green alga
- Strain: Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
- Source: Brixham Environmental Laboratory (In-house)
- Age of inoculum (at test initiation): 3 day old
Test type:
static
Water media type:
freshwater
Total exposure duration:
72 h
Test temperature:
23.9 to 24.0°C
pH:
7.3 to 9.4
Nominal and measured concentrations:
Nominal: 4.0, 8.8, 19, 42, 92, 200, 440 and 970 µg/L
Measured: 4.4, 9.4, 19, 43, 81, 210, 450 and 940 µg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Borosilicate glass conical flasks of 250 mL nominal capacity closed with polyurethane foam bungs. Each flask contained 100 mL of test solution. The cultures were incubated at 24 ± 2°C (the nominal test temperature), under continuous "cool-white" illumination, with orbital shaking at 160 rpm, in an orbital incubator.
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): No renewal
- Initial cells density: 10000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 3

GROWTH MEDIUM
- Standard medium used: no
- Detailed composition if non-standard medium was used: Refer 'Any other information on materials and methods including tables' for preparation details

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Light intensity and quality: 8380 lux

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: electronic particle counter
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
260 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: (95% CL: 93 to >970 µg/L)
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
56 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: (95% CL: 24 to 130 µg/L)
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
8.8 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: biomass and growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
19 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: biomass and growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
Reported statistics and error estimates:
The areas under growth curve and logarithmic growth rates were examined by one-way analysis of variance, and Dunnett's procedure was used to identify significant differences (P=0.05) from the solvent control. The mean areas under growth curve, mean growth rates and the significant differences expressed as percentages, which were transformed to probability scale and analyzed by linear regression against log concentration, to estimate the median effective concentration and its 95% confidence limits.
Validity criteria fulfilled:
yes
Conclusions:
72 h EbC50 (biomass): 56 µg/L (95% CL: 24 to 130 µg/L)
72 h ErC50 (growth rate): 260 µg/L (95% CL: 93 to >970 µg/L)
72 h NOEC (biomass and growth rate): 8.8 µg/L
72 h LOEC (biomass and growth rate): 19 µg/L
Executive summary:

The effect of the test substance on the growth of Selenastrum capricornutum was determined in a test during a 3-day exposure period according to OECD guideline 201. An initial concentration of 10000 cells/mL per test vessel was exposed to nominal concentrations of 4.0, 8.8, 19, 42, 92, 200, 440 and 970 µg/L of the test substance and culture medium control and a solvent control. Triplicate cultures of the culture medium control, solvent control and each concentration of test substance were employed. The test was performed in Borosilicate glass conical flasks of 250 mL nominal capacity closed with polyurethane foam bungs. Each flask contained 100 mL of test solution. The cultures were incubated at 24 ± 2°C (the nominal test temperature), under continuous "cool-white" illumination, with orbital shaking at 160 rpm, in an orbital incubator.

After 1, 2 and 3 days (24, 48 and 72 hours), samples were removed from each test and blank vessel. The appropriate blank particle count was subtracted from that of the test culture to obtain the cell density.

The results obtained from the analysis of growth rate and areas under growth curve are as follows:

72 h EbC50 (biomass): 56 µg/L (95% CL: 24 to 130 µg/L)

72 h ErC50 (growth rate): 260 µg/L (95% CL: 93 to >970 µg/L)

72 h NOEC (biomass and growth rate): 8.8 µg/L

72 h LOEC (biomass and growth rate): 19 µg/L

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guideline for the Testing of Chemicals Section 2: Effects on Biotic Systems, Number 221 (2006)
Deviations:
yes
Remarks:
The deviation had no adverse impact on the results or interpretation of the study.
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Deviations:
yes
Remarks:
The deviation had no adverse impact on the results or interpretation of the study.
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Sampling method: At test initiation (0 hour), and at day 3 and day 5 of the exposure, a sample was removed from each test concentration, the blank control and the solvent control and analyzed for the test substance. The aged solution for the abiotic stability control (1000 μg a.s./L) was sampled and analyzed at day 3 and 5. Samples analyzed at 0 hour were removed from the test and control solutions prior to division into the replicate test vessels. Samples analyzed at the end of each renewal period were removed from the composited replicate solutions of each test concentration and control.
Three quality control (QC) samples were prepared at each sampling interval at nominal concentrations of the test substance approximating the test concentration range and remained with the exposure solution samples throughout the analytical process.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A 20 mg a.s./mL primary stock solution was prepared prior to test initiation by adding 0.5035 g of the test substance (0.5000 g a.s.) to a 25-mL volumetric flask and bringing it to volume with dimethylformamide. Secondary stock solutions were prepared by adding variying volumes of the primary stock solution with 10 mL of DMF. The 0.1 mL of the resultant secondary stock solutions were made to a final volume of 1000 mL with 20X AAP nutrient medium.
- Controls: a dilution water control, an abiotic control and a solvent control
- Chemical name of vehicle: dimethylformamide
Test organisms (species):
other: Lemna gibba
Details on test organisms:
TEST ORGANISM
- Common name: duckweed
- Strain: Lemna gibba
- Source: Canadian Phycological Culture Centre (CPCC), Toronto, Canada
- Stock cultures were grown in 270-mL covered crystallizing dishes containing 100 mL of 20X AAP medium. Approximately 50 fronds were transferred weekly into fresh 20X AAP medium to maintain a continuous culture under testing conditions. The cultures were maintained in an environmental chamber with the following conditions: a temperature of 24 ± 2ºC and continuous illumination of approximately 650 to 880 footcandles (7000 to 9500 lux).
Test type:
static
Water media type:
saltwater
Limit test:
yes
Total exposure duration:
7 d
Test temperature:
23-24°C
pH:
7.7-9.4
Nominal and measured concentrations:
Nominal: 0, 10, 26, 64, 160, 400, 1000 µg a.s./L
Measured: 0, 7.9, 20, 49, 160, 340, 1000 µg a.s./L
Details on test conditions:
TEST SYSTEM
- Test vessel: 270-mL crystallizing dishes containing approximately 100 mL of test solution and covered with an inverted, sterile glass Petri dish
- Renewal rate of test solution: Test solutions were renewed on days 3 and 5.
- No. of organisms per vessel: 12
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per solvent control (replicates): 7

GROWTH MEDIUM
- Standard medium used: yes (20X AAP medium)

OTHER TEST CONDITIONS
- Photoperiod and Light intensity: Continuous fluorescent lighting at an intensity of 7000 to 8800 lux

EFFECT PARAMETERS MEASURED:
- Fronds were counted and observations were made on days 3 and 5, and at test termination (day 7). Frond density determinations were completed at test termination and the fronds from three of the four replicates (six of the seven solvent control replicates) were removed from each vessel, blotted dry, and transferred to pre-weighed aluminum pans. Fronds were dried in a Labline Imperial II oven at 72 to 73°C for five days prior to dry weight determination.

TEST CONCENTRATIONS
- Range finding study: A range-finding exposure was conducted with a blank control and nominal concentrations of 0.00010, 0.0010, 0.010, 0.10, and 1.0 mg a.s./L. Test solutions were renewed on days 3 and 5. Two exposure vessels were used for each concentration and the blank control. Following 7 days of exposure, fronds exposed to the 1.0 mg a.s./L treatment were observed to be curled. Fronds exposed to all remaining treatment levels (0.10, 0.010, 0.0010, and 0.00010 mg a.s./L) and the blank control were observed to be normal. Frond densities in the 0.00010, 0.0010, 0.010, 0.10, and 1.0 mg a.s./L treatment levels averaged 391, 381, 386, 425 and 123 fronds, respectively. Mean frond density in the blank control was 437 fronds.
The preliminary range-finding test with this test substance was conducted without the use of a co-solvent. Analytical sampling performed in conjunction with this exposure resulted in recoveries of approximately 20% of nominal concentrations. Additional solubility trials with this test substance were conducted to evaluate the use of a co-solvent (i.e., dimethylformamide, DMF). Based on these results, and in consultation with the Study Sponsor, DMF was used to solubilize the test substance for the definitive testing.
An initial definitive test was conducted at nominal concentrations of 12, 31, 77, 190, 480, 1200, and 3000 μg a.s./L, a blank control and a solvent (DMF) control. However, due to variable analytical results related to the solubility of the test substance at nominal concentrations >1000 μg a.s./L, the definitive test was repeated at nominal concentrations of 10, 26, 64, 160, 400, and 1000 μg a.s./L.

Reference substance (positive control):
no
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
260 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: frond density
Remarks on result:
other: 95% confidence limits
Remarks:
160-340 µg a.s./L
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
230 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: yield based on frond density
Remarks on result:
other: 95% confidence limits
Remarks:
160-330 µg a.s./L
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks:
based on frond density
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: dry weight
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 1 000 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: yield and growth rate
Remarks:
based on dry weight
Key result
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
49 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: frond density; yield and growth rate (based on frond density)
Key result
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
49 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: dry weight; yeild and growth rate (based on dry weight)
Key result
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
160 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: frond density; yield and growth rate (based on frond density)
Key result
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
160 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: dry weight; yeild and growth rate (based on dry weight)
Details on results:
A summary of inhibition for each endpoint following exposure of Lemna gibba to the test substance for 7 days is presented in the table (refer 'Any other information on results including tables').
Reported statistics and error estimates:
Frond density, dry weight biomass, and growth rate and yield based on frond density and dry weight data were evaluated for normality and homogeneity of variance (α = 0.05) using the Shapiro-Wilks’ and Bartlett’s Tests, respectively. Since all data sets were normal with homogeneous variances, the treatment groups were compared to the pooled control using Williams’ Test (α = 0.05). The results of the statistical analyses, as well as an evaluation of the concentration-response pattern, were used to determine the NOEC and LOEC relative to each parameter at 7 days. All calculations and statistical analyses were conducted using TOXSTAT Version 3.5.

Table 1: Inhibition summary for each endpoint following exposure of Lemna gibba to the test substance for 7 days

Mean, measured concentration (µg a.s./L)

 

 

% Inhibition relative to pooled control

 

Frond Density

 

Yield (Frond Density)

Growth Rate

(Frond Density)

 

Dry Weight

 

Yield (Dry Weight)

 

Growth Rate

(Dry Weight)

Untreated Control

 

-

-

-

-

-

-

Solvent Control

 

-

-

-

-

-

-

7.9

-7

-8

-2

0

-2

0

20

3

4

2

4

3

2

49

9

9

2

2

0

0

160

42

44

16

24

24

10

340

55

57

24

43

44

20

1000

70

73

37

38

39

17

Negative values indicate stimulation; positive values indicate inhibition.

Validity criteria fulfilled:
yes
Conclusions:
The most sensitive parameter was yield based on frond density with an EyC50 value of 230 μg a.s./L, a NOEC of 49 μg a.s./L, and a LOEC of 160 μg a.s./L based on mean, measured concentrations.
Executive summary:

The acute toxicity of the test substance to duckweed, Lemna gibba (L. gibba), under static-renewal conditions was determined in a 7-day exposure test. Test solutions were renewed on days 3 and 5. The test was conducted in accordance with the OECD Guideline 221 (2006) and U.S. EPA OPPTS Draft Guideline 850.4400 (1996).

The study was conducted with six concentrations of the test substance, a dilution water (twenty strength synthetic algal-assay-procedure nutrient medium, 20X AAP) control, an abiotic control and a solvent (dimethylformamide, DMF) control at a temperature of 23 to 24°C. Four replicates with 12 fronds per replicate were initiated for each test substance concentration and the dilution water control. Seven replicates with 12 fronds per replicate were initiated for the solvent control. A single test vessel containing no L. gibba was initiated at the highest test concentration for the abiotic stability control.

Nominal exposure concentrations of the test substance were 10, 26, 64, 160, 400, and 1000 μg active substance (a.s.)/L. Mean, measured concentrations were 7.9, 20, 49, 160, 340, and 1000 μg a.s./L, respectively, and ranged from 77 to 100% of nominal. The mean, measured concentration of the 1000 μg a.s./L abiotic control was 960 μg a.s./L.

Inhibition of frond density (number of fronds/100 mL) of L. gibba exposed to mean, measured test substance concentrations of 7.9, 20, 49, 160, 340, and 1000 μg a.s./L as compared to the pooled control was -7, 3, 9, 42, 55, and 70%, respectively, at the end of the 7-day exposure period. The 7-day “effective concentration” producing a 50% inhibition of growth (EC50), based on mean, measured concentrations of the test substance and frond density, was 260 μg a.s./L.

Inhibition of frond dry weight (biomass) of L. gibba exposed to mean, measured test substance concentrations of 7.9, 20, 49, 160, 340, and 1000 μg a.s./L was 0, 4, 2, 24, 43, and 38%, respectively, at the end of 7 days. The 7-day EbC50, based on mean, measured concentrations of test substance and dry weight, was > 1000 μg a.s./L.

Inhibition of yield based on frond density of L. gibba exposed to mean, measured test substance concentrations of 7.9, 20, 49, 160, 340, and 1000 μg a.s./L was -8, 4, 9, 44, 57, and 73%, respectively, at the end of 7 days. The 7-day EyC50, based on mean, measured concentrations of the test substance and yield based on frond density, was 230 μg a.s./L.

Inhibition of growth rate based on frond density of L. gibba exposed to mean, measured test substance concentrations of 7.9, 20, 49, 160, 340, and 1000 μg a.s./L was -2, 2, 2, 16, 24, and 37%, respectively, at the end of 7 days. The 7-day ErC50, based on mean, measured concentrations of the test substance, was > 1000 μg a.s./L.

Inhibition of yield based on dry weight of L. gibba exposed to mean, measured test substance concentrations of 7.9, 20, 49, 160, 340, and 1000 μg a.s./L was -2, 3, 0, 24, 44, and 39%, respectively, at the end of 7 days. The 7-day EyC50, based on mean, measured concentrations of the test substance and yield based on dry weight, was > 1000 μg a.s./L.

Inhibition of growth rate based on dry weight of L. gibba exposed to mean, measured test substance concentrations of 7.9, 20, 49, 160, 340, and 1000 μg a.s./L was 0, 2, 0, 10, 20, and 17%, respectively, at the end of 7 days. The 7-day ErC50, based on mean, measured concentrations of the test substance and growth rate based on dry weight, was > 1000 μg a.s./L.

Recovery was assessed for all mean, measured test concentrations (7.9, 20, 49, 160, 340, and 1000 μg a.s./L). Based on a > 7X increase in healthy frond count in 7 days, the effects upon growth and reproduction of L. gibba were found to be phytostatic within 7 days at mean, measured concentrations less than or equal to 1000 μg a.s./L.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
At test initiation (0 hour) and test termination (96 hours), a sample was removed from each test concentration and the controls and analyzed for the test substance. The abiotic solution (0.10 mg a.s./L) was sampled and analyzed at 96 hours only. Samples analyzed at 0 hour were removed from the test and control solutions remaining prior to division into the replicate test vessels. Samples analyzed at 96 hours were removed from the composited replicate solutions of each test concentration and control. Three quality control (QC) samples were prepared at each sampling interval at nominal concentrations of picoxystrobin approximating the test concentration range and remained with the exposure solution samples throughout the analytical process. Analysis of the QC samples was used to determine the precision and quality control maintained during the analytical process. All test and QC samples were diluted into the calibration standard range prior to analysis. The final matrix composition was 50:50 water:methanol.
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A 30 mg a.s./mL primary stock solution was prepared prior to test initiation by placing 0.7552
g of the test substance (0.7499 g a.s.) in a 25 mL volumetric flask and bringing it to volume with dime
thylformamide. Secondary stock solutions were prepared by adding variying volumes of the primary stock
solution with 25 or 10 mL of DMF. The 0.1 mL of the resultant secondary stock solutions were made to a
final volume of 1000 mL with AES medium.
- Controls: a dilution water control, an abiotic control and a solvent control
- Chemical name of vehicle: dimethylformamide
Test organisms (species):
Skeletonema costatum
Details on test organisms:
TEST ORGANISM
- Common name: marine diatom
- Strain: Skeletonema costatum (Strain CCMP 1332)
- Source: Bigelow Laboratories, West Boothbay Harbor, Maine
- Method of cultivation: Stock cultures were grown in 250 mL glass flasks each containing 100 mL of AES medium. The flasks were covered with stainless steel caps to permit gas exchange. Cultures were regularly transferred to fresh AES medium to provide three- to seven-day old cultures for testing.
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
19 to 20ºC
pH:
7.8 to 8.8
Conductivity:
43000 μmhos/cm
Nominal and measured concentrations:
Nominal: 0, 0.0010, 0.0026, 0.0064, 0.016, 0.040, 0.10 mg a.i./L
Measured: 0, 0.00090, 0.0023, 0.0053, 0.013, 0.035, 0.096 mg a.i./L
Details on test conditions:
TEST SYSTEM
- Test vessel: 250-mL Erlenmeyer flask containing approximately 100 mL of test solution and fitted with stainless steel caps to permit gas exchange
- Renewal rate of test solution: No renewal
- Control end cells density: 7.7 x 10e4 cells/mL per replicate
- No. of colonies per vessel:
- No. of fronds per colony:
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per solvent control (replicates): 6
GROWTH MEDIUM
- Standard medium used: yes, Artificially Enriched Seawater (AES) Nutrient Medium
OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: The initial pH of this medium was adjusted, if necessary, to 8.1±0.1 with dilute hydrochloric acid or sodium hydroxide prior to use.
- Photoperiod: 14 hours per day of cool-white fluorescent lighting and 8 hours of darkness
- Light intensity and quality: 3900 to 4700 lux

EFFECT PARAMETERS MEASURED:
- Healthy cell counts were recorded approximately 24, 48, 72 and 96 hours after test initiation. Healthy cell count (cell density), yield, and growth rate were recorded and expressed as percent inhibition relative to the blank control following exposure to the test substance for 96 hours.
RANGE-FINDING STUDY
- A range-finding exposure was conducted with a blank control and nominal concentrations of 0.00010, 0.0010, 0.010, 0.10, and 1.0 mg a.s./L. Two exposure vessels were used for each concentration and the blank control. Following 96 hours of exposure, cells exposed to all treatment levels tested and the blank control were observed to be normal. Cell densities in the 0.00010, 0.0010, 0.010, 0.10, and 1.0 mg a.s./L treatment levels averaged 114.38, 110.75, 76.25, 5.50, and 2.50 x 10e4 cells/mL, respectively. Mean cell density in the blank control was 88.00 x 10e4 cells/mL. The preliminary range-finding test with this test substance was conducted without the use of a co-solvent. Analytical sampling performed in conjunction with this exposure resulted in recoveries of approximately 20% of nominal concentrations. Additional solubility trials with this test substance were conducted to evaluate the use of a co-solvent (i.e., DMF). Based on these results, and in consultation with the Study Sponsor, DMF was used to solubilize the test substance for the definitive testing and the nominal
concentrations selected for the definitive test were 0.0010, 0.0026, 0.0064, 0.016, 0.040, and 0.10 mg a.s./L, a blank control and a solvent (DMF) control.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
0.004 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: cell density
Remarks on result:
other: 95% confidence limits 0.0037-0.0045 mg a.s./L
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
0.004 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: yield
Remarks on result:
other: 95% confidence limits 0.0034-0.0043 mg a.s./L
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
0.006 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits 0.0058-0.0069 mg a.s./L
Duration:
96 h
Dose descriptor:
other: EC25
Effect conc.:
0.003 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: yield
Remarks on result:
other: 95% confidence limits 0.0018-0.0033 mg a.s./L
Duration:
96 h
Dose descriptor:
other: EC25
Effect conc.:
0.004 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% confidence limits 0.0035-0.0041 mg a.s./L
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.002 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: cell density, yeild, and growth rate
Details on results:
A summary of algal growth inhibition following exposure of Skeletonema costatum to the test substance for 96 hours is presented in the table (refer 'Any other information on results including tables').
Reported statistics and error estimates:
Cell density, yield, and growth rate data were evaluated for normality and homogeneity of variance (α = 0.05) using the Shapiro-Wilks’ and Bartlett’s Tests, respectively. Since cell density and growth rate data were normal with homogeneous variances, the treatment groups were compared to the blank control using Williams’ Test (α = 0.05). Yield data were evaluated using Kruskal-Wallis’ Test. The results of the statistical analyses, as well as an evaluation of the concentration-response pattern, were used to determine the NOEC relative to each parameter at 96 hours. All calculations and statistical analyses were conducted using TOXSTAT Version 3.5.

Table 1. Summary of algal growth inhibition following exposure of Skeletonema costatum to the test substance for 96 hours

Mean, measured

concentration

(mg/L)

% Inhibition relative to

untreated control

Cell density

Yield

Growth rate

Untreated Control

-

-

-

Solvent Control

-

-

-

0.00090

8

8

3

0.0023

10

10

4

0.0053

72

76

45

0.013

92

97

86

0.035

96

102

114

0.096

96

102

116

 

Validity criteria fulfilled:
yes
Conclusions:
96 hr EC50: 0.0042 mg a.i./L (95% CL: 0.0037-0.0045 mg a.s./L) (cell density)
96 hr EC50: 0.0040 mg a.i./L (95% CL: 0.0034-0.0043 mg a.s./L) (yield)
96 hr EC50: 0.0063 mg a.i./L (95% CL: 0.0058-0.0069 mg a.s./L) (growth rate)
96 hr NOEC: 0.0023 mg a.i./L (cell density, yeild, and growth rate)
Executive summary:

The acute toxicity of the test substance to the marine diatom, Skeletonema costatum, under static conditions was determined in a 96-hour exposure test. The test was conducted in accordance with the draft U.S. EPA OPPTS Ecological Effects Test Guidelines. Additionally, a recovery phase was initiated after 96 hours of exposure to determine if the test substance was algistatic or algicidal.

The study was conducted with six concentrations of the test substance, a dilution water (Artificially Enriched Seawater (AES) nutrient medium) control, and a solvent (dimethylformamide, DMF) control at a temperature range of 19 to 20°C. Three replicates with an initial cell density of approximately 7.7 x 10e4 cells/mL per replicate were initiated for each test substance concentration and the dilution water control. Six replicates were established for the solvent (DMF) control. A single test vessel containing no S. costatum was initiated at the highest test concentration for the abiotic control.

Percent inhibition of cell density (number of cells/mL) of S. costatum exposed to mean, measured test concentrations of 0.00090, 0.0023, 0.0053, 0.013, 0.035, and 0.096 mg a.i./L was 8, 10, 72, 92, 96, and 96%, respectively, compared to the blank control at the end of the 96-hour exposure period. Mean, measured concentrations of test substance in the test concentrations ranged from 82 to 96% of the nominal test substance concentrations. Mean, measured concentrations of the test substance were used in the determination of EC50 values. The 96-hour "effective concentration" producing a 50% inhibition of cell density (EC50), compared to the blank control, based on mean, measured concentrations of test substance, was 0.0042 mg a.i./L. The highest mean measured concentration causing no statistically significant reduction when compared to the blank control in cell density (NOEC) was 0.0023 mg a.i./L.

Percent inhibition of yield of S. costatum exposed to mean, measured test concentrations of 0.00090, 0.0023, 0.0053, 0.013, 0.035, and 0.096 mg a.i./L was 8, 10, 76, 97, 102, and 102%, respectively, compared to the blank control at the end of 96 hours of exposure. The 96-hour EyC25, based on mean, measured concentrations of test substance, was 0.0029 mg a.i./L. The 96-hour EyC50, based on mean, measured concentrations of test substance, was 0.0040 mg a.i./L. Based on Kruskal-Wallis’ Test, the NOEC for yield after 96 hours of exposure was 0.096 mg a.i./L. However, 102% inhibition of yield was observed in this treatment level compared to the blank control. Since Kruskal-Wallis’ Test was not sensitive enough to determine a significant difference at any treatment level, the NOEC value was determined to be 0.0023 mg a.i./L as a conservative estimate, based on the observed dose response, the NOEC determined from cell count and consultation with the Study Sponsor.

Percent inhibition of growth rate of S. costatum exposed to mean, measured test concentrations of 0.00090, 0.0023, 0.0053, 0.013, 0.035, and 0.096 mg a.i./L was 3, 4, 45, 86, 114, and 116%, respectively, at the end of 96 hours of exposure. The 96-hour ErC25, based on mean, measured concentrations of test substance, was 0.0038 mg a.i./L. The 96-hour ErC50, based on mean, measured concentrations of test substance, was 0.0063 mg a.i./L. The NOEC for growth rate after 96 hours of exposure was 0.0023 mg a.i./L. The test substance was algistatic to S. costatum at nominal concentrations less than or equal to 0.10 mg a.i./L in a 4-day recovery phase.

The results are summarized as follows:

96 hr EC50: 0.0042 mg a.i./L (95% CL: 0.0037-0.0045 mg a.s./L) (cell density)

96 hr EC50: 0.0040 mg a.i./L (95% CL: 0.0034-0.0043 mg a.s./L) (yield)

96 hr EC50: 0.0063 mg a.i./L (95% CL: 0.0058-0.0069 mg a.s./L) (growth rate)

96 hr NOEC: 0.0023 mg a.i./L (cell density, yeild, and growth rate)

Description of key information

72-hour ErC50 (Pseudokirchneriella subcapitata): 0.260 mg/L; OECD 201; Reliability = 1

72-hour NOEC (Pseudokirchneriella subcapitata): 0.0.009 mg/L; OECD 201; Reliability = 1

96-hour EC50 (Skeletonema costatum): 0.006 mg/L; EPA OPPTS 850.5400; Reliability = 1

Key value for chemical safety assessment

EC50 for freshwater algae:
0.26 mg/L
EC50 for marine water algae:
0.006 mg/L

Additional information

Two algae toxicity tests were conducted in the freshwater species Pseudokirchneriella subcapitata and Anabaena flos-aquae. Acute 72 h ErC50 values were 0.260 mg a.s./L and >3 mg a.s./L, respectively. The most sensitive species Pseudokirchneriella subcapitata has an acute 72h ErC50 less than 1 mg a.s./L. The ErC50 from the 7-d static–renewal Lemna gibba toxicity test is 0.230, based on growth rate.  The ErC50 from the 96-h Skeletonema costatum test is 0.006 mg/L, based on growth rate. The ECx value for aquatic plants is less than 1.0 mg a.s./L.